ENCYCLOPEDIA

OF
FOOD
MICROBIOLOGY
Editor-in-Chief
RICHARD K. ROBINSON

Editors

CARL A. BATT

PRADIP D. PATEL

u
ACADEMIC PRESS
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 EDITORIAL ADVISORY BOARD

R G Board G W Gould
South Lodge 17 Dove Road
Northleigh Bedford MK41 7AA
Bradford-on-Avo? UK
Wi Its hi re
BA152RG UK
M Griffiths
Department of Food Science
R L Buchanan
University of Guelph
US Food and Drug Admiiistration
Guelph, Ontaro N1G 2W1
Center for F3od Safety a i d Applied Nutritior:
Canaca
200 C-Street, SW
Washington
20204 DC USA G Kalantzopoulos
Department of Food Science and Technology
D 0 Cliver Agricultural University of Athens
Department of PopLlation Health and Reproduction Botanikos 118 55
School of Veterinary Medicine Athens Greece
University of California, Davis
Davis CA 956 16-8743
USA T Keshavarz
Schoo of Biological and Health Sciences
B Colonna Ceccaldi University of Westminster
Industrial Microbiology 115 New Cavendish Street
Pernod Ricard London W1 M 83s
Centre de Recherche UK
120 Avenue du Marechal Foch
94015 Creteil Cedex, France P Kulkarni
University of Murnbai
M A Cousin Department of Chemical Technology
DepartTent of Food Science Nathalal Parikh Marg
1160 Smth Hall Matunga
Purdue University Mumbai-400 019
W Lafayette IN 47907 India
USA

C 0 Gill S Notermans
Agriculture and Agri-Food Canada Researcn Centre TNO Nutrition and Food Research Institute
6000 C & E Trail PO Box 360
Lacombe, Alberta 3700 AJ Zeist
T4L 1W 1 Canada The Netherlands
vi Editorial Advisory Board

Y Oda F M Rombouts
Department of Applied Biological Science LUW
Fukuyarna University PO Box 8129
Fukuyarna, Hiroshima 729-0292 6700 EV Wageningen
Japan The Netherlands

B Ozer A N Sharpe
Faculty of Agriculture PO Box 1224
Department of Food Science and Technology Alrnonte
University of Harran Ontario KOA 1AO
Sanliurfa Canada
Turkey
K Steinkraus
T A Roberts
Department of Food Microbiology
Food Safety Consultant
Cornell University
59 Edenharn Crescent
15 Cornell Street, lthaca
Reading RGl 6HU
N Y 14850, USA
UK

D Roberts M-L Tortorello

Food Hygiene Laboratories National Center for Food Safety and Technology
Central Public Health Laboratory US Food and Drug Administration
61 Colindale Avenue 6502 South Archer Road
London NW9 5HT Summit-Argo IL 60501
UK USA
 FOREWORD

Public concern about food safety has never been greater. In part this is due to the ever increasing demand
from consumers for higher and higher standards. But new food-borne pathogens like E. coli 0157 have
emerged in recent years to become important public health problems, and changes in production and
manufacturing sometimes reopen doors of opportunity for old ones. A powerful reminder that food scientists
have much unfinished business to attend to is provided by the succession of food scares that generate strong
stories for the media.
Experience tells us that science must underpin all approaches to food safety, whether through the application
and implementation of well-tried approaches or the development of new or improved methods. Microbiologists
have had a central role in this since the high quality work of pioneers like van Ermengem on botulism and
Gaffky on typhoid more than a century ago. The large amount of important data that has accumulated since
then joins with the current rapid rates of technological and scientific advance to make the need for a structured
and authoritative source of information a very pressing one. It is provided by this encyclopedia.
These are exciting times for food microbiologists. Expectations are high that as scientists we will soon
provide answers to the many problems still posed by microbes - from spoilage to food poisoning. Approaches
like HACCP are making everyone think hard about how best to apply the data we have to develop better
ways for reducing and eliminating food-borne pathogens. The pace of scientific developments continues to
accelerate and more and better methods are available for the detection and enumeration of microbes than
ever before. The microbes themselves continue to evolve and so present moving targets. The solid foundation
presented by the mass of information in this encyclopedia provides the launching pad and guide for meeting
these challenges.
It could be said that a penalty of working in food microbiology is that because the subject is broad-ranging,
mature and dynamic, its practitioners, teachers and students have to know about many things in breadth and
depth. For most of us, of course, this is not a penalty but an attractive bonus because of its intellectual
challenge. I am particularly pleased to be associated with the encyclopedia because it will help us all to meet
this test with confidence. I wish it every success.

Professor H Pennington
Department of Medical Microbiology
University of Aberdeen
 INTRODUCTION

The advent of antibiotics gave the general public, and many professional microbiologists as well, the feeling
that bacterial diseases were under control, and the elimination of smallpox and the control of polio suggested
that even viruses posed few problems. However, this complacency has received a nasty jolt over the last
decade, and the emergence of HIV and multiple-drug-resistant strains of bacteria has become a major concern
for the medical profession. The food industry has been similarly shaken by the appearance of new, and
potentially fatal, strains of Escherichia coli, a species that for over 100 years was regarded as little more than
a nuisance. Equally unexpected was the devastating impact of BSE, and fresh reports of the activities of so-
called ‘emerging food-borne pathogens’ are appearing with alarming regularity.
In some cases, it has been possible to understand, with the advantages of hindsight, why a particular species
of bacterium, fungus or protozoan has become a major risk to human health while, on other occasions, the
vagaries of nature have left the ‘experts’ totally bemused. However, even in these latter situations, control
over the threat posed to food supplies has to be instituted, but the ability of the food industry, in conjunction
with Public Health and other bodies, to develop effective responses can only be as good as the scientific
knowledge available. In the case of food microbiology, this background has to be derived from a wide range
of sources. Thus, agricultural practices may alter the biochemistry of a crop and, perhaps, its microflora as
well; the microflora of any given foodstuff and/or processing facility will have specific characteristics that
need to be understood before control is possible; techniques must be available to monitor a retail food for
microorganisms that would pose a risk to the consumer. As the procedures necessary to monitor these various
facets become ever more sophisticated, so fewer microbiologists can claim total competence, and the need for
a specialist source of outside knowledge increases.
It is this latter need that the Encyclopedia of Food Microbiology seeks to satisfy for, within this work, a
busy microbiologist can find details of all the important genera of food-borne bacteria and fungi, how the
same genera may react in different foods and under different environmental conditions, and how to detect
the growth and/or metabolism of the same organisms in foods using classical o r modern techniques. In order
to place this information into a broader context, the reader can explore the latest advice concerning food
standards/specifications, or the role of monitoring systems like HACCP in achieving product targets for
specific microorganisms; potential concerns over viruses and protozoa are also evaluated in the light of current
knowledge. Readers interested in fermented foods will find the pertinent information in a similarly accessible
form; indeed, purchasers of the print version of the encyclopedia will be entitled to register for access to the
on-line version as well. This form allows the user the benefit of extensive hypertext linking and advanced
search tools, adding value to the encyclopedia as a reference source, teaching aid and text for general interest.
It is inevitable, of course, that short articles written to a tight deadline may have omissions, but it is to be
hoped that such faults are minimal and, in any event, more than compensated for through the careful selections
of further reading. If this optimism is justified, then the major credit rests with the authors of each article.
They are all recognized as experts in their fields, and their willing participation has been much appreciated
by the editors. The role of the Editorial Advisory Board merits a special mention as well, for their constructive
xii Introduction

criticisms of the list of articles, their suggestions for authors and their expert refereeing of the manuscripts
has provided a solid foundation for the entire enterprise.
However, the finest manuscripts are of little value to the scientific community until they have been published,
and the editorial team at Academic Press - Carey Chapman (Editor-in-Chief),Tina Holland (Associate Editor),
Nick Fallon (Commissioning Editor), Laura O’Neill (Editorial Assistant), Tamsin Cousins (Production Project
Manager), Richard Willis (Freelance Project Manager), Emma Parkinson (Electronic Publishing Developer),
Peter Lord (Publishing Services Manager), Emma Krikler (Picture Researcher) - have been outstanding in
their support of the project. Obviously, each member of the team has made an important contribution, but it
must be recorded that the role of Tina Holland has been absolutely invaluable. Thus, not only has Tina co-
ordinated the numerous inputs from the editors, referees and authors, but even found time to help the editors
with the location of authors; the editors acknowledge this unstinting assistance with much gratitude.

R.K. Robinson, C.A. Batt, P.D. Pate1

Editors
 PREFACE

Although food microbiology and food safety have, in recent times, become major concerns for governments
around the world, equally importts the fact that, without yeasts and bacteria, popular meals like bread
and cheese would not exist. Consequently, a knowledge of the relationship between foodstuffs and the
activities of bacteria, yeasts and mycelial fungi has become a top priority for everyone associated with food
and its production. Farmers have concerns related to produce harvesting and storage, food processors have
to generate wholesome retail products that are both free from pathogenic organisms and have a satisfactory
shelf life and, last but not least, food handlers and consumers need to be aware of the procedures necessary
to ensure that food is safely prepared and stored.
In order for these disparate groups to operate successfully, accurate and objective information about the
microbiology of foods is essential, and this encyclopedia seeks to provide a source of such information. In
some areas, introductory articles are provided to guide readers who may be less familiar with the subject but,
in general, superficiality has been avoided. Thus, the coverage has been developed to include details of all the
important groups of bacteria, fungi, viruses and parasites, the various methods that can be employed for their
detection in foods, the factors that govern the behaviour of the same organisms, together with an analysis of
likely outcomes of microbial growth/metabolism in terms of disease and/or spoilage. A further series of articles
describes the contribution of microorganisms to industrial fermentations, to traditional food fermentations
from the Middle or Far East, as well as during the production of the fermented foods like bread, cheese or
yoghurt that are so familiar in industrialized societies. The division of these topics into 358 articles of
approximately 4000 words, has meant that the contributing authors have been able to handle their specialist
subject(s) in real depth.
Obviously, another group of editors might have approached the project in a different manner, but we feel
confident that this encyclopedia will provide readers at all levels of expertise with the data being sought. A
point enhanced, perhaps, by the inclusion at the end of each article of a list for further reading, comprising a
selection of review articles and key research papers that should encourage further exploration of any selected
topic. If this confidence is borne out in practice, then the efforts of the contributors, the members of the
Editorial Board and the editorial team from Academic Press will be well rewarded, for raising the scientific
profile of food microbiology is long overdue.

R.K. Robinson, C.A. Batt, P.D. Patel

Editors
 CONTENTS

VOLUME 1
A
ACCREDITATION SCHEMES see LABORATORY MANAGEMENT Accreditation Schemes
ACETOBACTER R K Hommel, P Ahnert 1
ACINETOBACTER P Kampfer 7
ADENYLATE KINASE M J Murphy, D J Squirrel1 16
AEROBIC METABOLISM see METABOLIC PATHWAYS: Release of Energy (Aerobic); Release of
Energy (Anaerobic)
AEROMONAS
Introduction IS Blair, M A S McMahon, D A McDowell 25
Detection by Cultural and Modern Techniques B Austin 30
AFLATOXIN see MYCOTOXlNS: Classification
ALCALIGENES T J Klem 38
ALE see LAGER
ALGAE see SINGLE-CELL PROTEIN: The Algae
ALTERNARIA S E Lopez, D Cabral 42
ANAEROBIC METABOLISMsee METABOLIC PATHWAYS: Release of Energy (Anaerobic)
ANTIMICROBIAL PACKAGING see CHILLED STORAGE OF FOODS: Packaging with Antimicrobial
Properties
ANTIMICROBIAL SYSTEMS see NATURAL ANTIMICROBIAL SYSTEMS: Preservative Effects
During Storage; Antimicrobial Compounds in Plants; Lysozyme and Other Proteins in Eggs;
Lactoperoxidase and Lactoferrin
ARCOBACTER IV Wesley 50
ARTHROBACTER M Gobbetti, E Smacchi 54
ASPERGILLUS
Introduction P-K Chang, D Bhatnagar, T E Cleveland 62
Aspergillus oryzae K Gomi 66
Aspergillus flaws D Bhatnagar; T E Cleveland, G A Payne 72
ATOMIC FORCE MICROSCOPY see MICROSCOPY Atomic Force Microscopy
ATP BIOLUMINESCENCE
Application in Meat Industry D A Bautista 80
xliv Contents

Application in Dairy Industry W Reybroeck 88

Application in Hygiene Monitoring M W Griffiths 94
Application in Beverage Microbiology A Thompson 101
AUREOBASIDIUM T Roukas 109

B
BACILLUS
Introduction M K Dah/ 113
Bacillus cereus C A Batt 119
Bacillus stearothermophilus P Kotzekidou 124
Bacillus anthracis L Baillie 129
Bacillus subtilis M K Dah1 135
Detection of Toxins S H Beattie, A G Williams 141
Detection by Classical Cultural Techniques I Jenson 149
BACTERIA
The Bacterial Cell R W Lovitt, C J Wright 158
Bacterial Endospores G W Gould 168
Classification of the Bacteria - Traditional V M de Ambrosini, C H Gusils, S N Gonzalez, 173
G Oliver
Classification of the Bacteria - Phylogenetic Approach E Stackebrandt 178
BACTERIAL ADHESION see POLYMER TECHNOLOGIES FOR CONTROL OF BACTERIAL
ADHESION
BACTERlOClNS
Potential in Food Preservation T O’Keeffe, C Hill 183
Nisin E A Davies, J Delves-Broughton 191
BACTEROIDES AND PREVOTELLA H J Flint, C S Stewart 198
BACTERIOPHAGE-BASED TECHNIQUES FOR DETECTION OF FOOD-BORNE PATHOGENS
R J Mole, V K Dhir, S P Denyer, G S A B Stewart (dec) 203
BEER see LAGER
BENZOIC ACID see PRESERVATIVES: Permitted Preservatives- Benzoic Acid
BEVERAGE MICROBIOLOGY see ATP BIOLUMINESCENCE:Application in Beverage
Microbiology
BIFIDOBACTERIUM D G Hoover 210
BIOCHEMICAL and MODERN IDENTIFICATION TECHNIQUES
Introduction D Y C Fung 218
Food Spoilage Flora (Le. Yeasts and Moulds) G G Khachatourians, D K Arora 228
Food-poisoning Organisms D Y C Fung 237
Enterobacteriaceae, Coliforms and E. coli R R Beumer, M C te Giffel, AGE 244
Microfloras of Fermented Foods J P Tamang, W H Holzapfel 249
BlOFlLMS B Carpentier, 0 Cerf 252
BIOPHYSICAL TECHNIQUES FOR ENHANCING MICROBIOLOGICALANALYSIS A D Goater,
R Pethig 259
BIOSENSORS
Scope in Microbiological Analysis M C Goldschmidt 268
BIO-YOGHURTsee FERMENTED MILKS: Yoghurt
BOTRYTIS M D Alur 279
BOVINE SPONGIFORM ENCEPHALOPATHY (BSE) D M Taylor, R A Somerville 283
 Contents xlv

BREAD
Bread from Wheat Flour R S Singhal, P R Kulkarni 288
Sourdough Bread B J B Wood 295
BRETANOMYCES J Jimenez, M Fidalgo, M Alguacil 302
BREVIBACTERIUM B Weimer 308
BREWER’S YEAST see SACCHAROMYCES: Brewer’s Yeast
BROCHOTHRIX R H Holley 31 4
BRUCELLA
Characteristics J Theron, T E Cloete 31 9
Problems with Dairy Products P Papademas 324
BURKHOLDERIA COCOVENENANSsee PSEUDOMONAS: Burkholderia cocovenenans
BUTTER see MILK AND MILK PRODUCTS: Microbiology of Cream and Butter
BYSSOCHLAMYS P Kotzekidou 328

C
CAKES see CONFECTIONERY PRODUCTS: Cakes and Pastries
CAMPYLOBACTER
Introduction M T Rowe, R H Madden 335
Detection by Cultural and Modern Techniques J E L Corry 341
Detection by Latex Agglutination Techniques W C Hazeleger, R R Beumer 347
CANDIDA
Introduction R K Hommel 352
Yarrowia (Candida) lipolytica G M Heard, G H Fleet 360
CANNING see HEAT TREATMENT OF FOODS: Principle of Canning; Spoilage Problems
Associated with Canning
CATERING INDUSTRY see PROCESS HYGIENE: Hygiene in the Catering Industry
CELLULOMONAS M I Rajoka, K A Malik 365
CEREALS see SPOILAGE OF PLANT PRODUCTS: Cereals and Cereal Flours
CENTRIFUGATION see PHYSICAL REMOVAL OF MICROFLORAS: Centrifugation
CHEESE
In the Marketplace A Y Tamime 372
Microbiology of Cheese-making and Maturation N Y Farkye 381
Mould-ripened Varieties A W Nichol 387
Role of Specific Groups of Bacteria M El Soda 393
Microflora of White-brined Cheeses B H Ozer 397
CHEMILUMINESCENT DNA HYBRIDIZATION see LISTERIA: Listeria monocytogenes - Detection
by Chemiluminescent DNA Hybridization
CHILLED STORAGE OF FOODS
Principles B P F Day 403
Use of Modified-atmosphere Packaging R E O’Connor-Shaw, V G Reyes 41 0
Packaging with Antimicrobial Properties D Collins-Thompson, Cheng-An Hwang 41 6
CIDER (HARD CIDER) B Jarvis 421
CITRIC ACID see FERMENTATION (INDUSTRIAL): Production of Organic Acids
CITROBACTER see SALMONELLA: Detection by Enzyme Immunoassays
CLOSTRIDIUM
Introduction H P Blaschek 428
Clostridium perfringens H P Blaschek 433
xlvi Contents

Detection of Enterotoxins of C. perfringens L Petit, M Gibert, M R Popoff 438

Clostridium acetobutylicum H Biebl 445
Clostridium tyrobutyricum M Wiedmann, K J Boor, H Eisgruber, K-J Zaadhof 451
Clostridium botulinum E A Johnson 458
Detection of Neurotoxins of Clostridium botulinum S Notermans 463
COCOA AND COFFEE FERMENTATIONS P Nigam 466
COFFEE see COCOA AND COFFEE FERMENTATIONS
COLORIMETRIC DNA HYBRIDIZATION see LlSTERlA: Detection by Colorimetric DNA
Hybridization; SALMONELLA: Detection by Colorimetric DNA Hybridization
COLOURS see FERMENTATION (INDUSTRIAL): Production of Colours/Flavours
CONFECTIONERY PRODUCTS - CAKES AND PASTRIES P A Voysey, J D Legan 474
CONFOCAL LASER MICROSCOPY see MICROSCOPY: Confocal Laser Scanning Microscopy
COSTS/BENEFITS OF MICROBIAL ORIGIN J E Hobbs, W A Kerr 480
CREAM see MILK AND MILK PRODUCTS: Microbiology of Cream and Butter
CRITICAL CONTROL POINTS see HAZARD APPRAISAL (HACCP): Critical Control Points
CRUSTACEA see SHELLFISH (MOLLUSCS AND CRUSTACEA): Characteristics of the Groups;
Contamination and Spoilage
CRYPTOSPORlDlUM R W A Girdwood, H V Smith 487
CULTURAL TECHNIQUES see AEROMONAS: Detection by Cultural and Modern Techniques;
BACILLUS: Detection by Classical Cultural Techniques; CAMPYLOBACTER: Detection by
Cultural and Modern Techniques; ENRICHMENT SEROLOGY An Enhanced Cultural Technique
for Detection of Food-borne Pathogens; FUNGI: Food-borne Fungi - Estimation by Classical
Cultural Techniques; LISTERlA: Detection by Classical Cultural Techniques; SALMONELLA:
Detection by Classical Cultural Techniques; SHIGELLA: Introduction and Detection by
Classical Cultural Techniques; STAPHYLOCOCCUS: Detection by Cultural and Modern
Techniques; VEROTOXIGENIC E. COLI AND SHIGELLA SPP: Detection by Cultural Methods;
VIBRIO: Detection by Cultural and Modern Techniques
CULTURE COLLECTIONS F M Dugan, J. S Tang 498
CURING see MEAT AND POULTRY: Curing of Meat
CYCLOSPORA A M Adams, K C Jinneman, Y R Ortega 502
CYTOMETRY see FLOW CYTOMETRY

D
DAIRY PRODUCTS see BRUCELLA: Problems with Dairy Products; CHEESE: In the Market
Place; Microbiology of Cheese-making and Maturation; Mould-ripened Varieties; Role of Specific
Groups of Bacteria; Microflora of White-brined Cheeses; FERMENTED MILKS: Yoghurt;
Products from Northern Europe; Products of Eastern Europe and Asia; PROBIOTIC BACTERIA:
Detection and Estimation in Fermented and Non-fermented Dairy Products
DEBARYOMYCES W Praphailong, G H Fleet 515
DESULFOVIBRIO M D Alur 520
DEUTEROMYCETES see FUNGI: Classification of the Deuteromycetes
DIRECT (AND INDIRECT) CON DUCT1M ETR IC/I M PEDI M ETRIC TECHNIQUES
Food-borne Pathogens D Blivet 524
DIRECT EPIFLUORESCENT FILTER TECHNIQUES (DEFT) 5 H Pyle 527
DISINFECTANTS see PROCESS HYGIENE: Testing of Disinfectants
DRIED FOODS M D Alur, V Venugopal 530
 Contents xlvii

E
ECOLOGY OF BACTERIA AND FUNGI IN FOODS
Influence of Available Water K Krist, D S Nichols, T Ross 539
Influence of Temperature T Ross, D S Nichols 547
Influence of Redox Potential and pH A Rompf, D Jahn 556
EGGS
Microbiology of Fresh Eggs N H C Sparks 563
Microbiology of Egg Products J Delves-Broughton, R G Board 569
ELECTRICAL TECHNIQUES
Introduction D Blivet 573
Food Spoilage Flora and Total Viable Count (TVC) G Salvat, D Blivet 578
Lactics and other Bacteria L Curda 580
ELECTRON MICROSCOPY see MICROSCOPY: Scanning Electron Microscopy; Transmission
Electron Microscopy
ELECTROPORATIONsee MINIMAL METHODS OF PROCESSING: Electroporation - Pulsed
Electric Fields
ENDOSPORES see BACTERIA: Bacterial Endospores
ENRICHMENT SEROLOGY
An Enhanced Cultural Technique for Detection of Food-borne Pathogens C de W Blackburn 589
ENTAMOEBA see WATERBORNE PARASITES: Entamoeba
ENTEROBACTER T W Huber 598
ENTEROBACTERIACEAE, COLIFORMS AND E, COLI
Introduction A Pandey, V K Joshi, P Nigam, C R Soccol 604
Classical and Modern Methods for Detection/Enumeration E de Boer 610
ENTEROCOCCUS G Giraffa 617
ENTEROTOXINS see BAClLLUS: Detection of Enterotoxins; STAPHYLOCOCCUS: Detection of
Staphylococcal Enterotoxins
ENTEROVIRUSES see VIRUSES
ENZYME IMMUNOASSAYS: OVERVlEW A Sharma 625
ESCHERlCHlA COLI
Escherichia coli C A Batt 633
Detection of Enterotoxins of E. coli H - Y Tsen 640
ESCHERlCHlA COLI 0157
Escherichia coli 0 1 57:H7 M L Tortorello 646
Detection by Latex Agglutination Techniques E W Rice 652
Detection by Commercial lmmunomagnetic Particle-based Assays P M Fratamico, 654
C G Crawford
EUKARYOTIC ASCOMYCETES see FUNGI: Classification of the Eukaryotic Ascomycetes
(Ascomycotina)

VOLUME 2

F
FATTY ACIDS see FERMENTATION (INDUSTRIAL): Production of Oils and Fatty Acids
FERMENTATlON (I NDUSTR IAL)
Basic Considerations Y Chisti 663
Media for Industrial Fermentations G M Walker 674
xlviii Contents

Control of Fermentation Conditions T Keshavarz 683

Recovery of Metabolites P A Pawar, M C Misra, N P Ghildyal, N G Karanth 690
Production of Xanthan Gum M K Gowthaman, M S Prasad, N G Karanth 699
Production of Organic Acids M Moresi, E Parente 705
Production of Oils and Fatty Acids P Nigam 71 8
Colours/Flavours Derived by Fermentation R G Berger 729
FERMENTED FOODS
Origins and Applications G Campbell-Platt 736
Fermented Vegetable Products G Oliver, M Nuhez, S Gonzalez 739
Fermented Meat Products M C Montel 744
Fermented Fish Products J H Al-Jedah, M Z Ali 753
Beverages from Sorghum and Millet J Dewar, J R N Taylor 759
Fermentations of the Far East IGandjar 767
FERMENTED MILKS
Range of Products E Litopoulou, N Tzanetakis 774
Yoghurt R K Robinson 784
Products from Northern Europe H Roginski 791
Products of Eastern Europe and Asia D Ozer, B H Ozer 798
FILTRATION see PHYSICAL REMOVAL OF MICROFLORAS: Filtration
FISH
Catching and Handling A Chattopadhyay 806
Spoilage of Fish J J Leisner, L Gram 813
FLAVOBACTERlUM M-L Garcia-Lopez, J-A Santos, A Otero 820
FLAVOURS see FERMENTATION (INDUSTRIAL): Production of Colours/Flavours
FLOURS see SPOILAGE OF PLANT PRODUCTS: Cereals and Cereal Flours
FLOW CYTOMETRY C N Jacobsen, J Jakobsen 826
FOOD POISONING OUTBREAKS S Notermans 835
FOOD PRESERVATION see BACTERIOCINS: Potential in Food Preservation; HEAT TREATMENT
OF FOODS; HIGH PRESSURE TREATMENT OF FOODS; LASERS: Inactivation Techniques;
MICROBIOLOGY OF SOUS-VIDE PRODUCTS; MINIMAL METHODS OF PROCESSING:
Electroporation - Pulsed Electric Fields; ULTRASONIC STANDING WAVES; ULTRA-VIOLET
LIGHT
FREEZING OF FOODS
Damage to Microbial Cells R S Singhal, P R Kulkarni 840
Growth and Survival of Microorganisms P Chattopadhyay 845
FUNGI
The Fungal Hypha J Silva, S Gonzalez, J Palacios, G Oliver 850
Food-borne Fungi - Estimation by Classical Cultural Techniques A K Sarbhoy, M Kulshreshtha 854
Overview of Classification of the Fungi B C Sutton 860
Classification of the Peronosporomycetes M W Dick 871
Classification of the Zygomycetes P M Kirk 882
Classification of the Eukaryotic Ascomycetes M A Cousin 887
Classification of the Deuteromycetes B C Sutton 893
Classification of the Hemiascomycetes A K Sarbhoy 898
FUSARIUM U Thrane 901
 Contents xlix

G
GASTRIC ULCERS see HELICOBACTER
GENET1C ENGINEERING
Modification of Yeast and Moulds R Sandhir, S K Garg, D R Modi 907
Modification of Bacteria E Johansen 917
GENETICS OF MICROORGANISMS
Fungi R Sandhir, S K Garg, D R Modi 92 1
Bacteria S K Garg, R Sandhir 929
GEOTRICHUM A Botha 940
GIARDIA R W A Girdwood, H V Smith 946
GLUCONOBACTER R K Hommel. P Ahnert 955
GOOD MANUFACTURING PRACTICE B Jarvis 961
GUIDELINES GOVERNING MICROBIOLOGY see NATIONAL LEGISLATION, GUIDELINES &
STANDARDS GOVERNING MICROBIOLOGY: Canada; European Union; Japan

H
HAFNIA ALVEI J Ridell 973
HANSENULA G Gellissen, C P Hollenberg 976
HARD CIDER see CIDER (HARD CIDER)
HAZARD APPRAISAL (HACCP)
The Overall Concept F Untermann 982
Critical Control Points S Leaper 990
Establishment of Performance Criteria T Mahmutoglu, f Bozoglu 992
Involvement of Regulatory Bodies 0 P Snyder, V K Juneja 1001
HEAT TREATMENT OF FOODS
Thermal Processing Required for Canning A Azizi 1008
Spoilage Problems Associated with Canning L Ababouch 1016
Ultra-high Temperature (UHT) Treatments M J Lewis 1023
Principles of Pasteurization R A Wilbey 1030
Action of Microwaves A Stolle, B Schalch 1036
Synergy Between Treatments E A Murano 1041
HELICOBACTER I V Wesley 1047
HELMINTHS AND NEMATODES K D Murre11 1052
HEMIASCOMYCETES- 1 AND 2 see FUNGI: Classification of the Hemiascomycetes
HEPATITIS VIRUSES see VIRUSES: Hepatitis Viruses
HIGH-PRESSURE TREATMENT OF FOODS M Patterson 1059
HISTORY OF FOOD MICROBIOLOGY N D Cowell 1066
HURDLE TECHNOLOGY L G M Gorris 1071
HYDROPHOBIC GRID MEMBRANE FILTER TECHNIQUES (HGMF) P Entis 1076
HYDROXYBENZOIC ACID see PRESERVATIVES: Permitted Preservatives - Hydroxybenzoic Acid
HYGIENE MONITORING see ATP BIOLUMINESCENCE: Application in Hygiene Monitoring
HYGIENIC PROCESSING see PROCESS HYGIENE: Overall Approach to Hygienic Processing

I
ICE CREAM A Kambamanoli-Dimou 1083
IMMUNOLOGICAL TECHNIQUES see MYCOTOXINS: Immunological Techniques for Detection
and Analysis
IMMUNOMAGNETIC PARTICLE-BASEDASSAYS see ESCHERICHIA COLI 0757: Detection by
I Contents

Commercial lmmunomagnetic Particle-based Assays; LISTERIA: Detection by Commercial

lmmunomagnetic Particle-based Assays; SALMONELLA: Detection by lmmunomagnetic
Particle-based Assays
IMMUNOMAGNETIC PARTICLE-BASED TECHNIQUES: OVERVIEW K S Cudjoe 1088
INACTIVATION TECHNIQUES see LASERS: Inactivation Techniques
INDIRECT CONDUCTIMETRIC/IMPEDlMETRlCTECHNIQUES see DIRECT (AND INDIRECT)
CONDUCTIMETRIC/IMPEDlMETRlC TECHNIQUES: Food-borne Pathogens;
Enterobacteriaceae, Coliforms and E, coli
INDUSTRIAL FERMENTATIONsee FERMENTATION (INDUSTRIAL): Basic Considerations; Media
for Industrial Fermentations; Control of Fermentation Conditions; Recovery of Metabolites;
Production of Xanthan Gum; Production of Organic Acids; Production of Oils and Fatty Acids;
Colours/Flavours Derived by Fermentation
INTERMEDIATE MOISTURE FOODS K Prabhakar 1095
INTERNATIONAL CONTROL OF MICROBIOLOGY B Pourkomailian 1101

K
KLEBSIELLA P T Vanhooren, S De Baets, G Bruggeman, E J Vandamme 1107
KLUYVEROMYCES C A Batt 1115

L
LABORATORY DESIGN M Ahmed 1119
LABORATORY MANAGEMENT- ACCREDITATION SCHEMES C Bowles 1128
LACTIC ACID BACTERIA see LACTOBACILLUS: Introduction; Lactobacillus bulgaricus;
Lactobacillus brevis; Lactobacillus acidophilus; Lactobacillus casei; LACTOCOCCUS:
Introduction; Lactococcus lactis Sub-species lactis and cremoris; PEDIOCOCCUS
LACTOBACILLUS
Introduction C A Batt 1134
Lactobacillus bulgaricus P C M Teixeira 1136
Lactobacillus brevis P C M Teixeira 1144
Lactobacillus acidophilus T R Klaenhammer; W M Russell 1151
Lactobaciius casei M Gobbetti 1157
LACTOCOCCUS
Introduction C A Batt 1164
Lactococcus lactis Sub-species lactis and cremoris P D Courtney 1166
LACTOFERRIN see NATURAL ANTIMICROBIAL SYSTEMS: Lactoperoxidase and Lactoferrin
LACTOPEROXIDASE see NATURAL ANTIMICROBIAL SYSTEMS: Lactoperoxidase and
Lactoferrin
LAGER ICampbell 1172
LASERS: INACTIVATION TECHNIQUES IA Watson, D E S Stewart-Tu11 1177
LATEX AGGLUTINATION TECHNIQUES see CAMPYLOBACTER: Detection by Latex
Agglutination Techniques; ESCHERICHIA COLI 0 157: Detection by Latex Agglutination
Techniques; SALMONELLA: Detection by Latex Agglutination Techniques
LEGISLATION see NATIONAL LEGISLATION, GUIDELINES 8, STANDARDS GOVERNING
MICROBIOLOGY Canada; European Union; Japan
LEUCONOSTOC A Lonvaud-Funel 1183
LIGHT MICROSCOPYsee MICROSCOPY Light Microscopy
LIPID METABOLISM see METABOLIC PATHWAYS: Lipid Metabolism
 Contents Ii

LlSTERlA
Introduction C A Batt 1195
Detection by Classical Cultural Techniques G D W Curtis 1199
Detection by Commercial Enzyme Immunoassays M Wagner, A Bubert 1207
Detection by Colorimetric DNA Hybridization A D Hitchins 1214
Detection by Commercial lmmunomagnetic Particle-based Assays B Kohn 1222
Listeria monocytogenes S E Martin, C W Fisher 1228
Listeria monocytogenes - Detection by Chemiluminescent DNA Hybridization A D Hitchins 1238
Listeria monocytogenes - Detection using NASBA (an Isothermal Nucleic Acid Amplification 1244
System) M R Uyttendaele, J M Debevere
LYSINS see MINIMAL METHODS OF PROCESSING: Potential use of Phages and/or Lysins
LYSOZYME see NATURAL ANTIMICROBIAL SYSTEMS: Lysozyme and other Proteins in Eggs

M
MALOLACTIC FERMENTATION see WINES: The Malolactic Fermentation
MANOTHERMOSONICATION see MINIMAL METHODS OF PROCESSING:
Manothermosonication
MANUFACTURING PRACTICE see GOOD MANUFACTURING PRACTICE
MATHEMATICAL MODELLING see PREDICTIVE MICROBIOLOGY AND FOOD SAFETY
MEAT AND POULTRY
Spoilage of Meat G-J E Nychas, E H Drosinos 1253
Curing of Meat K Prabhakar 1260
Spoilage of Cooked Meats and Meat Products I Guerrero, L P Chabela 1266
METABOLIC ACTIVITY TESTS see TOTAL VIABLE COUNTS: Metabolic Activity Tests
METABOLIC PATHWAYS
Release of Energy (Aerobic) A Brandis-Heep 1272
Release of Energy (Anaerobic) M D Alur 1279
Nitrogen Metabolism M D Alur 1288
Lipid Metabolism R Sandhir 1298
Metabolism of Minerals and Vitamins C Umezawa, M Shin 1313
Production of Secondary Metabolites - Fungi P Nigam, D Singh 1319
Production of Secondary Metabolites - Bacteria M D Alur 1328
METABOLITE RECOVERY see FERMENTATION (INDUSTRIAL): Recovery of Metabolites
METHANOGENS W Kim, W B Whitman 1334
MICROBIOLOGY OF SOUS-VIDE PRODUCTS F Cadin 1338
MlCROCOCCUS M-L Garcia-Lopez, J-A Santos, A Otero 1344
MICROFLORA OF THE INTESTINE
The Natural Microflora of Humans C L Willis, G R Gibson 1351
Biology of Bifidobacteria A Y Tamime 1355
Biology of Lactobacillus acidophilus W R Aimutis 1361
Biology of the Enterococcus spp. N Tunail 1365
Detection and Enumeration of Probiotic Cultures G Kalantzopoulos 1373
MICROSCOPY
Light Microscopy R W Lovitt, C J Wright 1379
Confocal Laser Scanning Microscopy D F Lewis 1389
Scanning Electron Microscopy U J Potter, G Love 1397
Transmission Electron Microscopy U J Potter, G Love 1407
lii Contents

Atomic Force Microscopy W R Bowen, M Hila/, R W Lovitt, C J Wright 1418

Sensing Microscopy M Nakao 1425
MICROWAVES see HEAT TREATMENT OF FOODS: Action of Microwaves
MILK AND MILK PRODUCTS
Microbiology of Liquid Milk B H Ozer 1436
Microbiology of Dried Milk Products D Muir 1441
Microbiology of Cream and Butter R S Singhal, P R Kulkarni 1445
MILLET see FERMENTED FOODS: Beverages from Sorghum and Millet
MINERAL METABOLISM see METABOLIC PATHWAYS: Metabolism of Minerals and Vitamins
MINIMAL METHODS OF PROCESSING
Electroporation - Pulsed Electric Fields M L Calderdn-Miranda, G V Barbosa-Canovas, 1456
B G Swanson
Manothermosonication J Burgos 1462
Potential use of Phages and/or Lysins J Jofre, M Munisa 1469
MODIFIED ATMOSPHERE PACKAGING see CHILLED STORAGE OF FOODS: Use of Modified
Atmosphere Packaging
MOLECULAR BIOLOGY IN MICROBIOLOGICAL ANALYSIS - DNA-BASED METHODS FOR THE
DETECTION OF FOOD-BORNE PATHOGENS L O’Connor, M Maher 1475
MOLLUSCS see SHELLFISH (MOLLUSCS AND CRUSTACEA): Characteristics of the Groups;
Contamination and Spoilage
MONASCUS L Martinkova, P Patakova 1481
MORAXELLA J-A Santos, M-L Garcia-Ldpez, A Otero 1487
MOULDS see BIOCHEMICAL IDENTIFICATION TECHNIQUES - MODERN TECHNIQUES: Food
Spoilage Flora (Le. Yeasts and Moulds); FUNGI; GENETIC ENGINEERING: Modification of
Yeast and Moulds; STARTER CULTURES: Moulds Employed in Food Processing
MPN see TOTAL VIABLE COUNTS: MPN
MUCOR A Botha, J C du Preez 1493
MYCELIAL FUNGI see SINGLE-CELL PROTEIN: Mycelial Fungi
MYCOBACTERIUM J B Payeur 1500
MYCOTOXINS
Classification L B Bullerman 1512
Occurrence M de Nijs, S H W Nofermans 1520
Detection and Analysis by Classical Techniques IA Ahmed 1526
Immunological Techniques for Detection and Analysis A Sharma, M R A Pillai 1532
Toxicology D Abramson 1539

VOLUME 3
N
NASBA see LISTERIA: Listeria monocytogenes - Detection using NASBA (an Isothermal Nucleic
Acid Amplification System)
NATAMYCIN see PRESERVATIVES: Permitted Preservatives- Natamycin
NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY
Canada B E Brown 1549
European Union B Schalch, H Beck 1561
Japan S Kumagai 1564
NATURAL ANTIMICROBIAL SYSTEMS
Preservative Effects During Storage V M Dillon 1570
 Contents liii

Antimicrobial Compounds in Plants C Umezawa, M Shin 1576

Lysozyme and Other Proteins in Eggs E A Chartec G Lagarde 1582
Lactoperoxidase and Lactoferrin B H Ozer 1587
NEISSERIA S A S Hanna 1591
NEMATODES see HELMINTHES AND NEMATODES
NISIN see BACTERIOCINS: Nisin
NITRATE see PRESERVATIVES: Permitted Preservatives - Nitrate and Nitrite
NITRITE see PRESERVATIVES: Permitted Preservatives - Nitrate and Nitrite
NITROGEN METABOLISM see METABOLIC PATHWAYS: Nitrogen Metabolism
NUCLEIC ACID-BASED ASSAYS
Overview M W Griffiths 1599

0
OENOLOGY see WINES: Specific Aspects of Oenology
OILS see FERMENTATION (INDUSTRIAL): Production of Oils and Fatty Acids; PRESERVATIVES:
Traditional Preservatives - Oils and Spices
ORGANIC ACIDS see FERMENTATION (INDUSTRIAL): Production of Organic Acids, e.g. Citric,
Propionic; PRESERVATIVES:Traditional Preservatives - Organic Acids

P
PACKAGING OF FOODS A L Brody 1611
PANARY FERMENTATION see BREAD: Bread from Wheat Flour
PANTOEA A Morin, Z Parveen 1623
PARASITES see CRYPTOSPORIDIUM; CYCLOSPORA; GIARDIA; HELMINTHES AND
NEMATODES: TRICHINELLA: WATERBORNE PARASITES: Entamoeba; Detection by Classic
and Modern Techniques
PASTEURIZATIONsee HEAT TREATMENT OF FOODS: Principles of Pasteurization
PASTRY see CONFECTIONERY PRODUCTS: Cakes and Pastries
PCR-BASED COMMERCIAL TESTS FOR PATHOGENS P A Bertram-Drogatz, F Wilborn,
P Scheu, A Pardigol, C Koob, C Gronewald, M Fandke, A Gasch, K Berghof 1630
PEDIOCOCCUS M Raccach 1641
PENlClLLIUM
Introduction J IPitt 1647
Penicillium in Food Production G Blank 1655
PERONOSPOROMYCETES see FUNGI: Classification of the Peronosporomycetes
PETRIFILM -AN ENHANCED CULTURAL TECHNIQUE R Jordano, L M Medina 1662
PHAGES see BACTERIOPHAGE-BASED TECHNIQUES FOR DETECTION OF FOOD-BORNE
PATHOGENS; MINIMAL METHODS OF PROCESSING: Potential Use of Phages and/or Lysins
PHYCOTOXINS A Sharma 1672
PHYLOGENETIC APPROACH TO BACTERIAL CLASSIFICATION see BACTERIA: Classification
of the Bacteria - Phylogenetic Approach
PHYSICAL REMOVAL OF MICROFLORAS
Filtration P Boyaval 1675
Centrifugation V V Mistry 1681
PlCHlA PASTORIS C Kalidas 1686
POLYMER TECHNOLOGIES FOR CONTROL OF BACTERIAL ADHESION D Cunliffe,
C A Smart, C Alexander 1692
POLYSACCHARIDES see FERMENTATION (INDUSTRIAL): Production of Xanthan Gum
liv Contents

POULTRY see MEAT AND POULTRY: Spoilage of Meat; Curing of Meat; Spoilage of Cooked
Meats and Meat Products
POUR PLATE TECHNIQUE see TOTAL VIABLE COUNTS: Pour Plate Technique
PREDICTIVE MICROBIOLOGY AND FOOD SAFETY T Ross, T A McMeekin,
J Baranyi 1699
PR ESERVATIVES
Classification and Properties M Surekha, S M Reddy 1710
Traditional Preservatives - Oils and Spices G-J E Nychas, C C Tassou 1717
Traditional Preservatives - Sodium Chloride M S Brewer 1723
Traditional Preservatives - Organic Acids M Stratford 1729
Traditional Preservatives - Wood Smoke L J Ogbadu 1737
Traditional Preservatives - Vegetable Oils V Venugopal 1743
Permitted Preservatives - Sulphur Dioxide K Prabhakar, K S Reddy 1750
Permitted Preservatives - Benzoic Acid L J Ogbadu 1754
Permitted Preservatives - Hydroxybenzoic Acid R S Singhal, P R Kulkarni 1757
Permitted Preservatives - Nitrate and Nitrite R S Singhal, P R Kulkarni 1762
Permitted Preservatives - Sorbic Acid L V Thomas 1769
Permitted Preservatives - Natamycin J Stark 1776
Permitted Preservatives - Propionic Acid R S Singhal, P R Kulkarni 1781
PROB IOTIC BACTERIA
Detection and Estimation in Fermented and Non-fermented Dairy Products W Kneifel, 1783
T Mattila-Sandholm, A von Wright
PROBlOTlCS see BIFIDOBACTERIUM; MICROFLORA OF THE INTESTINE: The Natural
Microflora of Humans
PROCESS HYGIENE
Designing for Hygienic Operation G C Gurakan, T F Bozoglu 1790
Types of Biocides J F Williams, S D Worley 1794
Overall Approach to Hygienic Processing M A Mostert, H L M Lelieveld 1802
Modern Systems of Plant Cleaning Y Chisti 1806
Risk and Control of Airborne Contamination G J Curie/, H M J van Eijk, H L M Lelieveld 1816
Testing of Disinfectants J F Williams, J R Bickert 1822
Involvement of Regulatory and Advisory Bodies R Cocker, H L M Lelieveld 1830
Hygiene in the Catering Industry.. N Johns 1845
PROPlONlBACTERlUM M Gautier 1850
PROPIONIC ACID see FERMENTATION (INDUSTRIAL): Production of Organic Acids, e.g. Citric,
Propionic ; PRESERVATIVES : Permitted Preservatives - Propio nic Acid
PROTEUS B W Senior 1857
PSEUDOMONAS
Introduction M A Cousin 1864
Pseudomonas aeruginosa M H J Bennik 1867
Burkholderia cocovenenans J Cox, E Kartadarma, K Buckle 1871
PSYCHROBACTER M-L Garcia-Lopez, M P Maradona 1875

Q
QUALITY ASSURANCE AND MANAGEMENT see HAZARD APPRAISAL (HACCP): The Overall
Concept
QUANTITATIVE RISK ANALYSIS S H W Notermans 1883
 Contents Iv

R
RAPID METHODS FOR FOOD HYGIENE INSPECTION M Upmann, C Bonaparte 1887
REDOX POTENTIAL see ECOLOGY OF BACTERIA AND FUNGI IN FOODS: Influence of Redox
Potential and pH
REFERENCE MATERIALS P H In’t Veld 1895
REGULATORY BODIES see HAZARD APPRAISAL (HACCP): Involvement of Regulatory Bodies
[1340] RHODOTORULA Y Yeeh 1900
RISK ANALYSIS see QUANTITATIVE RISK ANALYSIS

S
SACCHAROMYCES
Introduction Y Oda, K Ouchi 1907
Saccharomyces sake Y limura 1913
Saccharomyces cerevisiae 5 C Viljoen, G M Heard 1918
Saccharomyces: Brewer’s Yeast G G Stewart 1925
SAKE see SACCHAROMYCES: Saccharomyces sake
SALMONELLA
Introduction J Cox 1928
Salmonella enteritidis T S Hammack, W H Andrews 1937
Salmonella typhi J Cox 1943
Detection by Classical Cultural Techniques R M ArnaguaAa, W H Andrews 1948
Detection by Latex Agglutination Techniques J Cox 1952
Detection by Enzyme Immunoassays P Patel 1956
Detection by Colorimetric DNA Hybridization H-Y Tsen 1964
Detection by lmmunomagnetic Particle-based Assay K S Cudjoe 1968
SALT see PRESERVATIVES: Traditional Preservatives - Sodium Chloride
SAMPLING REGIMES & STATISTICAL EVALUATION OF MICROBIOLOGICAL RESULTS
G Hildebrandt 1976
SCANNING ELECTRON MICROSCOPY see MICROSCOPY: Scanning Electron Microscopy
SCHIZOSACCHAROMYCES G H Fleet 1984
SECONDARY METABOLITES see METABOLIC PATHWAYS: Production of Secondary
Metabolites - Fungi; Production of Secondary Metabolites - Bacteria
SENSING MICROSCOPY see MICROSCOPY: Sensing microscopy
SERRATIA F Rafii 1989
SHELLFISH (MOLLUSCSAND CRUSTACEA)
Characteristics of the Groups L Le Vay, B Egan 1993
Contamination and Spoilage C A Kaysner 2001
SHEWANELLA L Gram, B F Vogel 2008
SHIGELLA: Introduction and Detection by Classical Cultural Techniques K A Lampel,
R C Sandlin, S Formal 201 5
SINGLE-CELL PROTEIN
The Algae M Garcia-Garibay, L Gdmez-Ruiz, E Barzana 2021
Yeasts and Bacteria M Garcia-Garibay, L Gomez-Ruiz, E Barzana 2027
Mycelial Fungi P Nigam 2034
SODIUM CHLORIDE see PRESERVATIVES: Traditional Preservatives - Sodium Chloride
SORBIC ACID see PRESERVATIVES: Permitted Preservatives- Sorbic Acid
SORGHUM see FERMENTED FOODS: Beverages from Sorghum and Millet
Ivi Contents

SOURDOUGH BREAD see BREAD: Sourdough Bread

SOUS-VIDE PRODUCTS see MICROBIOLOGY OF SOUS-VIDE PRODUCTS
SPICES see PRESERVATIVES: Traditional Preservatives- Oils and Spices
SPIRAL PLATER see TOTAL VIABLE COUNTS: Specific Techniques
SPOILAGE OF PLANT PRODUCTS
Cereals and Cereal Flours D R Twiddy, P Wareing 2045
SPOILAGE PROBLEMS
Problems Caused by Bacteria K M J Hansen, D A Bautista 2051
Problems Caused by Fungi M 0 Moss 2056
STAPHYLOCOCCUS
Introduction S E Martin, J J landolo 2062
Staphylococcus aureus J Harvey, A Gilmour 2066
Detection by Cultural and Modern Techniques S R Tatini, R Bennett 2071
Detection of Staphylococcal Enterotoxins M S Bergdoll (dec) 2076
STARTER CULTURES
Uses in the Food Industry R C Wigley 2084
Importance of Selected Genera C R Dass 2095
Cultures Employed in Cheese-making T M Cogan 2100
Moulds Employed in Food Processing T Uraz, B H Ozer 2109
STATISTICAL EVALUATION OF MICROBIOLOGICAL RESULTS see SAMPLING REGIMES &
STAT1STICAL EVALUAT I0N 0F MICROBI0LOGICAL RESULTS
STERILANTS see PROCESS HYGIENE: Types of Sterilant
STREPTOCOCCUS
Introduction M Gobbetti, A Corsetti 2117
Streptococcus thermophilus G Zirnstein, R Hutkins 2127
STREPTOMYCES A Sharma 2134
SULPHUR DIOXIDE see PRESERVATIVES: Permitted Preservatives - Sulphur Dioxide

T
THERMUS AQUATICUS C K K Nair 2139
TORULOPSIS R K Hommel, H-P Kleber 2142
TOTAL COUNTS
Microscopy S R Tatini, K L Kauppi 2149
TOTAL VIABLE COUNTS
Pour Plate Technique J W Messer, C H Johnson 2154
Spread Plate Technique J W MeSser, E W Rice, C H Johnson 2159
Specific Techniques M G Williams, F F Busta 2160
Most Probable Number (MPN) M G Williams, F F Busta 2166
Metabolic Activity Tests A F Mendonca, V K Juneja 2168
Microscopy C D Zook, F F Busta 2176
TOXICOLOGY see MYCOTOXINS: Toxicology
TRANSMISSION ELECTRON MICROSCOPY see MICROSCOPY: Transmission Electron
Microscopy
TRICHINELLA H R Gamble 2181
TRICHODERMA D E Eveleigh 2187
TRICHOTHECIUM A Sharma 2189
 Contents lvii

U
UHT TREATMENTS see HEAT TREATMENT OF FOODS: Ultra-high Temperature (UHT) Treatments
ULTRASONIC IMAGING
Non-destructive Methods to Detect Sterility of Aseptic Packages L Raaska, T Mattila- 2195
Sandholm
ULTRASONIC STANDING WAVES
Inactivation of Food-borne Microorganisms using Power Ultrasound G D Betts, A Williams,
R M Oakley 2202
ULTRA-VIOLET LIGHT G Shama 2208

v
VAGOCOCCUS L M Teixeira, M da G S Carvalho, R R Facklam 2215
VEGETABLE OILS see PRESERVATIVES: Traditional Preservatives- Vegetable Oils
VEROTOXIGENIC E. COLl
Detection by Commercial Enzyme Immunoassays D W Pimbley 2221
VEROTOXIGENIC E. COLl AND SHlGELLA SPP.
Detection by Cultural Methods C Vernozy-Rozand 2232
VIBRIO
Introduction, Including Vibrio vulnificus, and Vibrio parahaemolyticus P M Desmarchelier 2237
Vibrio cholerae F Y K Wong, P M Desmarchelier 2242
Standard Cultural Methods and Molecular Detection Techniques in Foods K Venkateswaran 2248
VINEGAR M R Adams 2258
VIRUSES
Introduction D 0 Cliver 2264
EnvironmentallyTransmissible Enteric Hepatitis Viruses: A and E T L Crorneans, M D Sobsey 2267
Detection D 0 Cliver 2274
VITAMIN METABOLISM see METABOLIC PATHWAYS: Metabolism of Minerals and Vitamins

W
WATER QUALITY ASSESSMENT
Routine Techniques for Monitoring Bacterial and Viral Contaminants J Watkins, L Straszynski, 2281
D Sartory, P Wyn-Jones
Modern Microbiological Techniques C Fricker 2287
WATERBORNE PARASITES
Entamoeba W A Petri Jc J M Schaenman 2292
Detection by Conventional and Developing Techniques H V Smith, R W A Girdwood 2295
WINES
Microbiology of Wine-making G M Walker 2306
Malolactic Fermentation T F Bozoglu, S Yurdugul 231 1
Specific Aspects of Oenology P Nigam 2316
WOOD SMOKE see PRESERVATIVES: Traditional Preservatives- Wood Smoke
X
XANTHAN GUM see FERMENTATION (INDUSTRIAL):Production of Xanthan Gum
XANTHOMONAS A Sharma 2323
XEROMYCES BlOSPORUS FRASER A D Hocking, J I Pitt 2329

Y
YEASTS: Production and Commercial Uses R Joseph 2335
lviii Contents

YERSlNlA
Introduction P Kampfer 2342
Yersinia enterocolitica S Bhaduri 2350
YOGHURT see FERMENTED MILKS: Yoghurt

z
ZYGOMYCETES see FUNGI: Classification of the Zygomycetes
ZYGOSACCHAROMYCES J P Erickson, D N McKenna 2359
ZYMOMONAS H Yanase 2365

COLOUR PLATE SECTIONS:

Volume 1 between pages 358 and 359
Volume 2 between pages 1116 and 1117
Volume 3 between pages 1970 and 1971

APPENDICES:
Appendix 1: Bacteria and Fungi Ai
Appendix 2: List of Suppliers Aiii

INDEX li

I Accreditation Schemes see Laboratory Management:Accreditation Schemes.
ACETOBACTER
Rolf K Hommel, Cell Technologie Leipzig, Germany
Peter Ahnert, Department of Biochemistry, Ohio State University, Columbus, USA
Copyright 0 1999 Academic Press
Acetic acid bacteria have been used for making
vinegar, their best-known product, since Babylonian
times. For most of this time, vinegar was obtained by
fermentation from alcoholic solutions without under-
standing of the natural process. A number of research-
ers established the microbial basis of this process
in the beginning of the nineteenth century, such as
Kiitzing, Lafare and Boerhaave. In 1822 Persoon per-
formed the first biological study of surface films of
wine and beer and proposed the name Mycoderma.
Later Kiitzing (1837) isolated bacteria from naturally
fermented vinegar for the first time. Considering them
to be a kind of algae, he named them Ulvina aceti.
Pasteur established the causal connection between the
presence of Mycoderma aceti and vinegar formation
in the first systematic studies on acetic acid fer-
mentation. These discoveries and following studies
resulted in better understanding and new methods
(Pasteur method) of vinegar formation.
Characteristics of the Genus Acetobacter
The classification of protobacteria by DNA-rRNA
hybridization studies places acetic acid bacteria in the
rRNA superfamily IV (synonymous: alpha group).
Acetic acid bacteria, formerly classified into the family
Pseudomonadaceae, constitute the family Aceto-
bacteraceae consisting of only two closely related
genera, Acetobacter and Gluconobacter, each of
which is a separate rRNA branch. The family Aceto-
bacteraceae represents strictly aerobic chemo-
organotrophic bacteria, able to carry out a great
variety of incomplete oxidations and living in or on
plant materials, such as fruits and flowers. Some
members of this family are plant pathogens. None
display any pathogenic effect toward mammals,
including humans.
Based on physiological criteria the present nomen-
ACETOBACTER 1
clature of the genus Acetobacter subdivides it into
four species: A. aceti, A. liquefaciens, A. pasteurianus
and A. hansenii. DNA-rRNA hybridization studies
indicate the presence of three additional species: A.
diazotrophicus, A. methanolicus and A. xylinum.
Based on DNA-DNA hybridization a new species, A.
europaeus, has been proposed; strains of this species
show very low similarity to other species of the genus.
Acetobacter are Gram-negative rods. Old cells may
become Gram-variable. Cells appear singly, in pairs,
or in chains and they are motile by peritrichous flagella
or non-motile. There is no endospore formation.
Acetobacter spp. are obligate aerobes except for A.
diaztrophicus and A. nitrocaptans which belong to the
diverse group of free-living aerobic or microaerophilic
diazotrophs.
The metabolism is respiratory and never fer-
mentative. Single amino acids do not serve as sole
source of nitrogen and carbon. Essential amino acids
are not known. Depending on growth substrates,
some strains may require p-aminobenzoic acid, niacin,
thiamin, or pantothenic acid as growth factors. The
temperature range is 5-42°C with optima between 25
and 30°C.
Acetobacter strains show a moderate to high acid
tolerance. The pH range is between pH 4 and pH 7,
with optima between pH 5.4 and pH 6.3. Strains used
in making vinegar are more resistant toward acidic
pH. The minimum accepted by A. acidophilus is
pH2.6. The maximum is pH4.2. The internal pH
closely follows the external (A. aceti). At or below
pH 5.0 the membrane potential of a cell is normally
uncoupled, resulting in free proton exchange across
the cytoplasmic membrane, thus depriving ATP syn-
thesis of its driving force. However, the formation of
acetic acid (or other acids) proceeds via membrane-
bound dehydrogenases. These processes are closely
2 ACETOBACTER
connected to irreversible ATP-yielding reactions, suf-
ficient to keep the energy metabolism alive. In A. aceti,
a gene encoding citrate synthase is involved in acetic
acid tolerance. This enzyme is assumed to play a
central role in supplying sufficient ATP to protect the
cell against accumulation of acetic acid.
Ethanol concentrations higher than 8% and 10%
inhibit strains A. aceti and A. xylinum, respectively.
Some strains, for example spoilers of sakC, tolerate a
higher ethanol content. In general, the ethanol tol-
erance in Acetobacter is higher than in Gluconobacter.
The high direct oxidative capacity for sugars, alco-
hols and steroids is a special feature of Acetobacter.
This ability is used in vinegar fermentation, food
processing, chemical synthesis, and even in enantio-
selective oxidations, for example with A. past-
eurianus. Examples of other reactions are the
formation of 2,5-dioxogluconic acid by A. mel-
anogenum and A. carinus, the oxidations of ethane-
diol to glycolic acid, of lactate to acetoin, of glycerol
to dihydroxyacetone, for example polyols in which
two secondary cis-arranged hydroxyl groups in D-
configuration may be oxidized to ketoses. Two strains,
A. rancens and A. peroxidans, are reported to oxidize
n-alkanes, mainly by monoterminal attack yielding
the corresponding fatty alcohols and fatty acids.
Acetobacter are equipped with two sets of enzymes,
catalysing the same oxidation reactions. Enzymes in
the first set are bound in the cytoplasmic membrane,
the active site facing the periplasm. Enzymes in the
second set are located in the cytoplasm and are
NADP-dependent. The latter enzymes display neutral
or alkaline pH optima. Membrane-bound enzymes
show acidic optima. The high oxidative capacity of
Acetobacter is attributed to membrane-bound pro-
teins such as alcohol dehydrogenase, aldehyde
dehydrogenase, glucose dehydrogenase and sorbitol
dehydrogenase. The specific activities of these
enzymes are up to three orders of magnitude higher
than those of their cytoplasmic counterparts. Most
membrane-bound enzymes share the prosthetic group
pyrroloquinoline quinone (PQQ; Fig. 1). The sub-
strates do not need to be transported into the cell for
oxidation. Electrons generated are transferred by the
reduced form of PQQ either directly to a ubiquinone
( Q S ) of the respiratory chain or via a cytochrome
I
COOH
HOOC
0 industrially are highly variable, changing their pheno-
Figure 1 Structure of pyrroloquinoline quinone (PQQ).
c (subunit of some alcohol dehydrogenases) to the
terminal ubiquinol oxidase which is either cyto-
chrome al or cytochrome 0 . Energy is gained by these
oxidations but they are not contributing to carbon
metabolism. For instance, the oxidation of one mole
ethanol to one mole acetic acid yields six moles of
ATP. It is assumed that these systems function as
ancillary energy-generating pathways when the
energy demand of the cell is high. Nz-fixing cells of
A. diazotrophicus contain three-times higher enzyme
levels of quinoprotein glucose dehydrogenase than
under non-N2-fixing conditions. Flavin (FAD) is an
additional covalently bound prosthetic group present
in the membrane-bound gluconate dehydrogenase. It
is also linked directly to the respiratory chain.
For intracellular sugar metabolism, Acetobacter
possesses the hexose monophosphate pathway and a
complete tricarboxylic acid cycle. Glycolysis is absent
or rudimentary. In A. xylinum sugar metabolism pro-
ceeds (asin Gluconobacter)via the Entner-Doudoroff
pathway.
In addition to the typical growth substrates, such
as sugars or ethanol, A. methanolicus also accepts
methanol. The major assimilatory pathway of this
facultative methylotrophic bacterium proceeds via the
ribulose monophosphate cycle. In contrast, most of
the Gram-negative methanol-utilizing bacteria that
contain the ribulose monophosphate cycle are obli-
gate methylotrophs.
Some Acetobacter species, e.g. A. xylinum, syn-
thesize bacterial cellulose. The fibres may be regarded
as part of the glycocalyx and serve to maintain these
highly aerobic organisms at the liquid-air interface.
This exopolysaccharide, (f3-glul+4P-glu)n, is ex-
creted into the medium and then rapidly aggregates
as microfibrils yielding a surface pellicle. Bacterial
cellulose is produced either in static cultures, or in
submerged, fed-batch cultures with low share con-
ditions. Yields up to 28 g 1-' of dry polysaccharide
may be obtained after selection of high yielding
strains. The product, cellulose I form, is very pure.
It does not contain hemicelluloses, lignins or pectic
substances, and is therefore used mainly in medicine
as wound dressings for patients with burns, extended
loss of tissue, etc.
The majority of Acetobacter species have 1-8 plas-
mids varying in size from 1.5 to 95 kb. Isolates from
some German submerse vinegar processes have 3-1 1
plasmids, isolates from surface fermentation processes
3-7 plasmids (2-70 kb). Plasmid profile analysis has
become a powerful tool for controlling homogeneity,
stability, identity etc. of the microbial populations in
production processes. However, many strains used
typic and other properties in just a few generations.

This phenomenon could not be correlated with
plasmid profiles. Acetobacter contains four ribosomal
RNA operons on the chromosome.
Recombinant DNA techniques have been adapted
to Acetobacter. Host-vector systems and trans-
formation methods have been developed. Trans-
formation systems are available for A. aceti and A.
xylinum. For instance, bacteriophages specific for
Acetobacter lead to a complete stop of submerged
fermentation. Morphologically different phage types
are described which were isolated from vinegar fer-
mentations in Europe. They belong to the Bradley's
group A and to the Myoviridae. The high number of
phages in disturbed acetic acid fermentations suggests
their responsibility for production problems.
The classical, well known and well-studied, niches
of Acetobacter are in making vinegar and spoilage of
beer and wine (see below). Acetobacter spp. were
originally associated with plants and soils. Preferred
habitats, such as fruits and flowers, are rich in sugars,
alcohols and/or acids. Fermenting fruits, in particular,
are excellent sources of sugar and ethanol. Various
Acetobacter spp. have been isolated from apricots,
almonds, beets, bananas, figs, guavas, grapes, man-
darins, mangoes, oranges, pomegranates, pears,
peaches, persimmons, pineapples, plums, strawberries
and tomatoes. A. aceti, A. xylinum and A. past-
eurianus were predominantly associated with ripe
grapes. Local priorities have been found: A. past-
eurianus accounted for 75% of the strains in isolates
from Southern France. Especially high numbers were
found on damaged grapes. Acetobacter spp. have been
isolated from the immature spadix of the palm tree.
A. xylinum was present on the leaflets of the palm
tree and in the surrounding air. Acidic acid bacteria,
such as A. aceti and A. pasteurianus, hibernate in
dried and injured apples and in spring they are able
to spread to flowers. The nitrogen fixing A. diazo-
trophicus has settled the stem and roots of sugarcane
in Brazil. Different Acetobacter spp. have been isol-
ated from cocoa bean flora. Acetobacter is the causal
agent of bacterial rot in pears and apples, resulting in
different shades of browning and tissue degradation.
Pears are more susceptible to bacterial brown rot.
Acetobacter spp. have been isolated from decaying
apple tissue and from the larvae and adults of apple
maggots. Artificial inoculation of 100 cells may
induce apple brown rot. Although the optimum tem-
perature is about 25"C, rotting also proceeds at 4°C.
The pink disease of pineapple fruit is caused by A.
liquefaciens.Through the open flowers the bacterium
enters the internal nectary and placental regions,
invades the ovaries and starts to grow in the ripening
tissue. The dilution of nectar by rain during flowering
ACETOBACTER 3
is a prerequisite, because undiluted nectar is not a
growth substrate.
Methods of Detection
Strains of both Acetobacter and Gluconobacter are
present in the same habitat. Members of the latter
genus are normally co-isolated. For routine isolation
of Acetobacter from natural or artificial habitats,
culture media of low pH, containing 2-4% ethanol
as energy source, are recommended. Aerobic growth
is optimal between 25°C and 30°C. As low cell counts
are expected, enrichment cultures become necessary.
For such purposes beer has been recommended.
However, preservatives added to the beer may limit
success. Many specific enrichment cultures adapted
to individual sources are described in older literature.
Yeast water-glucose medium is recommended for
isolation and purification. It contains yeast water
(supernatant of autoclaved bakers' or brewers' yeast,
200 g I-'), and glucose (20 g I-'), p H 5.5-6.0. This
composition is also very useful for the enrichment of
solid media (agar concentration: 15-30 g I-').
Wort medium comprises malt powder diluted with
tap water to 8% soluble solids; for solid medium the
pH should be 5.5-6.
Peptone glucose agar comprises bacto-peptone or
bacto-tryptone ( 5 g I-'), glucose (20 g I-'), primary
potassium phosphate (1g I-') in tap water; agar con-
centration: 15-20 gl-'.
To enhance the growth of some strains, the addition
of yeast extract (3-Sg1-') may be useful. Further
enhancement may be achieved by the addition of
100ml of filtered and freshly prepared tomato juice
to 1 litre of culture medium.
An isolation procedure to differentiate between A.
pasteurianus, A. aceti and Gluconobacter oxydans
has been developed by Frateur, which uses three to
five different culture media for each species. It includes
enrichment in liquid media (30°C) and subsequent
agar plating.
Isolation of production strains from vinegar tends
to be difficult due to the strains being highly adapted
to the production conditions (see below). A mixture
( 4 ml) of vinegar to be tested and pasteurized vinegar
is added to tubes with 15ml of solid medium (yeast
water, 100 g I-' glucose, 30 g I-' CaC03, 20 g IF' agar).
Bacterial growth proceeds in the interphase. Alter-
natively, yeast extract-calcium lactate agar or wort
agar containing 1.5% ethanol or 5gl-l yeast extract,
and 25 g I-' agar may be used.
Acetobacter settling on flowers or fruits may be
efficiently enriched in a broth containing 50 g I-'
glucose, 10 g IF' yeast extract, and 0.1 mg I-' cyclohe---
imide at 30°C. The ring or pellicle formed after 2-8
4 ACETOBACTER

days is plated out on a solid medium which may Table 1 Common media for maintenance and cultivation of
Acetobacter
also servc for further purification of the acid-forming
colonies: 50 g I-'glucose, 10 g IF' yeast extract, 30 g I-' Medium Component Amount
C a C 0 3 and 25 g IF1 agar. Glucose-yeast extract agar Glucose
In cider making, various culture media are re- ('Acefobacter/G/uconobacter Yeast extract
commended for successful isolation of acetic acid bac- agar') CaCO,
teria from orchard soil, apples, pomace, juice, pH 7.5 f 0.2, 25°C Agar
Distilled/deionized
fermenting juice, cider or from the factory equipment. water
The media are based on apple juice-yeast extract
Glucose
made from apple juice with low tannin content, 'Acetobacter agar'
Yeast extract
pH4.8 and 3Ogl-l agar. The addition of 0.1 mgl-' CaCO,
actidione is recommended to suppress yeasts and Agar
moulds. Incubation is carried out at 28°C for 3-5 Tap water
days. Alternatively, a broth composed of 500ml of 'Acetobacter medium' Glucose
sweet cider, 500 ml deionized water, 12 g I-' yeast pH 7.0 f 0.2, 25°C Autolysed yeast
extract, and 2 g (NH4)#04( p H 5),yields good results. CaC03
Agar
Strains of A. diazotrophicus can be isolated by
Distilled/deionized
stepwise enrichment in different media, including water
semisolid ones. Acetobacter strains may be held in or
Mannitol agar Mannitol
on a variety of media, such as beer (without pre- (YPM agar: 12 g I-' agar) Yeast extract
servation agents) or wort. Recommended con- Peptone
servation media are summarized in Table 1. Optimum Agar
growth is obtained at 25-30°C. Agar cultures should DistiIled/deionized
be kept at 4°C and transferred monthly. Most strains water
stay alive lyophilized for several years and some for Yeast-glucose agar Glucose
longer than 10 years. pH 7.0 f 0.2, 25°C Yeast extract
Agar
Phenotypic identification of Acetobacteraceae is Distilled/deionized
based on general properties which are partially shared water
with Gluconobacter and some members of the genus
Potato glycerol agar Glycerol
Frateuria (superfamily I1 syn. gamma subclass). One 25°C Glucose
of the properties used to further identify Acetobacter Yeast extract
is the oxidation of acetate and lactate to COZ and Agar
H z O (overoxidation) at neutral and acidic pH. This Supernatant of
freshly sliced and
is detected on medium composed of ethanol ( 3 % ) ,
boiled potatoes
C a C 0 3 (20 g I-'),and agar (20 g I-'). The appearance (200 g 1-1)
of acetic acid reveals clear zones around the colonies
and overoxidation results in (re-)precipitation of
C a C 0 3 . Alternatively, bromcresol green (0.022 g I-') oxidation of acetic acid and lactic acid to C 0 2 and
may be added to a medium composed of yeast extract HZO
(30gl-'), ethanol (2%) and agar (2OgI-I). The colour n o H2S formation
shifts from green to yellow as acid is formed. Overox- growth factors may or may not be required
idation results in a change of the indicator to blue. specific ubiquinone types.
Bacteria belonging to Acetobacteraceae may be
Gram-negative or Gram-variable (namely older cells), The ubiquinones are Q9 and Q S with minor com-
are strictly aerobic and oxidize ethanol to acetic acid ponent of Q8( A . aceti, A. pasteurianus);some strains
have Qloor Qlowith minor component Q9 (A. meth-
in neutral or acidic media. Cells are ellipsoidal t o rod-
shaped (0.6-0.8 x 1-4 pm), have a respiratory type of
anolicus, A. diazotrophicus, A. xylinum, A.
metabolism, are oxidase negative and acidify glucose
liquefaciens). Acetobacter strains grow at p H 5 and
prefer ethanol or lactate over glucose for growth.
below p H 4.5. They d o not form endospores, liquefy
Further differentiation among Acetobacter species can
gelatin, reduce nitrate or form indole. The rapid
be achieved as shown in Table 2.
phenotypic idcntification of Acetobacter is based on
The phenotypic identification may be affected by
the following features:
spontaneously occurring mutations even in taxo-
nomically important properties. Mutants of A. aceti
0 peritrichous flagella exist that are unable to oxidize ethanol. In these cases
 ACETOBACTER 5

Table 2 Phenotypical differences among Acetobacter species (Reproduced with permission from Swings 1992)
Feature A. A. A. A. A. A. A.
aceti lique- Pasteur- hansenii xylinum methano- diazo-
faciens ianus licus trophicus

Formation of
Water-soluble brown pigments on GYC" medium +
y-Pyrones from D-fructose +
5-Oxogluconic acid from o-glucose -
2, 5-Dioxogluconic acid from o-glucose +
Ketogenesis from glycerol d
Growth factor required -
Growth on carbon sources
Ethanol +
Methanol -
Sodium acetate +
Growth on L-amino acids in the presence
of D-mannitol as carbon source
L-Asparagine d + - + - - +
L-Glutamine d + - + - - -
Formation of H2S - - - - - - -
Growth in presence of 30% D-glucose - - - - - - +
Ubiqinone type Qg Qio Qs ND Qio Qio Qio
G+C content (mol%) 56-60 62-65 53-63 58-63 55-63 62 61-63
~~ ~~~~~ ~~~~ ~

Symbols: +, 90% or more of the strains positive; (+), weakly positive reaction; d, 11-89% of the strains positive; -, 90% or more of
the strains negative; ND, not determined.
Glucose-yeast extract cycloheximide.

DNA-rRNA hybridization studies and polymerase
chain reaction (PCR) studies will be more useful for
exact taxonomic classification.
Importance to the Food Industry
Food Processes
Acetobacter spp. are used in different processes of
making foods and food additives. Well established are
the fermentations to produce one special product,
such as acetic acid or gluconic acid. These oxidation
reactions are backed by the high oxidative capacity
of enzymes bound in the cytoplasmic membrane with
the active centre directed into the periplasm. Other
processes also use such enzymes but are more complex
with regard to the microbial population and the sub-
strates used.
Vinegar is the most popular product of Acetobacter.
This process is discussed in detail in a separate article.
In acetic acid fermentation, mixtures of highly
adapted predominantly Acetobactsr spp. are used,
which are not derived from pure cultures. These
strains display an extremely strong tolerance to acetic
acid. The most important detectable species are A .
pasteurianus, A . lovaniensis, A . ascendens, A.
paradox, A . aceti, A . xylinum and A . orleanensis.
Difficulties may arise during isolation, subsequent cul-
tivation under artificial conditions, and preservation
due to the high adaptation as demonstrated with A .
polyoxogenes isolated in Japan and with A . acid-
ophilus. Both could not be maintained in strain col-
lections and were lost. A . europaeus isolated from
production facilities in Switzerland requires acetic
acid for growth on agar plates and tolerates 4-8'30.
Specialized industrial strains tolerate p H values down
to 2.6. DNA-DNA hybridization studies shows
nearly no difference between isolates from different
commercial processes (9O-lOO% hybridization).
However, a comparison of highly productive strains
from German plants and those from strain collections
showed large differences. Values below 45% were
obtained with definite strains ( A . pasteurianus, A .
aceti, G . oxydans).The DNA-DNA similarities of A .
europaeus strains isolated from industrial processes
versus strains from collections are below 22%.
Membrane-bound quinoproteins, i.e. alcohol and
aldehyde dehydrogenases, are the enzymatic basis of
acetic acid formation (Fig. 2). These enzymes are
more active and stable under acidic conditions than
those of Gluconobacter. Prevention of overoxidation
of acetic acid to COz and H20 requires a constant
high concentration of ethanol. Lack of ethanol and
oxygen damage acetic acid bacteria populations. Even
 Next Page
6 ACETOBACTER

naturally fermented by a high supply of air in up to

13 days. Yeasts, moulds and bacteria are involved.
Ethanol produced by yeasts is oxidized by Acetobacter
spp. Seeds are killed in the presence of acetic acid and
by a temperature of 50°C. The fruit mass is then
degraded and the typical flavour and brownish colour
of the bean are developed.
Nata is a dessert delicacy in Southeast Asia. This
gelatin-like, firm, creamy-yellow to pinkish substance
is composed of a form of cellulose formed by bacteria
Ethanol +--;?---<--Acetaldehyde --;;---<:--* Acetate from sugared fruit juices. Acetobacter hansenii is
Y s' / Y
NAD+ NAD(P)H NAD+ NAD(P)H responsible for this process. Nata is usually grown on
Figure 2 Scheme of ethanol oxidation by acetic acid bacteria. fruit juice and the floating mat is candied, while still
The formation of acetic acid via the quinoprotein alcohol dehydro- chewable, to produce gumdrops-like treats.
genase (ADH) and aldehyde dehydrogenase (ALDH) yields 6 mol A symbiosis culture of a yeast with Acetobacter (A.
of ATP per mol of ethanol. The cytoplasmic pyridine nucleotide- xylinum) is the so-called 'tea fungus'. In this process a
dependent counterparts are alcohol dehydrogenase (NAD(P)-
slightly sweet, alcoholic, aromatic and acidic beverage
ADH) and aldehyde dehydrogenase (NAD(P)-ALDH). The pre-
ferred direction of the reversible reaction of the NAD(P)-ADH (kombucha, Ma-Gu etc.) is made by fermenting
is indicated by the arrow. (Reproduced with permission from sweetened (sucrose, 5-150 g 1-') black tea. Health
Matsushita et al (1994)) effects attributed to it include in vitro antimicrobial
activity, improved athletic performance, and
the application of pure oxygen or oxygen-enriched enhanced sleep and pain thresholds. The yeasts yield
air harms the process. ethanol; the alcohol content never exceeds l o g I-'. A.
Aspergillus niger and Gluconobacter oxydans (syn. xylinum initially oxidizes ethanol to acetic acid and
Acetobacter suboxydans) are the preferred micro- glucose to gluconic acid. Acetic acid concentrations
organisms in the production of gluconic acid, which may rise to levels as high as 30 g 1-' if the tea is allowed
has useful properties, such as an extremely low tox- to ferment for up to 30 days. The usual concentration
icity, low corrosiveness and formation of water- of acetic acid is 10 g 1-' (after 3-5 days). Gluconic acid
soluble complexes with divalent and trivalent metal is also present in substantial quantities, about 20 g 1-'.
ions. On this basis gluconic acid has found many Different yeasts are involved, like Brettanomyces,
applications in the food, textile and tanning industries Candida, Pichia, Saccharomyces, Schizosaccharo-
and in medicine. According to German law gluconic myces, Torulaspora, Zygosaccharomyces, depending
acid is considered a food. The quinoprotein-depend- on origin and culture conditions. The production of
ent glucose oxidase also occurs in Acetobacter. Acet- cellulose by A. xylinum forms a compact, granular
obacter methanolicus possesses a similar enzyme. It and gelatinous surface film in which yeast and bac-
grows on methanol and oxidizes glucose that is not terial cells are housed. Both benefit from the floating
assimilated to gluconic acid. Glucose (150-250 g 1-') mat which eases aeration for these aerobic micro-
is nearly completely (98%) converted with a prod- organisms.
uctivity > 30 g 1-' h-'.
Acetobacter spp. are involved in a number of Food Spoiling
natural fermentations. A typical tropical beverage, Acetobacter may cause both considerable economic
palm wine, is made from the fermentation of palm profits and losses. The latter aspect results from the
sap. It is a result of a mixed alcoholic, lactic acid and spoiling activity in many products that provide suf-
acetic acid fermentation. The microbial consortium is ficient conditions for growth. The best example is the
complex. At first, yeasts and Zymomonas ferment the aerobic acidification of wine, the origin of vinegar
available sugar to ethanol, which is partly converted making. Acetobacter spp. are present in the wine
into acetic acid by Acetobacter spp., which appear must, on the surface of grapes, and on injured grapes.
after two to three days of fermentation and can be Acetic acid formation may only proceed when
isolated from the final product. adequate oxygen is available, i.e. on fruits, in juices
Acetobacter strains have been isolated from cocoa and in mashes. Under the anaerobic conditions of
wine, made by fermentation of cocoa seeds. Its alcohol wine making, vinegary spoilage is rare. Its occurrence
content of 9-12% is higher than that of palm wine. is indicated by increased concentrations of acetic acid,
Species of Acetobacter, A. rancens, A. xylinum, A. ethyl acetate and D-lactate. There is a positive cor-
ascendens, A. lovaniensis and others, are involved in relation between acetic acid and ethyl acetate. Sweet
cocoa seed fermentation. Seeds in the fruit mass are white wines spoiled by dextran-producing Aceto-

BACILLUS
Contents
Introduction
Bacillus cereus
Bacillus stearothermophilus
Bacillus anthracis
Bacillus subtilis
Detection of Toxins
Detection by Classical Cultural Techniques
Introduction
Michael K Dahi, Department of Microbiology,
University of Erlangen, Germany
Copyright 0 1999 Academic Press
The genus Bacillus is one of the preferentially used
organisms for producing metabolites and enzymes by
fermentation. This is partly due to the fact that most
(but not all) members of the genus are non-patho-
genic, excellent protein and metabolite secretors, and
easy to cultivate. Non-pathogenic Bacillus strains are
used both in foods and in industry. The numerous
products accepted as safe include enzymes for food
and drug processing, as well as foods produced from
these strains. The tremendous advances in molecular
biology have increased the use of Bacillus spp. in
heterologous gene expression. One member of the
genus, Bacillus subtilis, has been especially subjected
to intensive microbiological, biochemical and genetic
investigations. In the late 1950s, John Spizizen suc-
cessfully demonstrated the genetic transformation of
a particular B. subtilis isolate using purified DNA.
This laid the foundation for a series of intensive
studies of metabolism, gene regulation, bacterial dif-
ferentiation, chemotaxis and starvation. At present,
B. subtilis, together with Escherichia coli, is one of
the best understood prokaryotes, and the complete
sequence of the genome of B. subtilis is now available,
which facilitates further investigations into biological
molecular mechanisms. Insights made by analysing
molecular mechanisms in B. subtilis can easily be
transferred to related organisms, thereby helping to
advance applied research and food production.
BAC/LLUS/Introduction 113
The Genus Bacillus
At present the genus Bacillus encompasses more than
60 species. It is widespread in nature and can be
isolated from food, soil, water and even from euka-
ryotic organisms. Owing to the enormous genetic
diversity of this genus, it is difficult to define it con-
cisely. One of the most important properties for tax-
onomy is spore formation, since it is easily detectable.
However, the fact that sporulation depends on the
growing conditions complicates classification by this
characteristic. Spores can be detected microscopically
and provide a simple characteristic for the endospore-
forming family Bacillaceae. In this family, the genus
Bacillus incorporates many species of Gram-positive,
rod-shaped bacteria, which are able to grow under
aerobic and facultatively anaerobic conditions and
thus differ from Clostridium spp. which are strictly
anaerobic. However, aerobic endospore-forming bac-
teria are currently assigned to four genera in the family
Bacillaceae. Therefore, the genus Bacillus is basically
defined by morphological characteristics. Depending
on the type of spore formation observed, we dis-
tinguish between:
0 species producing oval endospores that distend the
mother cell
species producing oval endospores that do not
distend the mother cell
0 species producing spherical endospores.
Typically, Bacillus spores contain dipicolinic acid, but
the diversity of spore formation described above
makes a classification by this property difficult.
Numerical analysis of additional phenotypic features
leads to some idea of how the genus Bacillus might be
114 BACILLUSilntroduction

6. pantothenticus (37)
reorganized into several genera. There is no generally 6. lentus (36)
accepted definition of a prokaryote genus. Never- 6.badius (44)
6. smithii (39)
theless, it has been recommended that the maximum 6. azotoformans (39)
genetic diversity should not exceed a chromosomal 6. firmus (41)
6. circulans (39)
base composition range of 10-12% guaninekytosine 6. benzoevorans (41)
(G+C) content. Phylogenetically, Bacillus belongs to 6. simplex (41 )
(6. rnagaterium C)
the low G+C content group of bacteria, although, 6. maroccanus (ND)
6. psychosaccharolyticus(44)
depending on the species, the G+C content can vary 6. megaterium (37)
in the range from 33% ( B . anthracis) to 69% ( B . B. fastidiosus (35)
6. acidoterrestris (52)
tbermocatenulatus). Assuming the G+C content 6. coagulans (44)
definition is correct, the chromosomal base com- 6. mycoides (34)
6. medusa (ND)
position range in Bacillus would signify great phylo- 6. fhuringiensis (34)
genetic divergence, leading to the conclusion that 6. cereusB. anthracis (36)
6. pumilis (41)
Bacillus encompasses more than one genus. This 6. atrophaeus (42)
assumption is also supported by rRNA sequence 6. popilliae (41)
6. lentimorbus (38)
analysis, which reveals as much divergence as in the 6. amyloliquefaciens (43)
6. subtilis (43)
combined families of Enterobacteriaceae and Vib- 6. licheniformis (43)
rionaceae. In turn, the size of the genus Bacillus com- B. lautus (51)
B. sphaericus (37)
plicates the identification of new isolates. Therefore, B. fusiformis (36)
at least thirty phylogenetic tests must be carried out 6. insolitus (36)
6. globisporus (40)
before grouping an isolate in this genus, including 1 6 s 6. psychrophilus (42)
rRNA analysis (Fig. 1). 6. pasfeurii (38)
6. thermoglucosidasius(45)
6. stearothermophilus (52)
6. kaustophilus (53)
From Genomes to Proteomes 6. aicalophilus (37)
8.aneurinolyticus (42)
The composition and structural organization of
genomes are increasingly important in the clas-
. -. ..
c- - 6. brevis (47)
6. laterosporus (40)
6. cycloheptanicus (56)
sification and subdivision of bacteria into families and B. alvei (46)
6. gordonae (55)
genera. Owing to the rapid development of molecular 6. larvae (38)
methods and automatic DNA sequencing, a complete 6. pulvifaciens (44)
6.rnacerans (52)
genome can be determined within a short period. B. poiymyxa (44)
One of the most famous representatives of the genus 6. azotofixans (52)
6. pabuli (49)
Bacillus, B. subtilis, is used as a model organism in 6. macquariensis (40)
basic research and as a host for recombinant DNA in 6. amylolyficus (53)

applied research. In the early 1990s B. subtilis was
chosen for investigation of its complete genome
sequence. This collaborative project involved about
35 groups in Europe, the USA and Japan and was
completed in the autumn of 1997. The whole genome
is composed of 4 214 814 base pairs (which may vary
slightly due to error corrections) harbouring about
41 00 open reading frames, which reflect potential
protein-encoding genes. The genes present in the
genome have been classified according to functional
features (Table I). At present, the assignmcnt of genes
can be grouped into five categories based on sequence
homologies and functional analysis:
definite assignment of genes due to experimentally
identified functions (about 10%)
probable assignments due to high sequence hom-
ologies (about 50%)
possible assignments due to low sequence hom-
ologies (about 1 5 % )
putative assignments due to weak sequence hom-
ologies (about 10%)
Figure I Phylogenetic tree of Bacillus spp. according to
16s rRNA analysis. The G+C content is given in parentheses
in mole per cent. ND, not determined.
15% with n o counterpart found in the protein
sequence data base.
About half of the genes can be assigned to proteins
with a defined or probable function, and half have n o
clear function. Based on these genome sequence data,
a systematic function search programme has been
started. The analysis of the protein composition of B.
subtilis (the proteome) will provide further knowledge
of the organism and the genus Bacillus in general and
lead to the functional assignment of the proteins and
the corresponding genes.
Cell Wall Composition
In contrast to the Gram-negative bacteria, the Gram-
positive bacteria including Bacillus reveal a highly
varied peptidoglycan composition and structure.

Table 1 Bacillus subtilis genome summary
4099 ORFs are known at present
3044 ORFs have homologues (73.3%)
1326 ORFs contain superfamily assignments (32.3%)
1700 ORFs are assigned to functional categories (415%)
578 ORFs have known or homologous three-dimensional
structure (14.1 %)
540 ORFs have signal peptides (13.2%)
1140 ORFs have at least one transmembraneregion (27.8%)
75 1 ORFs have at least two transmembrane regions
(18.3%)
613 ORFs have at least three transmembrane regions
(15.0%)
152 ORFs contain more then 20% of low-complexity
sequence (3.7%)
185 ORFs contain coiled-coil regions (4.5%)
693 ORFs are all-alpha proteins (16.9%)
181 ORFs are all-beta proteins (4.4%)
1971 ORFs are alphdbeta proteins (48.1 %)
114 ORFs are irregular proteins (2.8%)
ORF, open reading frame, including all putative, potential and
defined genes.
About a hundred different types have been described.
Therefore, cell wall composition is often a useful
criterion in taxonomy. The murein sacculus of Bacil-
lus consists of peptidoglycan and is composed of up
to about thirty layers. The peptidoglycan is a hetero-
polymer of glycan cross-linked by short peptides.
Peptide chains are always composed of alternating L-
and D-amino acids. In the genus Bacillus, the murein
version of most species (with some few exceptions) is
of the direct-linked meso-diaminopimelic acid type.
Sporulation
Spore formation in Bacillus takes place when the
cell culture reaches the stationary growth phase. The
process of spore formation is an excellent model
system for studying the molecular biology of dif-
ferentiation. During the sporulation process, a vege-
tative cell (the progenitor) gives rise to two specialized
cells differing in cell type both from each other and
from the parent cell. Furthermore, in some cases this
process is associated with the synthesis of bio-
technologically important products such as insect
toxins and peptide synthetases creating peptide anti-
biotics.
The sporulation process is initiated at the end of
the exponential growth phase. The development of
the endospore formation involves an energy-intensive
pathway and requires the production of a complex
morphological structure. External (and presumably
also internal, however partially unknown) signals
force the cell to respond by inhibiting cell division and
initiating the sporulation process. Initially a complex
signal transduction system is turned on, the phos-
phorylay, which subsequently, at the end of the signal
BAClLLUS/lntroduction 115
cascade, activates the transcriptional regulator
protein SpoOA through phosphorylation. In contrast
to vegetative growth, sporulation gives rise to an
asymmetrically positioned septum which partitions
the developing cell into compartments of unequal
sizes. The smaller part is the forespore, which in
its subsequent development exhibits a biochemical
composition and structure completely different from
the remaining mother cell. During the sporulation
process several genes are sequentially activated; this
selected gene activation is induced by the com-
munication of mother cell and forespore, by signals
transferred across the septum. The subsequent specific
transcriptional regulation of spore genes is influenced
by the activation of different alternative sigma factors,
which confer promoter specificity to the RNA poly-
merase. In turn, the forespore transforms itself into
the endospore, and the mother cell dies by cell lysis.
The sporulation characteristics of various Bacillus
species are summarized in Table 2.
Isolation of Sporulating Bacteria
Spore-formers can be selectively isolated from natural
samples after incubation at 80°C for 10min. This
treatment effectively destroys vegetative cells, whereas
spores remain viable. The heat-treated probes can be
streaked onto plates of medium and further incubated
under aerobic conditions at their individual optimal
growing temperatures. The colonies obtained are
almost exclusively made up of the genus Bacillus.
Gene Transfer
Another interesting features of some members of the
Bacillus genus, which has been well analysed for B.
subtilis, is the development of natural competence for
DNA uptake. Before sporulation initiation, about 10-
20% of a cell culture express competence in the post-
exponential growth phase under defined growth con-
ditions. Such competent cells efficiently bind, process
and internalize available exogenous high-molecular-
weight DNA. The DNA can originate either from
chromosomal DNA or DNA fragments, which must
integrate themselves into the host chromosome in
order to survive there, or from plasmid DNA, which
can endure and replicate as extrachromosomal DNA
in the cytoplasm if it contains a functional origin of
replication. Several stages of the DNA transformation
process have been described, including binding, frag-
mentation, uptake, and (in the case of transforming
chromosomal DNA) also integration and resolution.
In several organisms, including B. subtilis, com-
petence has been used to genetically analyse and con-
struct stable and defined mutationally altered strains.
The latter can be obtained by allelic exchange based
116 BACILLUSIIntroduction

Table 2 Selected characteristics of representative species of the genus Bacillus

1 (A) Spores oval or cylindrical, facultative aerobes, casein and starch hydrolysis, no swollen sporangia and thin spore wall I
Thermophiles and acidophiles Spore position central or terminal
B. acidocaldarius Spore position terminal
MesoDhiles Spore position central
B. cereus Spore position central
Spore position central
Spore position central
B. subtilis Spore position central
I B. thuringiensis I Insect pathogen ] Spore position central I
I Sporangia distinctly swollen, thick spore wall I
B. stearothermophilus Thermophile Spore position terminal
B. polymyxa Mesophiles Spore position terminal
B. macerans Spore position terminal
B. circulans Spore position central or terminal
B. larvae Insect pathogens Spore position central or terminal
8. popilliae Spore position central

B. sphaericus Sporangia swollen Spore position terminal

I B. pasteurii 1 Sporangia not swollen 1 Spore position terminal I

on homologous recombination, introducing defined a sweeter taste than either glucose or sucrose. This
mutations, recombinant DNA or foreign genes enzymatic process therefore has become increasingly
flanked by DNA fragments with homologies to important for the industrial production of sugar from
genomic regions. This important feature of B. suhtilis starch, especially as a sweetening agent in soft drinks.
makes the strain suitable as a host for genes under In principle, these reactions can be catalysed sep-
regulatory promoter control for industrial use in arately by enzymes which operate sequentially in the
protein overproduction. conversion reactions. These reactions are composed
of three principal steps:
From Starch to Sugar
Thinning reaction, in which the starch poly-
Many bacilli produce extracellular hydrolytic
enzymes essential for the breakdown of poly-
saccharides o r oligosaccharides, nucleic acids, pro- Table 3 Examples of commercially produced enzymes from
teins and lipids. The resulting products can be used Bacillus spp.
as carbon sources, nitrogen sources, energy sources Bacillus species Enzyme
and electron donors. However, they also contain
B. amyloliquefaciens,B. Alpha-amylase
hydrolytic enzymes in the cytoplasm, which prepare licheniformis, B.
carbon sources to enter glycolysis by further hydro- stearothermophilus
lysation, phosphorylation and isomerization reac- B. coagulans Glucose isomerase
tions. The enzymes involved in sugar metabolism are B. stearothermophilus Glucose kinase
of commercial interest to the food industry and in B. stearothermophilus Glucose-6-phosphate
dehydrogenase
diagnostic medicine. B. amyloliquefaciens Metalloprotease
Bacillus species are used to manufacture com- B. cereus Phospholipase
mercially important enzymes (Table 3),for example B. stearothermophilus Phosphotransacetylase
for the production of glucose from corn, wheat or B. acidopullulyticus Pullulanase
potato starch. The resulting glucose can be attacked B. licheniformis, B. lentus, B. Serine protease
alcalophilus
by glucose isomerase to produce fructose, which has

saccharides are attacked by a-amylase, shortening
the chain and reducing viscosity.
0 Saccharification, which produces glucose from the
shortened polysaccharides catalysed by the glu-
coamylase.
0 Isomerization, which converts glucose into fruc-
tose, catalysed by the glucose isomerase.
The resulting fructose-containing syrup can be used
directly to sweeten food products.
Regulatory Aspects Studied in B. subfilis
Bacillus detects and responds to environmental
changes to survive adverse conditions by adapting to
changes in the composition of available nutrients.
This is also the case during fermentation in industrial
microbial enzyme production, where the meta-
bolization of nutrients and the secretion of end prod-
ucts result in the medium composition continuously
changing. These environmental signals subsequently
result in a shift of gene expression rates. Therefore, an
understanding of signal reception and the molecular
mechanisms of cellular response responsible for gene
expression is crucial to optimize enzyme production
and to cut production expenses. As mentioned above,
B. subtilis has been chosen as a representative of the
genus Bacillus for fundamental molecular biological
research, and numerous scientific projects deal with
aspects of regulatory mechanisms on the tran-
scriptional, translational and enzymatic levels. An
important network regulation mechanism on the tran-
scriptional level with a central regulatory component
is known as carbon catabolite repression (CCR). The
presence of different rapidly metabolized carbo-
hydrates leads to the sequential expression of different
specific sugar utilization systems. Glucose and fruc-
tose are preferentially metabolized. Therefore, a
change in the sugars used during fermentation or
food production will affect the concentration and
composition of the remaining nutrients and fer-
mentative products, thereby altering the taste of the
end product. In Bacillus CCR is essentially mediated
by the central regulatory component CcpA, after its
interaction with HPr (phosphorylated at serine pos-
ition 46 by an ATP-dependent HPr kinase). When it
is phosphorylated at a second site, namely histidine
position 15, HPr is one of the central components
in the phosphoenolpyruvate-dependent phospho-
transferase systems of sugar uptake systems, where it
transfers the phosphate from enzyme I to the sugar
permease. The component CcpA belongs to the LacI-
GalR regulator family. In the mediation of CCR for
transcriptional control, it acts as a repressor able to
BACILLUSIlntroduction 117
Flgure 2 Model of CcpA-dependent mediated carbon cat-
abolite repression in Bacillus. ATP, adenosine triphosphate; cre,
catabolite responsive element (DNA); El, enzyme I of the pho-
sphoenolpyruvate-dependent phosphotransferase system; Ell,
specific glucose permease; FDP, fructose diphosphate; His, his-
tidine residue; P, phosphoryl group; PEP, phosphoenolpyruvate;
Ser, serine residue.
bind cis-active DNA elements (cre)(Fig. 2) located in
a range of 200 base pairs downstream or upstream
from the transcriptional start sites of genes or operons
under catabolite control.
This newly discovered CCR mechanism differs
completely from the mechanism observed in Esch-
erichia coli, which presumably exists only in the
Enterobacteriaceae. The CcpA protein was detected
in eight different species of Bacillus, in Lactobacillus,
Lactococcus, Micrococcus, Mycobacterium,
Staphylococcus, Streptococcus and Streptomyces.
Therefore, it can be concluded that the CcpA-depend-
ent regulation of CCR is probably widespread in
Gram-positive bacteria and a universally used regu-
latory mechanism in bacteria.
Pathogenesis
Different variants of Bacillus thuringiensis, B. pop-
illiae, B. larvae, B. cereus, B. sphaericus and other
related species are pathogenic to insects. The use of
these strains for microbial insect control offers the
advantage of being safer than the more toxic chemical
control agents. Furthermore, they have relatively
slight effects on the ecological balance of the envir-
onment. The microbial insecticide comprises spores
and crystalline proteins which, when ingested by
larvae, cause gut paralysis, probably by upsetting the
ionic balance of the gut. The spore survives its passage
through the gut, penetrates the weakened midgut wall,
and multiplies in the haemolymph. Death results from
either intoxication or septicaemia. High selectivity
and the absence of harmful side effects on plants,
warm-blooded animals or humans give many of the
Bacillus products an advantage over other insecti-
cides. Several insect-specific pathogens are com-

118 BACILLUWlntroduction
mercially produced for use as microbial pesticides.
Bacillus thuringiensis produces insect larvicides. Cul-
tures of sporulated B. thuringiensis have been used
worldwide to control damage to crops, trees and
ornamental plants. During endospore formation, this
bacterium produces toxic protein crystals (Bt toxin)
that make it a good pesticide, which differs from B.
cereus (see below). Most of the toxin genes of B .
thuringiensis are located on conjugative plasmids,
which are transmissible by conjugation between B .
thuringiensis and B. cereus under laboratory con-
ditions. The resulting B. cereus transconjugants are
able to synthesize crystal proteins. Because the divid-
ing line between B . cereus and B. thuringiensis is so
dubious, these organisms could be considered to have
changed species to B. thuringiensis.
Bacillus popilliae causes a fatal illness called milky
disease in Japanese beetle larvae. After ingestion by
the larvae, B. popilliae germinates in the gut, begins
to multiply and invades the haemolymph. After about
10 days a typical milky appearance is observed due
to the massive numbers of bacteria.
Bacillus cereus strains are also often pathogenic
for insects. They produce phospholipase C, an ci-
exotoxin which permits the bacteria to pass through
the barrier of the intestinal epithelial cells. Subsequent
penetration into the haemolymph followed by multi-
plication kill the insect.
Certain strains of the soil saprophyte B . cereus are
also pathogenic for humans. Two toxins, one causing
diarrhoea and the other provoking vomiting, are pro-
duced in starchy foods, custards and dairy products
containing the common contaminant B. cereus.
Another member of the genus Bacillus with patho-
genic properties is B. anthracis. The three toxin genes
are located extrachromosomally on a large plasmid.
Bacillus anthracis causes the animal disease anthrax,
which spreads to humans primarily through minor
breaks in the skin or mucous membranes. Infection
sources can be milk, meat, wool or hairs from infected
animals. Cutaneous anthrax first appears as a papule
which develops into a vesicle and after 2-6 days into
a black eschar. This bacterium produces a potentially
lethal toxin and 5-20% of untreated cases are fatal.
Bacillus anthracis is similar in many respects to B.
cereus and B. thuringiensis. These taxa can be dis-
tinguished phenotypically by numerical taxonomy,
and there are some phenotypic tests such as sensitivity
to penicillin: B. cereus possesses a chromosomally
encoded p-lactamase, whereas B . anthracis is virtually
always penicillin-sensitive.
Next Page
Outlook
In the near future the demand of biotechnological
industry for new or improved products will lead to
the development of new genetically engineered strains
and new isolates from the environment, which will be
grouped in the genus Bacillus, increasing its enormous
phylogenetic diversity. The taxonomy of the genus
Bacillus is evolving; consequently, definitions for a
subdivision of the genus into phylogenetic and phene-
tic groups must be considered. Furthermore, the
genetic information about members of Bacillus will
dramatically increase. However, our knowledge of
chromosomal composition and the genes can only
give us a hint about their function when homologous
genes and proteins are known. Therefore, biochemical
research into protein functions will be a field of
increasing significance. In food production, as well as
in enzyme production involving bacilli, gene manipu-
lation will lead to strain constructions with new
(designed) features allowing an optimized and there-
fore less expensive production process. This can be
achieved by introducing and designing specific meta-
bolic pathways or heterologous enzyme over-
production.
See color Plate 2.
See also: Bacillus: Bacillus cereus: Bacillus stearo-
thermophilus; Bacillus anthracis; Bacillus subtilis: Detec-
tion of Toxins; Detection by Classical Cultural Techniques.
Further Reading
Cliver D (ed.) (1990) Food Borne Diseases. San Diego:
Academic Press.
Gerhardt P, Murray RGE, Wood WA and Krieg NR (eds)
(1994) Methods for General and Molecular Bac-
teriology. Washington: American Society for Micro-
biology Press.
Glick BR and Pasternak JJ (1998)Molecular Biotechnology,
2nd edn. Washington: American Society for Micro-
biology Press.
Hoch JA and Silhavy TJ (eds) (1995) Two Component
Signal Transduction. Washington: American Society for
Microbiology Press.
Hueck CJ and Hillen W (1995) Catabolite repression in
Bacillus subtilis: a global regulatory mechanism for the
Gram-positive bacteria? Molecular Microbiology 15(3):
39 5-40 1.
Kunst F, Ogasawara N, Moszer I et a1 (1997) The complete
genome sequence of the Gram-positive bacterium Bacil-
lus subtilis. Nature (London) 390: 249-256.
Piggot PJ, Moran CP and Youngman P (eds) (1994) Regu-
lation of Bacterial Differentiation. Washington: Ameri-
can Society for Microbiology Press.
Sneath PHA (ed.) (1982) Bergey’s Manual of Systematic
Bacteriology. Vol. 2. Baltimore: Williams & Wilkins.
 CAMPYLOBACTERAntroduction 335

Cakes see Confectionary Products: Cakes and Pastries.

Contents
Introduction
Detection by Cultural and Modern Techniques
Detection by Latex Agglutination Techniques

I Introduction Salmonella spp. in human gastroenteritis patients. In

these cases C. jejuni is the major species responsible,
M T Rowe and R H Madden, Department of with the incidence of C. coli being approximately
Agriculture for Northern Ireland and Queen’s University
l o % , but this figure may be higher depending on the
of Belfast, Northern Ireland
geographical location.
Copyright 0 1999 Academic Press
The incubation period for Campylobacter enteritis
is usually 1-7 days but can extend to 10 days. The
This article provides a synopsis of the current state of onset of symptoms is characterized by fever with
knowledge on those aspects of the genus Cam- confusion or delirium and general malaise followed
pylobacter which the authors consider relevant. These by severe abdominal cramping which in turn is fol-
include taxonomy, general physiology, ecology, lowed by profuse diarrhoea that becomes watery,
pathogenicity, typing of the genus, methods of control often containing blood and mucus. Although the diar-
and viable but non-culturable forms. It should be rhoea usually lasts for 2-7 days, abdominal pain and
appreciated that because of the relatively recent rec- cramping can persist for up to 3 months. Systemic
ognition of the importance of members of the genus as infection is uncommon but complications such as
food-poisoning agents and developments in molecular Guillain-Barr6 syndrome (GBS) and reactive arthri-
methods, the taxonomy of the genus, in particular, is tides (reactive arthritis and Reiter’s syndrome) can
evolving rapidly. arise. Fortunately, most Campylobacter enteritis cases
are self-limiting and fatality rates are low. The infect-
ive dose can be as low as 5-800 organisms with the
Introduction attack rate correlating with increasing dose.
Theodor Escherich in the 1880s made the first
recorded observation of spiral bacteria in the faeces
Guillain-Barre Syndrome
of patients with infantile diarrhoea but he was unsuc-
cessful in culturing them and regarded them as non- This disorder is rare and affects the peripheral nerves
pathogenic. The first isolation of Campylobacter spp. of the body. It can vary greatly in severity from the
was achieved by King in 1957 when he successfully mildest cases where clinical treatment is not sought
isolated ‘vibrios’ from blood samples of humans with to a complete paralysis that brings the patient close
diarrhoea. A major advance in the culture of these to death. Lipopolysaccharides isolated from C. jejuni
organisms was made by Martin Skirrow who devel- strains implicated in GBS have regions homologous
oped a selective medium that obviated the need for a to the human gangliosides GM1 and GDlb. The
laborious filtration stage, making possible the routine molecular mimicry between these regions may lead
isolation of these organisms. Currently Campylo- to autoimmunity and GBS. Miller-Fisher syndrome,
bacter spp. are isolated more frequently than similar to GBS, is an acute neuropathic disorder char-
336 CAMPYLOBACTERAntroduction

acterized by paralysis of the eye muscle, absence of General Physiology

reflexes and facial weakness.
Campylobacters have a distinctive morphology, being
Reactive Arthritides (Reactive Arthritis slender, spirally curvcd rods 0.2-0.5 km wide and
0.5-5 pm long. Species are highly motile by means of
and Reiter's Syndrome)
a single polar flagellum at one o r both ends, giving
Both syndromes are characterized by sterile inflam- rise to a characteristic corkscrew-like motion. The
mation of joints from infections originating from non- principal distinguishing feature of the physiology of
articular sites and are mediated by T cells. This may this genus is that they are microaerophilic with a
be triggered by enteritis involving Campylobacter spp. respiratory type of metabolism. Thus oxygen is
or other pathogens such as Salmonella or Yersinia. required for energy production but can only be tol-
Although viable organisms are not present, bacterial erated at levels below normal atmospheric pressure.
antigens are probably transported to the joints within This property was partly responsible for the genus
phagocytic cells. remaining undetected until relatively recently as it
could tolerate neither fully aerobic nor anaerobic con-
Taxonomy ditions, i.e. those normally employed to isolate organ-
The Campylobacter-like organisms are members of isms from animals and humans.
the family Spirillaceae, which comprises Gram-nega- Optimal oxygen concentrations have been quoted
tive curved rods and consists of the related genera as being 3-6% but media supplements can be used to
Anerobiospirillum, Arcobacter, Campylobacter and allow growth at higher concentrations, for example
Helicobacter. The unusual physiology of Campylo- FBP (ferrous sulphate, sodium metabisulphite and
bacter spp. led to them being discovered as a sig- sodium pyruvate) increases aerotolerance, allowing
nificant cause of food-borne disease only relatively growth at oxygen levels of 15-20%. The size and
recently. In 1984 Bergey's Manual listed five state of the inoculum will also dictate whcthcr growth
Campylobacter species; however, in 1996 a prob- in synthetic media takcs place with a heavy inoculum
ability matrix for the identification of campylobacters usually advised to ensure growth. Elevated levels of
(and related organisms) was published which listed COZ are also recommended with levels of 2-10%
eight more, with several subspecies also described. having been cited, and the growth of some species is
The use of DNA hybridization studies led to a dependent on the presence of hydrogen. Latterly it
major reorganization of campylobacters in 1991 with has been recommended that hydrogen is present in
the creation of the genus Arcobacter to which two the atmosphere used to incubate any clinical samples.
species of Campylobacter were assigned. At that time Despite their sensitivity to oxygen, C. jejuni and C.
11 species of Campylobacter were recognized and coli possess catalase, oxidase and superoxide dis-
both C. jejuni and C. coli were listed in the genus. It mutase activity; however, these enzymes appear to
is probable that more campylobacters are awaiting give limited protection from hydrogen peroxide and
discovery since techniques based on isolating and superoxide ions as shown by the increased growth
amplifying DNA from normal and pathological resulting from the addition of FBP supplement which
samples of the gastrointestinal tracts of animals and destroys these compounds. Blood also encourages
humans indicate the presence of species which have good growth and contains both catalase and super-
not yet been isolated in pure cultures. There is even a oxide dismutase.
possibility that campylobacters are present in humans Campylobacters are chemo-organotrophs which
as commensal organisms. neither ferment nor oxidize carbohydrates. Instead,
Differentiating between the two species most impli- energy is derived from either amino acids (aspartate
cated in food poisoning is usually simple since C. and glutamate can be utilized) o r tricarboxylic acid
jejuni can hydrolyse hippuric acid whilst C. coli (TCA)cycle intermediates. The amino acids are deam-
cannot. Thus the hippurate hydrolysis test is of great inated to provide TCA intermediates for subsequent
importance in food microbiology. Care must be exer- oxidation but n o complex molecules, such as proteins,
cised in its application, however, as inadequate buf- are utilized.
fering of the reaction mixture can lead to false- In terms of growth temperature, campylobacters
negative results. There have also been reports of hip- are mesophilic, as would be expected from their asso-
purate-negative C. jejuni isolates. Further inves- ciation with warm-blooded creatures. Growth tem-
tigations have shown the gene required for hippurate peratures range from about 25 to 45.S"C. The latter
hydrolysis to be present in such isolates but not to be temperature can be referred to as thermophilic in
transcribed. Thus genetic probes can help to identify medical circles and hence organisms growing at 42°C
these species correctly. are sometimes grouped under the general term of

thermophilic campylobacters. None of the genus arc
however true thermophiles.
Ecology
The normal habitats of Campylobacter spp. are
selected niches (intestinal tract, reproductive organs
and oral cavity) of warm-blooded animals. For those
organisms related to gastroenteritis the normal
habitat is the lower part of the gastrointestinal tract.
In this environment the organisms are exposed to
controlled temperatures in the range 37-41 "C and
hence the inability of campylobacters to grow below
30°C is of no consequence. The low oxygen tensions
found in the lumen of the gut mean that cam-
pylobacters do not require protective mechanisms to
counter the toxic effects of atmospheric levels of
oxygen, whilst the high nutrient levels are conducive
to the proliferation of these highly fastidious
organisms.
Campylobacters do not persist in the environment
due to their limited defences against oxygen, relatively
high minimum growth temperature and complex
nutritional requirements. Their spread is therefore
most likely to be by oral-faecal contamination. In the
case of foodstuffs they are relatively sensitive to heat,
hence normal cooking will kill them and transmission
will therefore be due to underprocessing or raw-
cooked contamination.
Species Other than C. jejuni and C. coli
Campylobacter upsaliensis
This species can be carried by healthy puppies and
kittens. In a survey of ribotypes isolated from humans
and dogs, the human strains were found to possess
a unique 16s ribotype and carried plasmids more
frequently than did canine strains. Besides human
enteritis, strains can cause abortion, bacteraemia and
haemolytic-uraemic syndrome. Transmission is much
more likely to be via contact with domestic pets rather
than food-borne.
Campylobacter sputorum
C. sputorum has been isolated from dairy cows and
calves. Although this species has not been associated
with human enteritis, it has been implicated in peri-
odontitis. Three biovars have been proposed and their
characteristic biochemical tests are as follows:
0 C. sputorum biovar sputorum: catalase-negative
C. sputorum biovar fueculis: catalase-positive
0 C. sputorum biovar puruureolyticus: urease-posi-
tive.
CAMPYLOBACTERAntroduction 337
If this proposal is adopted then this would require
the unification of biovars sputorum and bubulus into
biovar sputorum and the biovar name bubulus would
be discarded.
Campylobacter concisus
C. concisus has mainly been associated with peri-
odontitis but has been isolated from stool samples of
children suffering from enteritis. However, in one
study of children with and without enteritis there
was no significant difference in isolation rates of C.
concisus between the groups. Specific primers based
on 23s rDNA have been developed for this species.
Campylobacter fetus
This species has two subspecies: fetus and venerealis.
Both are primarily associated with animals. Although
better known as a cause of sporadic abortion in cattle,
they are also a rare cause of human disease. The
presence in the blood stream of C. fetus subsp. fetus
may be associated with cancer. Primers common to
both subspecies have been identified.
Campylobacter gracilis
C. gracilis may be involved in periodontal disease and
has been shown to cause infections in dental implants.
No conclusive evidence exists that it is a cause of
human enteritis.
Campylobacter helveticus
This species has been isolated from the faeces of
domestic cats and dogs. No conclusive evidence exists
that it is involved in human disease. A species-specific
recombinant DNA probe has been developed to help
determine its pathogenicity.
Campylobacter hyointestinalis
Two subspecies exist for this species: hyointestinulis
and lawsonii. The subspecies may be differentiated by
phenotypic tests, whole-cell protein and macro-
restriction profiling. Although this species was first
considered to be a pathogen of pigs, it has now been
established as an occasional human pathogen.
Campylobacter lari
This species has been isolated from mussels and
oysters. It is presumed that the shellfish become con-
taminated by the faeces of gulls feeding in the growing
waters. In one survey relatively extensive DNA poly-
morphisms were found within a single batch of shell-
338 CAMPYLOBACTERAntroduction
fish, indicating a high degree of genetic diversity
within the species. It has only been infrequently asso-
ciated with human enteritis and bacteraemia but there
is one published case where it was deemed to have
caused chronic diarrhoea and bacteraemia in a
neonate. Reactive arthritis may be a complication
following enteritis. Unique polymerase chain reaction
(PCR) primers for C. lari have been identified based
on a 23s rDNA sequence.
Pathogenicity
Campylobacter spp. can express virulence either dir-
ectly, by invasion of the epithelial cells of the gut and
releasing toxin or indirectly, by inducing an inflam-
matory response. Like many pathogens, the patho-
logical changes can be multifactorial in nature, with
a combination of determinants being involved. The
main factors described are motility, adhesion, inva-
sion, iron acquisition and toxin production.
C. jejuni has a polar flagellum (at one or both ends
of the cell) and is capable of rapid motility which,
when combined with a spiral morphology, gives the
organism a selective advantage in penetrating and
colonizing the thick viscous mucus barrier of intes-
tinal cells. Campylobacter spp. also exhibit chemo-
taxis regulated by the cheY gene. The flagella are
highly immunogcnic and can undergo both phase and
antigenic variations which help them to evadc the
immune response of the host. Two flagella genes have
been identified: flaA and flaB which are in a tandem
chromosomal arrangement separated by a short inter-
vening sequence. The flaA and flaB genes are of
approximately equal size (1.7kbp) with predicted
molecular masses of 59 588 and 59 909 respectively.
Flagella, in particular flagella with type A flagellin, do
appear to be necessary for invasion and intern-
alization but flagella may or may not act as adhesion
factors.
Carbohydrate moieties, probably a glycoprotein,
have been shown to be important for adhesion since
pre-treatment with L-fucose and D-mannose inhibits
adherence to INT 4 0 7 epithelial monolayers. A variety
of outer membrane proteins have also been described
that bind to eukaryotic cells. Campylohacter spp.
may also possess fimbriae (4-7nm in width) whose
synthesis is enhanced by bile salts. Non-fimbriated
mutants, however, are still able to adhere to and
invade INT 407 cells and colonize ferrets but with
ameliorated disease symptoms, which suggests some
role in virulence.
Although evidence of epithelial cell invasion in vivo
is sparse host cell invasion, which occurs within a
very short time period, has been observed experi-
mentally in macaque monkeys and in the colon of
patients. In addition to epithelial cell invasion the
organism may overcome the gut barrier by trans-
location (passing between cells) possibly via M cells.
Certainly Campylobacter spp. have been observed to
associate preferentially to intercellular junctions. The
ability to invade is strain-dependent. Using Hep-2
cells, n o correlation was found between invasiveness
and the type of symptoms observed, showing that
host factors such as immune status are important.
Campylobacter spp. d o not exhibit a positive Sereny
test and show variability in their ability to invade a
range of tissue cell lines, although invasion has been
shown to be more efficient when cells of human origin
are used.
Campylobacter spp. require iron for normal cell
division since in the absence of the metal ion cells
elongate, lack septa and become filamentous. C. jejuni
and C. coli d o not produce siderophores (iron chel-
ating agents) but are capable of utilizing siderophores
produced by other microorganisms, including entero-
bactin and ferrichrome. A transport system encoded
by the ceu operon may be involved in this process.
Most strains of C. jejuni and C. coli produce a cell-
associated haemolysin and possess a mechanism to
transport haemolysis products into the cell and release
iron from the complexes. C. jejuni also elaborates the
iron storage protein ferritin which helps protect the
bacterium against iron overload which may result in
iron-catalysed damagc to cellular components. The
organism also elaborates an iron-containing super-
oxide dismutase (SOD) which provides protection
against oxidative stress during invasion. C. jejuni pos-
sesses a fur gene which, if similar to fur genes in other
bacteria, is involved in the synthesis of SODS and
outer membrane siderophore receptors as well as
regulating other genes involved in pathogenesis.
The reported frequency of enterotoxin elaboration
amongst isolates varies greatly (Table 1). Differences
in methodology and in parameters, such as the
number of passages, strain storage and culture con-
ditions and polymyxin R treatment, probably
contribute towards this observed variation. Cam-
pylobacter appear to produce several types of cyto-
toxins, including a Shiga-like toxin (SLT), a cytolethal
distending toxin (CLDT), C. jejuni toxin (CJT; similar
to Vibrio cholerae toxin) and heat-labile toxin (similar
to Escherichia coli LT toxin). Some of the cytotoxins
which have a molecular weight range of 50000-
70000 are as yet poorly characterized but on tissue
culture cell lines the effects include cell rounding with
nuclear condensation, loss of cell monolayer adhe-
siveness and cell death within 24-48h. Cam-
pylobacter produce a cytotoxin (SLT)similar to Shiga
toxin, as evidenced by its neutralization by Shiga toxin
antibody and by a monoclonal antibody against the
 CAMPYLOBACTERAntroduction 339

Table 1 Incidence of enterotoxin production among strains of Typing of Campylobacter spp.

Campylobacter jejuni and C. coli
Number and origin of Enterotoxin- Method of detection
The identification of specific subspecies of pathogens
strains positive (%) is essential for effective epidemiology. However,
biotyping of campylobacters is restricted by their
25 clinical, Belgium 100 CHO
62 clinical, US 94 GM, ELISA"
limited range of biochemical activities, although
22 clinical, South Africa 77 Y-1 schemes were devised and applied. Serotyping has
44 clinical, Belgium 75 CHO also been developed and the method of Penner, based
32 clinical, Mexico 65 CHO, RlLT on heat-stable antigens, has been most widely applied.
80 clinical, Algeria 65 CHO However, this method has been limited by the avail-
12 clinical, diverse origin 50 CHO, GMi ELISA,
RlLT
ability of antisera and is generally only used by public
316 clinical, Canada 48 CHO, Y-1 health laboratories which have facilities to raise their
44 clinical, Costa Rica 47 Y- 1 own antisera. Coincidentally, the increasing import-
372 clinical, diverse origin 45 CHO ance of Campylobacter spp. as food-poisoning patho-
39 clinical, US 36 GM, ELlSA
gens occurred at a time when methods of genotyping
202 clinical, Sweden 3211gb CHO, GMi ELISAb
22 clinical, India 32 CHO, GM, ELlSA were becoming more widely available and, in fact,
22 clinical, US 0 CHO, GM1 ELISA the genomic sequence of C. jejuni has been recently
15 clinical, US 0 CHO, GM, ELlSA published.
47 carriers, India 12 CHO, GMi ELISA The lack of an established biotyping scheme for C.
30 carriers, Algeria 60 CHO
6 carriers, Mexico 16 CHO, RlLT
jejuni led to genotyping methods being applied and,
8 carriers, Mexico 0 CHO as methods evolved, or were invented, they have been
77 carriers, India 0 CHO, GM, ELISA, applied to investigate specific aspects of the epi-
RlLT demiology of this species. As with the development of
CHO, Elongation of Chinese hamster ovary cells; GM, ELISA, all tools, the ultimate aim of a given investigation will
ELISA with ganglioside GM, as the solid phase and antiserum determine which genotyping method is selected. Thus
against enterotoxin CT or LT as primary antiserum; Y-1, rounding a IJK Department of Health investigation into appro-
of mouse adrenal tumour cells; RILT, fluid accumulation in the priate methods of subtyping campylobacters aimed to
rat ileal loop test.
'The primary antiserum used with homologous serum against
define the most appropriate methods for use in clinical
enterotoxin of C. jejuni. laboratories. The group concluded that a two-stage
bDifferent results were obtained with the two methods. Adapted process was best, with biotyping used to screen incom-
from Wassenaar (1997). ing isolates into groups (using biochemical reactions
and Penner serotyping), then pulse-field gel elec-
B subunit of E. coli SLT-1. The titre produced by trophoresis (PFGE)being applied to define the specific
Campylobacter is however c 1000-fold less than that subtype.
produced by Shigella dysenteriae or E. coli 0157. In PFGE is based on the entire genome of the target
Chinese hamster ovary cells the CLDT of Cam- species being released from cells held in a gel and then
pylobacter caused accumulation of F-actin assemblies digested by a rare-cutting restriction enzyme in situ.
which resembled actin stress fibres and this was The very large fragments of DNA require the appli-
accompanied by a block in cell division which in vivo cation of an oscillating electrical field to migrate
would result in cytokinesis. Strains of C. jejuni have through the agarose gel and the subtyping is based
been shown to produce significantly more CLDT than on the pattern of DNA fragments which have their
C. coli. Published research on C. jejuni toxin currently molecular weight estimated from a comparison with
presents a confusing picture, particularly as regards a molecular-weight standard included in each gel.
its pathogenesis, chemical structure and genetic basis. The latter analysis can take place using commercial
It is however closely related to cholera toxin and LT software which analyses images of the gel patterns
toxin of E. coli since it can be neutralized by anti- and can subsequently produce similarity dendrograms
bodies raised against the latter two toxins and also based on numerical taxonomy principles.
by the fact that it binds strongly to the ganglioside However, PFGE is a relatively slow process: the
GM1 on target cells. Evidence from molecular studies electrophoresis takes about 24 h to complete, and for
suggests that all C. jejuni strains possess the toxin a full analysis it is recommended to use digestion with
gene but that it is not always expressed. A further more than one restriction enzyme. Hence, analytical
toxin, proteinaceous in nature, has been shown to methods aimed at specific regions of the genome have
be elaborated by C. jejuni and C. coli which causes been applied, such as ribotyping and PCR restriction
elongation of Vero and Hep-2 cells and rounding of fragment length polymorphism (PCR-RFLP). PCR-
CHO cells. RFLP typing has been applied based on ribosomal

340 CAMPYLOBACTERAntroduction
Table 2 Overview of typing methods applied to Campylobacter spp.
Method Advantages
Biotyping Simple, cheap, quick
Phage typing Relatively simple, reasonable discrimination
Serotyping Relatively simple, discrimination can be good
Flagellin typing Fast and uses widely available reagents
Pulsed-field gel Highly discriminatory
electrophoresis
Automated ribotyping Fast, intermediate level of discrimination
Amplified fragment length Fast, level of discrimination can be defined by
polymorphism analysis primers
RNA genes and also targeting the flagellin gene, of
which there are two copies, pa A and pa B. Whilst
individual groups report successes with this method,
the long-term stability of flagellin types has been
called into question.
An overview of some benefits and drawbacks of
the typing methods applied to Campylobacter spp. is
presented in Table 2.
Methods of Control
Pets, water and improperly handled and cooked foods
account for most cases of Campylobacter enteritis.
Untreated water and unpasteurized milk have been
responsible for those outbreaks with large numbers
of associated cases. However, undercooked poultry
products have mainly been responsible for the large
numbers of sporadic cases of campylobacteriosis. It
is probably in this latter area where adequate control
strategies still require most research effort. Since C.
jejuni is often found in high numbers (> 104cfu)per
processed carcass, perhaps the best approach is to
concentrate on devising methods which will ensure
that the birds arrive at the processing plant with
significantly reduced Campylobacter contamination.
This focuses attention on the rearing conditions. Cam-
pylobacter is rarely found in poultry feed or the hatch-
ery environment and generally colonizes the chicks
only after the second or third week. The most likely
vectors are flies, wild birds, rodents or contaminated
water. Three main strategies are currently being
employed: drinking water quality, vaccination and
competitive exclusion. Certainly some authors
contend that disinfection of drinking water is likely
to have the greatest impact on the prevalence of Cam-
pylobacter spp. Passive immunization of chicks has
resulted in reduced colonization by the organism but
the cost-effectiveness of this approach still has to
be determined. Competitive exclusion involves the
administration early in the chick’s life of a cocktail of
organisms that prevents subsequent colonization of
the bird when challenged with Campylobacter spp. A
Next Page
Disadvantages
Low discrimination
Phage sets not widely available
Antisera not widely available
Interlaboratory comparison of types difficult, long-
term stability of types in question
Slow, specific equipment required, interlab
comparison of types difficult
High capital and running costs
Needs expensive capital equipment, method still
undergoing development
three-strain mixture comprising Klebsiella pnew
moniae, Citrobacter diversus and Escherichia coli
(013:H-) provided 43-100% (average 78%)
protection.
Viable but Nan-culturable Forms
Campylobacter cells, in common with other genera
such as Vibrio, Salmonella and Shigella, have been
shown to metamorphose into a viable but non-cul-
turable (VNC) state when subjected to unfavourable
conditions such as would be experienced in water,
which generally has a low nutrient status. With Cam-
pylobacter the cells transform from a motile spiral
form to a coccoid VNC form which is incapable of
cell division in normal media entirely suitable for the
normal culturable form.
If the VNC form of Campylobacter is capable of
initiating an infection in humans or colonizing the gut
of domestic animals and poultry or indirectly via food
contact surfaces, then contaminated water must pose
a risk. There is still much controversy over the infect-
ivity of VNC Campylobacter cells. It must be stated
that such a phenomenon has been found with Vibrio
cholerae and other related enteric water-borne
pathogenic bacteria. Such authors suggest that the
VNC form is a degenerative state and that there
is a continuum of physiological states, with one
extreme being highly culturable and the other dead
cells. The VNC state is between these but tending
towards the latter state. Certainly more research
is needed to elucidate the role, if any, of the
VNC formof Campylobacter in the transmission of
disease and colonization of domestic animals and
birds.
See also: Campylobacter: Detection by Cultural and
Modern Techniques: Detection by Latex Agglutination
Techniques. Food Poisoning Outbreaks. Milk and
Milk Products: Microbiology of Liquid Milk.
 DEBARYOMYCES 515

D
Dairy Products see Brucella: Problems with Dairy Products; Cheese: In the Market Place; Microbiology
of Cheese-making and Maturation; Mould-ripened Varieties; Role of Specific Groups of Bacteria; Microflora of
White-brined Cheeses; Fermented Milks: Yoghurt; Products from Northern Europe; Products of Eastern Europe
and Asia; Probiotic Bacteria: Detection and estimation in fermented and non-fermented dairy products.

DEBARYOMYCES
W Praphailong, National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand
G H Fleet, Department of Food Science and Technology, The University of New South Wales, Sydney, Australia
Copyright 0 1999 Academic Press

Characteristics of the Genus and is negative. Lipid composition is characterized by the

Relevant Species
Species of Debaryomyces are commonly found in
soils, waters, plants, foods and clinical specimens.
Present taxonomic classification accepts 15 species
within the genus (Table l), although an additional
species Debaryomyces prosopidis has been proposed.
Debaryomyces hansenii (imperfect form Candida
famata) is, by far, the most significant species found in
foods. Of the other species, there are only occasional
reports on the isolation of D. polymorphus, D. etch-
ellsii, D. maramus and D. carsonii from foods. Species
of the genus undergo asexual reproduction by multi-
lateral budding, with cells occurring singly, in pairs,
short chains or small clusters. Pseudomycelium is
usually lacking, but primitive or even well-developed
pseudohyphae may be produced in some species.
Sexual reproduction characteristically occurs by con-
jugation between a mother cell and its bud, but occa-
sionally conjugation between separated cells is
observed. Variation in the morphology and number
of ascospores per ascus provides a good criterion for
differentiation between the species. Ascospores are
usually spheroidal to ovoidal in shape and are often
distinguished by a warty or roughened surface. The
ascospores of some species (e.g. D. occidentalis)have
a distinct equatorial ledge. The number of ascospores
per ascus varies from one to four depending on the
species and, with the exception of three species, they
are not usually liberated from the ascus.
The ability to ferment sugars varies from an absent,
weak to vigorous reaction, nitrate is not assimilated
but strains of some species assimilate nitrite. Ubiqui-
none Q-9 is present and the diazonium blue B reaction
presence of linoleic (C18 : 2) and linolenic (C18 :3 )
fatty acids. The mol% G+C content is in the range of
33-43%. Karyotyping of the genus is not complete,
but three to seven chromosomes arc generally present
within the species.
Inclusion of species within the genus has undergone
significant revision over the years. The first major
description of the genus by Lodder and Kreger-van
Rij in 1952 included only five species, namely D.
hansenii, D. kloeckeri, D. subglobosus, D. nicotianae
and D. vini. Lodder’s 1970 classification, based
largely on fermentation and assimilation tests, com-
bined the first four of these species into one species
D. hansenii, and introduced seven new species, D.
castellii, D. coudertii, D. marama, D. phaffii, D. can-
tarellii, D. tamarii and D. vanriji. Price and co-
workers in 1978 proposed a major revision of species
within the genus after a detailed study of DNA
sequence similarity by reassociation/hybridization
kinetics. In particular, they showed that several species
of Pichia were related to some Debaryomyces species.
Consequently, D. cantarellii and D. phaffii were
merged with Pichia polymorpha to become D. poly-
morphus. Pichia pseudopolymorpha became D.
pseudopolymorphus. Using data from partial sequen-
cing of ribosomal RNA subunits, Kurtzman and co-
workers and Yamada and co-workers have further
refined the description of the genus, and this has given
the current recognition of 15 species (Table 1). A
notable outcome from these studies was the close
similarity of Schwanniomyces occidentalis with Deba-
ryomyces species and its redefinition as D. occi-
dentalis with two varieties. However, some authors
do not agree with classification of Schwanniomyces
Table 1 Key properties" of species within the genus Debaryornyces

Species Mol% Fermentation Assimilation Ascospores

G+C 37°C NaCl Vit G su Tr su La Me Ra XY GI Er Shape NurnbeP LibC

(1 0%)
D. carsonii 36.8- + + - + V V + Spheroidal 1-4 +
39.7
D. castellii 37.1 - + - + + + + + Spheroidal 1-3(1)
D. coudertii 37.4 - + - - - - + + Spheroidal 1
D. etchellsii 38.5- + + - X wl- + + - Spheroidal 1-4
40.6
D. hansenii
var. hansenii 37.3- - + - wl- wl- wl- + V V + + X V Spheroidal 1-2(1)
38.6
var. fabryi 36.4- + + - wl- wl- wl- + V V + + X V Spheroidal 1-2(1)
36.8
D. rnararnus 39.1 - + - wl- + V V S + + + Ovoidal 142)
D. melissophilus 39.8 - + - - + - - - - + + Spheroidal 1-4
D. nepalensis 37.6- - + - wl- + V + + + + + Spheroidal 1
38.0
D. occidentalis
var. 35.2 + - - + + + V V + + + Spheroidal (L) 1-2(1)
occidentalis
var.persoonii 35.4 + - - + + + + V + - + - Spheroidal (L) 1-2(1)
D.polyrnotphus 35.7- + + + S S + V V + + + + Spheroidal 1-2(1)
35.9
D. 35.7 - + - + S + + + + + + + Spheroidal 1-4
pseudopolyrnorphus
D. robertsiae 42.7 - + + + + + + - + + + + Spheroidal 1-4 -
D. udenii 35.8 - + - ws wl- wl- + + + + + + Spheroidal 1-4 +
D. vanrijiae
var. vanrijiae 33.2- + + + wl- wl- + + + + + + Spheroidal 1-4
33.3
var. yarrowii 33.0 - + + wl- + + + + V Spheroidal 14
D. yarnadae 34.5 - + - S + + + W - Spheroidal 1-4
D. prosopidis 37-38 + + - - + + + + + Globose 1-2

Table adapted from Nakase et al(1998) with permission from Elsevier Science.
"Abbreviations: 37"C, growth at 37°C; NaCl (lo%), growth in 10% NaC1+5% glucose; Vit, growth in vitamin-free medium; G, glucose; Su, sucrose; Tr, trehalose; La, lactose; Me,
melibiose; Ra, raffinose; Xy, D-xylose; GI, gluconate; Er, erythritol; +, positive; s, positive but slow; x, positive or weak; w, weak; ws, weak and slow; wl-, weak or negative; v, variable;
-, negative.
bNumbersof ascospores per ascus; numbers in parentheses refer to the number of ascospores most frequently observed. (L) indicates ascospores with equatorial ledge.
"Ascospores liberated by lysis of asci.
 DEBARYOMYCES 51 7

occidentalis within the genus Debaryomyces because membrane bound ATPase that accomplishes an effect-
it has some quite distinct phenotypic properties. Also ive extrusion of Na- ions.
K'ingea robertsii was described as D. robertsiae. With the exception of D.hansenii, little is known
Other changes were the description of two former about the environmental factors which limit the
species of Pichia as D. carsonii and D. etschellsii and growth of species listed in Table 1. Other than D .
the removal of D. tamarii. The key properties that occidentalis, all grow in the presence of 10% NaCI.
differentiate species within the genus are shown in D. hansenii and D. etchellsii, at least, are also tolerant
Table 1. of very high concentrations of sugars and grow in the
presence of 60% (wh) sucrose. Growth of D. hansenii
is very weak at p H 2 . 5 but strong in the p H range
3.0-8.0. Many authors have made the qualitative
Physiological and Biochemical Properties
observation that D. hansenii exhibits faster growth at
Of the 15 species in the genus, only Debaryomyces l-5OC compared with other yeast species, and there
hansenii and D. occidentalis have attracted significant is a report of growth at -12.5"C. D.hansenii is not
study of their physiological, biochemical and molecu- particularly tolerant of preservatives or heat treat-
lar properties. These studies reflect the substantial ment. It is inhibited at p H 5 . 5 by 250-500mg1-' of
diversity in growth and metabolic behaviour of yeasts benzoic or sorbic acids and has a D value of 1 2 m i n
within the genus. at 48". Some strains have a strong tendency to floc-
Debaryomyces hansenii is considered to be non- culate and this could be a potential survival mech-
fermentative. It metabolizes sugars to pyruvate by the anism in hostile environments.
Embden-Meyerhof-Parnas (EMP) pathway and then In contrast t o D. hansenii, D. occidentalis is a
oxidizes pyruvate through the tricarboxylic acid vigorous fermenter of sugars under non-aerated con-
(TCA) cycle. Organic acids such as citric, lactic and ditions. It is a Crabtree negative yeast and, under
succinic are assimilated through the TCA cycle. The aerated conditions, it channels the sugars into the
pentose phosphate pathway also operates in this TCA cycle. Unlike D.hansenii, this species is not
yeast. Contrary to the general view, there are reports particularly tolerant of high s;lt or high sugar envir-
of some strains of D . hansenii and C. famata that onments. The most distinctive property of D. occi-
ferment glucose and other hexoses. Extracellular pro- dentalis is its efficient degradation of starch by the
tease and lipase production have been reported in production of extracellular a-amylases and a gluco-
some but not all strains. These enzymes have not been amylase that can by-pass the a-(l+b)-linked branch
isolated and characterized. Amylolytic and pec- points in amylopectin. Because of this property, there
tinolytic activities are absent. The most distinguishing has been substantial scientific and industrial interest
feature of D. hansenii is its ability to grow in the in this yeast. The kinetics of production and properties
presence of extremely high concentrations of salt of these amylolytic enzymes have been well char-
(KaC1).Although the growth response to NaCl varies acterized and their genes have been cloned and
with the strain, most grow in the presence of 15% sequenced. Techniques for manipulating the expres-
(wiv) NaCl and there are some strains that grow at sion of these genes and for transferring them to other
20-24% (wiv) SaC1. High salt tolerance has also yeast species have been developed.
been reported for D. etchellsii. Salt tolerance of D.
hansenii is greatest at p H values near 5.0 and
Significance in Foods
decreases at p H 3.0 and p H 7.0. The molecular basis
of salt tolerance in D.hansenii has been extensively Literature on the occurrence of Debaryomyces species
studied and is related to the ability of this yeast to in foods is largely unfocused and scattered over many
accumulate high intracellular concentrations of gly- years. It is difficult to track because of the numerous
cerol as an osmo-protectant or compatible solute. changes of name of the species. Most studies concern
Substantial amounts of this glycerol are excreted into D . hansenii and there are only occasional reports on
the extracellular medium, especially during the sta- the occurrence and significance of other species, such
tionary phase, but it is re-utilized when glucose sub- as D. etschellsii, D. polymorphus, D . maramus and
strate is exhausted. The pathway of glycerol D. carsonii in foods (Table 2). There is no reason to
production has been studied and it originates from explain why the other species listed in Table 1 (e.g.
glucose by the EMP pathway. Intracellular arabitol is D. occidentalis) are not found in food ecosystems,
also accumulated and excreted, but its production (via but more systematic and focused study will probably
the pentose phosphate pathway) appears constitutive reveal their presence.
and occurs in the absence of salt stress. It has been The early literature reveals the frequent isolation of
suggested that D. hansenii also has an appropriate D. hansenii from meat products, especially processed
51 8 DEBARYOMYCES

Table 2 Significance of Debaryomyces species in the food and beverage industries

Speoes
Debaryomyces hansenii (Candida
famata)
Debaryomyces etscheilsii
Debaryomyces polymorphus
Debaryomyces maramus
Debaryomyces carsonii
Debaryomyces occidentaiis
Significance
Occurrencelspoiiage: delicatessen, cured, fermented, minced meats; seafoods, fish sauces;
yoghurts, cheeses; brined vegetables; mayonnaise-based salads; silage
Biotechnological: starter cultures for meat sausage fermentation; starter cultures for maturation
of cheeses; biocontrol agent of bacterial and fungal spoilage; xylitol production
Occurrencelspoiiage: carbonated soft drinks; sugar syrups; brined vegetables: mayonnaise-
based salads, soy sauce; fermented meat products
Occurrencelspoiiage: carbonated soft drinks; fruit products delicatessen, cured and fermented
meats
Occurrencelspoiiage: meat products; cheese
Spoilage: salted fish paste
6iotechno/ogicai: amylase production; waste utilization; single-cell protein

products, such as frankfurters, bacon, hams and fer-
mented and unfermented sausages. In some cases,
presence of the yeast was associated with the devel-
opment of a slimy surface layer on the product.
Recent, more extensive studies have confirmed the
predominance of D. hansenii in meat products com-
pared with other yeasts, and these conclusions have
been extended to include seafoods such as fresh fish.
Populations in the range 104-10hcfu g-' (or even
higher in fermented salami) are often reported. D.
etschellsii, D. polymorphus and D . maramus are also
found in these products, but less frequently. The
impact of this yeast growth on the flavour of meat
products is not clear, but cannot be assumed to be
negative. Indeed, there is a positive correlation
between the desired flavour of some Italian salami
sausages and the presence of D. hansenii. Some, but
not all, of the strains of D.hansenii isolated from
meat products produce extracellular proteases and
lipases that could contribute to flavour development
by the breakdown of meat proteins and fats. The
ability of these enzymes to operate well at low p H
may be an appealing property. Consequently, con-
sideration has been given to the use of selected strains
of D. hansenii as starter cultures in the production of
fermented sausages. Factors thought to select for the
growth of D. hansenii in meat products include its
tolerance of salt, utilization of organic acids (e.g.
lactic), protease and lipase production, good growth
at low temperatures, and the ability of some strains
to utilize sodium nitrite which is added as a curing
agent in some products.
D. hanseniilC. famata have now emerged as the
most important yeasts in the dairy industry. Weakly
fermenting species have been linked to the spoilage of
yoghurts, but their greatest significance is in cheese
production, especially with the mould-ripened soft
cheeses such as Camembert, Brie and blue-veined var-
ieties. Many surveys of these and other types of
cheeses have revealed a consistently high incidence of
D. hansenii, often at populations of 106-10'cfug~' or
higher. The yeast originates as a natural contaminant
of the cheese brine and grows at both the outer and
inner parts of the cheese curd during the maturation
stage. Again, the ability of the yeast to tolerate the
high salt environment of the cheese, utilization of
lactic acid, protease and lipase production, growth at
low temperature and, possibly, production of polyols
such as glycerol are key factors that favour its growth
and contribution to the biochemistry of cheese mat-
uration. A clear link between such activity and a
sensory outcome remains to be established, but the
relationship is assumed to be positive since com-
mercial starter cultures of D . hansenii are available
for encouraging the maturation process. An important
property of these strains might be the ability of their
proteases and lipases to operate at high salt con-
centrations and low temperatures.
Debaryomyces species, especially D. etschellsii, are
frequently isolated from brines used to ferment prod-
ucts such as olives and cucumbers, and they are also
associated with traditional Japanese fermented prod-
ucts such as soy sauce and miso. Curiously, a high
proportion of killer strains of D. hansenii with broad-
spectrum killer activity has been isolated from the
latter ecosystems. Presumably, these yeasts grow on
the surface of brine solutions, utilizing lactic acid
produced by the lactic acid bacteria involved in these
fermentations. However, some strains of D . etschellsii
also ferment sugars. This latter property may also
explain the occasional association of D. etschellsii
with the spoilage of high sugar syrups. There are
occasional reports of the isolation of Debavyomyces
species from soft drinks, beer, wine and vegetable
salads but, generally they are not significant spoilers
of these products. An unusual but significant form of
spoilage has been reported for D . cavsonii, which
grew in a Japanese chickuwa fish paste, transforming
trans-cinnamic acid to styrene which gave the product
an unacceptable petroleum-like aroma.
4 s noted already, the association of D. hansenii
with foods does not necessarily have negative impli-

cations and there is significant interest in exploiting
this species as a starter culture in meat and cheese
production. In the case of cheese production, it has
been reported to have good biocontrol over spoilage
species of Clostridium. Also in the context of bio-
control, several papers in the late 1980s suggested
that D.hansenii was an effective natural antagonist
for controlling the fungal spoilage of various fruits by
species of Penicillium, Botrytis and Rhizopus.
However, later study revealed that the yeast is an
unusual strain of Candida guilliermondii and not D.
hansenii. Nevertheless, this work has stimulated inter-
est in the yeast as a potential novcl biocontrol agent.
In another industrial application, D. hansenii has
potential value in bioconversion of xylose into the
sweetener, xylitol. The enzymes, xylose reductase and
xylitol dehydrogenase, associated with this process
have been examined.
The amylases of D . occidentalis have potential
application in the production of sugar syrups for food
and beverage processing. The genes for these amylases
have been incorporated into brewing strains of S.
cerevisiae for the purpose of using these strains in the
production of low calorie or dextrin-free beers. D.
occidentalis could be used to process starchy waste
material into single-cell protein.
Debaryomyces spp. are not generally regarded as
pathogenic to humans and no food-borne disease out-
breaks have been attributed to these organisms.
However, D. hanseniilC. famata have been implicated
in isolated cases of septicaemia and skin and mucosal
surface infections where they are considered as weak
opportunistic pathogens, especially for immuno-
compromised patients.
Enumeration and Identification
Food sample ( l o g ) is suspended in 90ml of 0.1%
peptone water, homogenized for approximately 1min
and then diluted 10-fold, as necessary, in 0.1%
peptone water. Aliquots (0.1ml) of the dilution are
then spread inoculated over the surface of plates of
media such as malt extract agar, glucose-yeast extract
agar or tryptone glucose yeast extract agar. Bacterial
antibiotics, such as chloramphenicol, oxytetracycline,
chlorotetracycline, gentamicin and streptomycin can
be added to these media at concentrations up to
100 pgml-' to suppress bacterial growth. For the isol-
ation of Debaryomyces from products like cheese,
overgrowth of moulds (Penicilliumspp.) on the plates
can occur. Incorporation of the mould inhibitor,
biphenyl (50 mg 1-') into the medium can overcome
this problem. Plates are incubated a t 25°C for 4-7
days and yeast colonies counted. Virtually all yeast
species will grow on the media just described. There-
Next Page
DEBARYOMYCES 519
fore, it will be necessary to isolate and identify indi-
vidual colonies. The identification of Debaryomyces
spp. follows standard morphological biochemical and
physiological tests and keys as outlined in The Yeasts,
a Taxonomic Study, 4th edition, edited by CP Kurtz-
man and JW Fell, Elsevier Science (1998) (Table 1).
D.hanseniilc. famata, a t least, identifies very well in
the rapid computer-based Biolog (Biolog Inc
California) and ATB 32C (bioMtrieux) systems that
incorporate a range of these tests in kit form.
To avoid potential osmotic shock and stress, it has
been suggested that 5-10% NaCl be included in the
dilucnt and plating medium when isolating these
yeasts from high salt foods. However, wc and others
have not found these steps to offer any benefit.
No selective or differential media have been
reported for these yeasts. However, inclusion of 10-
1 5 % (wlv) NaCl into the medium formulation could
assist in selecting for the growth of these species,
except D. occidentalis. A differential medium based
on the hydrolysis of starch could be developed for the
isolation of D. occidentalis.
A PCR method that differentiates D.hanseniilC.
famata from Candida guilliermondii has been
reported and is based on amplification of the large
subunit rDNA between base positions 402 and 669.
A D. hansenii nucleic acid probe based on sequences
in the 18s rRNA has been reported. As yet, neither
of those molecular methods has been developed for
routine use.
See also: Fermented Foods: Fermentations of the Far
East. Fermented Milks: Yoghurt. Meat and Poultry:
Spoilage of Cooked Meats and Meat Products.
Further Reading
Andrews S, de Graaf H and Stamation H (1997) Opti-
misation of methodology for enumeration of xerophilic
yeasts from foods. International Journal of Food Micro-
biology 35: 109-1 16.
Dillon VM and Board KG (1991) Yeasts associated with
red meats. Journal of Applied Bacteriology 71: 93-108.
Dohmen RJ and Hollenberg CP (1996) Schwanniomyces
occidentalis. In: Wolf K (ed.) Nonconventional Yeasts in
Biotechnology. A Handbook. P. 117. Berlin: Springer-
Verlag.
Girio FM, Pelica F and Amaral-Collae MT (1996) Char-
acterisation of xylitol dehydrogenase from Debaryo-
myces hansenii. Applied Biochemistry and
Biotechnology 56: 79-87.
Kosse D, Ostenrieder I, Seiler H and Scherer S (1998) Rapid
detection and identification of yeasts in yogurt using
fluorescently labelled oligonucleotide probes In: Jakob-
sen M, Narvhus J and Viljoen BC (eds) Yeasts in the
 ECOLOGY OF BACTERIA AND FUNGI IN FOODWlnfluence of Available Water
ECOLOGY OF BACTERIA AND FUNGI IN FOODS
Contents
Influence of Available Water
Influence of Temperature
Influence of Redox Potential and pH
Influence of Available Water
K Krist, Meat and Livestock Australia, Sydney,
Australia
D S Nichols and T Ross, School of Agricultural
Science, University of Tasmania, Hobart, Australia
Copyright C 1999 .Academic Press
Introduction
Although there is a variety of resting or survival stages
of microorganisms that are resistant to drying, all
organisms need water to remain metabolically active.
The availability of water to a n organism in an envir-
onment is not simply a function of how much water
is present, but the degree to which it is adsorbed
to the insoluble components of the environment or
chemically associated with solutes in that envir-
onment. For this reason, the concept of water activity
( a & ) ,a measure of the availability of water to par-
ticipate in chemical reactions, was devised. Though
a,, is not a perfect predictor of the behaviour of micro-
organisms in a specified environment iknowledge of
the solutes and factors that contribute to the a , is also
required), it is widely used to describe the relationship
between the water in a n environment and its microbial
ecology.
The reduction of a,+to increase the microbiological
stability of foods has probably been used since
antiquity. The drying effect of the air and the sun
required no special technology and is still used today.
Similarly, the addition of salt or sugars requires no
special technology and has been used for centuries to
preserve food. Those techniques are still in use in
many parts of the world, using free energy and pro-
viding safe products. More recently, technology ie.g.
hurdle technology) has sought to maximize the poten-
tial of drying techniques while minimizing the severity
of treatments to develop shelf-stable products that are
less altered from the fresh state.
539
This article considers the microbial ecology of bac-
teria and fungi in relation to a,. a, and related terms,
are defined. Methods for manipulating a,, in foods are
discussed, and the effects of a,, on growth rate, lag-
phase duration, jield and death rate of micro-
organisms described. The physiology of the response
of microbial cells to a,, stress is also discussed.
Concept of Water Activity/Available
Water
Water activity can be affected by both solutes and
adsorption. The solutes effect is called osmotic poten-
tial. The adsorption effect is called matric water
potential but it is not widely considered in food micro-
biology. None the less, insoluble materials such as
wood, paper, metal and glass, and including foods,
adsorb water. The strength of the attachment is a
function of the physical and chemical properties of
the material. Those materials will tend to take water
up from, or release water to, the atmosphere until a n
equilibrium is reached between the atmosphere and
the material. Foods will tend to equilibrate with the
relative humidity of the container or environment
they are stored in. Thus, dry foods can take up water
from humid environments, o r moist foods will tend
to dry out in dry environments. If a food is allowed
to equilibrate with the humidity of the storage atmos-
phere, the matric a,. will affect the organism just as if
the osmotic a,. had been altered to the same relative
humidity.
The terms water activity, water potential, osmotic
pressure and solute concentration are often used inter-
changeably by microbiologists to refer to the avail-
ability of water to microorganisms. Although each of
these concepts is related, they are different. Solute
concentration is self-explanatory, although it may be
expressed in different ways (e.g. w h , w/v, molarity,
molality, etc.). High solute concentrations result in
decreased a,, , and less water available to micro-
540 ECOLOGY OF BACTERIA AND FUNGI IN FOODS/lnfluence of Available Water

organisms for metabolism. Solutes that alter a, are Increased osmotic pressure literally means that the
termed humectants. cell is subjected to an increased external pressure, or
alternatively, a decreased internal pressure. Increased
Water Activity extracellular osmotic pressure refers to a situation
A,%is a fundamental property of aqueous solutions. It where the availability of water to bacteria is
is defined as: decreased.
The term water potential, widely used by soil micro-
a ,---
P (Equation 1) biologists, also expresses the availability of water,
Po but is defined as the difference in free energy of the
environment being considered, and a pool of pure
where p = vapour pressure of the solution; po = vapour water at the same temperature: the terms water activ-
pressure of the pure water under the same conditions ity and water potential are measures of the energy of
of temperature, etc. And where: water. Water potential may be expressed in a number
-P = relative humidity of units, of which the most widely used is the bar
( IO6dyn cm-2). Water potential is always a negative
Po
value or zero.
The a,b of most solutions is temperature-dependent. As shown in Table 1, a,$ and water potential are
Equilibrium relative humidity, a measure widely used not directly proportional, however, a 0.01 decrease in
in meteorology and building environmental control, a , corresponds to a decrease of approximately 15 bar
is related to a, by the simple expression: water potential in the range of a , typical of foods.
Tables of a, for various solutes and solute mixtures
Equilibrium relative humidity ( % )
a, = (Equation 2) are available in the literature. The effect on a, of
100 solutions containing several solutes can be estimated
When solutes are dissolved in water, some of the from the concentration of each solute, using the fol-
water molecules become more ordered as they become lowing formula:
oriented on the surfaces of the solute molecule. This
reduces the vapour pressure of the solution, since on a,tota, = a , l x aw2x x ...............X awn (Equation 5 )
average the water molecules then have less entropy.
In turn, a,v is reduced. The a,v of a solution decreases where a,,l, a,$2,a,,3, a,,, are the a, calculated from the
with increasing solute concentration. The effect of concentration of each solute independently.
solute concentration on a., is expressed math- This equation can readily be applied to liquid foods,
ematically: e.g. broths, juices and syrups and can also be used
for solid foods by determining the concentration of
[Equation 3 ) solutes in the aqueous phase.
Water potential, y ~ ,is related to water activity by
where v = the number of ions generated by each mol- the equation:
ecule of solute (e.g. for non-electrolytes, v = l; for
NaC1, v = 2 ; for HzS04, v = 3 ) ; m=molaI con- (Equation 6 )
centration of the solute; cp = molal osmotic coefficient.
Equation 3 reveals that the a , at a given solute
concentration is dependent on the specific solute, where M = t h e molecular weight of water
because each solute has its own osmotic coefficient (0.018 kgmol-l) and all other terms are as previously
and will dissociate into a different number of ions. defined.
Osmotic Pressure
The osmotic pressure of a solution is related to its a,
and includes this term in its definition: Factors Affecting Water Activity
-RT In a, Addition of water or removal of solutes can increase
Osmotic pressure = - [Equation 4) a, In food microbiology, however, one is usually
V
interested in reducing a,%, to improve the micro-
where R = the universal gas constant biological stability of the product. The a,$ of an envir-
(8.314Jk-lmol-'); T =absolute temperature (K); V = onment can be reduced by the addition of solutes, or
partial molar volume of water and all other terms are water binding substances that decrease matric water
as previously defined. potential, or by the removal of liquid water.
 ECOLOGY OF BACTERIA AND FUNGI IN FOODS/Influence of Available Water 541

Table 1 Comparison of water activity (a,) and water potential ( y )values and concentration of solutes required to achieve them
a /: Water potential (bar)' NaCl concentration Sucrose concentration Other solutes (a,,/ at 25°C)
(g /-'I (9 i-') (9 I-')
0.995 -7 8.7 92
0.980 -28 35 342
0.850 -224 190 2050 (saturated)
0.843 -235 KCI (saturated. 357)
0.753 -390 260 (saturated)
0.577 -757 NaBr (saturated, 909)
0.328 -1534 MgClz (saturated,l667)
0.113 -3000 LiCl (saturated. 769)
0.1 00 -31 68
al bar = -1 00 J kg-'

Freezing in that the ionic species Na' is also a primary ion in

Liquid water can be removed, in effect, by freezing.
The preservative effects of freezing are due not only
to temperature depression, but also to the effect of
decreasing in the remaining liquid water. As the
water in the food freezes it increases the effective
concentration of solutes in the remaining liquid water.
Those organisms remaining in the liquid phase are
exposed to increasingly severe osmotic challenge as
freezing proceeds. The same ecological challenges
apply to bacteria naturally present in Arctic and Ant-
arctic environments. The physiology of the organisms
naturally present in those extreme environments is
instructive for understanding the effects on micro-
organisms present in frozen foods and is discussed
briefly later.
Drying
The removal of water by evaporation also increases
the concentration of the solutes in the remaining
water. As described below, the effect on a, of the
remaining free water will depend on the level and type
of solutes initially present.
Specific Solutes
The a,-modifying effects of several different solutes
are shown in Table 1. Addition of solutes increases
the osmotic potential of the water. As suggested by
Equation 3, the effect of specific ionic solutes is related
to their concentration, the number of ions that the
molecule dissociates into, its dissociation constant,
and also specific properties of the solute. Son-ionic
solutes also reduce water activity.
Generally, IiaC1, KCI, glucose and sucrose shon7
similar patterns of effect on microbial responses while
glycerol usually permits growth at lower a,$, although
there are specific exceptions, e.g. Staphylococcus
auyeus is more inhibited by glycerol than NaCI. Glp-
cero1 differs from other solutes in that it is able to
permeate the cell freely.
NaCl is somewhat unique in terms of humectants
cell function. Symporters are proteins that transport
selected substances across the cell membrane, in a
manner dependent on the co-transport of a second
substrate in the same direction. A number of sym-
porter systems are Na+-driven. Cytoplasmic levels of
Na' are also tightly regulated in most species, and
fluctuating external Na' levels challenge microbial
cells beyond the osmotic effect of a,,. Much of the
research in this area has been conducted using bac-
teria; however, the general principlcs also hold for
fungi.
Within Escherichia coli, an active extrusion mech-
anism is responsible for the regulation of intracellular
Na' concentration which enters via symporter
systems. The primary mechanism consists of a series
of membrane-associated transport proteins known as
antiporters. As protons flow into the cell (through the
antiporter channel) along the concentration gradient
established by respiratory chains, Na' is extruded
from the cytoplasm. Many marine and anaerobic bac-
teria rely heavily on Na' cycling, with additional Na*-
translocating respiratory chains and ATPases respon-
sible for Na' removal from the cell interior. Most, if
not all, symporters in these bacteria are coupled to
Iia' influx.
The linkage between Na'/H- antiporters results in
an increased interaction between p H and NaC1 in
marine and anaerobic bacteria, so that their growth
tends to be increasingly inhibited by S a C l as the p H
of the medium increases. This is an example of specific
effects of the humectant itself other than its direct
effect on a,.
Levels in np i c a l Foods
Representative a, of food5 are shown in Table 2.
Foods range from those with very little free water
(freeze-dried products, cereals, powdered products)
to almost completely free water ie.g. fresh meat and
produce, bottled water products). hlost fresh produce
has a,, close to 1.00 if the tissues are cut but may have
542 ECOLOGY OF BACTERIA AND FUNGI IN FOODS/lnfluence of Available Water

Table 2 Representative water activity of foods
Food Typical water
activity
Milk, fruit, vegetables 0.995-0.998
Fresh meat, fish 0.990-0.995
Cooked meat, cold smoked salmon 0.965-0.980
Liverwurst 0.96
Cheese spread 0.95
Caviar 0.92
Bread 0.90-0.95
Salami (dry) 0.85-0.90
Soft, moist pet food; chocolate syrup 0.83
Fruit cakes, preserves, soy sauce 0.80
Salted fish, honey 0.75
Dried fruit 0.60-0.75
Dried milk (8% moisture) 0.70
Cereals, confectionery,dried fruit, peanut butter 0.70-0.80
Ice at -40°C 0.68
Dried pasta, spices, milk powder 0.20-0.60
Freeze-dried foods 0.10-0.25
significantly lower surface water activity, e.g. on intact
fruits and vegetables due to the presence of the cuticle.
Meat carcass surfaces can also dry during processing,
lowering the a, sufficiently to inhibit microbial activ-
ity greatly. Thus, it is important to know not only the
type of food but also the form and packaging that it
is in to understand the microbial ecology.
General Reactions of Bacteria, Yeasts
and Mycelial Fungi
Most microorganisms are active over only a relatively
narrow range of a, and a," differences in the order of
0.001-2 are significant on the microbial ecology of
an environment. Thus, a, values in food microbiology
arc normally quoted to three significant figures.
Gram-negative bacteria, typically, are only able to
grow in environments of a, greater than about 0.95.
Many Gram-positive bacteria can withstand a, as low
as about 0.9, but few can grow at a, lower than
0.8. Some, specifically adapted to life in hyper-saline
environments, are active at a, as low as 0.75 and
might be found, e.g. in dried salted fish. Fungi are
generally more tolerant of reduced a, than are bac-
teria. Some yeasts and moulds are able to withstand
a, as low as 0.60. Growth rates of bacteria are typ-
ically faster than those of eukaryotes. Thus, despite
the fact that many yeasts and moulds are able to grow
on foods of high a, such foods are usually rapidly
dominated and spoiled by bacterial contaminants.
Fungi have a selective advantage at lower a, and are
more usually associated with the spoilage of reduced
a, products, e.g. bread, cheese, jams, syrups, fruit
juice concentrates, grains, etc. As indicated above, the
effect of a, depends on the major solutes responsible
for the reduced a,. Ionic solutes (salts) have a greater
inhibitory effect on microbial metabolism than non-
ionic solutes (e+ sugars).
Range of Growth
Each microorganism has a minimum and maximum
a, for growth. For many species, the maximum a, for
growth is effectively 1.000. Although growth could
not occur in pure water, some organisms are able to
grow in the presence of very low levels of nutrients.
Pseudomonads, and even algae, are able to grow in
some types of bottled water, indicating the need for
techniques to eliminate viable organisms from these
products during production. A range of terms used to
describe the response and tolerance of micro-
organisms to a, and specific solutes is shown in Table
3.
Table 3 Classification of microorganisms according to their
preferred water activity range for growth
Nomenclature Water activity range for growth
Haloduric Able to withstand, but not grow at, high con-
centrations of salt
Halophile Requiring salt for growth
Extreme hal- Requiring 1 5 2 0 % salt for growth
ophile
Osmotolerant Able to withstand, but not grow at, high con-
centrations of sugar
Osmophile Organisms that grow best, or only, under
high osmotic pressure, due to sugars
Xerophilic Requiring reduced water activity (as distinct
from requiring high osmotic pressure)
The a, range of growth is solute-dependent. Many
bacteria, for example, are more tolerant of reduced
a, if the solute is glycerol. This characteristic is not,
however, universal. Tolerance to a, is greatest when
all other factors in the environment are optimal for
growth. As other environmental factors become less
optimal the range of a, that supports growth is
reduced. Examples are presented in Figures 3 and 5
of the related entry 'Predictive microbiology'. The
effects, however, are not always intuitive.
Representative tolerance ranges under otherwise
optimal conditions for various microbial groups are
shown in Table 4.
Combinations of Factors
It is common in some foods for a variety of factors to
be used to control microbial growth. This approach
exploits the interaction of a, and other physico-
chemical parameters such as temperature and pH in
food environments. Such interactions form the basis
of the hurdle concept.
The physico-chemical factors of NaCl and tem-
perature have a close interaction, with the tem-
perature range for growth of most organisms
displaying a dependence on salinity. In general,
 ECOLOGY OF BACTERIA AND FUNGI IN FOODS/lnfluence of Available Water 543

Table 4 Representative tolerance ranges for various microbial 0.012

groups and species
0.010
Organism or group Lower a, limit (Solute)
a, 0.008
(Most) Gram-negative rods 0.95-0.96 (NaCI) I
e
Escherichia coli
Pseudomonas fluorescens
0.95-0.955 (NaCI)
0.97 (Sucrose)
2 0.006
2
Pseudomonas fluorescens 0.96 (NaCI) (3 0.004
Vibrio parahaemolyticus 0.96 (Glucose)
Vibrio parahaemolyticus 0.93 (NaCI) 0.002
(Most) Gram-positive bacteria 0.90-0.94 (NaCI)
Listeria monocytogenes 0.92-0.93 (NaCI) 0.000
....
0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99 1.00
Staphylococcus aureus 0.89 (Glycerol)
Water activity
Staphylococcus aureus 0.87 (Sucrose)
Staphylococcus aureus 0.86 (NaCI) Figure 1 Effect of water activity on the growth rate of bacteria.
Bacillus cereus 0.95 (Glucose) Curves A and D represent two organisms, each adapted to a
Bacillus cereus 0.94 (NaCI) different water activity range for growth. Curve B represents the
Bacillus cereus 0.92 (Glycerol) effect of a second suboptimal environmentalfactor on the growth
rate of organism A. The water activity range is unaltered, the
Yeasts 0.65-0.92 (NaCI) relative response remains the same, but the absolute growth rate
Zygosaccharomyces rouxii 0.65 (Sucrose) is reduced at all water activities. Curve C represents the effect of
Saccharomyces cerevisiae 0.90 (Sucrose) a different, non-ionic solute (or humectant) on the growth
Moulds 0.65-0.90 (NaCI) response of organism A. That humectant permits A to grow over
Penicillium chrysogenum 0.80 (KCI, glucose) a wider range of water activities. After Ross T (1999) Predictive
Wallemia sebi 0.75 (Glycerol) Food Microbiology Models in the Meat Industry. Sydney: Meat
Eurotium spp. 0.66 (Glucose and fructose) and Livestock Australia.

Algae 0.75-0.90
Most groups
0.90-0.95 (NaCI)
Dunaliella 0.75 (NaCI)
reduced a, confers enhanced heat resistance on micro-
bial cells. The basis for this behaviour is perhaps due
to the cross-protection that osmotic stress affords
against temperature stress, believed to be mediated by
a general stress response under the control of the Rpos
gene. (Interestingly, if grown at suboptimal salinities,
a number of marine bacteria exhibit a lowered
maximal temperature for growth compared to growth
at the optimal salinity.) The minimum temperature for
growth for many food-borne organisms is, however,
increased by decreasing a,.,. This raises the intriguing
possibility that the basis of these effects lies in the
energy of the water itself, i.e. if the kinetic energy of
water molecules mediates the lethal effect of tem-
perature, then the reduction of water energy by solutes
may have the same effect as reducing temperature.
The growth rate response of microorganisms to
water activity is illustrated in Figure 1. Growth rate
increases in proportion, approximately, with increas-
ing a,, above the minimum a, for growth, and up to
an optimum growth rate value. Beyond this value the
growth rate declines, usually rapidly as a function of
increasing a, until the maximum a, is reached.
Growth rate is a characteristic of the environment,
and is not affected by the previous history of the cell,
unlike lag time. The effect of a, on growth rate is
affected by the specific humectant.
There is no specific correlation between a, tolerance
and tolerance to other environmental factors. Thus,
the manipulation of a, in a product could have dif-
ferent consequences for the microbial ecology of the
foods at different temperatures. An illustration of the
selective effect of temperature and a, on different
organisms is presented in Figure 2.
Lag, Germination and Sporulation, Toxin
Production
The lag time is generally considered to be a period
of adjustment to a new environment, requiring the
synthesis of new enzymes and cell components to
enable the maximum rate of growth possible in that
environment. As indicated above, the growth rate
and, by inference the metabolic rate, is a function of
the environment. As such, the lag time observed upon
transfer of a cell to a new environment could be
expected to result from both the amount of adjust-
ment to that new environment and the rate at which
those adjustments could be made. In general, lag times
are longer at a, that are less optimal for growth and
where the difference between the old and new growth
environment is larger, especially when the new envir-
onment is less favourable for growth than the old.
Generally the limits for microbial sporulation are
the same as the limits for growth, although spo-
rulation may occur at a, slightly lower than that
required for growth. Spores can sometimes also ger-
minate at a, below those which permit growth. Toxin
production does not occur at a, below those which
permit growth, and is often prevented at a, con-
544 ECOLOGY OF BACTERIA AND FUNGI IN FOODShfluence of Available Water

3r
,
h

...........S.aureus h
h
....
...........S.aureus .... ...:
h

f. 4 - //\ 5.
-_---L.monocytogenes ----- L. monocytogenes .....
.'
.
!i -Pseudomonads
--- E. coli

Temperature ("C)

Figure 2 Comparison of the combined effect of environmental factors on growth rate of psychrotrophic spoilage pseudomonads,
Listeria monocytogenes, Escherichia coli and Staphylococcus aureus. (A) The predicted effect of temperature on rates of aerobic
growth at aw=0.990. (8)The predicted effect of temperature on rates of growth at aw=0.960.(C)Interactive effects of temperature
and water activity on the microbial ecology of foods. Dominance domains for selected microorganisms potentially present on raw
foods were estimated from predictive models for the aerobic growth of psychrotrophic spoilage pseudomonads, L. monocytogenes,
E. coli and S. aureus at many combinations of water activity and temperature. The shaded areas represent that combination of
factors in which the indicated organism would be expected to limit the acceptability of the product. The limits imposed for acceptability
were that the predicted increase in the pathogen should not exceed a factor of 10 after 7 days storage. The limits for pseudomonads
were that the increase in 7 days should be not more than 1000-fold, assuming an initial level of l000cfu cm-'. All organisms were
assumed to experience a lag time equivalent to one generation time at the nominated conditions. The part of the plot to the left of
the bold line shows those sets of conditions under which the required bacterial growth limits are exceeded. For all conditions the
organism closest to attaining the tolerance limit, and hence posing the greatest risk, is indicated. Note: The growth rate of
pseudomonadswas scaled to reflect the greater tolerance of this organism on the product, i.e. approx. 10 doublings of pseudomonads
but only approx. 3 doublings of pathogens are tolerable by the criteria described. After McMeekin and Ross (1996) with permission
from Elsevier Science.

siderably higher than those required to prevent as a function of a,,, until the lower a, limit for growth,
growth. approximately 0.95.5, is reached.
Yield
Inactivation
At a, less than the optimum, cell yield declines. The
decline is not always a direct function of the a,,,stress A t a, lower than the minimum for growth, the cell
applied and it appears that some bacteria, at least, either remains dormant or dies. Compounding this
can tolerate a range of a, without a change in yield. action, however, is the effect of a, on the cell and the
In E. coli, for example, in the a, range from approxi- environment itself. Reduced a, usually correlates with
mately 0.970 to 0.997 (using NaCl as the humectant), decreased chemical activity, with the result that the
yield declined slightly (620%) with decreasing a, preservative effect of low a, on foods may also pre-
compared to the optimum a, (ca. 0.995). At a, lower serve microorganisms present in the foods. This is
than approximately 0.970, yield declines dramatically particularly true for low a, (i.e. cO.7) products, in
 ECOLOGY OF BACTERIA AND FUNGI IN FOODS/lnfluence of Available Water 545
which microbial survival may be enhanced in com-
parison to that at higher a , .
Mechanisms
While the changes in cell physiology that accompany
osmotic stress are known in some detail, the physico-
chemical mechanisms that underlie the effects of those
responses are not well understood. One interpretation
of the effects of a, on the ecology of microorganisms
considers that a, creates a homeostatic burden. To
maintain homeostasis, the cell must expend energy,
whether to import or synthesize compatible solutes,
modify membrane components, etc. This energy is
unavailable for synthesis of new biomass and leads to
reduced yield. This hypothesis further proposes that
the cells' homeostatic demands ultimately consume
all the available energy and the cell is able only to
survive. Extending this paradigm, cell death could be
interpreted to result when the homeostatic demands
are unable to be met and the cell is unable to maintain
the functional integrity of those enzymes and path-
ways necessary for continued viability.
Effect of Water Activity on Intracellular
Structures and Chemical Composition of
Cells
To remain viable, microorganisms, like plant cells,
need to maintain a positive turgor pressure, possibly
to provide a stimulus for cell elongation and growth.
When a cell experiences an osmotic 'upshock' (i.e.
transfer to lower a,), the cell loses water due to
osmosis because the microbial cell membrane is per-
meable to water and relatively impermeable to solutes.
Water moves out of the cell to restore osmotic equi-
librium, resulting in shrinkage of the cells. In extreme
cases the cell membrane shrinks away from the cell
wall, a process termed plasmolysis. Microbial cells
must counteract the osmotic stress to restore the
turgid, pre-stress state and have evolved a number
of physiological responses to reduced a, including
changes in:
cell membrane composition
protein synthesis
adjustment of cytoplasmic a,,,.
The cell membrane is the main barrier to water
and solute exchange between the cytoplasm and the
external environment. It plays an important role in the
physiological response to osmotic stress, responding
with changes to both its lipid and protein components.
The synthesis of some proteins is induced by
osmotic stress. Increased levels of solute transport
proteins (porins) are likely during the osmoregulatory
response. Like porins, many other osmotically
induced proteins form the cellular machinery to facili-
tate a change in cytoplasmic a,".
Macromolecular conformation, and therefore func-
tion and activity, is affected by intracellular a, due,
in part, to the effects of humectants on the physical
structure of water. Some changes to membrane struc-
ture in response to a, stress appear to enable mem-
brane-bound enzymes to retain the conformation
required for catalytic activity. The role of compatible
solutes in optimizing molecular conformation is dis-
cussed below.
Cell Membrane Composition
The chemical composition of microbial cell mem-
branes is described clsewhere in this volume. In
response to high salinity there is an increase in the
proportion of negatively charged phospholipids, often
phosphatidylglycerol and/or glycolipids. This alter-
ation is needed to maintain the membrane in the
proper lipid bilayer phase for normal function.
Apart from the extreme halophiles of the Archaea
there does not appear to be a correlation between
microbial membrane composition and intrinsic a, tol-
erance. However, the effect of a, on a given membrane
composition does depend to a large extent on the
type of membrane (correlated with chemotaxonomic
grouping, e.g. Bacteria, Archaea, yeast, fungi) and to
a lesser extent, the nature of the humectant.
There are several elements common to cell mem-
brane responses to changing a,. The first of these is
membrane surface charge. The head groups of the
major microbial membrane lipids (phospholipids and
phosphoglycolipids) are negatively charged from the
associated phosphate residue. Certain phospholipid
classes also contain positively charged head-group
moieties, resulting in all polar lipid classes being either
anionic or zwitterionic. The membrane surface of
all microbes therefore possesses a net surface charge
dependent on the phospholipid classes present. Ionic
humectants may disrupt the membrane surface charge
by interaction with phospholipid groups, requiring a n
alteration in membrane composition. Many halo-
tolerant and modcrately halophilic bacteria respond
to reduced a , by increasing the proportion of anionic
phospholipids in the membrane at the expense of
zwitterionic components, believed to aid the mem-
brane in maintaining a functional bilayer phase.
The fatty acid composition of the cellular mem-
brane also influences functionality and is actively
modified in response to changing environmental
factors. In general, in response to decreasing a, most
hacteria increase fatty acid chain length and/or
decrease fatty acid unsaturation. Again, this is thought
to maintain the membrane in a functional bilayer
phase. In certain cases, the mechanism may involve
 Next Page

546 ECOLOGY OF BACTERIA AND FUNGI IN FOODSAnfluence of Available Water

direct inhibition of fatty acid biosynthetic enzymes by make additional physiological adjustments, especially
increased levels of NaCl. in regard to enzyme function. The enzymes of pro-
Archaeal membranes possess phosphorus-con- karyotes that use the salt-in-cytoplasm strategy have
taining lipid species as in other microorganisms but additional negative charge that makes them stable
consisting of a glycerol backbone with two ether- at high solute concentrations but unstable at low
linked C2"prenyl chains. This Domain contains all the concentrations.
extremely halophilic bacteria, with their membranes
characterized by diphytanylglycerol diethers. De-
Compatible Solutes
phosphorylated derivatives may be present with a
significant proportion of glycolipids. Extreme halo- The activity of water is significantly influenced by the
philes are characterized (but not exclusively) by the molecular structure of the solution. Water as a liquid
presence of neutral lipid Components, mostly iso- is characterized by a (relatively) high degree of
prenoid hydrocarbons, such as squalene. The resulting molecular motion resulting in a dynamic random dis-
membrane bilayer is more ordered and less flexible tribution of molecular orientation. The potential
than those formed from other lipid types. The C20 degree of hydrogen bonding between water molecules
phytanyl residues may be present as branched or ring- is therefore not fully realized, allowing water mol-
containing structures which act as similar adaptive ecules to pack together in a relatively tight manner or
responses to fatty acid structure within other micro- higher density. As the degree of molecular motion
organisms. It is believed that the close packing decreases (e.g. with lower temperature), a higher
exhibited by phytanyl residues in Archaeal mem- degree of hydrogen bonding between water molecules
branes is the basis for their resistance to extreme becomes possible and molecules adopt a more ordered
environmental conditions. array with a decreased density. With decreased tem-
While yeasts and fungi, as eukaryotes, contain perature this process continues until the ordered
many additional lipid types as storage and intra- molecular array of ice is achieved. Solute molecules
cellular membrane components, their cellular mem- decrease the activity of water by the same process.
brane is dominated by phospholipid species as for the The organic compounds synthesized or accu-
Bacteria. Thus, the common changes in fungal cell mulated by microorganisms to balance their intra-
membrane composition to changing a, are similar to cellular osmotic potential to that of their cnvironmcnt
those of the Bacteria, both in terms of polar lipid classshare the property that they do not affect the function
manipulation and adaptation of fatty acid of normal salt-sensitive enzymes. The use of com-
composition. patible solutes to counter osmotic stress is not limited
to microorganisms. Plants and animals also use the
Cytoplasmic Water Activity
organic-solute-in-cytoplasm strategy and employ the
Moulds and yeasts accomplish the restoration and same compounds as compatible solutes, suggesting
maintenance of turgor pressure by accumulation from that these compounds share fundamental properties
the environment, or by de novo synthesis, of intra- that make them suitable for this role.
cellular polyols to establish equivalent osmotic pres- The compatible solutes have low molecular weights
sure intracellularly as exists extracellularly. Bacteria and polar functional groups, properties which make
also accumulate or synthesize a range of compounds them highly soluble and facilitate their accumulation
for the same purpose. Compounds used in this way to high intracellular concentration. They are
share the property that they do not interfere with uncharged at normal cytoplasmic pH - an important
metabolic processes. As such, they have been termed property because high cytoplasmic ionic strength
compatible solutes. would be detrimental to enzyme function. These
Microorganisms adjust their cytoplasmic a, using requirements limit the range of compounds that can
one of two strategies: the salt-in-cytoplasm type and be utilized as compatible solutes. Classes of com-
the organic-osmolyte-in-cytoplasm type. Most, like pounds that are known to perform this function and
the yeasts and moulds, use the organic-osmolyte-in- specific examples are presented in Table 5.
cytoplasm strategy for osmoadaptation. In this strat- Compatible solutes do not hinder the function of
egy salts are excluded, while organic solutes are syn- normal (salt-sensitive)enzymes and, in fact, protect
thesized or accumulated from the environment. Some proteins from the denaturation that would otherwise
bacteria can also adjust their cytoplasmic water by occur in solutions of high ionic strength. That pro-
accumulating KC1 to high intracellular concentration. tection also extends to the denaturing effects of freez-
This is considered a primitive strategy because it does ing, heating and drying.
not provide a normal cytoplasmic environment. This The mechanism of this protective effect is unknown.
salt-in-cytoplasmstrategy requires that the cell should One observation, fundamental to attempts to resolve
 FERMENTATION (INDUSTRIAL)/Basic Considerations 663
1 Fatty Acids see Fermentation (Industrial): Production of Oils and Fatty Acids.
FERMENTATION (INDUSTRIAL)
Contents
Basic Considerations
Media for Industrial Fermentations
Control of Fermentation Conditions
Recovery of Metabolites
Production of Xanthan Gum
Production of Organic Acids
Production of Oils and Fatty Acids
Colours/Flavours Derived by Fermentation
Basic Considerations
Yusuf Chisti, Department of Chemical Engineering,
University of Almeria, Spain
Copyright 0 1999 Academic Press
Introduction
Fermentation processes utilize microorganisms to
convert solid or liquid substrates into various prod-
ucts. The substrates used vary widely, any material
that supports microbial growth being a potential sub-
strate. Similarly, fermentation-derived products show
tremendous variety. Commonly consumed fermented
products include bread, cheese, sausage, pickled vege-
tables, cocoa, beer, wine, citric acid, glutamic acid
and soy sauce.
Types of Fermentation
Most commercially useful fermentations may be clas-
sified as either solid-state or submerged cultures. In
solid-state fermentations, the microorganisms grow
on a moist solid with little or no ‘free’ water, although
capillary water may be present. Examples of this type
of fermentation are seen in mushroom cultivation,
bread-making and the processing of cocoa, and in the
manufacture of some traditional foods, e.g. miso (soy
paste), sakk, soy sauce, tempeh (soybean cake) and
gari (cassava), which are now produced in large indus-
trial operations. Submerged fermentations may use a
1
dissolved substrate, e.g. sugar solution, or a solid
substrate, suspended in a large amount of water to
form a slurry. Submerged fermentations are used for
pickling vegetables, producing yoghurt, brewing beer
and producing wine and soy sauce.
Solid-state and submerged fermentations may each
be subdivided - into oxygen-requiring aerobic pro-
cesses, and anaerobic processes that must be con-
ducted in the absence of oxygen. Examples of aerobic
fermentations include submerged-culture citric acid
production by Aspergillus niger and solid-state koji
fermentations (used in the production of soy sauce).
Fermented meat products such as bologna sausage
(polony), dry sausage, pepperoni and salami are pro-
duced by solid-state anaerobic fermentations utilizing
acid-forming bacteria, particularly Lactobacillus,
Pediococcus and Micrococcus species. A submerged-
culture anaerobic fermentation occurs in yoghurt-
making.
Fermentations may require only a single species of
microorganism to effect the desired chemical change.
In this case the substrate may be sterilized, to kill
unwanted species prior to inoculation with the desired
microorganism. However, most food fermentations
are non-sterile. Typically fermentations used in food
processing require the participation of several micro-
bial species, acting simultaneously and/or sequen-
tially, to give a product with the desired properties,
including appearance, aroma, texture and taste. In
non-sterile fermentations, the culture environment
664 FERMENTATION (INDUSTRIAL)/Basic Considerations

may be tailored specifically to favour the desired fermentation batch. The volume of the fermenting
microorganisms. For example, the salt content may broth increases with each addition of the medium, and
be high, the pH may be low, or the water activity may the fermenter is harvested after the batch time.
be reduced by additives such as salt or sugar. In continuous fermentations, sterile medium is fed
continuously into a fermenter and the fermented
Factors Influencing Fermentations product is continuously withdrawn, so the fer-
A fermentation is influenced by numerous factors, mentation volume remains unchanged. Typically, con-
including temperature, pH, nature and composition tinuous fermentations are started as batch cultures
of the medium, dissolved 0 2 , dissolved COZ, oper- and feeding begins after the microbial population has
ational system (e.g. batch, fed-batch, continuous), reached a certain concentration. In some continuous
feeding with precursors, mixing (cycling through fermentations, a small part of the harvested culture
varying environments), and shear rates in the fer- may be recycled, to continuously inoculate the sterile
menter. Variations in these factors may affect: the rate feed medium entering the fermenter (Fig. l ( D ) ) .
of fermentation; the product spectrum and yield; the Whether continuous inoculation is necessary depends
organoleptic properties of the product (appearance, on the type of mixing in the fermenter. ‘Plug flow’
taste, smell and texture); the generation of toxins; fermentation devices (Fig. l ( D ) ) ,such as long tubes
nutritional quality; and other physico-chemical that do not allow back mixing, must be inoculated
properties. continuously. Elements of fluid moving along in a
The formulation of the fermentation medium plug flow device behave like tiny batch fermenters.
affects the yield, rate and product profile. The medium Hence, true batch fermentation processes are rela-
must provide the necessary amounts of carbon, nitro- tively easily transformed into continuous operations
gen, trace elements and micronutrients (e.g. vitamins). in plug flow fermenters, especially if pH control and
Specific types of carbon and nitrogen sources may be aeration are not required. Continuous cultures are
required, and the carbon : nitrogen ratio may have particularly susceptible to microbial contamination,
to be controlled. An understanding of fermentation but in some cases the fermentation conditions may be
biochemistry is essential for developing a medium selected (e.g. low pH, high alcohol or salt content)
with an appropriate formulation. Concentrations of to favour the desired microorganisms compared to
certain nutrients may have to be varied in a specific potential contaminants.
way during a fermentation to achieve the desired In a ‘well-mixed’ continuous fermenter (Fig. 1(C)),
result. Some trace elements may have to be avoided - the feed rate of the medium should be such that the
for example, minute amounts of iron reduce yields in dilution rate, i.e. the ratio of the volumetric feed rate
citric acid production by Aspergillus niger. Additional to the constant culture volume, remains less than the
factors, such as cost, availability, and batch-to-batch maximum specific growth rate of the microorganism
variability also affect the choice of medium.
in the particular medium and at the particular fer-
mentation conditions. If the dilution rate exceeds the
Submerged Fermentations maximum specific growth rate, the microorganism
will be washed out of the fermenter.
Fermentation Systems Industrial fermentations are mostly batch oper-
Industrial fermentations may be carried out either ations. Typically, a pure starter culture (or seed),main-
batchwise, as fed-batch operations, or as continuous tained under carefully controlled conditions, is used
cultures (Fig. 1). Batch and fed-batch operations are to inoculate sterile Petri dishes or liquid medium in the
quite common, continuous fermentations being rela- shake flasks. After sufficient growth, the pre-culture is
tively rare. For example, continuous brewing is used used to inoculate the ‘seed’ fermenter. Because indus-
commercially, but most beer breweries use batch trial fermentations tend to be large (typically 150-
processes. 250m3), the inoculum is built up through several
In batch processing, a batch of culture medium in successively larger stages, to 5-10% of the working
a fermenter is inoculated with a microorganism (the volume of the production fermenter. A culture in rapid
‘starter culture’). The fermentation proceeds for a exponential growth is normally used for inoculation.
certain duration (the ‘fermentation time’ or ‘batch Slower-growing microorganisms require larger
time’), and the product is harvested. Batch fer- inocula, to reduce the total duration of the fer-
mentations typically extend over 4-5 days, but some mentation. An excessively long fermentation time (or
traditional food fermentations may last months. In fed- batch time) reduces productivity (amount of product
batch fermentations, sterile culture medium is added produced per unit time per unit volume of fermenter),
either continuously or periodically to the inoculated and increases costs. Sometimes inoculation spores,
 FERMENTATION (INDUSTRIAL)/Basic Considerations 665

-
Feed

- Final volume

- Initial volume

(6)

Feed
Feed Harvest
Recycle inocuium

(C)
- Harvest
Feed
Inoculum from
separate source
(D)
Harvest

Figure 1 Fermentation methodologies. (A) Batch fermentation. (B) Fed-batch culture. (C) Continuous-flow well-mixed fermentation.
(D) Continuous plug flow fermentation, with and without recycling of inoculum.

produced as seeds, are blown directly into large fer-
mentation vessels with the ingoing air.
Microbial Growth
Microbial growth in a newly inoculated batch fer-
menter typically follows the pattern shown in Figure
2. Initially, in the lag phase, the cell concentration
does not increase very much. The length of the lag
phase depends on the growth history of the inoculum,
the composition of the medium, and the amount of
culture used for inoculation. An excessively long lag
phase ties up the fermenter unproductively - hence
the duration of the lag phase should be minimized.
Short lag phases occur when: the composition of the
medium and the environmental conditions in the seed
culture and the production vessel are identical (hence
less time is needed for adaptation); the dilution shock
is small (i.e. a large amount of inoculum is used); and
the cells in the inoculum are in the late exponential
phase of growth. The lag phase is essentially an adap-
tation period in a new environment. The lag phase is
followed by exponential growth, during which the
cell mass increases exponentially. Eventually, as the
~ / ~ ~ ~ ~ ~ ~ Stationary
n t i a l
~ ~ Death
' qrowth Phase phase
~ ~
nutrients are exhausted and inhibitory products of
metabolism build up, the culture enters a stationary
phase. Ultimately, starvation causes cell death and
lysis, and hence the biomass concentration declines.
Exponential growth can be described by Equation
1:
dX
-= ,Ux- k d x (Equation 1)
dt
where: X is the biomass concentration at time t; p is
the specific growth rate (i.e. growth rate per unit
cell mass); and k d is the specific death rate. During
exponential growth, the specific death rate is neg-
ligible and Equation 1 reduces to Equation 2:
dX
-=px (Equation 2)
dt
For a cell mass concentration Xo at the beginning
of the exponential growth ( X , usually equalling the
concentration of inoculum in the fermenter), and
taking the time at which exponential growth com-
mences as zero, Equation 2 can be integrated to
produce Equation 3:
X
In- = p t (Equation 3)
XO
Using Equation 3 , the biomass doubling time, t d , can

be derived (Equation 4):

ln2
t d =- (Equation 4)
iu
Doubling times typically range over 45-160 min. Bac-
teria generally grow faster than yeasts, and yeasts
multiply faster than moulds. The maximum biomass
concentration in submerged microbial fermentations
0
Fermentation time is typically 40-50 kg m-3.
Figure 2 Typical growth profile of microorganisms in a sub- The specific growth rate p depends on the con-
merged culture. centration S of the growth-limiting substrate, until
666 FERMENTATION (INDUSTRIAL)/Basic Considerations

the concentration is increased to a non-limiting level O2 demand, or the fermentation will be 02-limited.
and p attains its maximum value pmax.The dependence O2 demand is especially difficult to meet in viscous
of the growth rate on substrate concentration typ- fermentation broths and in broths containing a large
ically follows Monod kinetics. Thus the specific concentration of 02-consuming cells. As a general
growth rate is given as Equation 5: guide, the capability of a fermenter in terms of 0 2
S supply depends on the aeration rate, the agitation
P = Pmax - (Equation 5 ) intensity and the properties of the culture broth. In
k, + S large fermenters, supplying 0 2 becomes difficult when
where k, is the saturation constant. Numerically, k, is demand exceeds 4-5 kg m-3h-’.
the concentration of the growth-limiting substrate At concentrations of dissolved 0 2 below a critical
when the specific growth rate is half its maximum level, the amount of O2 limits microbial growth. The
value. critical dissolved 0 2 level depends on the micro-
An excessively high substrate concentration may organism, the culture temperature and the substrate
also limit growth, for instance by lowering water being oxidized. The higher the critical dissolved 0 2
activity. Moreover, certain substrates inhibit product value, the greater the likelihood that 0 2 transfer will
formation, and in yet other cases, a fermentation become limiting. Under typical culture conditions,
product may inhibit biomass growth. For example, fungi such as Penicillium chrysogenum and Asper-
ethanol produced in the fermentation of sugar by gillus oryzae have a critical dissolved 0 2 value of
yeast can be inhibitory to cells. Multiple lag phases about 3.2 x kgm-3. For bakers’ yeast and Esch-
(or diauxic growth) are sometimes seen when two or erichia coli, the critical dissolved 0 2 values are
more growth-supporting substrates are available. As 6.4 x 10-’ kgm-3 and 12.8 x 10-jkg m-3 respectively.
the preferentially-utilized substrate is exhausted, the The aeration of fermentation broths generates
cells enter a lag phase while the biochemical machin- foam. Typically, 20-30% of the fermenter volume
ery needed for metabolizing the second substrate is must be left empty to accommodate the foam and
developed. Growth then resumes. Details of the kin- allow for gas disengagement. In addition, mechanical
etics of continuous culture, fed-batch fermentation, ‘foam breakers’ and chemical antifoaming agents are
product formation and more complex phenomena, commonly used. Typical antifoams are silicone oils,
such as the inhibition of growth by substrates and vegetable oils and substances based on low-molecular-
products, are given in the references listed under weight polypropylene glycol or polyethylene glycol.
Further Reading. Emulsified antifoams are more effective, because they
disperse better in the fermenter. Antifoams are added
Aeration and Oxygen Demand in response to signals from a foam sensor. The exces-
Submerged cultures are most commonly aerated by sive use of antifoams may interfere with some down-
bubbling with sterile air. Typically, in small fer- stream separations, such as membrane filtrations -
menters, the maximum aeration rate does not exceed hydrophobic silicone antifoams are particularly
1 volume of air per unit volume of culture broth. In troublesome.
large bubble columns and stirred vessels, the
Heat Generation and Removal
maximum superficial aeration velocity tends to be
c 0.1 m s-’. Superficial aeration velocity is the volume All fermentations generate heat. In submerged cul-
flow rate of air divided by the cross-sectional area tures, typically 3-15 kW m-3 comes from microbial
of fermenter. Significantly higher aeration rates are activity. In addition, mechanical agitation of the broth
achievable in airlift fermenters. In these, aeration gas produces up to 15 kW m-3. Consequently, a fermenter
is forced through perforated plates, perforated pipes must be cooled to prevent a rise in temperature and
or single-hole spargers located near the bottom of damage to the culture. Heat removal tends to be
the fermenter. Because 0 2 is only slightly soluble in difficult, because typically the temperature of the
aqueous culture broths, even a short interruption of cooling water is only a few degrees lower than that
aeration results in the available 0 2 becoming quickly of the fermentation broth. Therefore industrial fer-
exhausted, causing irreversible damage to the culture. mentations are commonly limited by their heat-trans-
Thus uninterrupted aeration is necessary. Prior to use fer capability. The ability to remove heat depends
for aeration, any suspended particles, microorganisms on: the surface area available for heat exchange; the
and spores in the gas are removed by filtering through temperature difference between the broth and the
microporous membrane filters. cooling water; the properties of the broth and the
The 0 2 requirements of a fermentation depend on coolant; and the turbulence in these fluids. The geom-
the microbial species, the concentration of cells, and etry of the fermenter determines the surface area that
the type of substrate. 0 2 supply must at least equal can be provided for heat exchange. Heat generation
 FERMENTATION (INDUSTRIAL)/Basic Considerations 667
due to metabolism depends on the rate of 0 2 con-
sumption, and heat removal in large vessels becomes
difficult as the rate of 0 2 consumption approaches
5 kg m-3h-'.
A fermenter must provide for heat transfer during
sterilization and subsequent cooling, as well as remov-
ing metabolic heat. Liquid medium, or a slurry, for a
batch fermentation may be sterilized using batch or
continuous processes. In batch processes, the medium
or some of its components and the fermenter itself
are commonly sterilized together in a single step, by
heating the medium inside the fermenter. Steam may
be injected directly into the medium, or heating may
take place through the fermenter wall.
Heating to high temperatures (typically 121°C)
during sterilization often leads to undesirable reac-
tions between components of the medium. Such reac-
tions reduce the yield, by destroying nutrients or by
generating compounds which inhibit growth. This
thermal damage can be prevented or reduced by ster-
ilizing only certain components of the medium in
the fermenter and adding other, separately-sterilized
components, later. Sugars and nitrogen-containing
components are often sterilized separately. Dissolved
nutrients that are especially susceptible to thermal
degradation may be sterilized by passage through
hydrophilic polymer filters, which retain particles of
0.45pm or more. Even finer filters (e.g. retaining
particles of 0.2 pm) are also available.
The heating and cooling of a large fermentation
batch takes time, and ties up a fermenter unpro-
ductively. In addition, the longer a medium remains
at a high temperature, the greater the thermal deg-
radation or loss of nutrients. Therefore, continuous
sterilization of the culture medium en route to a pre-
sterilized fermenter is preferable, even for batch fer-
mentations. Continuous sterilization is rapid and it
limits nutrient loss - however, the initial capital
expense is greater, because a separate sterilizer is
necessary.
Photosynthetic Microorganisms
Photosynthetic cultures of microalgae and cyano-
bacteria require light and COZ as nutrients. Micro-
algae such as Chlorella and the cyanobacterium
Spirulina are produced commercially as health foods
in Asia. Algae are also cultivated as aquaculture feeds
for shellfish.
Typically, open ponds or shallow channels are used
for the outdoor photosynthetic culture of microalgae.
Culture may be limited by the availability of light,
but under intense sunlight, photoinhibition limits
productivity. Temperature variations also affect
performance.
More controlled production is achieved in outdoor
tubular photobioreactors, bubble columns and airlift
systems. Tubular bioreactors use a 'solar receiver',
consisting of either a continuous tube looped into
several U-shapes to fit a compact area, or several
parallel tubes connected to common headers at either
end. The continuous looped-tube arrangement is less
adaptable, because the length of the tube cannot
exceed a certain value: photosynthetically-produced
0 2 builds up along the tube, and high levels of dis-
solved 0 2 inhibit photosynthesis. The parallel-tube
arrangement can be readily scaled up by increasing
the number of tubes. Typically, the tubes are 0.05-
0.08 m in diameter and the continuous-run length of
any tube does not exceed 5 0 m . However, greater
lengths may be feasible, depending on the flow vel-
ocity in the tube. The tubular solar receivers may be
mounted horizontally, or horizontal tubes may be
stacked in a ladder configuration, forming the rungs
of the ladder. The latter arrangement reduces the area
of land required.
The culture is circulated through the tubes by an
airlift pump or other suitable low-shear mechanism.
The maximum flow rate is limited by the tolerance of
the algae to hydrodynamic stress. The flow velocity is
usually 0.3-0.5 m c'.The tube diameter is limited by
the need to achieve adequate penetration of light. This
declines as the cell concentration increases, due to
self-shading. Closed, temperature-controlled outdoor
tubular systems attain significantly higher prod-
uctivity than open channels. The protein content of
the algal biomass, and the adequacy of the devel-
opment of colour (chlorophyll)affect the acceptability
of the product.
Among other types of culture system, airlift devices
tend to perform better than bubble columns because
only part of the airlift system is aerated and hence the
penetration of light is less affected by air bubbles.
Conventional external-loop airlift devices may not be
suitable because of the relatively high hydrodynamic
shear rates they generate. However, concentric-tube
airlift devices, with gas forced into the draft tube
(zone of poor light penetration), are likely to perform
well. Also, split-cylinder types of airlift system may
be suitable. However, the volume of the aerated zone
in any airlift device for microalgal culture should not
exceed approximately 40% of the total volume of the
circulating zones. This way the light blocking effect
of bubbles remains confined to a small zone.
Submerged-culture Fermenters
Types The major types of submerged-culture bio-
reactor are:
0 stirred-tank fermenter
bubble column
0 airlift fermenter
668 FERMENTATION (INDUSTRIAL)/Basic Considerations
Figure 3 Types of submerged-culture fermenter. (A) Stirred-tank fermenter. (B) Bubble column. (C) Internal-loop airlift fermenter.
(D) External-loop airlift fermenter. (E) Fluidized-bed fermenter. (F) Trickle-bed fermenter.
0 fluidized-bed fermenter
0 trickle-bed fermenter.
These are shown in Figure 3.
Stirred-tank Fermenter (See Fig. 3(A).) This is a
cylindrical vessel with a working height-to-diameter
ratio (aspect ratio) of 3-4. A central shaft supports
three to four impellers, placed about 1 impeller-diam-
eter apart. Various types of impeller, that direct the
flow axially (parallel to the shaft) or radially
(outwards from the shaft) may be used (Fig. 4). Some-
times axial- and radial-flow impellers are used on the
same shaft. The vessel is provided with four equally
spaced vertical baffles, that extend from near the walls
into the vessel. Typically, the baffle width is 8-10%
of the vessel diameter.
Bubble Column (See Fig. 3(B).)This is a cylindrical
vessel with a working aspect ratio of 4-6. It is sparged
at the bottom, and the compressed gas provides agi-
tation. Although simple, it is not widely used because
Liquid
- Liquid
overflow
Recycle
Product
(F)
of its poor performance relative to other systems.
It is not suitable for very viscous broths or those
containing large amounts of solids.
Airlift Fermenters (See Figs. 3(C) and 3(D).)These
come in internal-loop and external-loop designs. In
the internal-loop design, the aerated riser and the
unaerated downcomer are contained in the same shell.
In the external-loop configuration, the riser and the
downcomer are separate tubes that are linked near
the top and the bottom. Liquid circulates between the
riser (upward flow) and the downcomer (downward
flow). The working aspect ratio of airlift fermenters
is 6 or greater. Generally, these are very capable fer-
menters, except for handling the most viscous broths.
Their ability to suspend solids and transfer 0 2 and
heat is good. The hydrodynamic shear is low. The
external-loop design is relatively little-used in
industry.
Fluidized-bed Fermenter (See Fig. 3(E).) These are
 FERMENTATION (INDUSTRIAL)/Basic Considerations 669
(D) (E)
Figure 4 Impellers for stirred-tank fermenters. (A) Rushton disc turbine (radial flow). (B) Marine propeller (axial flow). (C)Lightnin’
hydrofoil (axial flow). (D)Prochem hydrofoil (axial flow). (E)lntermig (axial flow). (F)Chemineer hydrofoil (axial flow).
similar to bubble columns with an expanded cross
section near the top. Fresh or recirculated liquid is
continuously pumped into the bottom of the vessel,
at a velocity that is sufficient to fluidize the solids or
maintain them in suspension. These fermenters need
an external pump. The expanded top section slows
the local velocity of the upward flow, such that the
solids are not washed out of the bioreactor.
Trickle-bed Fermenter (See Fig. 3(F).)These consist
of a cylindrical vessel packed with support material
(e.g. woodchips, rocks, plastic structures). The sup-
port has large open spaces, for the flow of liquid and
gas and the growth of microorganisms on the solid
support. A liquid nutrient broth is sprayed onto the top
of the support material, and trickles down the bed. Air
may flow up the bed, countercurrent to the liquid flow.
These fermenters are used in vinegar production, as
well as in other processes. They are suitable for liquids
with low viscosity and few suspended solids.
Design Irrespective of their configuration, industrial
bioreactors for sterile operations are designed as pres-
sure vessels, capable of being sterilized in situ with
saturated steam at a minimum guage pressure of
0.11 MPa. Typically, the bioreactor is designed for a
maximum allowable working pressure of 0.28-
0.31 MPa (guage) and a temperature of 150-180°C.
The vessels are designed to withstand a full vacuum.
Modern commercial fermenters are predominantly
made of stainless steel. Type 316L stainless steel is
(F)
preferred, but the less expensive Type 304L (or 304)
may be used in less corrosive situations. Fermenters
are typically designed with clean-in-place capability.
A typical submerged-culture vessel has the features
shown in Figure 5. Sight glasses in the side and top of
the vessel allow for easy viewing. The top sight glass
can be cleaned during fermentation, using a short-dur-
ation spray of sterile water derived from condensed
steam. An external lamp is provided, to light the vessel
through the sight glass or a separate window. The vessel
has ports for sensors of pH, temperature and dissolved
0 2 . A steam-sterilizable sampling valve is provided.
Connections for the introduction of acid and alkali (for
p H control), antifoam agents, substrate and inoculum
are located above the liquid level in the bioreactor
vessel. Additional ports on the top support a foam-
sensing electrode, a pressure sensor and sometimes
other instruments. Filter-sterilized gas for aeration is
supplied through a submerged sparger. Sometimes COZ
or ammonia may be added to the aeration gas, for p H
control.
A harvest valve is located at the lowest point on the
fermenter. A mechanical agitator, entering from either
the top or the bottom, may be used. The agitator shaft
supports one or more impellers, of various designs
(Fig. 4). A high-speed mechanical foam breaker may
be provided at the top of the vessel, and waste gas
may exit through the foam breaker. Commonly, the
exhaust gas line also has a heat exchanger, to condense
and return water in the gas to the fermenter. The top
670 FERMENTATION (INDUSTRIAL)/Basic Considerations
11
-23
3 3. Nature and amount of suspended solids in broth.
4 4. Whether fermentation is aerobic or anaerobic,
Figure 4 A typical submerged-culture fermenter. (1) Reactor
vessel. (2) Jacket. (3) Insulation. (4) Protective shroud. (5) Inoc-
ulum connection. (6) Ports for sensors of pH, temperature and
dissolved 02.(7) Agitator. (8) Gas sparger. (9) Mechanical seal.
(10) Reducing gearbox. (11) Motor. (12) Harvest nozzle. (13)
Jacket connections. (14) Sample valve with steam connection.
(15)Sight glass. (16)Connections for acids, alkalis and antifoam
agents. (17) Air inlet. (18) Removable top. (19) Medium feed
nozzle. (20) Air exhaust nozzle (connects to condenser, not
shown). (21) Instrumentation ports for foam sensor, pressure
gauge and other devices. (22) Centrifugal foam breaker. (23)
Sight glass with light (not shown) and steam connection. (24)
Rupture disc nozzle. Vertical baffles are not shown. Baffles are
mounted on brackets attached to the wall. A small clearance
remains between the wall and the closest vertical edge of the
baffle.
of the fermenter is either removable or provided with
a manhole. A port on the top supports a ‘rupture disc’
that is piped to a drain. The disc is intended to protect
the vessel in the event of a pressure build-up. The
fermentation vessel is jacketed for heat exchange, and
the jacket may be covered with fibreglass insulation
and a protective metal shroud. Additional surfaces
for heat exchange, typically coils, may be located
inside the vessel.
The equipment for fermenting slurries containing
undissolved solid substrates is identical to that used in
submerged-culture processes. Commonly-used slurry
fermenters include stirred tanks, bubble columns, and
airlift vessels.
Selection Considerations in selecting industrial fer-
menters are:
1. Nature of substrate solid, liquid, suspended
slurry, water-immiscible oils).
2. Flow behaviour (rheology), broth viscosity and
type of fluid (e.g. Newtonian, viscoelastic,
pseudoplastic, Bingham plastic).
and O2 demand.
5. Mixing requirements.
6. Heat-transfer needs.
7. Shear tolerance of microorganism, substrate and
product.
8. Sterility requirements.
9. Process kinetics, batch or continuous operation,
single-stage or multistage fermentation.
10. Desired process flexibility.
11. Capital and operational costs.
12. Local technological capability and potential for
technology transfer.
Solid-state Fermentations
Substrate Characteristics
Water Activity Typically, solid-state fermentations
are carried out with little or no free water. Excessive
moisture tends to aggregate the substrate particles,
and hence aeration is made difficult. For example
steamed rice, a common substrate, becomes sticky
when the moisture level exceeds 30-35%w/w. Per-
centage moisture by itself is unreliable for predicting
growth: for a given microorganism growing on dif-
ferent substrates, the optimum moisture level may
differ widely. This water activity correlates with
microbial growth. The water activity of the substrate
is the ratio of the vapour pressure of water in the
substrate to the saturated vapour pressure of pure
water at the temperature of the substrate. Water activ-
ity equals 1/100th of the relative humidity (RH%) of
the air in equilibrium with the substrate. Typically,
water activities of <0.9 do not support bacterial
growth, but yeasts and fungi can grow at water activ-
ities as low as 0.7. Thus the low-moisture environment
of many solid-state fermentations favours yeasts and
fungi.
The water activity depends on the concentrations
of dissolved solutes, and so sometimes salts, sugars or
other solutes are added to alter the water activity.
Different additives may influence the fermentation
differently, even though the change in water activity
produced may be the same. Furthermore, the
 FERMENTATION (INDUSTRIAL)/Basic Considerations 671
fermentation process itself leads to changes in the
water activity, as products are formed and the
substrate is hydrolysed, e.g. the oxidation of carbo-
hydrates produces water. During fermentation,
the water activity is controlled by aeration with
humidified air and, sometimes, by intermittent spray-
ing with water.
Particle Size The size of substrate particles affects
the extent and the rate of microbial colonization, air
penetration and C 0 2 removal, as well as the down-
stream extraction and handling characteristics. Small
particles, with high surface-to-volume ratios, are pre-
ferred because they present a relatively large surface
for microbial action. However, particles that are too
small and shapes that pack together tightly (e.g. flat
flakes, cubes) are undesirable because close packing
reduces the interparticle voids that are essential for
aeration. Similarly, too many fine particles in a batch
of larger particles will fill up the voids.
Substrate pH The p H is not normally controlled in
solid-state fermentations, but initial adjustments may
be made during the preparation of the substrate. The
buffering capacity of many substrates effectively
checks large changes in pH during fermentation. This
is particularly true of protein-rich substrates, espe-
cially if deamination of the protein is minimal. Some
pH stability can be obtained by using a combination
of urea and ammonium sulphate as the nitrogen
source in the substrate. In the absence of other con-
tributing nitrogen sources, an equimolar combination
of ammonium sulphate and urea is expected to yield
the greatest pH stability.
Aeration and Agitation
Aeration plays an important role in removing COZ
and controlling temperature and moisture. In some
cases, an increased concentration of CO2 may be
severely inhibitory, while an increase in the partial
pressure of O2may improve productivity. Deep layers
and heaps of substrate may require forced aeration
and agitation. Forced aeration rates vary widely, a
typical range being (0.05-0.2) x m3kg-' min-'.
Occasional turning and mixing improve 0 2 transfer
and reduce compaction and mycelial binding of the
substrate particles. However, excessive agitation is
undesirable because continuous agitation damages the
surface hyphae - although mixing suppresses spor-
ulation, which is often unwanted. The frequency of
agitation may be purely experience-based, as in the
occasional turning of a fermenting heap of cocoa
beans, or it may be adjusted in response to a tem-
perature controller.
Heat Transfer
The biomass levels in solid-state fermentations, at 10-
30 kg m-3, are lower than those in submerged cultures.
However, because there is little water, the heat gen-
erated per unit of fermenting mass tends to be much
greater in solid-state fermentations than in submerged
cultures, and again because there is little water to
absorb the heat, the temperature can rise rapidly. The
cumulative metabolic heat generation in fer-
mentations producing koji, for the manufacture of a
variety of products, has been noted at 419-2387kJ
per kilogram solids. Higher values, up to
1 3 398 kJ kg-', have been observed during com-
posting. Peak heat generation rates in koji processes
lie in the range 71-159 kJ kg-'h-' but average rates
are more moderate, at 25-67kJkg-'h-'. The peak
rate of production of metabolic heat during the fer-
mentation of readily oxidized substrates, such as
starch, can be much greater than that associated with
typical koji processes.
The substrate temperature is controlled mostly
through evaporative cooling - hence drier air provides
a better cooling effect. The intermittent spraying of
cool water is sometimes necessary to prevent dehy-
dration of the substrate. The air temperature and
humidity are also controlled. Occasionally, the sub-
strate-containing metal trays may also be cooled (by
circulating a coolant), even though most substrates
are relatively dry and porous, and hence are poor
conductors. The intermittent agitation of substrate
heaps further aids heat removal. However, despite
much effort, temperature gradients in the substrate
do occur, particularly during peak microbial growth.
Koji Fermentations
Koji fermentations are widely practised, typical
examples of solid-state fermentations. Koji comprises
soybeans or grain on which mould is growing, and
has been used in oriental food preparation for thou-
sands of years. Koji is a source of fungal enzymes,
which digest proteins, carbohydrates and lipids into
nutrients which are used by other microorganisms in
subsequent fermentations. Koji is available in many
varieties, which differ in terms of the mould, the
substrate, the method of preparation and the stage of
harvest. The production of soy sauce, miso and sakt.
involves koji fermentation. Koji technology is also
employed in the production of citric acid in Japan.
The production of soy sauce (shoyu in Japanese) koji
is detailed below, as an example of a typical industrial
solid-substrate fermentation.
The koji for soy sauce is made from soybeans and
wheat. Soybeans, or defatted soybean flakes or grits
are moistened and cooked (e.g. for 0.25 min or less,
672 FERMENTATION (INDUSTRIAL)/Basic Considerations

at about 170°C) in continuous pressure cookers. The
cooked beans are mixed with roasted, cracked wheat,
the ratio of wheat to beans varying with the variety
of shoyu. The mixed substrate is inoculated with a
pure culture of Aspergillus oryzae (or A. sojue), the
fungal spore density at inoculation being about
2.5 x 10' spores per kilogram of wet solids. After a 3-
day fermentation, the substrate mass becomes green-
yellow because of sporulation. The koji is then har-
vested, for use in a second submerged fermentation
step. Koji production is highly automated and con-
tinuous - processes producing up to 4150 kg h-' of
koji have been described. Similar large-scale oper-
ations are used to produce koji for miso and saki. in
Japan.
Solid-state Fermenters
Solid-state fermentation devices vary in technical
sophistication, from very primitive banana-leaf wrap-
pings, bamboo baskets and substrate heaps to the
highly automated machines used mainly in Japan.
Some 'less sophisticated' fermentation systems, e.g.
the fermentation of cocoa beans in heaps, are quite
effective at large-scale processing. Also, some of the
continuous, highly mechanized processes for the fer-
mentation of soy sauce, that are successful in Japan,
are not suitable for less highly developed locations in
Asia. Thus, fermentation practice must be tailored to
local conditions.
The use of pressure vessels is not the norm for solid-
state fermentation. The commonly used devices are:
tray fermenter
static-bed fermenter
tunnel fermenter
0 rotary disc fermenter
0 rotary drum fermenter
agitated-tank fermenter
continuous screw fermenter.
These are described below. Large concrete or brick
fermentation chambers, or koji rooms, may be lined
with steel, typically Type 304 stainless steel. For more
corrosion-resistant construction, Type 304L and
316L stainless steels are used.
Tray Fevmenter This is a simple type of fermenter,
widely used in small- and medium-scale koji oper-
ations in Asia (see Fig. 6). The trays are made of
wood, metal or plastic, and often have a perforated
or wire-mesh base to achieve improved aeration. The
substrate is fermented in shallow (60.15m deep)
layers. The trays may be covered with cheesecloth to
reduce contamination, but processing is non-sterile.
Single or stacked trays may be located in chambers in
which the temperature and humidity are controlled,
or simply in ventilated areas. Inoculation and occa-
sional mixing are done manually, although the hand-
ling, filling, emptying and washing of trays may be
automated. Despite some automation, tray fermenters
are labour-intensive, and require a large production
area. Hence the potential for scaling up production is
limited.
Static-bed Fermenter This is an adaptation of the
tray fermenter (Fig. 7). It employs a single, larger and
deeper, static bed of substrate located in an insulated
chamber. 0 2 is supplied by forced aeration through
the bed of substrate.
Tunnel Fermenter This is an adaptation of the static-
bed device (Fig. 8).Typically, the bed of solids is quite
long but no deeper than 0.5m. Fermentation using
this equipment may be highly automated, by way
of mechanisms for mixing, inoculation, continuous
feeding and harvest of the substrate.
Rotary Disc Fermenter The rotary disc fermenter
consists of upper and lower chambers, each with a
circular perforated disc to support the bed of substrate

Exhaust t f Conditioned

Figure 6 Tray fermenter. Figure 8 Tunnel fermenter.


FERMENTATION (INDUSTRIAL)/Basic Considerations 673
, t
Figure 9 Rotary disc fermenter.
+
Filling port
Motor
Figure 10 Rotary drum fermenter.
(Fig. 9). A common central shaft rotates the discs.
Inoculated substrate is introduced into the upper
chamber, and slowly moved to the transfer screw.
The upper screw transfers the partly fermented solids
through a mixer to the lower chamber, where further
fermentation occurs. The fermented substrate is har-
vested using the lower transfer screw. Both chambers
are aerated with humidified, temperature-controlled
air. Rotary disc fermenters are used in large-scale koji
production in Japan.
Rotary Drum Fermenter The cylindrical drum of
the rotary drum fermenter is supported on rollers,
and rotated at 1-5r.p.m. around the long axis (Fig.
10).Rotation may be intermittent, and the speed may
vary with the fermentation stage. Straight or curved
baffles inside the drum aid in the tumbling of the
substrate, hence improving aeration and temperature
control. Sometimes the drum can be inclined, causing
the substrate to move from the higher inlet end to the
lower outlet during rotation. Aeration occurs through
coaxial inlet and exhaust nozzles.
Agitated-tank Fermenter In this type of fermenter,
either one or more helical-screw agitators are
Next Page
-f Exhaust
Air
Motor
Figure 11 Agitated-tank fermenter.
mounted in cylindrical or rectangular tanks, to agitate
the fermented substrate (Fig. 11). Sometimes, the
screws extend into the tank from mobile trolleys,
that ride on horizontal rails located above the tank.
Another stirred-tank configuration is the paddle fer-
menter. This is similar to the rotary drum device, but
the drum is stationary and periodic mixing is achieved
by motor-driven paddles supported on a concentric
shaft.
Continuous Screw Fermenter In this type of
fermenter, sterilized, cooled and inoculated sub-
strate is fed in through the inlet of the non-aerated
chamber (Fig. 12). The solids are moved towards
the harvest port by the screw, and the speed of
rotation and the length of the screw control the
fermentation time. This type of fermenter is suit-
able for continuous anaerobic or microaerophilic
fermentations.
 GENETIC ENGINEERING/Modification of Yeasts and Moulds 907
1 Gastric Ulcers see Helicobacter.
GENETIC ENGINEERING
Contents
Modification of Yeasts and Moulds
Modification of Bacteria
Modification of Yeasts and
Moulds
R Sandhir, Department of Biochemistry, Dr Ram
Manohar Lohia Avadh University, Faizabad, India
S K Garg and D R Modi, Department of Microbiology,
Dr Ram Manohar Lohia Avadh University, Faizabad,
India
Copyright 0 1999 Academic Press
The beneficial activities of yeasts and other fungi are
of great economic importance. They have long been
exploited as food, in food processing, and in brewing.
During the 20th century, as the fermentation industry
has developed, they have yielded an increasing range
of valuable products, including antibiotics, vitamins
and various enzymes. However, some fungi can also
cause immense economic losses. Recently, with the
advent of recombinant DNA technology, fungi have
been exploited to produce hormones and proteins
which were hitherto only available from mammals.
The performance of fungus in an industrial process
depends on its genotype under the conditions of the
fermentation process. To maximize yield in a process,
it is essential that new strains are continuously devel-
oped. This involves using genetic techniques com-
bined with selection procedures designed to screen for
the desired improvement. This article describes the
methods and techniques employed for establishing
novel genotypes in various yeasts and moulds.
Techniques for Natural Strain Selection
In nature, there is a great diversity of fungal strains.
The successful isolation of a fungal strain from nature
is dependent on well-planned and often ingenious
screening procedures. The ‘seek and discard principle’
I
is frequently called screening and even today
represents the beginning of any process of strain
improvement. More often the search for strains pro-
ducing desired products entails making dilute solu-
tions of soil or decaying plant material and carrying
out isolations in a way that indicates the desired
properties. The organisms that are obtained may be
moulds or yeasts but can equally well be actino-
mycetes. Sometimes what is sought, as in the pro-
duction of single-cell protein, is an organism capable
of utilizing a particular type of substrate, such as
petroleum hydrocarbons or starch. Although exten-
sive tests are required to ensure that the protein is of
high quality and that the toxic substances are absent,
the first step is isolation of organisms from appro-
priate sites that are able to utilize the selected sub-
strate. The starch-utilizing Fusarium that is used to
produce mucoprotein, for instance, was isolated from
starch-rich effluents.
An enormous range of substances occur in the soil,
and the organisms capable of degrading them are
also present, but in correspondingly smaller numbers.
Their numbers can be accordingly increased prior to
attempting isolation by enriching the soil sample with
an appropriate substrate and then incubating. The
agar medium on which diluted samples are spread
will contain a proper carbon source, for example
starch, if starch-utilizing organisms are being sought.
In this instance, not only is the growth of starch-
metabolizing organisms encouraged, but their pres-
ence is indicated by a clear circular zone around the
colonies, which results from diffusion of amylase from
the colonies and the hydrolysis of opaque starch. The
Aspergillus species used for amylase production is an
example of an organism isolated using this technique.
Organisms that produce enzymes such as pectinase,
908 GENETIC ENGINEERING/Modification of Yeasts and Moulds
cellulase and protease that degrade other macro-
molecules have been detected and isolated by similar
methods. Production of organic acids by micro-
organisms, such as citric acid, can be detected by
including calcium carbonate in the medium, which
must not, however, contain salts such as ammonium
chloride, where the uptake of cation causes the pro-
duction of mineral acid. Organisms producing an
excess of a vitamin can be detected through stimu-
lation of growth of an auxotroph (tester organism)
which is unable to synthesize the vitamin; the test
organism is seeded into or on to a layer of agar added
after the growth of the organism being screened. The
production of an antibiotic by a microorganism can
be recognized by the presence of a clear zone around
the colony, thereby indicating its inhibitory effect. The
colony can then be isolated and its ability to inhibit
the growth of selected pathogens determined.
Although, in the past, this procedure has yielded many
useful antibiotics, it now tends to result only in the
rediscovery of antibiotics that are already known.
Methods of Genetic Improvement
In biotechnology, as soon as a microorganism is avail-
able that forms a certain useful product, the aim exists
to increase its yield. This can be achieved, on the one
hand, by changing or improving the growth con-
ditions (process optimization) and/or, on the other
hand, by genetic manipulation.
Mutagenesis and Selection
Mutagenesis and selection of improved strains have
been the most extensively used methods for intro-
ducing beneficial changes into industrially important
fungi. Mutations are heritable changes which occur
in the genome as a result of one or many types of
events and may involve individual nucleotide bases or
large regions of chromosomes. For a specific gene, the
frequency of spontaneous mutation is very low and
would be expected to occur 1 in 10’-106 nuclei. Con-
sidering the whole genome, such spontaneous muta-
tions are much less rare and provide the basis for the
overall genetic variation found in different isolates or
strains of fungus. The low frequency of spontaneous
mutations of individual genes is clearly not adequate
to meet the constant demands of improved strains
for a fermentation process. To meet this need, it is
necessary to induce mutations using a mutagenic
agent which can be either a physical or chemical agent.
Under optimal conditions, a mutagen will increase
the mutation frequency of a particular gene by at least
one order of magnitude. The physical and chemical
mutagens used frequently for strain improvement in
fungi and their effects on the DNA molecule are sum-
marized in Table 1.
The primary effect of a mutagen is to induce a
lesion or modification of the base sequence of the
DNA molecule. A mutation arises if the alteration
to the molecule is not repaired by the host repair
mechanisms. Our understanding of these repair pro-
cesses is based on studies in Escherichza colt and
Saccharomyces cerevisiae. In the former case, it is
apparent that the repair of damaged DNA is import-
ant in the maintenance of viability because more than
1% of the genome is composed of genes concerned
with the function. The mutations that constitute the
steps in strain improvement programmes commonly
do not affect the morphology, and increase the
product yield by not more than 10-15%. Sometimes
a mutation may result in a much larger increase in
yield, but such a mutation is often accompanied by
changes in morphology and behaviour that require
extensive modification of the medium and fer-
mentation process. Such infrequent major mutations
tend to be less useful than minor mutations, the cumu-
lative effect of which may be more impressive. Inter-
estingly, the haploid status of most fungi ensures that
mutations are expressed soon after they are produced.
The synthesis of primary metabolites (such as an
amino acid) is controlled in such a way that it is
produced in an amount required by the organism.
This is because of the inhibition of the enzyme activity
and repression of the enzyme synthesis by the end
product and such a control is referred to as feedback
Table 1 Mutagens effective against fungi and their mode of
action
Mutagen Action responsible for mutagenesis
Physical mutagens
X-rays Single- and double-strand breaks in
the phosphodiester backbone.
Point mutations because of
deamination and dehydroxylation
of bases
Ultraviolet light Pyrimidine dimer formation
Chemical mutagens
Nitrous acid Reacts with primary amino group to
form hydroxyl group (adenine to
hypoxanthine, guanine to xanthine
and cytosine to uracil, causing
alterations in subsequent
replication)
Alkylating agents Causes alkylation of N and 0 atoms
(dimethylsulphonate, of bases. Alkylation leads to
etc.) mispairing of alkylated bases
Acridines Acridine mustard is comprised of an
(acridine mustard) acridine ring and alkylating moiety.
The former intercalates in base
pairs, causing disruption of base
pairing
 GENETIC ENGINEERING/Modification of Yeasts and Moulds 909
control. It is obvious that a good commercial mutant
should lack the control system and lead to over-
production of the product. Mutagenesis has been suc-
cessful in improving the yield of most of the
antibiotics, including penicillin. Mutation frequency
has been enhanced in microorganisms by inducing
mutations in DNA repair mechanisms.
Strains giving improved performance have to be
selected following mutagenesis and this is governed
by the type of mutation desired. A variety of selection
methods are available for nutritionally defective
mutants, resistant mutants, temperature-sensitive and
similar types of mutations. The original approach was
random screening. Samples of the cells that had been
treated by the mutagen were spread on the agar
medium and the colonies arising from the single cells
were isolated. The isolates were then grown in liquid
medium and the culture filtrate assayed for the
required product. Such a procedure is tedious and
labour-intensive, since only a small percentage will
show improvement in yield. A reduction in labour
can be achieved where it is possible to detect improved
performance on agar media, employing methods
similar to those employed for initial detection of prod-
ucts of interest. Alternatively, indirect screening
methods are sometimes possible, where what is esti-
mated is not the amount of desired product, but some
readily determined attribute likely to be correlated
with the amount of desired product. For example,
sensitivity to sodium monofluoroacetate is correlated
with accumulation of citric acid. This is because the
compound inhibits the enzyme aconitate hydratase
which converts citric acid to isocitric acid prior to
its further metabolism. Hence, a mutant with a low
amount of this enzyme will be more sensitive to the
sodium monofluoroacetate and will also be one in
which citric acid tends to accumulate.
Recombination
Recombination offers an alternative approach for
genetic modification and is achieved through either
sexual or asexual reproductive processes. Employing
genetic recombination, one can obtain the desirable
features of two different strains involved in recom-
bination. Recombination between whole genomes or
involving small fragments of DNA provides add-
itional approaches to the generation of strains with
novel phenotypes that can be included in the strain
improvement programmes. Whole genome recom-
bination occurs as a result of sexual and parasexual
processes, and recombination involving small frag-
ments is achieved through transformation methods in
vivo.
Sexual Hybridization Fungi exhibit a true sexual
process of fusion of two parental cells to form a
heterokaryon, containing nuclei of both parental
types in a common cytoplasm. These nuclei exist
separately, so recombination does not occur at this
stage; complementation can, however, be demon-
strated at this stage. The duration of the hetero-
karyotic stage varies from ephemeral to indefinite
(depending on the species) and is succeeded by fusion
of two nuclei to give a true diploid cell, usually within
a specialized structure. In yeasts the diploid stage may
persist, but in filamentous fungi the formation of
diploid nucleus is followed by meiosis with the pro-
duction of haploid spores. The four products of
meiosis are retained in a structure called a tetrad.
Most fungi of industrial importance reproduce only
by asexual sporulation. However, the notable excep-
tions to the rule are S. cerevisiae and Agaricus bi-
sporus, the common white mushroom. Other sexually
reproducing fungi of industrial interest are Claviceps
purpurea and Mucor species. In both of them, the
reproductive process cannot be easily manipulated
in the laboratory. Spore germination is poor in A.
S , about 70% of the spores are self-fertile,
~ Z S ~ O Y Uand
due to the presence of nuclei of both the mating
types in a usually binucleate spore. In spite of this,
homokaryons of both mating types can be obtained,
and mated to yield new dikaryotic strains that give
fruiting bodies. However, strains employed in brewing
often sporulate poorly or not at all; spores, if
obtained, may be difficult to germinate, and mating
of the resultant haploids may not be easy. Despite all
this, mating has been used in strain improvement. For
example, mating among haploids obtained from a
diploid lager yeast gives some diploids with better
flocculation and lower diacetyl production, both
desirable features that occur with the parent strain.
Here the beneficial results have been obtained through
the elimination of undesirable dominant genes in the
original diploid; the introduction of new genetic
material requires mating with the haploid progeny of
a diploid strain.
Parasexual Hybridization Many species of Asper-
gillus, Penicillium and other fungi of genetic import-
ance lack a sexual cycle. They are, however, able to
undergo a parasexual cycle. The first step in this
cycle is the formation of a heterokaryon which is
accomplished by fusion of protoplasts of the two
strains. The next step, vegetative diploidization, is
normally a rare event, but the frequency can be greatly
increased by exposing to camphor or ultraviolet radi-
ation. Other treatments can be used to increase both
the frequency of recombination between homologous
chromosomes and of haploidization, the return to
910 GENETIC ENGINEERING/Modification of Yeasts and Moulds

haploid strain. The strains are selected by growing
the protoplasts in the presence of required nutrients
and forcing them to grow under conditions which
support the development and growth of the hetero-
karyon and selection against the two parental strains.
Nutritionally complementing auxotrophic strains are
generally used and a minimal medium provides the
required selective conditions to promote hetero-
karyosis. The parasexual cycle has proved useful in
bringing about genetic recombination between closely
related industrial strains, for example strains of Pen-
icillium chrysogenum with high yields of penicillin
and ancestral strains with lower yields but superior
growth and sporulation have been obtained. Vege-
tative compatibility hinders heterokaryon formation
between distantly related strains; although protoplast
fusion can to some extent by-pass the barrier, there has
been rather little success in obtaining recombinants
between such strains.
The major differences between the sexual and para-
sexual processes are summarized in Table 2. The in
vivo gene manipulations have been successfully used
to increase enzyme production, for overproduction of
primary and secondary metabolites.
Table 2 Comparison of sexual and parasexual mechanisms of
recombination
Sexual Parasexual
Specialized cells are involved Vegetative cells are involved in
in hyphal anastomosis hyphal anastomosis
(plasmogamy), rarely
vegetative cells
Heterokaryon formation
controlled by compati bility
genes in vegetative
(plasmogamy)
Recombinant DNA Technology
The more recent technology of DNA recombination
in vitro provides a new and more direct method for
manipulating the fungal genome. The methods of
strain improvement discussed in previous sections are
uncertain in their effects. A mutation that has brought
about the desired genetic change in a strain may at
the same time cause other mutations that are dis-
advantageous. Although gene cloning is more time-
consuming as well as labour-intensive, its value has
been recognized, and now figures in the strategies of
strain improvement adopted for many products by
most major companies involved in fungal fer-
mentation technology. The various steps involved in
recombinant DNA technology are described and illus-
trated in Figure 1.
Obtaining DNA SequencedGene of Interest The
first step in gene cloning is isolation of the desired
gene. This involves extraction of DNA from the donor
animal, plant or microbe, and treatment in various
ways with restriction enzymes to obtain the fragment
of DNA or the desired gene. If the gene to be cloned
in fungi is of mammalian or eukaryotic origin, it may
contain one or more introns (non-coding intervening
sequences). The nucleotide sequence of such a gene is
transcribed into RNA molecules that need to be edited

Heterokaryon formation
controlled by
incompatibility genes
Reverse
transcription
\

i r
Restriction
endonuclease
I Restriction
endonuclease

plasmogamy DNA fragmenticDNA

Diploid phase is restricted to a
single nuclear generation
The diploid nucleus IS
spatially separated in a
specialized cell
Diploid nucleus is a somatic
nucleus and persists
through many mitotic Annealing and ligation

Chromosomes are involved in

multiple cross-over during
meiosis
divisions and can be
maintained by employing
suitable selection
Cross-over during mitotic
divisions is rare, and
normally involves one arm of
‘0’ ,
;rimbinant
molecule

Haploidization is achieved by
meiosis, the meiotic
products being
incorporated into spores
a pair of chromosomes
{Transformation
Mitotic non-disjunction results
in aneuploidy, ultimately
yielding haploid status
~ ~~

Products of meiosis are readily Host (yeast/fungus)

Recombinants occur among
recognized and isolated vegetative cells and are
identified by use of suitable
i
Selection /screening
markers
Figure 1 Scheme for gene cloning in yeasts and fungi.
 GENETIC ENGINEERING/Modification of Yeasts and Moulds 91 1
before being passed from nucleus to cytoplasm for
synthesis of protein. Prokaryotes do not have the
necessary mechanisms for processing eukaryotic RNA
and therefore have to be provided with DNA
sequences complementary with mRNA. Fungi, being
eukaryotes, have the mechanisms required for RNA
processing and production of heterologous
(mammalian) proteins. Therefore, a gene having
introns can be cloned in fungi. However, the fungal
genome, having few introns, does not have identical
mechanisms for processing DNA as mammals or other
eukaryotes and hence complementary DNA (cDNA)
is still a better choice. This involves isolation of
mRNA for the desired gene and copying to double-
stranded cDNA using the enzyme reverse tran-
scriptase. The amount of DNA obtained for cloning
can be amplified using the technique of polymerase
chain reaction.
Introduction of DNA into Fungal Vectors A pre-
requisite for gene cloning is the availability of a suit-
able vector - a DNA molecule that will survive and
replicate in the selected host and into which the gene
to be cloned can be inserted. Numerous plasmids for
fungal transformation have been constructed. The
basic components of these plasmids are first, a gene
that can be used for selection of fungal trans-
formations, and second, bacterial plasmid sequences
that can be used for selection and propagation of the
plasmid in E. coli. The number of different selectable
markers available for fungi is now fairly large and
these selective genes can be used in many species, thus
providing substantial flexibility in designing plasmids
for specific purposes. Selective markers can be divided
into at least three functional groups: genes that com-
plement pre-existing mutations and lead to pro-
totrophic growth (auxotrophic markers); genes that
provide a new functian and lead to drug resistance or
growth on previously non-utilizable nutrient source
(drug-resistant or added-function markers) and DNA
fragments that give rise to selectable mutations when
integrated into the genome of the recipient strain at
specific locations (mutagenic markers). Each type of
marker has specific uses. One of the most commonly
used marker in S. cerevisiae is the URA3 gene which
encodes orotidine-5’-phosphate decarboxylase, an
Table 3 Selectable markers used in Saccharomyces cerevisiae
Marker Function
URA3 Orotidine-5’-phosphate decarboxylase
HIS3 Imidazole glycerol phosphate dehydratase
LEU2 p-lsopropylmalate dehydrogenase
TRPl N-(5’-phosphoribosyl) anthranilate isomerase
TUN@ UDP-N-acetylglucosamine-1 -P transferase
SUP4 Tyrosine-tRNA
enzyme of uracil biosynthesis. Acquisition of a func-
tional URA3 gene by the cells that were previously
mutant at this locus allows them to grow in the
absence of exogenous uracil. Examples of other fre-
quently used selectable markers in S. cerevisiae that
can be selected in a similar way are given in Table 3.
TUN@ confers resistance to the antibiotic tun-
icamycin, an inhibitor of glycosylation. Lack of SUP4
(an ochre chain termination suppressor) can be
selected in suitable host backgrounds. One such host
has an ochre chain termination mutation in the CAN1
gene. The wild-type gene encodes a permease that
causes the uptake of the toxic arginine analogue cana-
vanine and, consequently, causes cell death in the
presence of canavanine. So CAN1 mutant cells are
resistant to canavanine, as they are unable to take it
up. However, the chain termination suppressor SUP4
causes the production of functional permease in the
CAN1 background (because it suppresses the CAN1
mutation) and, therefore, causes the death of CAN1
cells in the presence of canavanine. Loss of SUP4
abolishes canavanine uptake and canavanine resist-
ance, which can be readily selected. Cells containing
SUP4 can also be identified by the use of hosts with
a chain termination mutation in the ADE2 gene for
phosphoribosyl amino-imidazole carboxylase, a com-
ponent of the arginine biosynthesis pathway. The
mutation causes the cells to accumulate a red pigment,
whereas colonies are of the usual colour if they are
wild-type or if the ochre mutation is suppressed by
SUP4. Cells that have lost SUP4 therefore acquire the
red pigment and are visibly distinguishable from the
others. If host strain without LEU2 is chosen, and a
medium lacking leucine is employed, only cells trans-
formed by the vector having LEU2 marker will grow.
Several selectable markers are available for fila-
mentous fungi and the common examples are given
in Table 4. The argB marker is involved in arginine
biosynthesis and needs a host deficient in arginine
biosynthesis. The other markers are responsible for
conferring resistance to variety of antibiotics.
The yeast S. cerevcsiae has been extensively used
for gene cloning, and a variety of vectors have been
employed. Most vectors used in yeast are shuttle
vectors as they are able to replicate in a second species,
Pathwaylpheno type
de novo synthesis of pyrimidines
Histidine biosynthesis
Leucine biosynthesis
Tryptophan biosynthesis
Glycosylation; confers tunicamycin resistance
Ochre suppressor
912 GENETIC ENGINEERING/Modificationof Yeasts and Moulds

Table 4 Selectable markers used in filamentous fungi presence of a centromeric sequence allows the plasmid
Marker Function Pathwaylphenotype to be partitioned by the spindle apparatus of the cell,
in the same way that the endogenous chromosomes
argB Ornithine Synthesis of arginine
transcarbamylase
are partitioned. This also results in the reduction of
(from Aspergillus copy number per cell, but confers a much greater
nidulans) stability during cell division, allowing the plasmid to
hph Hygromycin Hygromycin resistance be maintained in the absence of selection.
phosphotransferase An example of YEP plasmid is shown in Figure 3.
(from Escherichia col/J
Resistance to fungicide
An episomal plasmid is a plasmid that can replicate
benA p-Tubulin (from A.
nidulans) benomyl independently but can also be integrated into the
amdS Acetamidase (from A. Growth on acetamide as chromosome. YEps may do this as a result of sequence
nidulans) sole carbon or nitrogen homologies between the selectable marker gene of
source the vector and mutant allele of the host, allowing
ble Bleomycin-binding Bleomycin resistance
protein (from (binds to bleomycin and
recombination. Most of the features of YEp are
Sfreptoalloteichus inhibits its DNA- similar to those of YCp plasmids, except for the
hindustanus) damaging activity) absence of the centromeric sequence and its replace-
ment with a different origin of replication. YEp has
been constructed from a naturally occurring episomal
E. coli. Initial cloning is done in E . coli and then the plasmid, 2 p plasmid of S. cerevisiae using restriction
recombinant DNA is transformed into yeast cells. The enzyme and DNA ligase. YEps are very effective in
plasmid vectors most commonly used in yeast can be bringing out transformation and occur in high copy
divided into two categories: yeast centromeric plasmid numbers. Hence, these can have high rates of mRNA
(YCp) and yeast episomal plasmid (YEp) vectors. An transcription and a high output of protein specified
example of a YCp plasmid is given in Figure 2. It by the cloned gene. Apart from a naturally occurring
contains prokaryotic ampicillin and tetracycline origin of replication, YEps also have a naturally
resistance genes, and an origin of replication from E. occurring cis-acting sequence, REP3, which is the site
coli, all in a region of the plasmid derived from of action of proteins that help in partitioning the
pBR322. These will allow it to function as a shuttle plasmid during cell division. This gives YEp plasmids
vector. Many of these vectors are based on pBR322 a mitotic stability similar to YCp vector, but with a
in this way, but other plasmids have also been used, somewhat high copy number. The 2p plasmid has the
including pUC and bluescript species. YCp vectors genes essential for DNA replication and maintenance
also have a selectable marker and an origin of rep- of numerous copies - up to about 100 - in the yeast
lication for yeast, ARSI. ARS, autonomously rep- cell. About half of the plasmid, however, is not con-
licating sequences, are the sequences that confer on cerned with these functions and can be dispensed
the plasmid the ability to replicate without integration within vector construction. In the yeast Kluy-
into another replicon. The distinguishing feature of veromyces lactis, vectors equivalent to S. cerevisiae
YCp plasmids is the presence of a chromosomal cen-
tromeric sequence, CEN4 in the present example. The Te1

Tet

EC Or LEU2

EC Or1 URA3

CEN
Figure 2 Yeast centromeric plasmid, YCp50 (7.9kb). The
vector has a suitable origin (Escherichia coli origin: EC Ori) and
selectable markers (Amp and Jet) that allow it to be used as a
shuttle vector in E. coli. The vector also contains a yeast select-
able marker (URA3), a centromeric sequence (CEN) and an
autonomously replicating sequence (ARS7).
REP3
Figure 3 Structure of yeast episomal plasmid, YEpl3 (10.8 kb).
The YEp is derived from 2 pm plasmid of Saccharomyces cer-
evisiae and Escherichia coli plasmid pBR322. The vector has E.
coli origin of replication (EC Ori) and selectable markers (Amp
and Jet) that allow it to be used as a shuttle vector in E. coli. The
vector contains a yeast selectable marker (LEU2), partitioning
sequence (REP3) and an autonomously replicating sequence
(ARS).
 Amo -
GENETIC ENGINEERING/Modification of Yeasts and Moulds 913

Chromosomal DNA

Figure 4 Structure of yeast artificial chromosome (YAC). The

vector has two telomeres that constitute the ends of the vector
once it is linearized with BamH1 after introducing the desired
gene into the cloning site (SUP4).
- V

Mutated gene
Figure 5 Gene inactivation by homologous plasmid integration.
The constructed plasmid contains a DNA fragment from within
the gene, shown as a filled rectangle; the deleted regions of the
gene are shown by the open area within the rectangle. Chromo-
somal recombination by a single homologous recombination
YEP have been obtained from 1 . 6 circular
~ plasmid, event (X) leads to duplication of two partially deleted copies of
and also from linear killer plasmids. the genes separated by plasmid DNA sequence, leading to gene
inactivation.
Another vector used is yeast artificial chromosomes
(YAC), which resemble chromosomes in that they are
linear DNA with a centromere and a telomere at each
end - a sequence that prevents attack by endo- orotidine-5’-phosphate decarboxylase, which can be
nuclease. An example of YAC is shown in Figure 4. selected by resistance to 5’-fluoroorotic acid.
The vector contains HIS3, URA3, TRPl and SUP4 The transforming DNA is integrated into the
markers. CEN4 is a centromeric sequence and TEL chromosomes by the process of homologous and het-
is a telomeric sequence derived from the ends of erologous integration during vegetative growth.
ribosomal RNA encoding molecules from the mac- Homologous integration of chromosomal DNA
ronucleus of Tetruhymena. In addition, there is a resembles that of yeast. The relative frequencies with
yeast origin of replication, a prokaryotic origin of which homologous and non-homologous recom-
replication, and an ampicillin resistance gene. The bination events occur depend on the species and the
cloning strategy involves cutting the YAC DNA with length of the DNA molecule. In A. nidulans and P.
the restriction enzyme, BamH1, in the present chrysogenum, over half of the integration events are
example, to generate linear DNA with telomeres at homologous, while in Neurospora cvassa such events
the two ends, followed by treatment with another can be less than 5%. Homologous integration is
restriction enzyme (Smal) to accommodate the important for manipulation involving gene disruption
foreign DNA. YACs are large, stable and ideal for and deletion. Multiple transformation events are quite
cloning large DNA sequences. YACs are around 20 kb common in filamentous fungi and can be a way of
in size and can carry inserts up to 500 kb. increasing gene dosage in commercial applications.
Another type of vector used in fungi are integvating Since the integrating vectors give low-frequency trans-
vectors, which do not have their own origin of rep- formants, by analogy with yeasts, replicating plasmids
lication but rather become integrated into the nuclear are expected to give better results. Naturally rep-
DNA by homologous recombination and replicate licating vectors are rare in fungi; some species of
along with the nuclear genome. As illustrated in Neurospora contain mitochondrial plasmids of
Figure 5, homologous integration of a circular unknown function, but these have not proved useful
plasmid carrying an intergenic DNA fragment leads in the construction of autonomously replicating
to duplication in which the two partially deleted vectors, A plasmid of Phanerochaete chrysosporium,
copies of the genes are obtained separated by plasmid pME, has recently been detected and has been incorp-
DNA sequences. Such an event most often leads to a orated into a stable, replicating vector with kan-
loss of gene function that, in some cases, can be used amycin resistance marker which is maintained
for selection of the integration event and thus for extrachromosomally in the absence of selection. The
transformation. For example, integration of an native circular plasmid exists in low copy number,
internal fragment of Aspergillus nidulans pyrG gene, and is relatively difficult to detect.
as shown in Figure 5, is expected to lead to loss of In the absence of natural plasmids, and by analogy
914 GENETIC ENGINEERING/Modification of Yeasts and Moulds

with yeasts, it has been possible to construct rep- by digestion of cell wall with appropriate enzymes, in
licating vectors using chromosomal sequences, carry- the presence of a suitable osmotic buffer to prevent
ing putative origins of replication, autonomously lysis. Calcium chloride and PEG facilitate uptake of
replicating sequences. A replicating vector of Podo- transforming DNA by the protoplasts. However, the
spora anserina was constructed by adding telomeric use of PEG for transformation is disadvantageous as
sequences from Tetrahymena thermophila ribosomal it causes cell fusion and so diploids and polyploids
DNA (a self-replicating element in protozoans). This may result. Interestingly, it has been found that
plasmid becomes linearized in Podospora following lithium ions render fungal walls permeable to DNA,
transfer, and replicates at low copy number without so the use of protoplasts is not always necessary.
integration. More recently, a sequence isolated from Recently, electroporation (entry of DNA under the
A. nidulans, AMAl, confers the ability to replicate influence of electric charge) has also been used for
autonomously to the plasmids that carry this gene, successful transfer of DNA to protoplasts. A yet more
and this leads to an increase in transformation fre- efficient method, the Shotgun approach, involves
quency 100-fold or more compared to integrating bombardment of DNA-coated tungsten particles dir-
vectors. Although present in 10-30 copies per cell, ectly into the mycelium.
these vectors are unstable and are rapidly lost without The filamentous fungi, such as A. nidulans, N .
selection. An ARS-based vector that replicates crassa and P. chrysogenum, are also of considerable
autonomously has been reported for corn smut patho- interest. Transformation can be carried out with
gen Ustilago maydis. protoplasts made from spores or hyphae, by digestion
After a suitable vector is decided, the vector is of the cell wall/coat with suitable enzymes. DNA is
opened with the help of restriction enzyme, an enzyme added in the presence of calcium ions and PEG. The
that recognizes a specific nucleotide sequence and cuts latter causes protoplast fusion and the DNA is taken
the molecule at that point and no other. A vector has up at the same time. However, electroporation can
to have a cloning site containing sequences that can also be used for transformation. The protoplasts are
be cut with the restriction enzymes to permit insertion then regenerated. Ensuring the stable maintenance of
of DNA to be cloned. Usually a polylinker cloning extrachromosomal DNA is difficult, because of the
site is employed: this is a short synthetic double- filamentous nature of the fungi. As a result of this
stranded sequence that consists of a number of sites filamentous habit, cells that have lost the selectable
on which different restriction sites can act, giving marker can be maintained by those cells that have
experimental versatility. A portion of opened vector retained it. Thus using the system that results in inte-
is mixed with mammalian DNA or the gene of interest gration of incoming DNA may be more convenient.
that has to be inserted, and allowed to anneal, fol- After transformation, the cells carrying the desired
lowed by treatment with DNA ligase which rejoins plasmids are selectedhcreened using the suitable
the cut DNA vector. markers and looked-for expression. The fate of the
incoming DNA depends upon whether or not a suit-
Entry and Expression of Recombinant DNA in Fungal able origin of replication is present. ARS elements
Cells Once the recombinant DNA is constructed, it have been isolated, such as UARSZ from U. maydis,
has to be transformed into a suitable host. The thick which allows maintenance as a plasmid.
cell walls of yeasts and other fungi are a major obs- When plasmid has to be inserted, a plasmid-free
tacle to the entry of DNA into the cell. There are a host has to be employed, so that the plasmids do not
number of ways available for introducing exogenous compete with the vector. The clones containing the
DNA into fungi. One of the easiest ways is simple desired vector are selected using markers, since all the
treatment of the cells with lithium acetate and poly- treated host cells may not acquire plasmid. LEU2 and
ethylene glycol (PEG), together with transforming HIS are selectable markers and code for enzymes that
DNA and extra nonspecific carrier DNA (for example, are essential for leucine and histidine biosynthesis,
from calf thymus) to.increase the total amount of respectively. In some cases, antibiotic resistance
DNA present. Although this method is relatively markers have also been used.
straightforward, the efficiency of transfer can be Various factors determine the effectiveness with
rather low - approximately IO3 colonies per micro- which a gene is expressed, but of special importance
gram of plasmid - although higher frequencies have is the nature of the promoter. Controlled expression
been claimed and may represent differences between can be directed conveniently from the alcA promoter,
strains. for alcohol dehydrogenase, in response to carbon sub-
A second and often more efficient approach of strate. There are several controllable promoters avail-
introducing exogenous DNA into S. cerevisiae is the able for expression in yeast. In order to have good
transformation of spheroplasts, which are obtained expression, the vectors generally have promoter from
 GENETIC ENGINEERING/Modification of Yeasts and Moulds 915
host cell, e.g. promoters for galactose epimerase, acid
phosphatase from S. cerevisiae, lactase from K. lactis.
Secretion of overexpressed proteins by fusion with
a secretion signal may be more useful as it facilitates
purification and the proteins produced will be min-
imally exposed to host proteases. In addition, it may
help to bring about correct glycosylation and may
also enhance protein folding. A number of secretion
signals from yeast and fungi can be used; the most
commonly used are those for the mating pheromone
alpha-peptide and for invertase. Aspergillus spp. have
been used to secrete proteins/enzymes into the
medium and hence downstream processing is eco-
nomical in such cases.
Applications of Recombinant DNA Technology
Production of Mammalian Proteins by Fungi The
bacterium E . coli was not only the first host for gene
cloning, but also the first microbe to be used for
commercial production of mammalian proteins. In
spite of these successes, E. coli is not ideal for pro-
duction of heterologous proteins (production that
would not be made by the species in question), espe-
cially of mammalian origin. As a polypeptide is syn-
thesized it folds and disulphide bridges are formed
to give a protein-precise three-dimensional structure.
The folding and disulphide bridge formation can
occur in prokaryotic cells giving a protein with the
same amino acid sequence, but different properties.
In mammalian cells a peptide can be modified in a
variety of different ways following its synthesis. Hence
the active protein is sometimes a protein in which part
of the original polypeptide chain has been removed by
proteolytic cleavage. Alternatively, the active product
may result from enzymatic addition of groups such as
oligosaccharides, phosphates or sulphates to specific
amino acids. Bacterial cells are incapable of these
post-translational modifications. Furthermore, E. coli
usually retains the proteins it makes within the cell
instead of exporting them into the culture medium.
This means that the downstream processing is more
difficult, especially since E . coli produces endotoxins
(toxins associated with the cell) and pyrogens (fever-
inducing agents) which must be completely eliminated
during purification. These shortcomings have lead to
research aimed at finding ways of using other species
as hosts for gene cloning.
The first fungus with which success was achieved
was S. cerevisiae. Another yeast, K. lactis, has been
found to be an efficient host for synthesizing and
exporting mammalian protein chymosin. Bovine chy-
mosin or rennin is used in the cheese industry, and
because of its high value and supply problems, it has
been the target for microbial expression for some
time. As a result of difficulties encountered in earlier
attempts at expression in bacterial and yeast systems,
A . awamori has been successfully developed as a host,
using a wide range of genetic manipulation tech-
niques. The preprochymosin gene is expressed using
transcription signals derived from host glucoamylase
gene to produce recombinant chymosin.
Fungal Enzymes A wide-range of fungal ( A . niger,
A . oryzae) enzymes like amylases and cellulases are
used in food industry. Most of these are secreted
enzymes of low value, made in large quantities by
strains already improved by conventional procedures.
Nevertheless, many of the genes encoding for these
enzymes have been isolated, and attempts have been
made to improve their yield by transformation. Many
oversecreting recombinant strains of fungi have been
produced using recombinant DNA technology, secret-
ing high amounts of enzymes with applications espe-
cially in food biotechnology (Table 5).
Some of the genes encoding useful enzymes, such
as the a-amylase (taka-amylase) of A. oryzae and the
glucoamylase of A. nzgerlA. awamori, already possess
some of the strongest fungal promoters known, par-
ticularly when they are derived from commercially
improved strains, and these promoters have been
exploited for construction of hybrid expression
systems. Fungal lipases such as those from Humicola
and Rhizopus species are now used in detergents.
Their production has been enhanced by expression
in A. oryzae, using the a-amylase promoter of this
organism.
Strain Improvement Recombinant DNA technology
has been widely used to improve strains. For example,
in brewing yeast, it has been advantageous in the
improvement of fermentation and post-fermentation
stages in beer production. Some of the strain improve-
ments achieved in brewing yeasts are shown in Table
6. The process can be made more efficient by reducing
Table 5 Enzymes produced by fungi having applications in
food biotechnology
Enzyme Source Applications
a-Amylase Aspergillus niger, Starch conversions
A. oryzae
Glucose oxidase A. niger Food processing
Rennet Mucor spp. Milk coagulation
Lipases M u c o ~Aspergillus, Dairy industry
Penicillium spp.
Hemicellulase A. niger Bakery; gum
Pectinases Aspergillus, Clarifying fruit juices
Rhizopus spp.
Invertase Yeasts Confectionery
Proteases (acid, Aspergillus sp. Breakdown of
neutral, alkaline) proteins (baking,
brewing and
dairy)

916 GENETIC ENGINEERING/Modification of Yeasts and Moulds
the residence time in beer vessels. Product quality
can be improved by inactivating the genes which are
responsible for production of off-flavours or reducing
susceptibility to contaminants. Yeast capable of
secreting enzymes that would otherwise be added to
fermentation medium will make the process more
efficient and economical. The yield of a particular
product can also be improved using in vitro gene
manipulation techniques. Employing such
approaches, the yield of antibiotic production can be
greatly improved by recombinant DNA technology.
The commercial p-lactam antibiotic producers P.
chrysogenum and Cephalosporium acremonium have
been the main targets for this work. This is achieved
by inserting additional copies of such genes.
Table 6 Strain improvement of brewing yeast
Properties Effed
Amylosis Low-carbohydrate beer
Proteolytic yeast Aids colloidal stability
Reduced phenolic compounds Reduced off-flavours
Reduced diacetyl production Reduces fermentation time
Catabolite derepression Improved fermentation
Zymocin production Protection against
contamination
Therapeutics A number of therapeutic products
have been produced in fungal systems, such as cyto-
kines (interleukin-2).
Risks to Consumers and/or Ecology of
Non-target Species
Considering the benefits of recombinant DNA tech-
nology, any preconceived hazard cannot be ignored.
The introduction of modern biotechnology involving
genetic manipulation in the last two decades has gen-
erated some concern. This highlights the need to
ensure that what sometimes seem to be specific
hazards from an organism used in biotechnology are
adequately assumed and the risks are controlled. The
public perception of microorganisms is that they cause
disease. The impact of a laboratory infection can
stretch beyond the immediate surroundings where
industrial processes are employed, and it can have a
serious impact on the whole plant, on the marketing
of the products, on innocent bystanders, and on the
environment. Therefore, a microorganism used in bio-
technology should be safe and cause no mortality. A
pathogenic organism should not produce an allergic
response or endotoxin or a toxic reaction. For
example, an E. coli strain producing botulism toxin
could conceivably be very dangerous; insertion of the
penicillinase gene into Streptococcus pyogenes (still
universally sensitive to penicillin) could make the
Next Page
treatment of streptococcal infections much more dif-
ficult.
It has been observed that single-cell protein con-
sumption caused ill health in consumers. Physical
contamination or escape from the laboratory of
recombinant organisms may pose a health hazard. Let
us take a hypothetical situation; if an interleukin-2-
producing microorganism escapes from a laboratory
it may produce an immunological imbalance in the
host it enters. The modified organisms released should
also have no adverse effect on the environment. The
growth of a microorganism depends on the substrate
present in a particular ecological niche. If the organ-
ism shows change in the substrate utilization, it will
grow in a different ecological niche from its natural
niche and may have a deleterious effect on a given
habitat. If an antibiotic-resistant microorganism is
used, there is a risk that it may transfer antibiotic
resistance genes to other organisms by the processes
of conjugation, transformation and transduction.
Therefore, a genetically engineered organism should
only be released after thorough assessment of the
risks.
See also: Aspergillus: Introduction. Ecology of Bac-
teria and Fungi in Foods: Influence of Available Water;
Influence of Temperature; Influence of Redox Potential
and pH. Escherichia coli: Escherichia coli. Fusarium.
Genetic Engineering: Modification of Bacteria. Gen-
et ics of Microorganisms: Fungi. Kluyveromyces.
Nucleic Acid-based Assays: Overview. Penicillium:
Introduction. Yeasts: Production and Commercial Uses.
Further Reading
Arora DK, Elander RP and Mukherji I<G (1989) In: Hand-
book of Applied Mycology. Vol. 4. New York: Marcel
Dekker.
Bennett J W and Lasure LL (1991) In: Gene Manipulations
in Fungi. San Diego, CA: Academic Press.
Berry DR (1988)In: Physiology oflndustrialFungi. Oxford:
Blackwell Scientific.
Hambleton P, Melling J and Salusbury TT (1994) In: Bio-
safety in Industrial Biotechnology. London: Blackie Aca-
demic & Professional.
Moo-Young M (1985) In: Comprehensive Biotechnology:
The Principles of Biotechnology: Fundamentals. Vol. 1.
Oxford: Pergamon Press.
Peberdy JF, Caten CE, Ogden JE and Bennett JW (1991) In:
Applied Molecular Genetics of Fungi. Oxford: Cam-
bridge University Press.
Puhler A (1993) In: Genetic Engineering of Micro-organ-
isms. Germany: VCH.
Sussman M, Collins CH, Skinner FA and Stewart DE (1988)
The release of genetically engineered microorganisms In:
Proceedings of the 1st International Conference on the
 HAFNIA ALVEI 973

HAFNIA ALVEI
Jouko Ridell, Department of Food and Environmental Hygiene, University of Helsinki, Finland
Copyright 0 1999 Academic Press

Characteristics of the Species growth temperature characteristics - a minimum

growth temperature of 0.2-3.7"C (mean 2.6"C) and
Hafnia alvei is the only species in the Hafnia genus. a maximum growth temperature of 41-43°C. In con-
The species has been known by the names Entero- trast, the diarrhoeagenic 'H. alvei' strains have much
bacter hafnieae, Bacterium cadaveris, Bacillus asi- higher mesophilic growth temperatures - minimum
aticus and B. paratyphi alvei. Hafnia is the old name growth temperatures of 10.2-1 1.5"C and maximum
for Copenhagen. The epithet alvei is chosen because growth temperatures of about 46°C.
the bacterium has been isolated from the intestinal H. alvei occurs in many natural environments such
tract of honey bees. In spite of the great developments as surface waters, soil, sewage and vegetation. H.
in bacterial taxonomy during recent years, many alvei has been reported to be one of the major species
Enterobacteriaceae species are still genetically diverse. which contribute to the microbial ecology of silage.
H.alvei consists of two distinct genetic groups which It has been isolated from the polluted waters of fish
are about 50% related at the DNA level. The groups farms as well as from the unpolluted northern rivers
are not reliably distinguishable by the methods used of Finland. Based on its ubiquitous distribution in
currently in the routine laboratory (Table 1). The first nature it is a frequent inhabitant in the intestinal
genetic group contains a biochemically less active contents and faeces of fish, insects, birds and
subgroup, H. alvei biogroup 1, which is also known mammals. Consequently, H. alvei is a common con-
as Obesumbacterium proteus (genetic group 1).This taminant of meat, dairy products and fish, and all
phenotypically distinct group of Hufnia species is environments where they are handled.
found as a contaminant of breweries. Yokenella H. alvei is one of the most numerous Entero-
regensburgei is a phenotypically similar species which bacteriaceae species in minced meat at retail level.
has been separated from H.alvei in 1985 (Table 2). About two-thirds of minced meat samples collected
H. alvei occurs in many natural environments and it from retail stores contained H. alvei. The initial
is frequently found in food samples. It is known to be numbers of H. alvei in meat are usually low, but
an opportunistic human pathogen causing a variety because the refrigeration temperatures used for meat
of extraintestinal diseases. True H. alvei is almost usually exceed the minimum growth temperature of
certainly not a diarrhoeal pathogen. Instead, DNA- this species, the numbers increase rapidly during
based methods showed that the diarrhoeagenic H. storage. The moist anaerobic environment in vacuum
alvei strains were not true H. alvei but were related packages is favourable for the growth of H. alvei. In
to the Escherichia coli-Shigella group. The inability vacuum-packed beef the numbers of H. alvei reach
of the present identification schemes to differentiate their peak (usually 103-105cfu g-*)in about 3 weeks -
true H. alvei and these diarrhoeagenic strains explains depending on temperature - and thereafter usually
largely why H. alvei has been thought to be an emer- decrease slowly. In the meat of stressed cattle - called
ging diarrhoeal pathogen. At present the diar- DFD-meat (dark, firm, dry) - which has a high pH,
rhoeagenic 'H. alvei' lacks a valid name. H.alvei may reach higher levels and even contribute
H. alvei is a Gram-negative facultatively anaerobic to the spoilage of the meat. Factors affecting the
rod, and it conforms to the general definition of development of the H. alvei population include the
Enterobacteriaceae. It is only about 20% related to type of package, composition of the atmosphere, pH
the other species of the family. Most strains are motile. and composition of the competing microflora. The
H. alvei is able to grow at a wide temperature range. inhibition of the growth of H. alvei - and other
Both genetic groups have similar psychrotrophic Enterobacteriaceae species as well - is caused by the
974 HAFNIA ALVEI

Table 1 Phenotypic characteristics of Hafnia alvei Methods of Detection

~

Test %+ Sign
No specific selective agars are available commercially
Indole 0 for the isolation of H. alvei. H. alvei grows readily on
Methyl red (35°C) 40
Voges-Proskauer (35°C) 59
the nonselective and selective isolation media used for
Citrate (35°C) 6 enteric bacteria, such as violet red bile (VRB), eosin
Hydrogen sulphide (Triple Sugar Iron) 0 methylene blue (EMB), MacConkey and xylose-
Urea hydrolysis 2
lysine-deoxycholate (XLD) agars. Most of the strains
Phenylalanine deaminase 0
Lysine decarboxylase 100 grow even on the highly selective isolation media such
Arginine dihydrolase 7 as Salmonella-Shigella agar. Most of the H. alvei
Ornithine decarboxylase 100 strains are lactose-negative and on the above-men-
Motility 94
Gelatin hydrolysis 0
tioned isolation media they produce colourless col-
Growth in KCN 97 onies. O n less inhibitory agars, such as VRB, the
Malonate utilization 83 colonies are usually relatively large, translucent, cir-
D-Glucose acid 100
cular, and low convex, with a smooth surface.
D-Glucose gas 98
For isolation purposes, MacConkey or VRB agar
Acid from: containing sucrose and sorbitol may be used. The
Lactose 3"
Sucrose 12 colourless colonies may be further tested with bio-
D-Mannitol 100 chemical tests. The L-prolineaminopeptidase test is
Dulcitol 2 useful to screen the colonies; H. alvei and Serratia
Salicin 16
Adonitol 0
spp. are the only Enterobacteriaceae species to give a
myo-Inositol 0 positive reaction.
D-Sorbitol 0 Reliable differentiation of H. alvei and most other
L-Arabinose 95 enteric bacteria can usually be made by routine bio-
Raffinose 4
L-Rhamnose 92 chemical tests performed either separately or included
Maltose 100 in the commercial identification systems. It is note-
D-Xylose 99 worthy that the incubation temperature affects the
Trehalose 99
results of some biochemical tests (methyl red, Voges-
Cellobiose 67
a-Methyl-d-glucoside 0 Proskauer and citrate). H. alvei can be differentiated
Melibiose 0 from species of Enterobacter and Serratia based on
D-Arabitol 0 positive reaction in lysine, arginine and ornithine
Mucate 0
Esculin hydrolysis 5 decarboxylase tests and a negative reaction in sorbitol
Tartrate (Jordan) 58 and inositol fermentation tests. However, the limited
Acetate utilization 90 set of tests - 10-20 - which are used in routine
Lipase (corn oil) 0
clinical laboratories or included in the commercial
DNAse 0
Oxidase 0 biochemical panels may not always be sufficient. In
ONPG test (O-Nitrophenyl- 67 particular, the differentiation of H . alvei and diar-
galactoside) rhoeagenic ' H . alvei' possessing the attachment efface-
Nitrate to nitrite 100
2-Ketogluconate util. 100
ment gene (eaeA+ 'H.alvei') as well as H. alvei
3-Hydroxybenzoate util. 0 biogroup 1 and true 0. proteus (genetic group 2 ) is
Minimum growth temperature 2.6"C difficult. Other species difficult to distinguish from
Maximum growth temperature 42°C
H. alvei include k: regensburgei and inactive (non-
+ = 90-1 00% positive; [+I = 75-89% positive; v = 26-74% lactose-fermenting) Escherichia coli ( E . coli 2). From
positive: [-I = 11-25% positive; - = 0-1 0% positive.
the diagnostic standpoint, reliable identification of
a 10-15% of the strains ferment lactose slowly.
the diarrhoeagenic eaeA+ 'H. alvei' strains is essential.
This is not achieved by API20E alone or by the trad-
itional set of biochemical tests. Additional tests such
competing microflora which is usually composed of as growth at 4"C, 2-ketogluconate and 3-hydroxy-
lactobacilli. The effect is mediated by low pH, bac- benzoate assimilation tests are needed. DNA-based
teriosins and other antagonistic effects of the lacto- methods such as polymerase chain reaction detection
bacilli population which gradually overwhelm the of the eaeA gene offer a reliable alternative for
meat surface inside the vacuum package. detection.

Table 2 Differential characteristics of Hafnia alvei and biochemically similar species
Test H. alvei H. alvei biogroup 7
Voges-Proskauer (35°C) [+I
L-Arabinose fermentation +
L-Rhamnose fermentation +
Maltose fermentation +
ONPG test (O-Nitrophenyl- +
galactosidase)
3-Hydroxybenzoate util. -
2-Ketogluconate util. +
Growth at 4°C +
+ = 90-1 00% positive: [+] = 75-89% positive; v = 26-74% positive: [-] = 11-25% positive: - = 0-1 0% positive; NA= data not available.
Importance to the Food Industry
H. alvei produces biogenic amines, cadaverine and
putrescine, and may sometimes contribute to the
spoilage of meat. It is probable that only refrigerated
meat with an exceptionally high p H (DFD-meat)
offers a suitable environment for H. alvei to reach
population densities which produce sensory changes.
H. alvei is one of the bacterial species which is respon-
sible for the production of histamine in fish. Tuna
fish, in particular, has been associated with histamine
food poisoning.
In clinical laboratories H. alvei may disturb the
detection of Salmonella. Many strains grow in the
isolation broths used to enrich Salmonella and
produce colonies resembling those of Salmonella on
routine isolation media. Moreover, H. alvei cultures
can sometimes be agglutinated by Salmonella O-anti-
sera. Because of their psychrotrophic nature, H. alvei
organisms may disturb the cold enrichment of Yersinia
enterocolitica in phosphate-buffered saline. In food
laboratories, the occurrence of H. alvei in food and
water samples may lead to difficulties in interpreting
the results of the coliform test - an indicator bacteria
analysis which is based on lactose fermentation.
Although most H. alvei strains are lactose-negative,
some strains have a plasmid-mediated ability to
ferment lactose with variable speed. This results in
colonies of variable size and makes the agar plates
difficult to read.
H. alvei can cause fatal extraintestinal infections in
farmed fish affected by predisposing factors such as
overcrowding. In rainbow trout H. alvei has been
reported to cause haemorrhagic septicaemic disease,
which is associated with high mortality. In sherry
salmon H. alvei has caused kidney infections. H. alvei
can infect honey bees affected by a parasitic disease.
Thus, it appears that H. alvei can cause opportunistic
infections in animals in a similar manner as in
humans.
Next Page
HAFNIA ALVEI 975
eaeA+ ‘H. alvei’ Escherichia coli: Yokenella
inactive regensburgei
Importance to the Consumer
H. alvei is an opportunistic human pathogen which
can cause a variety of extraintestinal diseases in sus-
ceptible individuals with a severe underlying con-
dition. H. alvei has been associated - either in pure
or mixed growth - with wound infections, abscesses,
septicaemia, pneumonia, etc. Predisposing factors
include malignancy, immunodeficiency or any debili-
tating disease. For healthy persons H. alvei apparently
does not cause any risk concerning extraintestinal
diseases.
H. alvei has been considered to be an emerging
human diarrhoeal pathogen. The main reason for the
belief is based on studies concerning the diar-
rhoeagenic eaeA+ ‘H. alvei’ strains isolated in Ban-
gladesh. These strains caused diarrhoea with similar
mechanisms and comparable symptoms to entero-
pathogenic E. coli - a causative agent of tourist diar-
rhoea and diarrhoea of children in developing
countries. However, taxonomic methods determining
the structure of rRNA genes and total DNA homology
revealed that these eaeA+ ‘H. alvei’ strains were not
true H. alvei, but closely resembled the E. coli-Shi-
gella group, although the taxonomic position of this
group remained unclear. At the moment the geo-
graphic distribution and clinical importance of the
diarrhoeagenic ‘H. alvei’ is not known.
From time to time H. alvei has been reported to be
a causative agent of diarrhoea based on isolation from
diarrhoeal faeces without further studies concerning
causal relationships. However, it is a fact that true
H. alvei is more often isolated from diarrhoeal than
normal human faeces. This epidemiological asso-
ciation of H. alvei with diarrhoeal symptoms is care-
fully documented but has not been explained. N o
virulence mechanisms have been found in spite of
careful studies, and it is possible that the phenomenon
has nothing to do with virulence. For instance, the
increased intestinal motility during diarrhoea may
facilitate the detection of food-originating bacteria
 ICE CREAM 1083

ICE CREAM
A Kambamanoli-Dimou, Department of Animal Production, Technological Education Institute, Larissa, Greece
Copyright 0 1999 Academic Press

Ice cream is made from a liquid mix which is based hazard of staphylococcal enterotoxin in ice cream
on milk, cream, water, milk solids-not-fat, milk fat or may be present if whey powder is used as a source
other fat as may be legally required, sugar, emulsifyingof milk solids. Careful control and storage of these
and stabilizing agents, flavours and colours. This wide powders under dry and cool conditions are necessary.
variety of ingredients, the possible variations in their Dry sugars used as ingredients should be almost
microbiological standard and quality, as well as the sterile if properly prepared, processed and stored.
conditions and methods used to prepare the final Sugar syrups also are used as sweetening ingredients.
product, greatly affect the quality of the ice cream. The contamination of sugars or sugar syrups is
Consequently, the microbiological quality of ice limited, mainly consisting of small numbers of osmo-
cream depends on many factors, the most important philic microorganisms. Certain yeasts and moulds
of which are described here. would be the principal flora. Some species of bacteria
have also been suggested as possible spoilage prob-
Raw Materials (Major Components and lems, including species of Bacillus and Leuconostoc.
Osmophilic yeasts may be able to grow in these syrups
Additives)
and moulds may grow on the surface if contamination
Any of the wide variety of ingredients that may be should occur, so it is suggested that tests for yeasts
used to produce ice cream may contribute micro- should be carried out on sugars and sugar syrups.
organisms to the product and affect its quality. The Butter and butter oil (anhydrous milk fat) are made
heat treatment process gives only an adequate reduc- from pasteurized cream, in which pathogenic and
tion in bacterial numbers as well as the destruction of most spoilage organisms have been destroyed. Rela-
pathogenic organisms. It cannot entirely correct the tively small numbers of mesophilic bacteria, coliforms
hygienic quality of poor ingredients. So carefully and lipolytic organisms, particularly the Pseudo-
selected ingredients are essential for the manufacture monas sp. responsible for butter spoilage, as well as
of ice cream of the highest quality. moulds and yeasts, can be found. Butter commonly is
Liquid milk, cream, skimmed milk and con- kept refrigerated, and during commercial storage is
centrated skimmed milk may contain appreciable kept at about -20°C at which temperature no micro-
numbers of bacteria, including some that are patho- bial growth can take place. For these reasons bacteria
genic (e.g. Mycobacterium spp., Streptococcus spp.). usually do not grow in butter, and when they do, their
Adequate heat treatment by the supplier, together growth is not extensive. The flavour of good butter is
with handling and storage under sanitary conditions so delicate, however, that small amounts of growth
(kept under refrigeration and used promptly) leads may cause appreciable damage to the flavour. When
to raw materials of a satisfactory quality. The main satisfactory hygienic conditions are applied during
organisms present in these dairy materials are a few the production of the above ingredients and they are
spore-forming bacilli, micrococci, and psychrotrophic properly handled (storage temperatures of no more
and thermoduric microorganisms, which may some- than -20°C for butter, and dry, refrigerated conditions
times spoil the mix but are not a major health hazard. for butter oil), combined with tests for the above-
Milk powders may contain large numbers of spore- mentioned microorganisms, a high microbiological
forming bacilli or may be contaminated by sal- quality will be ensured.
monellae. Numerous outbreaks of food poisoning Fats other than milk - commonly vegetable fats -
attributed to salmonellae or staphylococci from milk may also be used. The high temperature used during
powder provide evidence that these pathogens do on their processing and their low moisture content give
occasions survive in the final product. A special raw materials containing very few microorganisms.
1084 ICE CREAM
Dry, refrigerated conditions should be used for their
storage.
Stabilizers do not constitute an important source of
bacteria if they have been packaged under hygienic
conditions, as they are usually produced by methods
involving high temperatures. Gelatin as an animal
product may be a hazard if produced under insanitary
conditions, so it should be obtained from a reputable
supplier and kept under cool and dry conditions.
Emulsifiers should not present any problem except
for eggs, which may be contaminated by Salmonella
spp., and pasteurization is required to avoid any
hazard arising from their use.
Many other foodstuffs are added to ice cream,
either mixed with it or as coatings. These may be
flavouring materials such as vanilla, chocolate and
cocoa, fruit (canned, fresh or frozen) and nuts, as well
as colours. All of them are potential sources of hazard,
particularly if they are added after the heat treatment
of the mix. Yeasts and moulds will predominate in
fresh fruits, while canned fruits, because of their heat
treatment, should be of a satisfactory microbiological
standard. Nuts may be contaminated by moulds and
may possibly contain mycotoxins. Coconuts can also
contain salmonellae. For these reasons, it is much
better for all these materials to be used after their heat
treatment (roasted nuts, for example, or pasteurized
chocolate), especially if they are added to the mix
after its pasteurization. Also, they should be stored
in cool, dry conditions. The examination of these
materials should include a visual inspection and the
enumeration of mesophilic bacteria, coliforms, yeasts
and moulds. Colours manufactured and handled care-
lessly may cause microbiological problems, but this
can be avoided if they are obtained from a good
supplier and stored properly.
All these potential hazards highlight the necessity
of using high-quality raw materials, purchased from
a reputable supplier, and carefully stored under con-
ditions that will not allow the proliferation of micro-
organisms. In addition, it is suggested that
appropriate microbiological tests should be carried
out on raw materials, and the use of strict stock
rotation is essential.
Hygiene During Production
After the high-quality raw materials have been chosen,
the mix is ready for processing. This begins with
combining the ingredients into a homogeneous sus-
pension that can be pasteurized, homogenized,
cooled, aged, flavoured and frozen. This is a complex
operation comprising a series of steps which all have
some effect on the microbiological quality of the final
product, so they must be carefully controlled to
produce a product that will safeguard the consumer's
health.
After the ingredients have been weighed or meas-
ured, they are blended to make a liquid mix. This
mixture is then subjected to a heat treatment process,
which is specified by legal requirements in most coun-
tries. This pasteurization renders the mix substantially
free of vegetative microorganisms, killing all of the
pathogens likely to be present. The ice cream mix is
always homogenized, often as a step in the pas-
teurization process. The homogenizer is a complex
piece of equipment and must be carefully cleaned and
disinfected each time it is used, or the mix may be
seriously contaminated. It is therefore suggested that
homogenization of the ice cream mix is carried out
before it is finally heat-treated, where this is possible.
Cooling of the mix to about 4°C is followed by an
ageing period; then the mix is passed to the freezer
where it is subjected to considerable agitation and
reduction in temperature, as well as incorporation of
air. On leaving the freezer the ice cream will normally
be packaged (in family packs, individual retail packs,
or any other forms), frozen hard in wind tunnels at
-40°C or in hardening rooms, and then kept at a
temperature of about -30°C until and during dis-
tribution. Some ice cream is sold direct from a dis-
pensing freezer as 'soft-serve' ice cream in cones, or
in various types of made-up desserts in restaurants
and cafks, or from vehicles complete with their own
electricity generation equipment.
Ice cream mixtures must not be kept for more than
1h at a high temperature (more than 7.2"C) before
being pasteurized, in order to avoid the proliferation
of organisms in the ingredients. During pas-
teurization, time as well as temperatures should be
strictly observed in order to avoid on the one hand
excessive heat treatment, which may lead to undesir-
able flavour changes, and on the other hand, to ensure
the destruction of pathogenic organisms and the
adequate reduction in bacterial numbers. The cooling
of the ice cream mix to about 4°C must be rapid and
the mix must be kept at that low temperature until it
is frozen, otherwise the proliferation of any viable
organisms may occur. This can lead to a product
with a high microbial count and possibly to a disease
outbreak. The same danger exists if the cooling system
stops during the ageing of the mix. In this case, the
mix must be discarded because, although a new pas-
teurization will kill the organisms, it will not destroy
possible toxins already present. Normally the mix
should be frozen within 24 h of heat treatment, as
undue prolongation of storage may lead to pro-
liferation of psychrotrophic organisms, with a serious
risk of spoilage of the mix.
The incorporation of air into the ice cream mix

~~~
during the freezing process may cause air-borne con-
tamination, so air filters are required to prevent the
ingress of organisms. For some products the ice cream
is blended with water ice on leaving the freezer, and
may be frozen on a stick or in a cone (single portions)
and covered with a chocolate or flavoured couverture,
together with broken biscuits or nuts. All this hand-
ling has to be done under sanitary conditions in order
to minimize the microbiological contamination of ice
cream.
In addition to the careful operation of the equip-
ment, the proper cleaning and sanitizing of the plant
and equipment is of great importance. Any equipment
that comes into contact with the ice cream or ice
cream ingredients must be carefully and effectively
cleaned and sanitized immediately after use. This is
usually done at the end of each day’s operation;
however, if scale builds up during the operation, it
may be necessary to clean the plant thoroughly before
further processing the same day. Not only is the pro-
cessing equipment important, but ancillary equip-
ment, in particular at the final sales point, must be
kept in a satisfactory hygienic condition. Poor clean-
ing and sanitizing of the plant and equipment may
lead to pockets of ice cream residues where intense
proliferation occurs, which results in recontamination
of the pasteurized mix with a large number of bac-
teria. For soft-serve freezers manufacturers typically
provide specific instructions for cleaning and sani-
tizing, and these should be followed closely.
Clean surroundings are essential if equipment is to
be kept in hygienic condition. All rooms, especially
toilets and locker rooms, must be kept as clean and
sanitary as the area immediately surrounding the
packaging equipment. Surrounding activities (e.g.
sewerage plants, rubbish tips) often represent poten-
tial sources of contamination, and birds, rodents and
insects are all important vectors of such con-
tamination. In addition to preventing access of pests
to the process area, it is important that the factory
yard is kept free of food waste, rubbish and spilled
material that might attract birds, rodents and insects.
All such material should be kept in lidded containers
and removed on a daily basis. Pet animals such as dogs
and cats similarly have no place in a food production
factory.
Operations must be segregated to minimize the
chances of pathogenic microorganisms being carried
from raw materials to finished products. Persons
handling raw milk or cream must not be allowed
access to rooms where pasteurized products are
exposed unless those persons have first changed their
clothes completely and have disinfected themselves.
Room air pressures should be maintained at suc-
cessively higher levels from the mix room to the pro-
ICE CREAM 1085
cessing room, to the freezing operation and to the
packaging operation. Thus, the flow of air will be
away from the most critical area, the packaging room.
The supply of hot and cold water must be unrestricted
and facilities for disposal of both liquid and solid
wastes must be adequate. All water that is used in
food formulation, or will be used on (or could gain
access to) food contact surfaces, should be of potable
quality and be stored in enclosed tanks and distributed
in piping that is completely segregated from other
pipe systems. Potable water may be derived from
public mains supplies or other sources such as bore-
holes which must be protected against contamination
by surface water or underground contamination from
drains, seeping from farm or industrial tips and
similar potentially hazardous areas. Whatever the
origin of water it should be routinely examined micro-
biologically at the point of entry to the site and at the
point of use, particularly if there is on-site storage.
The hygienic standards of the workforce are crucial
to the ice cream manufacturer. No worker should be
allowed to perform tasks in the plant who has not
been adequately taught the necessities of personal
hygiene and approved practices within the plant.
Every employee must dress in a clean uniform, wear
a hair restraint, wash and sanitize hands, disinfect
footwear on entry to the process area, and refrain
from touching any product contact surface without
properly sanitizing the hands, or gloves if worn.
Proper sanitary practices are essential to the ice cream
plant. No one should be allowed to enter the pro-
cessing environment who is not familiar with the
required sanitary procedures or who does not
conform to the required dress and personal hygiene
measures. Freedom from chronic contagious diseases
should be confirmed yearly by medical examination.
Provided that preparation of ice cream is conducted
in a closed processing cycle with modern industrial
equipment of high hygiene standards, the oppor-
tunities for contamination by human contact are few.
The packaging materials may occasionally cause
trouble, but there should be no problem if they have
been handled and stored under hygienic conditions.
Tests carried out during the production process
should be indicative of the standard of hygiene.
Microbial Changes During Storage
Ice cream is a perfect substratum for the proliferation
of microorganisms because of its composition. It con-
tains sugar, proteins and oxygen, all the essential
components that microbes need, as well as a suitably
high pH. The only factor not available is high tem-
perature. If a rise in temperature does occur, all the
conditions are in place for the development and
1086 ICE CREAM

proliferation of microbes that may have survived the mercial establishments but at home, where a com-
heat treatment, or come from post-pasteurization con- bination of faulty practices may occur. The use of raw
tamination or insanitary processing and packaging. milk or cream, the addition of raw eggs containing
Ice cream may be sold direct from the freezer as a Salmonella to the mix and the use of contaminated
‘soft-serve’ product, or it may be further reduced in equipment, in addition to inadequate heat treatment
temperature and frozen in wind tunnels at -40°C or and contamination from infected persons, give rise
in hardening rooms, to produce ‘hard’ ice cream, to products with high microbial loads, especially of
which will be stored at a temperature of about -30°C pathogenic bacteria, which, if they are present, may
until it is sold. Deep-freezing stabilizes the microbial survive for many months in ice cream.
content of ice cream: microorganisms found in it no
longer proliferate. Some sensitive species (Gram-
Problems at Point of Sale
negative) die and their populations reduce. Even if
the period between freezing and final sale is several Even when the greatest care has been taken to produce
months, there will be little change, if any, in the an ice cream of the highest quality, it is still liable
microbial content of the ice cream. Extensive research to contamination at the point of sale. The largest
has shown that both Mycobacterium and Salmonella, proportion of microbiological problems, in general,
as well as many other less harmful but often more are due to poor techniques of selling and serving at
resistant types, can survive at the low temperature of the final point of distribution.
storage for very long periods. They do not multiply, Although much ice cream is retailed in its final
provided that the temperature is low enough for the packaging, a significant quantity is portioned from
ice cream to remain hard; in effect, the microbial bulk packs at point of sale. The method of sale has a
quality of ice cream is ‘locked in’ by the hardening major bearing on the amount of contamination to
process. It is, therefore, essential that the bac- which the product is subjected. Ice cream sold pre-
teriological content of the ice cream from the freezer packed as a single retail portion, and which has only
is as low as possible, as neither the final hardening to be handled by the consumer in its wrapping, should
process nor the low temperature storage can be relied have the least contamination of all. Greater degrees
upon to reduce the numbers appreciably, and patho- of contamination may occur in ice creams served in
genic organisms should be absent. cones or other individual portions scooped from bulk
If the mix is frozen promptly spoilage is impossible, ice cream in restaurants or coffee shops, or from
for microorganisms cannot grow in the frozen vehicles complete with their own electricity generation
product. However, if there is a delay between pas- equipment. Here there is a possibility of considerable
teurizing and freezing, spoilage can occur, as well as contamination, unless all the equipment used (servers,
in cases of melting and refreezing of the product etc.), the method of dispensing and the personal
resulting from temperature variations or failure of hygiene of the operator are all of a very high standard.
the freezing systems. Such delays are unusual in the The equipment (servers, wafer holders and so on) has
manufacture of hardened ice cream. Special care is to be kept free of all residues of ice cream, which
needed for mix with soft-serve ice cream that has to might otherwise melt and allow the growth of bacteria
be transported, often for long distances, by trucks to to recommence. Wherever possible these items of
retail soft-serve stores or stands where it is kept soft- equipment should be kept in running cold water. If
frozen and dispensed to consumers. Both con- they have to be kept in a jug of water, this water must
tamination and temperature abuse of the mix may be regularly changed to avoid it becoming a source of
easily occur. Furthermore, refrigeration space is contaminating bacteria. The personal hygiene of the
usually limited, and adequate facilities for cleaning server is also of the utmost importance. This applies
and sanitizing the freezer and the associated equip- even if pre-wrapped ice cream is being sold. Clean-
ment are often lacking or are, at best, marginal. liness of hands, clothing and habits must be above
Under these conditions, especially if wrong prac- reproach, and the operators must be trained in the
tices had occurred previously, the ice cream is over- best ways of maintaining this, and in the distribution
loaded with microbes which can lead to quality of the individual portions of ice cream.
deterioration or even to cases of food poisoning. Food Soft-serve ice cream sold directly from a dispensing
poisoning is known to have followed the consumption freezer can become contaminated very easily unless
of ice cream contaminated by microbes, such as stringent precautions are taken. The product is usually
Staphylococcus, Salmonella, Shigella, Listeria and manufactured at the point of sale, which may be a
Streptococcus group A organisms. With few excep- specialist outlet or cafi., a non-food outlet such as a
tions, outbreaks that have occurred in recent years filling station, or a mobile outlet or kiosk. Soft-serve
have been caused by ice cream made not in com- ice cream may be manufactured from a conventional
 Next Page

ICE CREAM 1087

mix produced on the premises, from an ultra-high cream contaminated by the manufacturer who was a
temperature processed, aseptically packaged mix, or urinary excretor of Salmonella typhi. There has been
from a spray-dried powder mix. Powder mixes may a case of Shigella dysentery caused by an ice cream
be formulated for reconstitution in either hot or cold that was accidentally touched by a monkey. Also,
water. Hot-water mixes are preferable with respect to outbreaks involving salmonellae and staphylococci
hygiene, but cold-water mixes are often considered have been reported. The personal hygiene and habits
more convenient. Attention must be paid to the recon- of vendors at sale points are important. Education, in
stitution of the mixes; this must be done under sat- addition to medical inspection, is absolutely necessary
isfactory hygienic conditions to prohibit the and no employee must be allowed to work without
proliferation of Salmonella, which may survive if full medical clearance.
mixes are not prepared with care. Finally, birds, rodents, insects and pet animals have
Generally, special dispensing freezers should be no place at the retail selling point.
To some extent ice cream retains a reputation as a
kept in constant operation and placed inside the shop
high-risk food. This is unjustified for commercially
with the taps facing the interior; they must not be
produced ice cream in developed countries which
directly exposed to the sun, dust or flies. A strict
have legislated microbiological standards for ice
cleaning and disinfecting regimen for the freezer must
cream, as well as for the conditions and methods to
be instituted, adhered to and performed thoroughly be used for heat treatment and subsequent storage
on a daily basis. In particular, ice cream must not be and sale.
allowed to remain in the freezer overnight, and the
personal hygiene of the operator must be of a very
high standard. It must be recognized that maintenance See also: Eggs: Microbiology of Fresh Eggs. Food
of the necessary hygiene standards can be more dif- Poisoning Outbreaks. Freezing of Foods:Growth and
ficult in an environment primarily concerned with Survival of Microorganisms. Heat Treatment of Foods:
retailing than in one wholly concerned with manu- Principles of Pasteurization. Milk and Milk Products:
facturing. Particular difficulties may be encountered Microbiology of Liquid Milk; Microbiology of Cream and
in outlets that are predominantly non-food, such as Butter. Salmonella: Introduction. Process Hygiene:
filling stations, and those with inherently limited facil- Overall Approach to Hygienic Processing; Hygiene in the
ities, such as kiosks. Catering Industry.
Special self-pasteurizing dispensing freezers are
now available which have a heat treatment process as
part of their operation, and provided the process is
used each and every day, they do not normally need
daily cleaning. At the end of each day’s operation (or Further Reading
other convenient time) the freezer is switched to the
Frazier W C and Westhoff D C (1988) Food Microbiology,
‘self-pasteurize’ mode, and all the mix, and every part 4th edn. New York: McGraw-Hill.
of the freezer that can come into contact with ice International Commission on Microbiological Spe-
cream or mix, are raised to a temperature above that cifications for Food (1980) Microbial Ecology ofFoods.
required for pasteurization of the mix, and held at Vol. 2. Food Commodities. London: ICMSF/Academic
that temperature for the legally required time. The Press.
freezer and its contents are then cooled rapidly to Jervis DI (1992) Hygiene in milk product manufacture. In:
about 4°C and held at that temperature. Tests have Early R (ed.) The Technology of Dairy Products. P. 272.
Chichester: John Wiley.
shown that there is little or no increase in the bacterial
Mantis AI (1993) Hygiene and the Technology of Milk and
content of the product over a period of more than a its Products. Thessaloniki: Kiriakidis.
week. It is to be recommended, however, that these Marshall R and Arbuckle WS (1996) Ice Cream, 5th edn.
freezers are cleaned out and disinfected at regular New York: Chapman & Hall.
intervals of, say, a week. It must be emphasized that Rothwell J (1985) Microbiology of frozen dairy products.
self-pasteurizing freezers are not intended to process In: Robinson RK (ed.) Microbiology of Frozen Foods.
unpasteurized mix. P. 209. London: Elsevier.
Rothwell J (1990)Microbiology of the icecream and related
Probably the most serious and dangerous sources
products. In: Robinson RK (ed.) Dairy Microbiology.
of contamination are operation and serving. Many Vol. 2, p. 1. London: Elsevier.
major food poisoning outbreaks have been caused by Varnam A and Sutherland J (1993)Milk and Milk Products.
human contamination. Cases of typhoid fever, includ- Food and Commodities Series no. 1. London:
ing deaths, have been reported to be caused by ice Chapman & Hall.
 KLEBSIELLA 1107

KLEBSIELLA
P T Vanhooren, S De Baets, G Bruggeman and E! J Vandamme, Department of Biochemical and Microbial
Technology, University of Gent, Belgium
Copyright 0 1999 Academic Press

Introduction
In the ninth edition of Bergey’s Manual of De- Klebsiella sp. are chemoorganotrophic, having both
terminative Bacteriology, the genus Klebsie1lL;l is a respiratory and a fermentative type of metabolism.
taxonomically classified in subgroup 1:family Entero- Glucose is fermented with the production of acid and
bacteriaceae, which belongs to group 5, describing gas (more COZ is produced than H2).Most strains
the facultatively anaerobic Gram-negative rods. Kleb- produce 2,3-butanediol as a major end product of
siella bacteria occur worldwide and are commonly glucose fermentation, whereas lactic acid, acetic acid
found in soil, carbohydrate-rich waste water, effluents and formic acid are formed in smaller amounts
from paper, pulp and textile mills, cooling water, and ethanol in larger amounts than in typical mixed
aerosols, woodland, on fruits, sugar cane, grains and acid bacterial fermentations. No special growth
on vegetables such as radish, lettuce, carrots and factor requirements are known. Some strains have
tomatoes. Drinking water reservoirs, made from the ability to fix molecular nitrogen under anaerobic
redwood (Californian sequoia), also harbour Kleb- conditions.
siella sp. and it is a frequent contaminant of water Klebsiella strains may be lysogenic, but phages have
supplies in general. These bacteria also occur in the been isolated from stools and sewage and used in
human respiratory tract, in faeces and in clinical spe- phage typing. Many Klebsiella strains produce bac-
cimens. As such, they can contaminate food and con- teriocins, klebecin being an example.
tribute to disease and spoilage. Certain species ( K . Klebsiella species are cytochrome oxidase negative
pneumoniae, K. oxytoca and occasionally others) are and catalase positive. Other typical Entero-
opportunistic pathogens. They are frequently the bacteriaceae taxonomical tests vary among the
cause of hospital acquired (nosocomial) infections species: indole, methyl red, Voges-Proskauer and
in urological, neonatal, intensive care and geriatric Simmons citrate. They are usually lysine decarb-
patients. Often, they display multiple antibiotic resist- oxylase positive and ornithine decarboxylase nega-
ance and resistance to biocides. Some strains could be tive. Several species hydrolyse urea and most reduce
exploited in industrial fermentation processes for the nitrates (except K . pneumoniae subsp. o z a e m e . ) They
synthesis of (fine) chemicals. are arginine dihydrolase negative and H2S is not pro-
duced. Most species ferment all commonly tested
Characteristics of the Genus K/ebsie//a carbohydrates, except dulcitol and erythritol; they
also grow in the presence of KCN. The mol% G+C
and Relevant Species
of the DNA is 53-58 ( T J .
Successful genetic recombinations have been
General Physiological Properties of Klebsiella reported in Klebsiella and K. pneumoniae has been
Strains
used by several workers for detailed genetic analysis
Klebsiella is a non-spore-forming, non-motile, fac- of the genes involved in N2-fixation (the Nif-genes).
ultative anaerobic Gram-negative straight rod, 3.3- These genes are clustered near the His-region in the
1.0pm in diameter and 0.6-6.0pm in length. The genome, but can be mobilized and transferred to other
rods are arranged singly, in pairs or in short chains. Gram-negative bacteria. Strains from clinical origin
The cells are capsulated. The optimal temperature for or those from nosocomial infections harbour R-
growth is 37°C. plasmids, which determine resistance to a variety of
1108 KLEBSlELLA

antibiotics, such as penicillins, cephalosporins, saccharides: D-glucose, D-mannose, D-galactose, L-

aminoglycosides, tetracyclines, chloramphenicol, rhamnose, L-fucose. In addition, non-carbohydrate
sulphonamides and trimethoprim. They are good constituents such as acetate, pyruvate or succinate
recipients of R-plasmids, making them a culprit in can occur in the capsules. For example, types 1, 6,
serious nosocomial epidemic diseases. The cell wall 16, 54, 58 and 6 3 contain L-fucose; their structure is
of Klebsiella consists of the typical Gram-negative represented in Figure 2.
bacterial type with two distinct layers; the pep- Klebsiella strains with the capsular (K-antigen j
tidoglycan layer and the 'outer membrane layer'. The types 1, 2 and 3 are a major cause of respiratory
latter consists of a complex of lipopolysaccharide tract infections (pneumonia), whereas those with the
(LPS), phospholipid and protein. The LPS structure capsular (K-antigen)types 8,9,10 and 24 are involved
determines the 0-antigen type, of which a few in infections of the urinary tract.
examples are given in Figure 1.
Klebsiella Strain Differentiation Methodology
The Klebsiella Polysaccharide Capsule Because of the ubiquitous distribution and the
A polysaccharide capsule surrounds the cell wall (opportunistic) pathogenicity of Klebsiella sp., sci-
structure of Klebsiella species and makes up the K- entists and field workers have become increasingly
antigen. Serological typing is based on the exam- aware of the potential health hazard and the need for
ination of the K-antigens, since the number of 0- monitoring the organism in environmental, food and
antigen types is lower than that of the K-antigen types clinical sources. Its clinical significance has been well
and also because 0-antigen determination can be established, but its potential as a public health
masked by the heat-stable K-antigens. problem in natural, industrial and food environments
Especially, K. pneumoniae and K. oxytoca have a needs more attention. In this respect, the need for
thick polysaccharide capsule, which gives rise to large a quick, cheap and reliable methodology to screen,
mucoid colonies, especially when grown on carbo-
Serotype Structure
hydrate rich agar media. 1 -4)-P-D-GlCAP-(l +4)-a-~-Fucp-(l+3)-P-D-GlCp-(l~
The K-antigens of Klebsiella bacteria have been 2 3
divided into 82 different types and most of these \/
C
capsular polysaccharides have been characterized. / \
CH3 COOH
The K-antigens are constituted of repeating units.
Most K-antigens contain only one charged mono-
6 +3)-cc-~-Fucp-(l+3)-P-o-Glcp-(l+3)-~-D-Manp-(l-,4)-a-~-GlcAp-(l~
saccharide, either D-ghcuronic or D-galacturonic 4 6
acid, and two to four of the following mono- \/
C

Serotype Structure
/\
CH3 COOH
a
l,6 -13) Gal ( 1 3

a a
3 -3) Man ( 1 3 3 ) Man (1-2) Man ( 1 2 2 ) Man (122) Man (13

a Q a
5 -3) Man (1-2) Man (1-3) Man (1-2) Man (122) Man (13
4 -2) Rib (1-4)
P a
Gai ( 1 3
- 54 +4)-a-~-GlcAp~(l+3)-a-~-Fucp-(l-+3)-P-~-Glcp-(l~
a P a a 4
7 -12) L-Rha (1-12) Rib (1-3) i-Rha (1-3) L-Rha (1- t
1
216OAc 2 OAc 216OAc P-D-GICP
I I Acetyl residue at 0-2of L-fucosyl on every other repeating unit
l a l a l a
6 -3) Gal (1-3) Gal (1-3) Gal ( I +
56 +3)-a-~-Glcp-(l-,4)-P-~-GlcAp-(l i 4 ) - a - ~ - F u c p - ( l +
214 OAc 214 OAc 214 OAc 214 OAc 3 2 3
\/ T

9 '3)
I
Gal (1-3)
I
Gal (1-3)
I

Gal (1+3) Gal (1-

I
:.":, 1
a-D-Glcp
T3 CH3 COOH
11 Acetyl residue on every repeating unit
Gal
a P Q P a
10 -3) L-Rha (1-3) L-Rib (1-4) L-Rha (1-3) Rib (1-4) L-Rha (1- 63 +3)-a-o-Galp-(l +3)-n-D-Galp-(li3)-a-~-Fucp-(l
Figure 1 Repetitive units (0-antigen) of the 0-specific poly- Figure 2 Structure of the L-fucose-containing tri- and tetra-
saccharide part of the lipopolysaccharide of Klebsiella species. saccharide repeating units of Klebsiella types 1, 6, 16, 54, 58 and
Redrawn from Orskov and 0rskov (1984). 63.
 KLEBSIELLA 1109

culture and identify Klebsiella to the species and sub- Serological tests based on the K-antigen can be used
species level remains a goal to be achieved. to confirm the identification results. A total of 62
Within the genus Klebsiella, the following species serotypes among 72 different serological Klebsiella
are currently distinguished: K. oxytoca; K. planticola; strains could be distinguished, based on a unique
K. pneumoniae subsp. pneumoniae; K. pneumoniae agglutination pattern with plant lectins.
subsp. ozaenae; K. pneumoniae subsp. rhino- Based on an extensive survey of about 160 strains
scleromatis; K. terrigena. of Bacillus, lactic acid bacteria, Enterobacteriaceae
K. pneumoniae subsp. pneumoniae is considered as and Staphylococcus, it can be concluded that gas
the type species. As to the differentiation of the family
chromatographic analysis of cellular fatty acid com-
Enterobacteriaceae from other families and genera, position was not sufficiently specific to the species
classified in Bergey’s group 5, the reader is referred to
level in several cases; characterization of food-borne
the ninth edition of Bergey’s Manual. bacteria, including Klebsiella sp. by the analysis of
As to the differentiation of about 30 genera (> 115 their cellular fatty acids should thus only be used to
species and subspecies) in the family Entero- complement other taxonomical methods.
bacteriaceae, about 50 differential biochemical char- Recently, several molecular identification tech-
acteristics can be verified; these can also be found in niques have been proposed for a wide range of med-
Bergey’s Manual. Due to the large number of data to ically and food-related bacteria, including Klebsiella
be compared in such an identification attempt, one SP.
tries to identify members of this family directly to the A fluorescence-based polymerase chain reaction-
genus or species level using the above battery of about single strand conformation polymorphism (PCR-
50 tests. Additional tests are then available to dif- SSCP) analysis of the 16s rRNA gene has been
ferentiate between certain species and subspecies; for described to identify a broad range of Gram-positive
Klebsiella specieshbspecies, these are summarized in and Gram-negative bacteria: 178 bacterial strains,
Table 1. representing 51 species in 21 genera were examined.
The usefulness of the pyrrolidonyl-arylamidase All strains gave species-specific patterns, except Shi-
activity test to differentiate among Enterobacteriaceae gella which resembled E . coli. This sensitive technique
and non-fermentative Gram-negative rods has also can be applied on very low numbers of bacteria, i.e.
been studied. Positive results were uniformly obtained IO colony forming units (cfu).
with Citrobacter, Klebsiella, Enterobacter and Ser- Two 16s DNA targeted oligonucleotides were used
ratia species. Negative results were displayed by as PCR primers for the specific detection of Salmonella
Escherichia coli, Proteus, Salmonella, Shigella, serotypes in food. Some of the primers (16s 111) also
Pseudomonas and Flavobacterium species, indicating hybridized with Klebsiella and Serratia sp., however.
its value as a complementary differentiation test.
The Biolog System has been evaluated for the idem- Recommended Methods for Detection
tification of 55 Gram-negative taxa (789 strains), and Enumeration of Klebsiella
likely to be encountered commonly in clinical labora-
tories. It performed best with oxidase-positive fer-
Detection and Enumeration
menters, but although for 39 of the 55 taxa an
identification rate of 70 % was achieved, problems Conventionally, eventual resuscitation (2 h at 17-
were encountered, particularly with the identification 25°C) in tryptic soy broth and (subsequent) plating
of capsulated strains of Klebsiella. on violet red bile glucose agar (VRBG) allows an
The new BBL CrystalEnterichIon Fermenter efficient presumptive enumeration of Entero-
(Crystal, Becton Dickinson Microbiology Systems) bacteriaceae, in foods or in other substrates. Other
identification system for Gram-negative rods has been isolation media commonly used in this respect are:
compared with the well-known API 20 E or API 20 NE Simmons citrate agar, MacConkey agar and eosin
(Bio-Merieux) system. More than 100 clinical isolates methylene blue agar. Incubation is at 35°C (clinical
were studied, including six K . oxytoca strains and ’12 samples) and 10°C (environmental samples) for 24-
K. pneumoniae strains; it was concluded that the 48 h. Raised mucoid colonies are selected and further
Becton Dickinson Crystal test allowed a quicker, differentiation and confirmation of Klebsiella is then
easier and more accurate identification of Gram-nega- based on the battery of tests mentioned above.
tive clinical isolates compared to the API system. The detection and isolation from sources such as
A remaining problem is to distinguish K. pneu- faeces, soil, water and food can be facilitated by use
moniae strains from non-motile Enterobacter aero- of the standard selective media (see Table 2). Specific
genes strains; however, the latter liquefy gelatin very Klebsiella enumeration is also of great importance
slowly and are urease negative. to environmental microbiologists investigating the
1110 KLEBSIELLA

Table 1 Differential biochemical characterization of Klebsiella spp.

Test Klebsiella Klebsiella Klebsiella Klebsiella Klebsiella Klebsiella
oxytoca planticola pneumoniae pneumoniae pneumoniae subsp. terrigena
subsp. ozaenae subsp. rhinoscleromatis
pneumoniae
-
Gram stain (24 h) - - -
Oxidase (24 h) - - -
Indole production - [-I - -
Methyl red [+I + [-I d
Voges-Proskauer + + + +
Citrate (Simmons) + + + d
Hydrogen sulphide production - - -
Urea hydrolysis + + -
Phenylalanine deaminase - - -
Lysine decarboxylase + + +
Arginine dihydrolase - - -
Ornithine decarboxylase - - [-I
Motility - - -
Gelatin hydrolysis, 22°C - - -
KCN, growth + + +
Acid production
D-Adonitol + + +
L-Arabinose + + +
Cellobiose + + +
Dulcitol [-I d [-I
p-Gentibiose + + +
D-Glucose + + +
Glycerol + + +
myo-Inositol + + [+I
Lactose + + +
Maltose + + +
D-Mannitol + + +
o-Mannose + + +
D-Melezitose - - +
Melibiose + + +
a-Methyl-o-glucoside + + +
Mucate + + +
Raffinose + + +
L-Rhamnose + + +
Salicin + + +
D-Sorbitol + + +
Sucrose + + +
Trehalose + + +
D-Xylose + + +
Tartrate, Jordans + + +
Aesculin hydrolysis + + +
Pectate hydrolysis - - -
Utilization
Acetate d [+I [-I
Gentisate - - +
m-Hydroxybenzoate - - +
Malonate + + +
D-Glucose, gas production + + [+I
Growth 10°C + - +
Lactose, gas production 44°C - + -
Nitrate reduction + + +
Deoxyribonuclease, 25°C - - -
Lipase - - -
ONPG" + + +
Pigment - - -
 KLEBSlELLA 1111

Table 1 contd
Test Klebsiella Klebsiella Klebsiella Klebsiella Klebsiella Klebsiella
oxytoca planticola pneumoniae pneumoniae pneumoniae subsp. terrigena
subsp. ozaenae subsp. rhinoscleromatis
pneumoniae
Flagella arrangementb P - - - - -
Catalase production (24 h) + + + + + +
Oxidation-fermentationc F F F F F F
-, 0-10% positive; [-I, 11-25% positive; d, 26-75% positive; [+I, 76-89% positive: +, 90-100% positive. Results are for 48 h
incubation.
“ONPG, 0-nitrophenyl-p-D-galactopyranoside.
bP, peritrichous.
‘F, fermentative.

effects of pulp and paper mill and cannery effluents
in receiving waters.
The search for improved selective media and diag-
nostic tests goes on and is outlined below. A synthetic
medium, based on myo-inositol as the sole carbon
source has also been proposed for selection of Kleb-
siella (and Serratia). As a further elaboration, a Mac-
Conkey-inositol-carbenicillin agar medium (MCIC)
was proposed, the selectivity of this medium being
based on the high resistance of the capsulated Kleb-
siella cells towards carbenicillin. Based on its oligo-
trophic characteristics, the development of a synthetic
medium was claimed for the detection of K . pneu-
moniae; it only contains, apart from agar, I g l - l
KN03,2 g I-’ KH2P04, 20 g 1-l sucrose, but it is sup-
plemented with 1 0 pg I-’ carbenicillin.
A highly selective, differential medium for the enu-
meration and isolation of Klebsiella species has been
devised: MacConkey-inositol-potassium tellurite
(MCIK) agar. With pure cultures, 100% recovery
of Klebsiella was observed, and with environmental
samples recovery of Klebsiella was as good as or better
than on MCIC agar. MCIK agar was subsequently
field tested for its ability to selectively enumerate
Klebsiella species from the cold waters (1-6°C) of the
Saint John River Basin (New Brunswick, Canada)
which include fresh and marine waters. Results of this
study indicate that 77% of the typical colonies on
MCIK agar were Klebsiella species, but the total Kleb-
siella population enumerated was greatly under-
presumptive identification of urinary tract bacterial
isolates, including Klebsiella, in specimens from a
rehabilitation centre. Recently, a new chromogenic
plate medium (CHROMagar Orientation) for the
visual differentiation and presumptive identification
of Gram-negative bacterial species and enterococcal
isolates was evaluated. Similarity in colour resulted
in failure to discriminate between Klebsiella, Entero-
bacter and Citrobacter species, but these species could
be readily differentiated from other members of the
Enterobacteriaceae. These data indicate an urgent
need for the development of a simple, reliable and
specific detection and enumeration methodology for
Klebsiella sp.
Media Composition Suited for Cultivation of
Klebsiella Strains
The composition of media suitable for cultivation of
Klebsiella strains is listed in Table 2.
The media are routinely sterilized by autoclaving
(20min at 121°C and 2.1 atm). The carbon source is
Table 2 Composition of media for cultivation of Klebsiella
strains
Concn. (g 1”)
Nutrient broth (Oxoid) - pH 7.4
‘Lab-Lemco’ beef broth 1.o
Yeast extract 2.0
Peptone 5.0
NaCl 5.0

estimated; the MICK medium seems to be more Klebsiella medium - pH 7.0

specific for its target organisms but appears to lack Glucose 100.0
Soya peptone 10.0
sensitivity. Yeast extract 0.5
Various selective media have been assessed as to MgSO,. 7H20 0.5
their ability to detect and differentiate K. oxytoca and K2HPO4 0.7
E. coli in drinking-water samples. Only two media, NaH2P04 0.7
membrane lauryl sulphate agar and deoxycholate agar Orskov and Orskov medium - pH 7.0
allowed differentiation, with K. oxytoca only able to Glucose 15.0
Bacteriologicalpeptone 7.0
grow at 37°C and not at 44°C.
NaCl 5.0
The CPS ID2 medium (Bio-Merieux) enabled the
1112 KLEBSIELLA
thereby separated from the nitrogen source to prevent
Maillard reactions. Solidified media are obtained by
adding 20 g 1-1 of agar before sterilization.
Culture Maintenance
Klebsiella strains can be easily maintained in meat
extract agar stabs at room temperature. They can also
be preserved either by storage in broth, containing
10% glycerol at -80°C or by lyophilization.
Procedures Specified in
National/lnternational Regulations or
Guidelines
No official guidelines/regulations seem to exist at
national or international level with regard to specific
Klebsiella detection, enumeration or threshold
numbers. The reader is referred to water and food
quality guidelines, related to Enterobacteriaceae or
coliforms.
Medical Aspects of Klebsiella Bacteria
As indicated above, Klebsiella bacteria are present in
the respiratory tract and faeces of about 5 % of normal
individuals. They cause a minor proportion (about
3 %) of bacterial pneumonias and can cause extensive
haemorrhagic necrotizing consolidation of the lung.
They occasionally produce urinary tract infection,
septicaemia, bacteraemia with focal lesions and men-
ingitis in debilitated patients. K. pneumoniae and K.
oxytoca, especially, cause hospital-acquired infec-
tions. Two other Klebsiella subspecies are associated
with inflammatory conditions of the upper respiratory
tract:
K. pneumoniae subsp. ozaenae has been isolated
from the nasal mucosa in ozena, a fetid, progressive
atrophy of mucous membranes;
K. pneumoniae subsp. rhinoscleromatis has been
isolated from rhinoscleroma, a destructive gran-
uloma of the nose and pharynx.
K. pneumoniae is resistant to penicillin and ampicillin;
resistant strains usually produce R-plasmid encoded
p-lactamase. Broad-spectrum third generation
cephalosporins such as cefotaxime, or amino-
glycosides are used to combat normal strains
(community-acquired), whereas hospital-acquired
strains are multiple antibiotic resistant. Often, K.
pneumoniae infections commonly occur following
antibiotic treatment.
Environmental Relevance of Klebsiella
Bacteria
Since Klebsiella species are widely distributed in the
environment and in water systems, and since often
little or no differences can be detected between envir-
onmental and clinical strains, there is increasing
concern about the potential health hazard related
to Klebsiella. There is, therefore, a growing need to
monitor these organisms in the environment, espe-
cially in (drinking) water systems, soils, aerosols,
cooling waters, biofilms and industrial effluents.
Relevance of Klebsiella in the Food
Sector
General Aspects
The relevance of Klebsiella in foods as a contaminant
or spoilage organism is only recently being addressed.
Even in standard texts on food microbiology, there is
little or no mention of problems related to Klebsiella
food spoilage, contamination or transmission to
humans. This is in sharp contrast with its ubiquitous
presence in the daily human environment, and the
pathogenic character of the clinical Klebsiella isolates,
which are taxonomically very similar to the envir-
onmental strains.
As indicated already, Klebsiella species are oppor-
tunistic pathogens, that can give rise to bacteraemia,
pneumonia, urinary tract and several other types of
human infection. The origin and transfer of the infec-
tion is not always clear, since Klebsiella spp. are widely
distributed in nature, occurring in soil, water, grain,
fruits, vegetables, biofilm etc. Many of these envir-
onmental strains belong to the species K. terrigena
and K . planticola. Since they also occur, albeit in
low numbers, in the human respiratory and intestinal
tracts, these seem to form the main reservoir for
human-to-human infection (via food or otherwise),
particularly in hospitals, where the hands of personnel
and aerosol formation are the main factors in trans-
mission. Outbreaks particularly occur in urological
patients and in neonatal and intensive care units.
Enterotoxin-producing Klebsiella strains have also
been described.
K. pneumoniae strains are mainly isolated in asso-
ciation with several pathological processes in humans
(respiratory and urinary tract infections) and in
animals (metritis in mares and mastitis in cows) and
as such can also enter the food chain.
Equally, the importance of the occurrence of Kleb-
siella species in the food sector is difficult to judge.
Klebsiella species are usually not selectively cultured
from foodstuffs, but are present when total counts or

the presence of Enterobacteriaceae are tested. It is
assumed that K. terrigena, isolated mainly from
aquatic and soil environments, and K. planticola,
mainly isolated from botanical, aquatic and soil envir-
onments, are saprophytic strains, that are easily trans-
mitted to food. K. oxytoca is present in the intestinal
tract of humans and animals, and has also been isol-
ated from botanical and aquatic environments and as
such it can also contaminate food and infect humans,
indicating again an urgent need for specific Klebsiella
detection. In this respect, a bioluminescence-based
detection of lux’ K. oxytoca strains was developed
and their survival in the barley rhizosphere was
studied; it was found that K. oxytoca specifically
survived on the plant roots during the whole vege-
tative period and that it could not be isolated from
the soil.
Drinking Water Quality
Although it has been claimed that the attachment of
bacteria, i.e. Klebsiella sp., to surfaces via capsule
formation is a cause of increased bacterial disinfection
resistance (for instance survival in chlorinated water
supplies), it has also been found that resistance to
chlorine was not related to the presence of poly-
saccharide, but to the formation of cell aggregates.
Indeed, K. pneumoniae and K. oxytoca grown in low-
nutrient media were found to be more resistant to
chloramine than cells grown in rich media, and to
form large aggregates/flocs of 10-io3 cells per milli-
litre. This formation of flocs and aggregates allows
the cells to survive chlorination and to enter the water
distribution system. Indeed, Klebsiella sp. is one of
the principal microorganisms involved in bacterial
regrowth within chlorinated drinking water systems.
The regrowth of this organism is of particular import-
ance since, as a coliform, it will make compliance
to water quality guidelines difficult, and it may be
involved in opportunistic infections.
The growth kinetics of coliform bacteria, including
K. pneumoniae, have been studied under conditions
relevant to drinking water distribution systems. It
was found that most of them - and Klebsiella in
particular - could develop in unsupplemented mineral
salts medium and in the unsupplemented distribution
water. This proves that environmental coliforms, and
equally K. pneumoniae, can develop under the con-
ditions found in operating municipal drinking water
systems. In this context, the ability of K. pneumoniae,
Entero bacter aerogenes, Agro bacterium tumefaciens,
Bacillus subtilis and Pseudomonas strains to grow
and maintain motility and viability in drinking water
has been studied. Plate counts dropped below the
detection limit within 7 days for all strains mentioned,
except for Bacillus and Pseudomonas strains.
KLEBSIELLA 1113
The drinking water quality in a major South African
metropolitan area, in collecting water samples from
private houses, apartments and public places was
assessed over a period of two years. Entero-
bacteriaceae bacteria were found in 33% of the
samples, as well as Bacillus sp. Klebsiella was also
frequently found. The age of the plumbing system
was clearly correlated with poorer microbiological
quality of the potable water. Among 62 trademarks
of bottled drinking water, a sampling of 158 bottles
revealed the presence of K . oxytoca, along with other
coliforms, in three bottles.
The quality of packaged ice, sold in retail estab-
lishments in Iowa, USA has also been studied. A total
of 18 samples were analysed in relation to the drinking
water standards of the US-EPA. Only one sample
exceeded the health standard and contained K. pneu-
moniae. Several samples had heterotrophic plate
counts, which exceeded the recommendation
(< 500 cfu ml-l) of the Packed Ice Association.
Although such ice consumption does not represent an
immediate threat to personal or public health, the
potential for disease transmission exists in a sector,
which is in this respect self-regulated.
It is clear that Klebsiella species comprise a large
part of the coliforms, usually detected as indicators
of water quality; in most instances however, they are
not differentiated.
Food Quality
As to their occurrence in foods, the microbiology of
contaminated foods in health-care facilities in the
USA was surveyed and the importance of microbial
surveillance, quality assurance and employee edu-
cation was stressed. K. pneumoniae is mentioned as
one of the encountered contaminants, together with
E. coli, Yersinia intermedia, Aeromonas hydrophila,
Enterobacter agglomerans, E. cloaca, Campylobacter
jejuni, Acinetobacter anitraturn, Streptococcus vir-
idans, Serratia liquefaciens, Staphylococcus sp., Sal-
monella sp., Corynebacterium sp., Lactobacillus s ~ . ,
Listeria sp. and others.
High numbers of Klebsiella species were found in
samples of the local food, pap ‘akamu’, prepared in
Nigeria from cereals (maize, guinea corn and millet);
Klebsiella sp., Enterobacter sp. and Staphylococcus
sp. were the most common bacteria found. These data
indicate the widespread occurrence of Klebsiella sp.
in these indigenous foods, in combination with other
opportunistic pathogens.
K. pneumoniae subsp. pneumoniae (using the API
20 E system) was also found to be present in the
industrial fermentation process of ‘Saccharina’ pro-
duction (fermented fodder) from sugar cane.
‘Coliforms’ were enumerated in fresh and processed

1114 KLEBSIELLA
mangoes (puree and cheeks) in order to establish the
source of the organisms in the production chain, to
determine whether they have any public health sig-
nificance, and to devise methods for their control.
Products from four processors were tested on two
occasions, The retail packs of cheeks-in-puree having
the highest coliform counts were those in which raw
puree was added to the cheeks. Coliform counts in
these samples ranged between 1.4 x 1O3 and
5.4 x IO4cfu g-'. Pasteurization reduced the coliform
count of raw puree to 5 cfug-'. Around 47% of the
73 colonies, isolated as coliforms on the basis of
their colony morphology on violet red bile agar, were
identified as K. pneumoniae, using the ATB 23 E Iden-
tification System. Klebsiella strains were tested for
growth at 10°C, faecal coliform response and fer-
mentation of D-melezitose; these tests are used com-
monly to differentiate the three phenotypically similar
strains K. pneumoniae, K. terrigena and K. planticola.
Results indicated that 41% of the isolates gave reac-
tions typical of K. pneumoniae. A further 44% of
strains gave an atypical reaction pattern and were
designated 'psychrotrophic' K. pneumoniae. K. pne-
umoniae counts of 2.1 x lo3-4.9 x lo4cfu g-' were
predicted to occur in the retail packs of mango cheeks-
in-puree produced by the processors, who constituted
this product with raw puree. In view of the oppor-
tunistic pathogenic nature of K. pneumoniae, its pres-
ence in these products is considered undesirable and
steps, such as pasteurization of puree, should be taken
in order to inactivate it.
Recently attempts have been made to correlate the
presence of selected pathogens (Campylobacter jejuni,
C. coli, Salmonella, K. pneumoniae and E. coli
0157:H7) in fresh hand-picked blue crab meat and
general microbial quality to sanitation practices by
the processors (Chesapeake Bay region, USA). K.
pneumoniae was isolated from 51 samples out of
the 240 (21%) (0.3-4.3 most probable number
(MPN)g-I), followed by C. jejuni (36 out of 240), C.
coli (14 out of 240). Salmonella and E . coli 0157:H7
were not detected in any of the 240 samples analysed.
The foregoing data indicate again that Klebsiella
sp. is frequently present as a contaminant in water
and food, often in high numbers. They are commonly
lumped within the group of the Enterobacteriaceae or
coliforms, with most attention always focused on the
well known food pathogen members of the group. It
is only recently that Klebsiella is being selectively
searched for and 'looked after' as a genushpecies,
relevant to food microbiologists too.
Next Page
Industrial Aspects of Klebsiella Bacteria
Production of 2J-butanediol
Most Klebsiella species are saprophytic, some are
pathogenic and only a few are of industrial use. Under
controlled fermentation conditions, K. oxytoca
strains produce high levels of 2,3-butanediol, an inter-
esting chemical feedstock or liquid fuel, from sugary
substrates such as glucose, xylose and whey permeate.
Due to its toxicity to the producer cells, only moderate
concentrations (approximately 100 g 1-I) can be
obtained in even optimized fermentation processes.
This, together with the high boiling point and hygro-
scopicity of 2,3-butanediol, makes recovery costs
high. 2,3-Butanediol can be chemically converted into
butadiene, the raw feedstock for synthetic rubber, or
into other derivatives such as ethylmethylketone (used
as a solvent, fuel additive) and tetramethylether
(antifreeze) or into polyester plastics.
Biofilm Formation by Klebsiella sp.
As a result of its capacity to form capsules, Klebsiella
species are often a main cause of (undesirable)
biofilm formation and fouling in cooling water
systems, piping and other industrial equipment.
The biofilm-forming capacity of several Klebsiella
species, isolated from pulp and paper mill water,
and of Klebsiella terrigena BCCM strains has been
studied in vitro by the authors in 2 litre lab
fermenters. The capsular polysaccharide from one
isolate was recovered (up to 4.6gl-'), its rheological
properties were identified as pseudoplastic and its
sugar composition was identified as: L-fucose, L-
rhamnose, D-galactose, D-glucose, D-mannose and
D-glucuronic acid. Enzymes which can efficiently
hydrolyse and remove biofilm have been looked for.
The Klebsiella Capsule as a Source of Unusual
Sugars
The Klebsiella capsule, as described above, often con-
tains unusual sugar moieties such as L-fucose and
L-rhamnose, and the authors have cultivated such
capsular bacteria on a large scale, as a source of
these specialty sugars, which are otherwise difficult to
obtain.
Klebsiella as a Vitamin Producer in Fermented
Foods
Recently, the formation of vitamin B12 was dem-
onstrated by strains of K. pneumoniae, isolated from
Indonesian tempeh samples, during controlled solid-
state tempeh fermentation. The absence of entero-
toxins in these strains was confirmed by using PCR
techniques, and it was even suggested that these safe

LABORATORY DESIGN
M Ahmed, Food Control Laboratory, Dubai, United Arab Emirates
Copyright 0 1999 Academic Press
Introduction
Food microbiology laboratories play an important
role in the control of food hygiene, quality and safety.
The potential hazards associated with pathogenic
microorganisms in these laboratories together with
the development of strict legislation to promote health
and safety at work have led to higher standards of
laboratory design. Contamination of samples within
the laboratory through air and other sources has been
a major problem associated with microbiological
analysis. The laboratory design must meet the require-
ments to avoid contamination. Cleanliness, ven-
tilation, accessibility, storage, waste disposal, security,
fire protection and emergency precautions must all be
considered at the initial stage of design.
Even though the final design of the laboratory is
made by architects and engineers, involvement of
microbiologists is essential when taking important
decisions that affect the working environment and
conditions. Microbiologists should work in close
association with the architect and explain all the tech-
nical and safety requirements of each room. They
should also follow the building through the different
stages of construction to ensure that all the require-
ments included in the design are fulfilled.
Study Report
Microbiological laboratories can be broadly classified
into three categories:
1. Hygiene control laboratories performing limited
microbiological tests to evaluate sanitation and
hygiene procedures followed in food production
plants, restaurants and catering establishments.
2. Quality control laboratories involved in the testing
of imported and locally produced foods as well as
hygiene control, which perform a wide range of
analyses and carry out work-related research and
investigations.
3 . Research laboratories involved in carrying out
LABORATORY DESIGN 1119
research and development (R&D) but not involved
in quality control.
Before designing a laboratory, a study report should
be prepared by a consultant with good knowledge and
experience of designing laboratories. The technical
experts of the consultant should meet the man-
agement, microbiologists and other technical staff and
discuss in detail their requirements. Due consideration
should be given to their views and recommendations
while preparing the study report, which should consist
of the following:
scope and objectives of the laboratory
organization chart indicating the various functions
of the laboratory
number of technical, administrative and support
staff
expected number of samples to be analysed
details of technical facilities required
service requirements
the interrelationships, if any, between the functions
of the laboratory and other disciplines (chemistry,
biochemistry, nutrition, etc.)
budget requirements.
The study report should also address the scientific
and technical developments in the area and make
provisions for future expansion of the laboratory.
The recommended organization of a food micro-
biology laboratory suitable for routine quality control
analysis is shown in Figure 1. The laboratory consists
of a general microbiology unit, with culture tech-
niques and media preparation sub-units, and an
advanced microbiology unit, with rapid diagnostic
techniques and instrumental techniques sub-units.
The administration sample management, quality
management, R&D and training management, and
calibration and maintenance constitute other func-
tions. These may be common to a laboratory con-
sisting of multiple disciplines such as chemistry,
biochemistry and nutrition. The major activities of
different functions in the laboratory are listed in Table
I.
1120 LABORATORY DESIGN

FOOD MICROBIOLOGY
LABORATORY
DISCIPLINES
(Head of Laboratory)

1 Quality Management kl Administration I

'if-
Research and Training
Management

Calibration and
Maintenance
Sample
Management

1 Advanced Microbiology Unit

(Head of Unit)

I I

Techniques Techniques
(In-Charge) (in-Charge)
Isolation and Media preparation
identification and sterilization
lmpedimetry

I
Food poisoning
1analysis preparation
DNA hybridization Turbidimetry
I Certification and Sterilization of
monitoring glasswareiutensils
Bioluminescence
- Standardization
Decontamination
of materials
Polymerase chain
Other techniques reaction

Figure 1 Food laboratory organization chart.

Building Layout carrying out routine quality control analysis of a wide

range of samples in addition to conducting a limited
Many types of laboratory layouts are possible,
depending on scope of work, space and budget. The number of applied research projects is shown in
building layout for a food microbiology laboratory Figure 2*

Table 1 Major activities of a food microbiology laboratory

Unitlfunction
General microbiology
Advanced microbiology
Quality management
Calibration and maintenance
R&D, training management
Sample management
Administration
Sub-unit Major activity
Culture techniques Certification and monitoring programmes, food poisoning -emer-
gency analysis, standardization
Media preparation and ster- Preparation and sterilization of media, glassware, sample uten-
ilization sils, decontamination and washing of used materials
Rapid diagnostic techniques Application development and implementation of immunoassay,
DNA hybridization, API, etc.
Instrumental techniques Application development and implementation of impedimetry,
turbidimetry, bioluminescence, PCR, etc.
Quality management Implementation of quality assurance system (IS0 9002/ISO
Guide 25), internal quality control, proficiency testing, audits,
etc.
Calibration and maintenance Equipment and building maintenance, calibration of equipment,
maintenance of services
R&, training management Planning, budgeting of R&D work, coordination with different
units, training requirements and their planning and sched-
uling, management of external training programmes
Sample management Receipt, identification, registration, preparation of composite
samples, assigning code numbers, distribution of samples to
different functions
Administration Secretariat, personnel management, budget/accounts,
purchase/stores, library and housekeeping

28 29 5 6
2
7
A n M /I/I
3 E
4 7
26 24
27
u U
22
25 23
General Considerations
The microbiology laboratories have a unique con-
tamination problem and should have a central air
conditioning system if possible. This system should
be divided into zones depending on the type of work
carried out in different rooms to facilitate exchange
of fresh air and to take necessary precautions against
environmental contamination. The incoming air is
filtered through 0.2ym filters to reduce the risk of
environmental contamination of the laboratory. The
humidity must be kept low to reduce problems with
hygroscopic materials such as media and chemicals,
and to avoid growth of moulds on laboratory sur-
faces. Air conditioning also stabilizes room tem-
peratures, enabling incubators to function more
efficiently. Temperatures and relative humidity should
be comfortable for workers and suitable to the
requirements of the laboratory equipment. Normally
an ambient temperature of 21-23°C and a relative
humidity of 40-50% are recommended.
Lighting It is recommended that laboratory lighting
be maintained at an average intensity of 0.5-1 klx
(50-1 00 footcandles). Dependence on natural sun-
light during the day is discouraged: direct exposure to
sunlight is known to alter the performance of media,
chemicals and reagents. Likewise, analytical work
must not be performed in direct sunlight since final
results are affected.
Storage Sufficient storage space should be provided
for equipment, materials and samples. The laboratory
wall space should be utilized for additional shelving,
protected by glass-enclosed cabinets to provide a dust-
free environment for storage of media, chemicals and
other materials. Samples should be stored in refriger-
ators, freezers or at room temperature according to
the procedures outlined in the operational manual.
LABORATORY DESIGN 1121
‘ 8 ’
- 9 10 11 12
L 7 ’
M M f- As, M
d w W w
20 3 16L 3 15
- 19 ia 14 13
21 17
3
Future Expansion Future expansion of activities,
increases in workload and staff should be considered
when designing a laboratory. The design should
include provision for a minimum of 25% of expan-
sion. The design should also be flexible to allow room
functional changes and allocation of new activities.
Allocation of Space The design should allow
maximum utilization of laboratory space. The sub-
unit of media preparation and filling, decon-
tamination of used material and cleaning of glassware
should be separated from the analytical area. Within
the analytical area, isolation and identification of
pathogenic materials should be carried out in a sep-
arate room if possible.
Safety The safety of the laboratory personnel should
be taken care of in the design. The laboratory should
be equipped with fire extinguishers and alarms, a
sprinkler system, eye-wash stations and safety
showers. Fire and smoke detectors are also recom-
mended. A comprehensive safety programme should
be a vital part of all laboratory procedures.
Access Two exits should be provided for the build-
ing for prompt exit in the event of fire or other emer-
gencies. Entrances should be designed to minimize
pedestrian traffic.
Security A security system must be provided to
restrict entry into the laboratory building. Laboratory
rooms should be separated from offices by another
security system, apart from the general security
system, to restrict unauthorized entry into the labora-
tory rooms, to avoid contamination and for effective
operation.
1122 LABORATORY DESIGN

The Building Programme
The building programme is a written document that
describes and quantifies the design goals for a build-
ing. The goal of a good programme is to define a
building that will have ample space, meet the technical
requirements of the user, function safely and meet the
owners’ budget. The design team with the assistance
of the laboratory management and technical staff will
develop the building programme from the analysis of
data collected on the following:
0 the range of analyses to be carried out
0 the number and types of personnel who will occupy
the building
the interrelationships of functions and personnel
0 the expected workload.
The programme should describe the architectural,
mechanical, electrical, plumbing and fire protection
criteria for the functions to be accommodated. It
should also identify areas of special concern for safety
such as high hazard areas containing flammable, toxic
or pathogenic materials, and should also address the
problem of waste removal.
The main tasks and sequence of a building pro-
gramme are as follows:
appropriate places in each room (Figs 3, 4). Cen-
tralized services for gases, deionized or distilled water,
etc., are preferred.
Electrical Connections
It is essential to determine the total electrical load of
each room. In order to achieve this, the equipment to
be placed in each room must be decided upon and
its power requirements (voltage, current rating, etc.)
listed and supplied to the consultant or contractor.
Equipment such as autoclaves and washing machines
may require three-phase connections. These have to
be identified and separated from equipment of low-
energy consumption. It is recommended that items of
high electrical rating are placed in different rooms
to balance the power consumption. Proper earth
--
--
1

0 analyse the study report

r
0 interview management, technical staff and other
users
0 establish space standards
list various activities and room types required for
such activities
draw a layout diagram for different room types 3
determine the number and area of each room type
develop room data sheets specifying details of
functions
establish building net and gross areas
describe basic mechanical, electrical, electro-
m Bench

mechanical and plumbing systems

describe the services ic Double electric point
estimate the cost of construction. TA Telephone connection
C= Data communication
c-t Cold water tap
E
Supply of Services
Proper supply of services such as electrical con-
-
- G
Medical mixing tap
Demineralizedidistilled water
LPG
nections, gases, hot water, demineralized or distilled 0- CA Compressed air
water, compressed air, vacuum, telephone and data 0- N Nitrogen
networks, fire protection systems, smoke detection
Figure 3 Room data sheet - culture techniques. General
system and alarms, emergency showers, sprinklers, laboratory furniture: 1, window bench, 75cm high; 2, island
eye-wash stations, etc. is essential for efficient running bench, 90 cm high; 3, stool; 4, slab sink; 5, safety storage cabinet;
of a laboratory. The services should be installed in 6, biohazard safety cupboard.
 LABORATORY DESIGN 1123

for the purpose. The piped supply runs along the

corridors, with branches into the laboratory rooms.
Each branch should be equipped with a valve enabling
the supply to be shut off in an emergency. Stainless
steel tubing with Swagelock@ fittings is recommended
for piping the gases. Welding should be avoided. Pres-
sure checks and certification from the contractor duly
approved by the consultant and approval from the
civil defence authority are required prior to the actual
supply of gas. All the lines must be accessible for
5 future leak checks. Gases such as nitrogen and COz
may be required only for anaerobic work stations. If
the use of such gases is limited to one or two rooms,
the cylinders may be housed in a purpose-built cabinet
near to the point of use or within the laboratory, as
they are not inflammable or hazardous.
Compressed Air and Vacuum
Laboratories requiring compressed air may be sup-
plied from a centrally located compressor connected
to the laboratory by a system o n copper or high-
i
( Double electric point pressure plastic pipes. The air should be dried to a
TA Telephone connection dewpoint of - 15°C and freed from oil droplets with
C c Data communication
the aid of filters. The pressure in the system as far as
the branches into the laboratories should be 7 bar,
3--t Cold water tap
which in the laboratories should be reduced to a

-
-it

0- G
Medical mixing tap
Demineralizedidistilled water
LPG
working pressure of 3 bar. Vacuum may be supplied
through a central system if it is required in many
rooms; otherwise, small vacuum pumps may be used.
0- CA Compressed air
Hot and Cold Water
3- N Nitrogen
The building should be provided with a supply of
Figure 4 Room data sheet - instrumental techniques. General
laboratory furniture: 1, window bench, 75 cm high; 2, wall bench,
process water and also drinking water if necessary.
90cm high; 3, wall bench with sink, 90cm high; 4, island bench, The process water should be equipped, downstream
90cm high; 5, stool; 6, slab sink; 7, safety storage cabinet; 8, of the meter, with a break installation. The pressure
adjustable chair. measured at the highest tap should be 2.5 bar. The
pipes should be laid in such a way that water nowhere
connections must be provided with bonding resistance becomes stagnant. Wherever necessary, hot water
per earth of less than 1ohm. The minimum resistance should be provided from closed-in boilers. The
of the earthing net should be 1.2 ohm. Circuit break- minimum temperature of the water should be 60°C.
ers must be installed at each workbench. Medical mixing taps with a lever should be provided
for mixing cold and hot water, in order to avoid
Gases contamination from the hands of the microbiologist.
Most microbiological laboratories require the fol-
Demineralized and Distilled Water
lowing gases:
A supply of demineralized or distilled water should
liquefied petroleum gas (LPG)
be available in all the laboratories. Demineralized
nitrogen
water can be prepared with the aid of an automatic
carbon dioxide double-column demineralization system housed in a
0 oxygen.
centrally located room. Distilled water can be pre-
The rooms and the locations in each room requiring pared with the aid of electrically operated distillation
supply of different gases should be identified and equipment. In both cases, the water should be trans-
listed. It is possible to provide all laboratory rooms ported through plastic tubing to the laboratories. The
with a supply of piped gases; the gases are supplied demineralized water should have specific conductance
in bulk cylinders and stored in an outhouse built less than 5 pa cm-'.
1124 LABORATORY DESIGN
Telephone and Data Network Connections
Rooms and laboratories requiring telephones must be
identified and the appropriate connections provided.
Most modern laboratories use client-server tech-
nology to manage sample information and analytical
data. Several laboratory information management
systems (LIMS)are commercially available, and could
be customized. Data network connections are pro-
vided in the laboratories to facilitate data entry and
necessary approvals. They should be located pref-
erably on the side workbenches at a height of 75cm
or near the desks, slightly away from the working
area. Network connections are also required on island
benches where analytical instruments with data sta-
tions are located, to hook up with LIMS for direct
transport of instrumental data.
Fire Protection, Smoke Detectors and Alarms
For the purpose of preventing fires, and quelling any
fires that break out, it is necessary to draw up plans
for fire prevention and firefighting systems. The
laboratory building should be divided into com-
partments separated by fireproof walls and ceilings.
The floor surface area of a compartment may vary
from 500m’ to 750m2, or as necessary to achieve a
logical arrangement of the compartments. In accord-
ance with internationally accepted test methods, the
fire retardance of floors and ceilings should not be
less than 1h. All electric and other cables should be
passed through fireproof blocks. All spots within the
building should be within reach of the jet of a fire
hose connected to the process water mains. The reels
should be hung on the walls of the corridors, and the
hoses should not exceed 30 m in length. In addition to
the fire hoses, fire-extinguishers should be distributed
throughout the building. Their filling should be in
accordance with the type of fires they are likely to be
used against. The building should be equipped with
an automatic fire alarm system. Ionization detectors
should be mounted in all rooms or spaces where fires
may start, and are mandatory in rooms where people
are at work. A (repeater)fire alarm should accompany
each fire-hose reel. The system should be combined
with an acoustic alarm system (hooters or sirens) and
should be fully independent of the building control
system. An emergency power supply system is needed
to illuminate and mark escape routes, enabling people
to leave the building in the shortest possible time in
emergencies. It should be equipped with a no-break
unit. The emergency power supply must serve all the
electrical equipment that must be kept in operation
in emergencies.
Eye-washes and Emergency Showers
All laboratory rooms should be provided with eye-
wash stations if possible. Emergency showers are
required in laboratories where hazardous chemicals
or other materials are being used, and must be easily
accessible.
Design of Furniture and Choice of
Finishes
Laboratory furniture normally consists of work-
benches, cupboards, wall units, desks and drawers.
Prefabricated furniture units are available.
Benches
Workbenches can be wall-mounted or island type.
The framework should comprise a mild steel tubular
framework based on a modular construction with an
epoxy-based plastic coating, and should incorporate
adjustable levelling jacks, pipe clips and cableways.
The bench top should be set at a height of 90-95 cm
for normal work in a standing position. The desk tops
or ‘sit down’ benches can be at a height of 65-79 cm as
needed to accommodate microscopes, plate counting,
computer usage or paperwork. The low-level benches
should be mounted on the window side walls to
accommodate microscopes, network computers, etc.
It is also necessary to keep instruments on low-level
island benches for easy access to the reverse of the
instrument. Services such as electrical sockets and gas
connections on island benches meant for installing
instruments should run at the side of the bench for
optimum utilization of the bench space. The storage
cabinets and drawers should be suspended from the
bench connections, and there should be a combination
of cabinets and drawers on each bench. Cabinets may
be built with WBP grade plywood with an inert and
corrosion-resistant finish with minimum seams (e.g.
seamless melamine). Drawers may be constructed
with corrosion-resistant faced plywood. The cabinets
and drawers on the workbench should be fixed in
such a way that adequate legroom remains. Ample
space should be allowed for refrigerators and writing
desks when installing wall-mounted workbenches.
Bench Tops
The bench tops may be constructed from solid mela-
mine, WBP plywood with a seamless melamine finish,
WBP plywood with stainless steel top and edges, or
solid hardwood with a laminate finish. The bench
tops should have a smooth surface and be easily dis-
infected. Cracks and crevices should be minimal as
they provide an opportunity for the build-up of debris
which may contribute to cross contamination of

samples. Stainless steel tops must be provided on the
benches in the washing room.
Sinks
At either end of the benches (apart from benches
meant for installing instruments) stainless steel sinks
should be mounted with 60 cm side adjoining them,
a 50 cm side jutting out, and a depth of 25 cm. Medical
mixing taps (cold and hot water) and a deionized or
distilled water tap should be mounted above the sinks,
as required.
Seating
Laboratory stools and chairs of adjustable or fixed
heights should be provided. Stools should be used at
the workbenches, and chairs may be used at computer
desks.
Wall-mounted Cupboards
Cupboards with sliding glass doors may be mounted
on the walls for storing reagents, media, etc. Other
cupboards may be used for books, catalogues and
instrument files.
Laminar Flow and Biohazard Safety Cabinets
Safety cabinets should comply with standards set by
organizations such as the British Standards Insti-
tution, the Standards Association of Australia and the
National Sanitation Foundation of the USA. Care
must be taken in siting equipment that might generate
air currents, e.g. fans and heaters. The safety cabinets
should be installed in proper sites in the laboratory.
Safety cabinets are intended to protect the worker
from airborne infection. Work should be done in the
middle to the rear of the cabinet, not near the front
and workers should not remove their arms from the
cabinet until the procedure is completed. After each
set of manipulations, aerosols should be swept into
the filters. The operator’s hands and arms may be
contaminated and should be washed immediately
after ceasing work. Bunsen burners and micro-incin-
erators should not be used as they disturb airflow.
Facilities for Incubation and Refrigeration
lncubators
Incubators and incubator rooms must be properly
constructed and controlled. It is best to obtain the
largest possible models to prevent crowding of the
interior. Small incubators suffer wider temperature
fluctuations when their doors are open than do larger
models. Incubator rooms, if used, must be well insu-
lated, equipped with properly distributed heating
LABORATORY DESIGN 1125
units and have appropriate air circulation. They
should be installed by specialist suppliers. The rooms
should be supplied with stainless steel shelves suitable
for holding Petri dishes, flasks, etc. Wooden shelves
are not recommended because of the problem of
mould growth in a humid atmosphere. The recom-
mended temperatures for incubators in food labora-
tories are 15-20°C, 30-37°C and 55°C. Cooled
incubators must be fitted with a refrigeration system
and heating and cooling controls, which must be
correctly balanced.
Incubators should be kept in rooms where tem-
peratures are within the range 16-27°C. The incu-
bator temperature must not vary by more than 2 1°C.
Chamber temperature must be checked twice daily
(morning and afternoon). The thermometer bulbs and
stem must be submerged in water or glycerol to the
stem mark. For best results use a recording thermo-
meter.
Water Baths
Water baths should be of an appropriate size for
the required workload with a suitable water level
maintained. When the level of water in the bath is at
half to two-thirds the level of the column of liquid in
the tube, convection currents keep the constituents of
the tube well mixed and hasten reactions such as
agglutination. Water baths should be equipped with
electrical stirrers to prevent temperature stratification.
They must also be lagged to prevent heat loss,
although the walls are fitted with sloping lids to
prevent heat loss and dripping of condensed water
on materials. To avoid choke deposits on tubes and
internal surfaces, distilled or deionized water should
be used. Only racks made with stainless steel, heat-
resistant rubber, plastic, plastic-coated substances or
corrosion-proof materials should be used. The tem-
perature of the water bath must be monitored and
recorded daily using a certified thermometer.
Refrigerators
A refrigerator maintained at 0-4°C for storing
untested food samples is required. Another refriger-
ator to cool and maintain the temperature of media
and reagents may also be used. The temperature of
the refrigerator should be checked and recorded daily,
and it should be cleaned monthly or more often when
required. Refrigerated rooms, if used, must be well
insulated and equipped with a distributed cooling
system. A continuous temperature monitoring and
recording system equipped with an alarm must be
used. The temperature at different points should be
recorded daily. Stainless steel shelves should be
installed for storing samples. Stored materials should
be identified and dated, and stored in such a way that
1126 LABORATORY DESIGN
cross contamination does not occur. Expired materials
should be discarded at regular intervals, e.g. quarterly.
Freezers
A freezer or a freezer room to maintain the tem-
perature of frozen food items at - 18°C is required.
The temperature should be recorded daily. A record-
ing thermometer with an alarm system is highly desir-
able. The freezer should be defrosted and cleaned
twice a year. Materials should be identified and dated,
and outdated materials should be discarded quarterly.
A separate freezing space should be identified for
storing freeze-dried bacterial cultures.
Clean/Dirty Sterilization Facilities
Sterilization facilities are required for sterilizing pre-
pared media, diluents, etc., and used glassware, Petri
dishes, flasks, tubes, etc. prior to washing or disposal.
The use of heat, particularly moist heat, is the most
desirable and widely used method of sterilization in
the microbiology laboratory. When using heat ster-
ilizing techniques, it is necessary to know the dif-
ference between dry and moist heat and the limitations
of each. Moist heat leads to the destruction of micro-
organisms through the irreversible denaturation of
enzymes and structured proteins. The temperature at
which denaturation occurs varies with the latent heat
of steam. With dry heating, the primary lethal process
is considered to be oxidation of cell constituents.
Thus, sterilization methods involving dry heat require
higher temperatures and longer exposure time than
are required with moist heat.
Hot-air Oven
Sterilization by hot-air oven is achieved by the slow
penetration of heat into the materials. The efficiency
of this process can be increased by the use of cir-
culating fans. Modern equipment has electronic con-
trols which can be set to raise the temperature to the
required level, heat for a specified time and switch off
automatically. These ovens are fitted with solenoid
locks to prevent the oven being opened before the
cycle is completed. This protects the staff from acci-
dental burns and safeguards the sterility of the mater-
ials. The load should be packed in the oven chamber
in such a way that sufficient space remains between
the articles for circulation of hot air. The high tem-
perature needed to achieve dry heat sterilization has
a damaging effect on many materials. This method
should therefore be used only for thermostable mater-
ials that cannot be sterilized by steam owing to dele-
terious effects or failure to penetrate. Materials that
can be sterilized by this method include heat-resistant
articles such as glass Petri dishes, flasks, pipettes,
metallic objects and coated materials.
The performance of a hot-air oven should be tested
quarterly with commercially available spore strips or
spore suspension. The temperature should be moni-
tored with a certified thermometer, accurate in the
temperature range of 160-180°C.
Autoclaves
The minimum recommended standard for ster-
ilization by autoclaves is the exposure to steam at
approximately 1 bar pressure, equivalent to 121"C,
for 15 min. Saturated steam is a much more efficient
means of destroying microorganisms than either
boiling water or dry heat. Air has an important influ-
ence on the efficiency of autoclaving. If about 50%
of the air remains in the autoclave, the temperature
of the steam-air mixture at 1 bar is only 112°C. As
successful autoclaving depends on the removal of all
the air from the chamber, the materials to be sterilized
should be packed loosely. There are two types of
laboratory autoclaves:
pressure cooker models
0 gravity displacement models.
The pressure cooker is a simple benchtop autoclave
consisting of a vertical metal chamber with a strong
metal lid which can be fastened down and sealed with
a suitable gasket. The lid is fitted with an aidsteam
discharge trap, a pressure gauge and a safety valve
(Fig. 5). Steam is generated from the water in the
bottom of the autoclave by an external immersion
heater or a steam coil.
The gravity displacement autoclave, widely used in
microbiological laboratories, consists of a chamber
surrounded with a jacket containing steam under
pressure, which heats the chamber wall. The steam
enters the jacket from the main supply which is at
high pressure, thus forcing the air and condensate to
flow out of the drain by gravity displacement (Fig. 6).
In modern autoclaves, air and steam are removed by
*
Safety valve
~ iPressure /gauge
Airisteam discharge valve
Chamber
Figure 5 Pressure cooker autoclave.
 Next Page

LABORATORY DESIGN 1127

Combined pressure
and vacuum gauge Valve
Pressure
gauge Safety
valve Cotton-wool
Jacket
\
Q 1 , ,
filter

f
.-2 3 Tochamber
Chamber Door

Vave Strainers I I /’
or steam ejector
11 7Nr-;-ptee
,, ,/
Thermometer

lire- Valve

Figure 6 Gravity displacement autoclave.

vacuum pumps and flexible thermocouple probes are
fitted in the chamber so that the temperatures at
various parts of the load may be recorded.
The performance of the autoclave should be
checked monthly using spore strips or suspension.
Log books and other records should be maintained
for each run, listing the items sterilized, temperature,
pressure and time.
Washing Machines
A washing machine may be used for cleaning and
drying glassware and other heat-resistant articles. The
machine should be capable of washing, rinsing and
drying cycles. A log book should be maintained with
the details of the programmes used and the materials
washed.
Safety Glasses or Goggles
Safety goggles are essential for viewing ultraviolet
cabinets and other equipment that may emit UV
radiation.
Masks
Face masks with various filters are available for use
in laboratories. Appropriate filters are required for
working with pathogenic microorganisms and spores,
acid fumes, solvent vapours, etc.
Clothing for Entering Freezers or Cold Rooms
Special clothing is available to protect staff entering
freezers or cold rooms, and must be worn if staff
intend to work for long periods in such rooms.

Personnel Requirements
Lockers
Lockers are needed to hold the personal belongings
of the staff. They should be spacious enough to hold
laboratory coats, etc. They may be kept in a staff
room.
Laboratory Coats
Laboratory coats must be composed of 100% cotton
materials. Polyester or polyester blends must not be
used as they easily catch fire. Coats should be long-
sleeved and knee-length. They should be washed and
decontaminated at least once a week.
Gloves
Latex, rubber, leather and heat-resistant gloves are
available for use. Gloves, Hot Hand@ Protector pads
must be used when handling hot beakers, conical
flasks, etc.
First Aid
A first-aid box and fireproof blankets must be kept
in a conspicuous place near the door for use in an
emergency.
See also: Good Manufacturing Practice. Laboratory
Management: Accreditation Schemes. Process
Hygiene: Designing for Hygienic Operation.
 MEAT AND POULTRY/Spoilage of Meat 1253

M
I Malolactic Fermentation see Wines: The Malolactic Fermentation. I

I Manothermosonication see Minimal Methods of Processing: Manothermosonication. I

~~

Manufacturing Practice see Good Manufacturing Practice.

1 Mathematical Modelling see Predictive Microbiology and Food Safety. I

MEAT AND POULTRY

Contents
Spoilage of Meat
Curing of Meat
Spoilage of Cooked Meats and Meat Products

I Spoilage of Meat
George-John E Nychas and Eleftherios H
Drosinos, Department of Food Science and
Technology, Laboratory of Microbiology and
Biotechnology of Foods, Agricultural University of
Athens, Greece
Copyright 0 1999 Academic Press
Introduction
Spoilage of meat is an ecological phenomenon,
encompassing the changes of the meat ecosystem
during the development of its microbial association.
The establishment of a particular microbial asso-
ciation of meat depends on the ecological factors that
persist during processing, storage, transportation and
in the market. In meat, five categories of ecological
determinants influence the development of the initial
and transient microbial associations and determine
the rate of attainment of a climax population by the
ephemeral spoilage microorganisms (those that fill the
niche available by adopting R-ecological strategy as
a result of enrichment disturbance of an ecosystem).
These are (1)the intrinsic factors associated with the
physico-chemical attributes and structure of meat (e.g.
pH, water activity, buffering power, the presence of
naturally occurring or added antimicrobial com-
ponents, Eh and redox poising capacity, and nutrient
composition - carbohydrate content and, in par-
ticular, the concentration of glucose); (2) the pro-
cessing factors; ( 3 ) extrinsic parameters that have
selective influences, such as temperature, relative
humidity and the composition of the gaseous atmos-
phere obtaining during distribution and storage; (4)
the implicit factors (intrinsic biotic parameters) that
play an important role in the genesis of spoilage asso-
ciations; and ( 5 ) the emergent effects due to those
factors that interact to produce effects greater than
would be expected from their action in isolation. In
essence all of the above determinants constitute the
dimensions of a particular ecological niche - an n-
dimensional hypervolume. Indeed, the ecosystem
approach is pertinent to an analysis of changes occur-
ring in meat or meat products. In practice, therefore,
meat technologists attempt to modify some or all of
1254 MEAT AND POULTRY/Spoilage of Meat

the dimensions noted above in order to extend the Table 1. The genera of bacteria and yeasts most frequently
found on meats and poultry
shelf life of meat or to create new products. ~~

Genus Fresh Processed VP/MAP Poultry

meat meat
Typical Microflora of Fresh or Frozen
Meat Bacteria
Acinetobacter xx X X xx
Aeromonas xx X X
Contamination and Spoilage Alcaligenes X X

The microbiology of carcass meats is greatly depend- Alteromonas X X X X

Arthrobacter X X X X
ent on the conditions under which animals are reared, Bacillus X X X
slaughtered and processed. Thus the physiological Bacteroides X

status of the animal at slaughter, the spread of con- Brochothrix X X xx X

tamination during slaughter and processing, the tem- Campylobacter xx

perature, and other conditions of storage and Carnobacterium X X xx X

Chromobacterium X
distribution are the most important factors that deter- Citrobacter X X
mine the microbiological quality of meat. The char- Clostridium X X X
acteristic microbial associations developing on meat Corynebacterium X X X X

and in meat products are the result of the determinants Enterobacter X X X X

noted above on the growth of microbes initially Enterococcus xx

Escherichia X X
present in the fresh meat or, more generally, intro- Flavobacterium X xx
duced during processing. As the inherent anti- Hafnia X X
microbial defence mechanisms of the live animal are Janthinobacterium X

destroyed at slaughter, the resultant meat is liable to Klebsiella xx

rapid microbial decay. Unless effectively controlled, Kluyvera xx
Kurthia X X
the slaughtering process may cause extensive con- Lactobacillus X xx xx
tamination of the cut face of muscle tissue with a Leuconostoc X X X
vast range of both Gram-negative and Gram-positive Listeria X

bacteria as well as yeasts (Table 1). Some of these Micrococcus X X X X

microorganisms will be derived from the animal’s Moraxella xx X

Neisseria X X X
intestinal tract, and others from the environment with Pantoea X
which the animal had contact at some time before or Pediococcus X X X
during slaughter. Studies on the origin of the con- Planococcus X

taminants have shown that the source of Entero- Plesiomonas X

bacteriaceae on meats is associated with work surfaces Providencia X

Proteus X X
and not with direct faecal contamination. Moreover,
Pseudomonas xx X X xx
psychrotrophic bacteria are recovered from hides and Psychrobacter X
work surfaces within an abattoir as well as from Serratia X X X X

carcasses and butchered meat at all stages of pro- Shewanella X

cessing. Streptococcus X xx X X

Streptomyces X

Microorganisms of the Spoilage Association Staphylococcus X

Vibrio X

Although a range of microbial taxa are found in meat Weissella X X

(Table l ) ,its spoilage in developed countries is caused Yeasts

by the selection of relatively few of these organisms Candida xx xx X

(Table 2). It is evident from Table 3 that chill storage Debaryomyces X X

Rhodotorula X xx
and the gaseous composition around meat packed Saccharomyces X
in vacuum or in modified atmospheres exert strong Torulaspora xx
selectivity on its microflora. Selective factors favour Trichosporon X X

the growth of particular organisms and, as a con- Key: x, known to occur; xx, most frequently reported.
sequence, a characteristic microbial association is VP/MAP, meat stored under vacuum or modified-atmosphere
present at the time of spoilage and it will manifest packaging.
characteristic spoilage features. For example, with the
advent of supermarkets in the late 1950s, storage of
meat aerobically at chill temperatures and high rela-
tive humidity became a major selective factor and
 MEAT AND POULTRY/Spoilage of Meat 1255

Table 2 Psychrotrophic bacteria associated with chilled meats Table 3 Specific spoilage flora on fresh meat stored at 0-4°C
and meat products in different gas atmospheres
Gram-negative bacteria Gram-positive bacteria Gas composition Specific spoilage flora
Aerobes Catalase reaction weak Air Pseudomonas spp.
Pseudomonas spp. Brochothrix > 50% C02 mixed with O2 Brochothrix thermosphacta
rRNA homology, group I: thermosphacta > 50% COP Enterobacteriaceae
P fluorescens < 50% C o n mixed with O2 Brochothrix thermosphacta, lactic
biovars I, (I, 111, IV, V acid bacteria
(includes 7 clusters) > 50% CO? Lactic acid bacteria
I? lundensis, I? fragi 100% con Lactic acid bacteria
Shewanella putrefaciens Vacuum pack Lactic acid bacteria, Brochothrix
Alteromonas spp. thermosphacta
Alcaligenes spp., ~~

Achromobacter spp.
Flavobacterium spp. Under Aerobic Conditions Although the Gram-
Moraxella spp. negative aerobic psychrotrophic bacteria of meat
Psychrobacter spp.
include a number of well-defined species (see Table
I? immobilis,
P phenylpyruvica 2), it is now well established that under aerobic
Acinetobacter spp. storage three species of Pseudomonas - P. fragi, P.
A. lwoffi, A. johnsonii fluorescens and P. lundensis - are the most important.
Facultative anaerobes Catalase reaction Off odours are present when the population of
Photobacterium spp. negative pseudomonads is of the order of l o 7per square centi-
Vibrio spp. Lactobacillus spp. metre and slime when these organisms reach 10' per
Aeromonas spp. L. sake square centimetre. In practice off odours become
Plesiomonas spp. L. curvatus
Serratia spp. L. bavaricus evident when the pseudomonads have exhausted the
S. liquefaciens Carnobacterium spp. glucose and lactate present in meat and begin to
S. marcescens C. divergens metabolize the amino acids.
Citrobacter spp. C. piscicola Although rarely, if ever, contributing significantly
C. freundii, C. koseri Leuconostoc spp. to the spoilage flora on meat and meat products, the
Providencia spp. L. carnosum
I? alcalifaciens, F! stuarti/, L. gelidum
Enterobacteriaceae have been considered as indicators
P rettgeri L. amelibiosum of food safety. With ground beef, Pantoea agglo-
Hafnia spp. L. mesenteroides subsp merans, Escherichia coli and Serratia liquefaciens
Hafnia alvei mesenteroides were the major representatives of this family (see
Pantoea agglomerans Weissella spp. Table 2).
Enterobacter spp. W. hellenica
W paramesenteroides
Brochothrix thermosphacta has been detected in
E. cloacae,
E. aerogenes Lactococcus raffinolactis the aerobic spoilage flora of chilled meat but it is not
E. agglomeransl Clostridium estertheticum considered to be important in spoilage except possibly
Erwinia herbicola of lamb. This organism has been isolated from beef
complex carcasses during boning, dressing and chilling. More-
Klebsiella spp.
over, lairage slurry, cattle hair, rumen contents, soil
K. pneumoniae
Kluyvera spp. from the walls of slaughterhouses, the hands of
Proteus spp. workers, the air in the chill room, the neck and skin
I? vulgaris, I? mirabilis of the animal as well as the cut muscle surfaces have
been shown to be contaminated with this organism.
Brochothrix thermosphacta is one of the main - if not
the most important - cause of spoilage which can be
Pseudomonas spp. are considered to be the main
spoilage organisms. Gram-positive bacteria (lactic recognized as souring rather than putrefaction. This
type of spoilage is commonly associated with meat
acid bacteria and Brochothrix thermosphacta) are the
packed under modified atmospheres.
main spoilage organisms in chill meat stored in a
modified atmosphere. To date studies on the con- Under Vacuum or Modified-atmosphere Packaging
tribution of yeasts to the spoilage of meat, whole or Conditions The atmosphere may be modified by
minced, has attracted little attention even though they vacuum packaging or storage of meat in atmospheres
are common contaminants. Yeasts do not outgrow containing a mixture of gases (Nz, COz and 0 2 ) . Meat
bacteria on meat or meat products unless a bac- in a vacuum pack or modified atmosphere (protective
teriostatic agent is included, such as sulphite in British atmosphere) has an extensive shelf life when com-
fresh sausages, or the water activity is reduced. pared with meat stored aerobically. Shelf life is
1256 MEAT AND POULTRY/Spoilage of Meat
determined by the choice of atmosphere, storage
temperature and meat type. As the bacterial popu-
lation of meat (particularly the aerobes, e.g.
pseudomonads) is restricted by the relative high con-
centration of COZ and the oxygen limitation, the
spoilage of meat stored under vacuum or modified
atmosphere occurs later than that of meat stored aer-
obically. In meat samples stored under vacuum or
modified-atmosphere packaging lactic acid bacteria
are recognized as important members of the spoilage
association (Table 3). Many of the isolates could not
be identified with existing species of Lactobacillus
(see Table 2 ) . It is now recognized that many of
these isolates belong to a recently defined genus,
Carnobacterium.
It needs to be stressed that each of the atmospheres
in Table 3 selects a microbial flora dominated by
Gram-positive bacteria (principally Brochothrix ther-
mosphacta and lactic acid bacteria) rather than the
Gram-negative ones that develop on meat stored aer-
obically at chill temperatures. As the former grow
much more slowly than the latter, the shelf life of meat
is extended. It needs to be stressed also that there are
differences in the metabolic attributes of these two
groups of spoilage organisms. These are manifested
at different times and in different ways as judged by
odours coming from the meat.
Another cold-tolerant microorganism, Clostridium
estertheticum, causes distension or explosion of packs
of vacuum-packaged meat. The optimum growth tem-
perature of these organisms is 20°C. It is tempting to
speculate that the production of a spore protects this
organism from those factors in meat processing that
kills psychrophiles lacking this means of protection.
Spoilage in Frozen Meat Studies of microbial
growth at subfreezing temperatures clearly indicate
that microbial growth does not occur in meat eco-
systems with a temperature below -8°C. Thus, the
main determinant for the storage period of a properly
frozen meat ecosystem is the physical, chemical or
biochemical changes which are unrelated to micro-
biological proliferation. Therefore, frozen storage life
is limited by changes in other qualities such as appear-
ance or taste which are unrelated to microbiological
activity.
The key problem with frozen meat is the enu-
meration of the microbial populations of such eco-
systems. Microorganisms are injured by exposure to
reduced temperatures leading to sublethal damage,
the effects of which in microbial populations include
(1)increased lag times and (2)the inability to develop
quantitatively on selective media that do not exert
any inhibitory effect on undamaged populations of
the same taxon. These effects - especially the pro-
longed lag phase - are less noticeable when the meat
ecosystem is refrozen and analysed again after a short
period of storage. The appropriate resuscitation of
frozen meat flora prior to its enumeration is crucial;
resuscitation of the injured flora may take place in the
meat ecosystem during thawing, or in nonselective
culture media. Studies on the effect of different envir-
onmental stresses on the enumeration and the recov-
ery of microorganisms are focused on pathogenic
microorganisms; in which case the important feature
is to ascertain the presence or absence of the patho-
genic bacterium. The results obtained have a cardinal
role in the evaluation of microbiological hazards.
Roles of Microbes and Enzymes in
Spoilage
The role of the microbial flora is cardinal for the
spoilage of meat. The metabolic activity of the organ-
isms selected in a meat ecosystem leads to the mani-
festation of changes or spoilage of meat. This
manifestation is related to the level of (1)the popu-
lation and (2) the substrates in meat. Under both
aerobic and vacuum or modified-atmosphere pack-
aging the corresponding flora catabolize glucose for
growth. By the end of this phase changes and sub-
sequently overt spoilage are due to catabolism of
nitrogenous compounds and amino acids as well as
secondary metabolic reactions. The contribution of
indigenous meat enzymes to spoilage is negligible
compared with the action of the microbial flora. Post-
mortem glycolysis ceases after the death of the animal
when ultimate p H reaches a value of 5.4-5.5. During
storage, however, there is a proteolytic activity by
indigenous enzymes. The activity of these enzymes
has a role in the conditioning (ageing) of meat. Added
enzymes in meat may be used to artificially ameliorate
its organoleptic properties. Enzymes used for their
tenderizing effects are proteolytic and of bacterial,
fungal or plant origin.
Chemistry of Spoilage
The critical physico-chemical changes occurring
during spoilage take place in the aqueous phase of
meat. This phase contains glucose, lactic acid, certain
amino acids, nucleotides, urea and water-soluble pro-
teins which are utilized by most of the bacteria of
the meat microflora. The concentration of these low-
molecular-mass compounds is sufficient to support
massive microbial growth. Glucose is the prime nutri-
ent in a meat ecosystem and it is catabolized initially
during microbial growth. This substrate is attacked
by almost all groups of spoilage bacteria, under aerobic
and anaerobic conditions (Table 4). Until spoil-
age is evident organoleptically, the major detectable
 MEAT AND POULTRY/Spoilage of Meat 1257

Table 4 Substrates used for growth of major meat spoilage microorganisms

Microorganism
Pseudomonas spp.
AcinetobacterlMoraxella
Shewanella putrefaciens
Brochothrix fhermosphacfa
Enferobacter spp.
Lactobacillus spp.
Substrates used for growth
Aerobic Anaerobic
Glucose, giucose 6-phosphate, lactic acid, Glucose, lactic acid, pyruvate, gluconate,
pyruvate, gluconate, 6-phosphogluconate, amino acids
amino acids, creatine, creatinine, citrate,
aspartate, glutamate
Amino acids, lactic acid, glucose Glucose, amino acids
Glucose, lactic acid, pyruvate, gluconate, Formate
propionic acid, ethanol, acetate, amino acids
Glucose, amino acids, ribose, glycerol Glucose
Glucose, glucose 6-phosphate, amino acids, Glucose, glucose 6-phosphate, amino acids
lactic acid
Glucose Glucose, lactic acid, amino acids

effect of bacterial growth is a reduction of the glucose
concentration. This does not alter the organoleptic
properties of meat. When glucose or its oxidative
products are reduced to non-substrate levels, lactic
acid is catabolized. It needs to be stressed that when
this second major carbon and energy source is
exhausted the microbial association is at its climax
stage.
Under Aerobic Conditions The relative potential of
bacteria depends upon which species predominate,
and upon their ability to form malodorous com-
pounds such as H2S, volatile amines, esters and
acetoin. Pseudomonas spp. are important because of
their dominance in the aerobic climax associations at
chill temperatures. The key chemical changes asso-
ciated with the metabolic attributes of pseudomonads
have been studied extensively in broth and in model
systems such as meat juice (Table 5). Among these
major attributes are (1)the sequential catabolism of
D-glucose and L- and D-lactic acid with D-glucose
used in preference to lactate, and (2)the oxidization of
glucose and glucose 6-phosphate via the extracellular
pathway causing a transient accumulation of D-glu-
conate and an increase in the concentration of 6-
phosphogluconate. The increase in the concentration
of D-gluconate led investigators to propose a method
for controlling the microbial activity in meat by the
addition of glucose to meat and its transformation to
gluconate. The rationale for this suggestion is the fall
in pH due to the accumulation of oxidative products.
The transient pool of gluconate and its inability to be
catabolized by all the taxa of the association may
offer a selective determinant on the meat ecosystem.
Another important feature is the catabolism of
creatine and creatinine by Pseudomonas fragi under
aerobic conditions. The phenomenal release of
ammonia and the increase in pH are inextricably
linked with the catabolism of these substrates.
Ammonia, which is the major cause of the increase of
pH, can be produced by many microbes, including
pseudomonads during their amino acid metabolism.
A list of other volatile compounds found in spoiled
meat is given in Table 6. Pseudomonad species
growing on the surface of meat will preferentially
consume glucose until the rate of diffusion of glucose
from underlying tissues becomes inadequate to meet

Table 5 Metabolic activity of pseudomonads in meat juice at 0-4°C

Substrate Pseudomonas fragi Pseudomonas lundensis Pseudomonas fluorescens

D-Glucose C C C
D-Glucose 6-phosphate C C -
D-Gluconate f f f
6-Phospho-~-gluconate f f
L-lactic acid C C C
D-lactic acid C C C
Pyruvate flc flc flc
Acetic acid C nd nd
Amino acids C C C
Creatine C
Creatinine C -
Proteolysis + nd +
Ammonia f f f

Key: The substrate was catabolized (c) or formed (f) during growth; - neither catabolized nor formed; +, positive; nd, no available
data.
1258 MEAT AND POULTRY/Spoilage of Meat

Table 6 End product formation of Gram-negative bacteria (e.g. thermosphacta) usually occurs in meat during its
Pseudomonas spp., Shewanella putrefaciens, Moraxella etc) storage under modified atmosphere packaging.
when grown in broth, sterile meat model system and in naturally
spoiled meat
Among these, the physiological attributes of the lactic
acid bacteria and B. thermosphacta have been studied
Sulphur compounds: extensively. Environmental determinants such as the
sulphides, dimethylsulphide, dimethyldisulphite,
methylmercaptan, methanethiol, hydrogen sulphide,
oxygen tension, glucose concentration and the initial
dimethyltrisulphide pH have a major influence on the physiology of these
Esters: organisms, and hence on the end products formed.
methyl esters (acetate), ethyl esters (acetate) Brochothrix thermosphacta has a much greater spoil-
Ketones: age potential than the lactobacilli and can be import-
acetone, 2-butanone, acetoin/diacetyl
Aromatic hydrocarbons:
ant in both aerobic and anaerobic spoilage of meat.
diethylbenzene, trimethylbenzene, toluene This organism utilizes glucose and glutamate but no
Aliphatic hydrocarbons: other amino acid during aerobic incubation. It pro-
hexane, 2,4-dimethylhexane, methyl heptone duces a mixture of end products including acetoin,
Aldehydes: acetic, iso-butyric and iso-valeric acids, 2,3-but-
2-methyl butanal
Alcohols:
anediol, diacetyl, 3-methylbutanal, 2-methylpropanol
methanol, ethanol, 2-methylpropanol, 2-methylbutanol, and 3-methylbutanol during its aerobic metabolism
3-methylbutanol in media containing glucose, ribose or glycerol as the
Biogenic amines - other compounds: main carbon and energy source (Table 7). The precise
cadaverine, ammonia, putrescine, methylamine, proportion of these end products is affected by the
trimethylamine
concentration of glucose, pH and temperature.
Lactobacillus spp. constitute only a small pro-
their demand; when high numbers ( l o 8 per cm2) are portion of the initial spoilage bacterial population of
reached and glucose becomes depleted at the meat meat. When oxygen is in low concentration, as in
surface, pseudomonads start proteolysis and use vacuum-packed meats, the developing microflora is
nitrogenous compounds and free amino acids as their usually dominated by Lactobacillus spp. These fer-
growth substrate with production of malodorous sul- mentative organisms probably grow faster than
phides and esters (Table 6). would-be competitors because they are unaffected by
The Enterobacteriaceae can be important in spoil- p H and antimicrobial products such as lactic acid,
age if the meat ecosystem favours their growth. This Hz02 and antibiotics. These organisms utilize glucose
group utilize mainly glucose and glucose 6-phosphate for growth and produce lactic acid. When carbo-
as the main carbon sources; the exhaustion of these hydrates are exhausted, amino acids are utilized with
substances allows amino acid degradation. Moreover,
some members of this family produce ammonia, vola- Table 7 End products of homofermentative lactic acid bacteria
tile sulphides including H2S and malodorous amines (HO), heterofermentative lactic acid bacteria (HT) and
from amino acid metabolism (Table 6). Brochothrix thermosphacta (BT) when grown in broth, sterile
Acinetobacter and Moraxella constitute a major meat model system and in naturally spoiled meat
part of the aerobic spoilage population. These organ- Aerobic In different gaseous
isms are of low spoilage potential. They utilize amino atmospheres
acids as their growth substrate but do not form mal- Acetoin - HO, HT, BT Acetoin - HO
odorous by-products from amino acid degradation; Acetic acid - HO, HT, BT Acetic acid - HO, HT, BT
they rather enhance the spoilage activities of pseudo- L-Lactic acid - HO, HT, BT L-Lactic acid - HO, HT, BT
monads and Shewanella putrefaciens by restricting D-Lactic acid - HO, HT D-Lactic acid - HO, HT
the availability of 0 2 to these organisms. When O2 Formic acid - HO, HT, BT Formic acid - HO, HT, BT
Ethanol - HO, HT, BT Ethanol - HO, HT, BT
limits growth, pseudomonads attack amino acids, COP - HO, HT, BT
even when glucose is present, with the subsequent HzOZ - HO, HT
production of malodorous substances. Under anaer- iso-Butyric acid - BT
obic conditions S. putrefaciens will generate H2S, iso-Valeric acid - BT
resulting in the greening of meat due to sulph- 2-Methylbutyric acid - BT
3-Methylbutanol - BT
myoglobin formation. 2-Methylbutanol - BT
2,3-Butanediol- BT
Under Vacuum or Modified-atmosphere Packaging Diacetyl - HO, HT, BT
Conditions A shift from a diverse initial flora to one 2-Methylpropanol - BT
dominated by Gram-positive facultative anaerobic 2-Methylpropanal -BT
Free fatty acids - BT
microflora (lactic acid bacteria and Brochothrix
 Next Page

MEAT AND POULTRY/Spoilage of Meat 1259

the consequent production of volatile fatty acids given metabolites with the microbial spoilage of meat.
which impart a ‘dairy’ or ‘cheesy’ odour to the The idea for these methods is that as the bacteria
vacuum-packaged meat. Because with meat stored grow on meat they utilize nutrients and create by-
under modified atmosphere increased concentration products. The quantitative determination of these
of COz inhibits growth of aerobic flora - and glucose metabolites could provide information about the
assimilation by pseudomonads - the cheesy odours degree of spoilage. The identification of the ideal
are found mainly in samples stored in gas mixtures metabolite, fulfilling the criteria noted above, has
containing COZ,where they are probably produced by proved a difficult task for the following reasons:
Brochothrix thermosphacta and lactic acid bacteria.
1. Most metabolites are specific to certain organisms
These also form diacetyl, acetoin and alcohols from
(e.g. gluconate to pseudomonads).
glucose under aerobic conditions or low partial pres-
2. Although the metabolites are the product of the
sure of oxygen (Poz).In addition, alcohols (ethanol
metabolism of a specific substrate, the absence of
and propanol) are present at only trace levels at the
the given substrate or its presence in low quantities
beginning of storage but their concentrations increase
does not preclude spoilage.
significantly before the onset of spoilage, making them
3. The rate of microbial metabolite production and
promising compounds as indicators of spoilage (Table
the metabolic pathways of spoilage bacteria are
7). affected by the environmental conditions (e.g. pH,
Evaluation of Spoilage oxygen tension, temperature).
4. The accurate detection and measurement of meta-
Enumeration of bacterial population by culture tech-
bolites require sophisticated procedures.
niques (agar media) and rapid methods (malthusian)
5 . Many of them provide retrospective information.
in food is used as indicator of its hygiene. As the
spoilage of meat is caused by specific spoilage bac-
teria, different selective media should be applied.
Role of Cooking in Susceptibility to
Because the correlation between the population of
specific spoilage bacteria and the sensorial mani- Spoilage
festation of spoilage is imprecise, it is difficult to use Cooking raw meat results in the death of its microbial
bacterial levels as an estimate of spoilage. association. Subsequent recontamination of the
The time-consuming microbiological analyses can cooked meat and temperature abuse lead to the devel-
be replaced by assessment of the chemical, enzymatic opment of a new spoilage association. As the antag-
and physico-chemical changes associated with micro- onists belonging to the initial microflora of raw meat
bial growth on meat. For this reason a number of are absent, pathogens that contaminate cooked meat
chemical and physical methods have been proposed have a rich substratum for their proliferation. The
for the estimation of bacterial spoilage in meats. microbiological stability of cooked meat products
However, there is as yet no single test available to depends on extrinsic factors, mainly the packaging
assess meat quality. Spoilage is a subjective evaluation method and storage temperature, as well as on intrin-
and therefore a sound definition is required to develop sic ones, e.g. product composition.
a suitable method. The lack of a general agreement
on the signs of incipient spoilage in meat and the
Special Problems Associated with Meat
changes in the technology of meat preservation (e.g.
vacuum or modified-atmosphere packaging) make it Production of biogenic amines by microbial flora is a
difficult to identify spoilage indicators. problem in stored meat. Amines have been detected in
The spoilage indicators or microbial metabolite fresh meat stored under aerobic or vacuum/modified-
should meet the following criteria: atmosphere packaging conditions. Among them,
1. The indicator should be absent or initially at low putrescine and cadaverine show a constant increase
levels in meat. during storage. Concentrations of spermine, sperm-
2. It should increase proportionally with the storage idine and tryptamine remain steady, and a small
period. increase in tyramine concentration is usually observed
3. It should be produced by the dominant flora and after long storage periods. As lactic acid bacteria and
have a good correlation with sensory evaluation. Brochothrix thermosphacta do not produce amines,
the formation of these compounds has been attributed
As noted earlier, physico-chemical analyses of meat to Enterobacteriaceae. However, tyramine could also
can be used instead of microbiological ones for the be formed by some strains of the genus Lactobacillus.
evaluation of spoilage. For this reason, numerous The limiting factors of meat stored under modified-
attempts have been made since the 1970s to associate atmosphere packaging is another issue. Concerns have
 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1549
NASBA see Listeria: Listeria monocytogenes - Detection using NASBA (an Isothermal Nucleic Acid
Amplification System).
1 Natamycin see Preservatives:Permitted Preservatives - Natamycin.
NATIONAL LEGISLATION, GUIDELINES &
STANDARDS GOVERNING MICROBIOLOGY
Contents
Canada
European Union
Japan
Canada
Bruce E Brown, B. E. Brown Associates, Ottawa,
Ontario, Canada
Copyright 0 1999 Academic Press
Introduction
The regulation of the microbiological safety and
quality of foods in Canada operates in a complex
jurisdictional context involving federal, provincial
and municipal authorities and division of respon-
sibilities between the federal departments of agri-
culture, fisheries and health. In addition, each of the
10 provinces have departments of agriculture and
health that also regulate the microbiological safety
and quality of food. In recent years under an initiative
entitled ‘The Canadian Food Inspection System’
efforts are being made to harmonize legislation, regu-
lations and guidelines and integrate inspectional ser-
vices at the federal, provincial and municipal levels.
The Federal Acts and regulations involving the
hygienic practices for production and manufacturing
premises as well as the microbiological safety and
quality of food products are administered by the
departments of Health Canada, Agriculture and Agri-
Food Canada, and Fisheries and Oceans Canada. The
recent creation of the Canadian Food Inspection
Agency which united the inspection resources of the
three departments may well influence regulations,
guidelines, etc. However, the mandate of each of the
three departments to develop legislation and regu-
lations and to designate enforcement policy remains
unchanged.
Health Canada
Sections 4 , 5 , 6 and 7 of Part I of the Food and Drugs
Act are the primary national legislation governing the
overall safety and quality of food. The microbiological
safety and quality regulations fall under Sections 4, 6
and 7.
Section 4 states that no person shall sell any article
of food that: (a) has in or on it any poisonous or
harmful substance; (b) is unfit for human con-
sumption; (c) consists in whole or in part of any filthy,
putrid, disgusting, rotten, decomposed or diseased
animal or vegetable substance; (d) is adulterated; or
(e)was manufactured, prepared, preserved, packaged
or stored under unsanitary conditions. Foods con-
taining pathogens in numbers that would constitute
a direct hazard to health or their toxins would be
considered not to be in compliance with Subsections
4(a) and 4(b) and possible 4(e). Spoilage (i.e. micro-
biological quality) can be considered to contravene
Subsection 4(c). Subsection 4(e) and Section 7 deal
with the hygienic conditions in which foods are pro-
cessed, and opens the door for the sanitary inspection
1550 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada
of premises where food is manufactured, prepared,
preserved, packaged or stored. Subsection 6.1 permits
the establishment of regulatory microbiological stand-
ards as being necessary to prevent injury to the health
of the consumer or purchaser of the food. The term
‘sell’ is defined to include offer for sale, expose for sale,
have in possession for sale and distribute, whether
or not the distribution is made for consideration.
‘Unsanitary conditions’ means such conditions or cir-
cumstances as might contaminate with dirt or filth,
or render injurious to health, a food, drug or cosmetic.
The Act also permits the establishment of regu-
lations for carrying out the purposes and provisions
of the Act, and examples of subject matter for regu-
lations include:
setting the sale or conditions of any food, drug,
cosmetic or device
prescribing standards of composition, strength,
potency, purity, quality or other property of any
article of food, drug, cosmetic or device
respecting the importation of foods, drugs, cos-
metics and devices in order to ensure compliance
with the Act and regulations
respecting the method of manufacture, preparation,
preserving, packing, storing and testing of any
food, drug, cosmetic or device in the interest of, or
for the prevention of injury to, the health of the
purchaser or consumer
the keeping of books and records by persons who
sell food, drugs, cosmetics or devices that are con-
sidered necessary for the proper enforcement and
administration of the Act and regulations.
Microbiological Standards
The Food and Drug Regulations currently contain a
number of regulatory microbiological standards. The
standards have been developed on the basis of data
gathered over the years as an aid to the administration
of Sections 4 to 7 (inclusive) of the Act and relate to
the microbiological safety and general cleanliness of
food.
Most of the standards are specific to a micro-
organism or a group of microorganisms, while in
others the organism is not specified but implied. There
are two types of standards specific to microorganisms.
One requires prohibition, that is zero tolerance, while
the other permits some acceptable level. An example
of a prohibition standard can be found in regulation
B.08.014A which states that no person shall sell milk
powder, whole milk powder, dry whole milk, pow-
dered whole milk, skim milk powder or dry skim milk
unless it is free from bacteria of the genus Salmonella,
as determined by official method MFO-121, Micro-
biological Examination of Milk Powder, November
30,198 1. The official method is part of the regulation
and specifies the method that must be used to establish
compliance with the regulation, the sampling plan and
compliance criteria (Table 1). Regulation B.08.016
which states that flavoured milks may contain not
more than 50000 total aerobic bacteria per cubic
centimetre, is determined by official method MFO-7,
Microbiological Examination of Milk, November 30,
1981 is an example of a standard in which an accept-
able level is permitted.
The standards are classified with respect to three
degrees of risk, referred to as Health 1, 2 and Sani-
tation. The degree of risk is reflected in the sampling
plan and compliance criteria part of the official
method. Two-class plans are used where there is a
Health 1 risk (Table 1) and three-class plans for
Health 2 and Sanitation risks (Tables 2 and 3). The
sample size ( n )designates the number of sample units
to be taken and examined from a lot. The acceptance
number (c) is the maximum allowable number of
sample units that may exceed the level or con-
centration designated as acceptable, the m value. The
lot is unacceptable and can be considered to be in
violation of the respective regulatory standard when
this number is exceeded. The acceptable con-
centration of microorganisms ( m ) expressed
, per gram
or millilitre in a three-class plan separates sample
units of acceptable microbiological quality from those
classed as marginally acceptable, and in a two-class
plan separates acceptable sample units from unaccept-
able. In a three-class plan unacceptable concentrations
of microorganisms are represented by M which sep-
arates sample units of marginally acceptable quality
from those of defective quality. The lot is unacceptable
and in violation of the regulatory standard if one or
more sample units exceed the M value.
Health 1 indicates that there is a direct risk to
human health and appropriate action, for example a
product recall, should be taken to limit exposure in
the population. Follow-up action should ensure that
the cause has been determined and appropriate cor-
rective action has been taken. For Health 2 the hazard
Table 1 Microbiological standards for Salmonella considered
as a Health 1 risk
Product Regulation Method Criteria”
n c m
Chocolate 8.04.010 MFO-11 10 0 0
Cocoa 8.04.01 1 MFO-11 10 0 0
Milk powder B.08.014 MFO-12 20 0 0
Egg products 8.22.033 MFO-6 6 0 0
Frog legsb 6.22.033 MFO-10 10 0 0
a n , sample size; c, acceptance number; m, acceptable
concentration of microorganisms per gram.
This regulation and hence standard is due to be repealed.
 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1551
Table 2 Health 2 risk microbiological standards
Product Microorganism
Cheese Escherichia coli
made from pasteurized milk Staphylococcus aureus
Cheese Escherichia coli
made from unpasteurized milk Staphylococcus aureus
an, sample size; c, acceptance number; m, acceptable concentration of microorganisms per gram; M , unacceptable concentration
of microorganisms per gram.
identified represents a risk to human health only if
present in sufficient numbers and appropriate action,
such as product recall, should be taken to limit expos-
ure in the population to the product if the unaccept-
able level (M value) is exceeded. If the acceptance
number ( c )for levels between the acceptable level ( m )
and the unacceptable level ( M )is exceeded, corrective
action should be taken to bring about compliance. In
Sanitation the hazard identified is an indication of
a breakdown in hygienic practice. A review of the
manufacturer's good hygienic practices (GHP) and/or
the hazard analysis critical control point (HACCP)
system is appropriate where M or c/m values are
exceeded.
The sampling plans for Salmonella in Table 1, con-
sidered a Health 1 risk, are two-class plans. The lot
from which the sample units were drawn to be classed
is judged not to be in compliance with the specific
regulation if Salmonella is found in any of the sample
unit. Lots in violation of Health 1 risk standards are
generally ordered to be destroyed and the legal owner
could be prosecuted. Salvage operations may be per-
mitted if it can be established that the treatment would
decrease the hazard to acceptable levels. In such cases,
the verification sampling and acceptance criteria may
well exceed that of the particular regulation. In an
investigation of a suspected outbreak of a food-borne
illness, for example salmonellosis, sample numbers
may well exceed the values designated in the
regulation.
There are regulatory standards in which the micro-
organisms of concern are implied rather than stated.
Clostridium botulinum is the microorganism of
concern in regulation B.27.002 which requires a low-
acid food packaged in a hermetically sealed container
to be commercially sterile, unless it is kept refrigerated
at a temperature not exceeding 4°C or frozen and is so
labelled. Commercial sterility is defined in regulation
B.27.001 as 'the condition obtained in a food that has
been processed by the application of heat, alone or in
combination with other treatments, to render the food
free from viable forms of microorganisms, including
spores, capable of growing in the food at temperatures
at which the food is designed normally to be held
Regulation Method Criteria"
n C m M
6.08.048 MFO-14 5 2 io2 2x103
5 2 io2 io4
6.08.048 MFO-14 5 2 5 x 1 0 ~2 x 1 0 3
5 2 103 io4
during distribution or storage'. Under the regulation,
a hermetically sealed container means a container
designed and intended to be secure against the entry
of microorganisms, including spores. A low-acid food
is a food, other than an alcoholic beverage, where any
component of the food has a p H greater than 4.6 and
a water activity greater than 0.85.
Brucella and Mycobacterium bovis, and more
recently Salmonella and Listeria are the organisms of
concern in B.08.002.2. This regulation requires the
normal lacteal secretion obtained from the mammary
gland of the cow, genus Bos, or of any other animal,
or a dairy product made with any such secretion, to
be pasteurized by being held at a temperature and for
a period that ensure the reduction of the alkaline
phosphatase activity so as to meet the tolerances spe-
cified in official method MFO-3, Determination of
Phosphatase Activity in Dairy Products, dated
November 30, 1981.
Regulation B.21.025 deals with marine and fresh-
water animals, or marine and freshwater animal prod-
ucts, that are packed in a container that has been
sealed to exclude air and that are smoked or to which
liquid smoke flavour or liquid smoke flavour con-
centrate has been added. Under the regulation these
products must be heat processed after sealing at a
temperature and for a time sufficient to destroy all
spores of the species Clostridium botulinum (i.e. com-
mercially sterile). The only exception to the heat pro-
cessing requirements are where the contents of the
container comprise not less than 9% salt, the contents
of the container are customarily cooked before eating,
or the contents of the container are frozen and the
product so labelled. The specific organism of concern
is C. botulinum type E, which can grow - albeit
slowly - at normal refrigeration temperatures.
Microbiological Guidelines
Guidelines take three forms - microbiological guide-
lines, codes of hygienic practice and field compliance
guides. These have been developed after consultation
with the Canadian food industry, are published and
copies are available upon request. Microbiological
guidelines were developed from surveys conducted
1552 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada

Table 3 Sanitary risk microbiological standards

Product Standard Regulation Method Criteria"
n C mC M"

Flavoured milks ACCb B.08.016 MFO-7 5 2 5x104 1o6

B.08.018
8.08.026
Milk for manufacture ACC B.08.024 MFO-7 5 0 2 x loE -
Cottage cheese Coliforms 8.08.054 MFO-4 5 1 10 103
Ice cream ACC 8.08.062 MFO-2 5 2 105 1OE
Coliforms 5 2 10 1o3
Ice milk ACC B.08.072 MFO-2 5 2 105 1O6
Coliforms 5 2 10 103
Mineral or spring water Coliforms B.12.001 MFO-9 10 1 0 per 1000 ml 10 per 100 ml
Prepackaged ice Coliforms B.12.005 MFO-15 10 1 < 1.8 per 100 ml 10 per 100 ml
Water in sealed containers ACC 8.12.004 MFO-15 5 2 10' 1o4
Coliforms 10 1 < 1.8 per 100 ml 10 per 100 ml
an, sample size; c, acceptance number; m, acceptable concentration of microorganisms; M, unacceptable concentration of
microorganisms.
ACC, Aerobic Colony Count.
Per millimetre unless otherwise stated.

on specific products or groups of products across Volume 1 of the compendium is devoted to the
Canada. While guidelines are not regulatory stand- official microbiological methods (MFO). These are
ards, they are used in judging compliance with Sec- cited in the respective Food and Drug Regulations,
tions 4 and 7 of the Act. Even though a given guidelineare an integral part of the standard and must be used
may embody the same limiting criteria that would be by the regulatory agencies to determine compliance.
employed in a standard, it is generally based on fewer Health Protection Branch methods (MFHPB),used in
data than are used in developing a standard. However, the guidelines, are found in volume 2 of the com-
guidelines serve as useful indicators of levels that pendium. Both the official and HPB methods have
should be achievable using GHP. Guidelines can be been fully validated by interlaboratory studies.
readily modified, if necessary, as additional data Laboratory procedures (MFLP) are given in volume
become available. 3 . These have been validated in at least one HPB
The microbiological guidelines that are currently inlaboratory, apart from the laboratory that originated
force are given in Table 4. The same three levels of the method. These methods include those undergoing
concern or risk (Health 1, 2 and Sanitation) are development, newly developed rapid methods or
applied in the guidelines. methods for newly emerging pathogens. As for a regu-
In addition to Salmonella, the microorganisms con- latory standard, the sampling plans and compliance
sidered to be a Health 1 risk are Campylobacter coli, criteria are an integral part of each method and form
C. jejuni, Yersinia enterocolitica, Pseudomonas aeyu- part of the respective guideline.
ginosa, and Aeromonas hydrophila. The micro- The Code of Practice for the General Principles of
organisms considered to be a Health 2 risk are Food Hygiene developed by the Codex Alimentarius
Escherichia coli, Staphylococcus aureus, Bacillus Commission has been modified to reflect current Can-
cereus, and Clostvidium pevfringens. Aerobic colony adian good hygienic practices. The Canadian version
count, coliforms and yeast and mould counts form of this code of practice is intended to provide guidance
the basis for a sanitation or hygiene hazard. to the Canadian food industry on hygienic food hand-
All the methods cited in the standards and ling practices in order to comply with Sections 4 and
guidelines are contained in the Compendium of 7 of the Food and Drugs Act. The Code provides to
Analytical Methods, published by Polyscience Pub- both the regulatory inspector and the food industry a
lications for Health Canada. The compendium pro- template for the sanitary or hygienic inspection of
vides a ready reference of the food microbiological food processing and manufacturing premises.
methods used by the Health Protection Branch (HPB) The recommended Canadian Code of Hygienic
of Health Canada to determine compliance of the Practice for Low-acid and Acidified Low-acid Foods
food industry with standards and guidelines, to assess in Hermetically Sealed Containers (Canned Foods)
the quality of foods with respect to their micro- was adapted directly from the Codex Alirnentarius
biological content, and to support investigations of Recommended International Code of Hygienic Prac-
food-borne diseases and consumer complaints. tice for Low-acid and Acidified Low-acid Canned
 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1553

Foods. The Canadian Code is a guideline for com- foods is included as an appendix to the Field Com-
mercial processors who thermally process these prod- pliance Guide.
ucts for compliance with the regulations B.27.001 to The definition for recall under the Food and Drugs
B.27.006, inclusively. While Clostridium botulinum Act with respect to a product, other than a medical
is the primary microorganism of concern, the code device, means a firm’s removal from further sale or
also addresses microbiological spoilage. use, or correction, of a marketed product that violates
A good example of a field compliance guide is legislation administered by the Health Protection
that for ready-to-eat (RTE) foods contaminated with Branch. Three types or classes of recalls are des-
Listeria monocytogenes. An RTE food is defined as ignated. Class I is a situation in which there is a
one requiring no further processing before con- reasonable probability that the use of, or exposure to,
sumption. Of primary concern are RTE foods that a non-compliant product will cause serious adverse
have been subjected to some form of processing not health consequences or death. Class I1 is a situation
only to render them ready-to-eat but also to extend in which the use of, or exposure to, such a product
their shelf life. Such RTE foods can support the may cause temporary adverse health consequences
growth of L. monocytogenes even when maintained or where the probability of serious adverse health
under conditions of commercial refrigeration. The consequences is remote. Class I11 is a situation in
field compliance guide combines inspection, envir- which the use of, or exposure to, a product is not
onmental sampling and end product testing. The likely to cause any adverse health consequences.
results of the inspection should show the inspector
whether or not GHPs in place are adequate to control Agriculture and Agri-Food Canada
the potential for contamination of the product with
L. monocytogenes. However, if the inspector does not This department administers a number of acts and
consider them to be adequate, environmental sam- associated regulations. Only the Canadian Agri-
pling should be conducted. If the environmental sam- cultural Products Act, Health of Animals Act and the
pling indicates that there is a probability of finished Meat Inspection Act and their associated regulations
product becoming contaminated with pathogenic have microbiological standards or specifications dir-
microorganisms, then the finished product should be ectly applicable to foods. It should be noted that the
sampled in accordance with Table 5 and analysed. administration of many of the Acts by this department
The results of that analysis will determine the choice is limited to foods that are imported, exported or
of enforcement action as set out in Table 6. If envir- traded interprovincially. The Food and Drugs Act and
onmental sampling indicates little or no probability regulations have no such limitation.
for product contamination no further action is taken Canadian Agricultural Products Act
by the inspector other than to encourage strict imple-
mentation of GHPs, i.e. compliance with the Code of The relevant regulations under the Canadian Agri-
Practice for the General Principles of Food Hygiene. cultural Products Act are:
For the purposes of this guide, an RTE food is Livestock and Poultry Carcass Grading Regulations
considered capable of supporting growth of L. mono- 0 Egg Regulations (upgraded 18/03/98)
cytogenes if, in a naturally contaminated lot of the 0 Processed Egg Regulations
RTE food under consideration, L. monocytogenes can 0 Dairy Regulations (upgraded 15/04/98)
be detected by direct plating after the food has been 0 Fresh Fruit and Vegetable Regulations (updated
stored at 4°C or less until the end of its stated shelf 0 1/04/98)
life; OY if, in an inoculated batch representative of the 0 Honey Regulations (updated 01/04/98)
RTE food, L. monocytogenes increases in number by 0 Maple Products Regulations (updated 01/04/98)
at least 1 log after it has been stored at 4°C or less 0 Processed Products Regulations (updated
until the end of its stated shelf life, as determined 15/04/98)
by the direct plating method. The guide encourages 0 Licensing and Arbitration Regulations (updated
manufacturers to consider performing challenge tests 04/03/9 8).
not only under normal conditions of storage and
distribution, but also under conditions of mild tem- The Act stipulates that no person shall market a food
perature abuse (e.g. 7-10°C). A challenge test involves product in import, export or interprovincial trade as
the incubation of samples of the RTE food inoculated food unless the food product, including every sub-
with a known concentration of a cocktail of at least stance used as a component or ingredient thereof,
five strains of L. monocytogenes for periods of time ( a ) is not adulterated
to reflect the desired product shelf life. A guide for the (b) is not contaminated
challenge testing of L. monocytogenes on refrigerated (c) is sound, wholesome and edible
1554 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada

Table 4 Microbiological guidelines

Food Method Guideline Risk Criteria"
n C m M
Cocoa MFHPB-18 ACCb includes aerobic spore- Sanitation 5 2 105 106
form ers
MFHPB-22 Yeasts and mouldsb,d Sanitation 5 2 2x10~ io4
MFHPB-19 Coliformsb Sanitation 5 2 < 1.8 10
Chocolate MFHPB-18 ACC' includes aerobic spore- Sanitation 5 2 3x104 IO6
formers
MFHPB-19 Coliforms' Sanitation 5 2 < 1.8 10'
Instant infant cereal and MFHPB-18 ACC' Sanitation 5 2 103 104
powdered infant formula
MFHPB-19 Escherichia coli" Health 2 10 1 < 1.8 10
MFHPB-20 Salmonella Health 1 20 0 0 0
M FHPB-2 1 Staphylococcus aureus' Health 2 10 1 10 lo2
MFHPB-42 Bacillus cereusc Health 2 10 1 io2 io4
MFHPB-23 Clostridium perfringens" Health 2 10 1 i o 2 103
Fresh and dry pasta MFHPB-18 ACC Sanitation 5 2 5x104 l o 6
MFHPB-22 Yeasts and moulds" Sanitation 5 2 2x103 104
MFHPB-19 Escherichia coli" Health 2 5 2 < 1.8 103
M FHPB-21 Staphylococcus aureus Health 2 5 2 sXio 104
MFHPB-20 Salmonella Health 1 5 0 0 -
Bakery products' MFHPB-18 ACC Sanitation 5 2 5x104 106
MFHPB-19 Coliforms Sanitation 5 2 50 104
MFHPB-19 E. coli Health 2 5 1 < 1.8 103
MFHPB-22 Yeasts and moulds Sanitation 5 2 5xio2 104
M FHPB-21 S. aureus Health 2 5 2 ioz 104
MFHPB-20 Salmonella Health 1 5 0 0 -
Heat-treated fermented MFHPB-19 E. coli' Health 2 5 1 io 103
sausage
MFHPB-21 S. aureuse Health 2 5 1 50 io4
Raw fermented sausage MFHPB-19 E. coli" Health 2 5 1 10' 103
MFHPB-21 S. aureus Health 2 5 1 2.5~10' lo4
Heat-treated and raw MFHPB-20 Salmonella Health 1 5 0 0 -
fermented sausage
MFLP-46 Campylobacter coli or C. jejunig Health 1 5 0 0 -
MFLP-48 Yersinia enterocoliticag Health 1 5 0 0 -
Non-fermented meat MFHPB-19 Escherichia coli Health 2 5 2 loo 103
products (ready-to-eat)h
MFHPB-21 Staphylococcus aureus Health 2 5 2 loo io4
MFHPB-20 Salmonella Health 1 5 0 0
Deboned poultry products MFHPB-18 ACC Sanitation 5 3 104 106
(precooked)
MFHPB-19 E. coli' Health 2 5 2 io 103
MFHPB-21 S. aureus Health 2 5 1 100 io4
MFHPB-20 Salmonella Health 1 5 0 0 -
MFLP-46 Campylobacterjejuni or C. colig Health 1 5 0 0 -
MFLP-48 Yersinia enteroco/iticag Health 1 5 0 0 -
Dry mixes (gravy, sauce, MFHPB-18 ACC Sanitation 5 3 104 106
soup) heat and serve
MFHPB-19 Coliforms' Sanitation 5 3 10
MFHPB-22 Yeasts and moulds Sanitation 5 3 500 io4
MFHPB-19 E. coli' Health 2 5 2 io 103
MFHPB-21 S. aureus Health 2 5 2 100 io4
MFHPB-23 Clostridium perfringens Health 2 5 2 100 103
MFHPB-20 Salmonella Health 2 5 0 0 -
Soya bean products MFHPB-18 Psychrotrophic bacteria Sanitation 5 2 io5 io7
(ready-to-eat)
MFHPB-19 Escherichia colik Health 2 5 2 100 103
MFHPB-21 Staphylococcus aureus Health 2 5 2 100 io4
MFHPB-20 Salmonella Health 1 5 0 0 -
MFLP-48 Yersinia enterocolitica' Health 1 5 0 0 -
 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1555

Table 4 Microbiological guidelines (Continued)

~

Food Method Guideline Risk Criteria"

n C m M
Spices (ready-to-eat only) MFHPB-23 Clostridium petfringens Health 2 5 2 104 106
MFHPB-42 Bacillus cereus Health 2 5 2 io4
106
MFHPB-19 E. coli Health 2 5 2 100 103
MFHPB-21 S. aureusm Health 2 5 2 100 i o 4
MFHPB-20 Salmonella Health 1 5 0 0 -
MFHPB-22 Yeasts and moulds" Sanitation 5 2 100 104
Bottled water and ice" MFLP-61B Pseudomonas aeruginosa Health 1 5 0 OperlOO -
ml
MFLP-58B A. hydrophila Health 1 5 0 0per100 -
ml
Alfalfa and bean sprouts" MFHPB-19 ColiformsP Sanitation 5 2 io3 io5
MFHPB-20 Salmonella Health 1 5 0 0 -
a For definitions of n, c, m and M see previous tables.
From HPB Data-Gathering Survey Results.
From Microorganisms in Foods 2. Sampling for Microbiological Analysis: Principles and Specific Applications. International
Commission on Microbiological Specifications for Foods (ICMSF).
M adjusted for uniformity.
e M value adjusted for uniformity. For E. coli M = 103,for S. aureus, M = 104,and for yeasts and moulds M = 104.
Products that are microbiologically sensitive, i.e. containing eggs or dairy products. This food category consisted previously of
only cream pies but has been extended to other bakery products. Cream pies probably represent the worst case of this product
type.
Designates an optional analysis. It is not expected that these determinations will be done routinely.
Guidelines for this food category are new. Only organisms indicating a health concern are provided. The limiting values are
consistent with those for other products.
' M value adjusted for uniformity. For E. coli M = 103, for S. aureus M = 104.
' Values of m and M modified according to Health Canada monitoring results.
M value adjusted for uniformity. For E. coli M = lo3,for S. aureus M = lo4.
' Designates an optional analysis. It is not expected that these determinations will be done routinely.
Values are proposed by the International Commission on Microbiological Specifications for Foods (ICMSF).
Includes mineral or spring water, or water in sealed containers, or pre-packaged ice. The microbiological standards for ACC and
coliforms in these products (see Table 3) are under review.
Based on data-gathering survey results.
High coliform counts that do not confirm as faecal coliforms or E. coli should be investigated to determine if Klebsiella pneumoniae
is present. Take action appropriate for a Health 2 risk if K. pneumoniae levels exceed those for coliforms.

(d) is prepared in a sanitary manner
(e) where irradiated, is irradiated in accordance with
Division 26 of Part B of the Food and Drugs Act
and the Food and Drug Regulations
(f) meets all other requirements of the Food and
Drugs Act and the Food and Drug Regulations.
For the purposes of this Act, the term 'contaminated'
means containing a chemical, drug, food additive,
heavy metal, industrial pollutant, ingredient, med-
icament, microbe, pesticide, poison, toxin or any
other substance not permitted by, or in an amount
in excess of limits prescribed under, the Canadian
Environmental Protection Act, the Food and Drugs
Act or the Pest Control Products Act, or containing
any substance that renders the food inedible. Para-
graphs (b),(c), (d) and (f) address the microbiological
safety and quality of foods. These general provisions
are repeated in the regulations for each specific food
group. The regulations for the various food groups
may contain microbiological standards as well as dir-
ections with respect to sanitary preparation.
Processed Eggs Regulations The regulations contain
a general stipulation that no processed egg shall be
marked with a departmental inspection legend unless
the processed egg tests negative for salmonellae and
other pathogenic organisms of human health sig-
nificance as determined by a method approved by the
Minister. All establishments involved in the handling
and processing of eggs and egg product for import,
export or interprovincial trade are subject to inspec-
tion by Agriculture and Agri-Food Canada and the
product packaging must bear the inspection legend.
Unlike the microbiological standards under the Food
and Drug Regulations the specifics of the method and
sampling plan to be used are not given. In addition
to this general stipulation, there are a number of
microbiological standards for specific product types.
Frozen egg, frozen egg mix, liquid egg, liquid egg
1556 NATIONAL LEGISLATION, GUIDELINES 81STANDARDS GOVERNING MICROBIOLOGY/Canada

Table 5 Sampling plans for analysing ready-to-eat (RTE) foods for Listeria monocytogenes (LM)
Food product category Sampling Analysis Type of analysis

1. RTE foods causally linked to listeriosis Five sample units (100 g or ml 5 x 10 g or 2 x 25 g analytical ENRICHMENT
(this list includes soft cheese, liver each) taken at random from unitsa are either analysed ONLY
pate, coleslaw mix with shelf life > 10 each lot separately or composited
days, jellied pork tongue)
2. All other RTE foods supporting growth Five sample units (100 g or ml 5 x 5 analytical unitsa are either ENRICHMENT
of LM with refrigerated shelf life > 10 each) taken at random from analysed separately or ONLY
days (e.g. vacuum-packaged meats, each lot cornposited
modified atmosphere-packaged
sandwiches, cooked seafood,
packaged salads, refrigerated sauces)
3. RTE foods supporting growth of LM Five sample units (100 g or ml 5 x 10 g analytical unitsaare DIRECT
with refrigerated shelf-life 610 days each) taken at random from analysed separately PLATING
and all foods not supporting growthb each lot Where enrichment is necessary‘ ENRICHMENT
(e.g. cooked seafood, packaged 5 x 5 g analytical unitsa are
salads, ice cream, hard cheese, dry analysed separately or
salami, salted fish, breakfast and composited
other cereal products)
aThe designated analytical unit is taken from each sample unit.
Foods not supporting growth of LM include the following:
pH 5.0-5.5 and a, < 0.95
pH < 5.0 regardless of a,
a, 60.92 regardless of pH
frozen foods
The pH and a, determinations should be done on three out of five analytical units. The food is presumed to support the growth of
L. monocytogenes if any one of the analytical units falls into the range of pH and a, values which are thought to support the
growth of the organism.
For the last category, if GMP is inadequate and L. monocytogenes has been found in the environment of the finished product area,
or where examination of GMP status is not possible, the method to isolate L. monocytogenes from foods and environmental samples
(MFHPB-30) and the method for enumeration of L. monocytogenes (MFLP-74) may be used as appropriate.

mix, frozen egg product or liquid egg product that is
marked with an inspection legend shall, in addition
to meeting the general requirements for salmonellae,
have a coliform count of no more than 1 0 per gram,
and a total viable bacteria count of no more than
50 000 per gram.
Dried egg, dried egg mix or dried egg product that
is marked with an inspection legend shall, in addition
to meeting the requirements for salmonellae, have a
coliform count of no more than 1 0 per gram, and a
total viable bacteria count of no more than 50000
per gram in the case of whole egg, whole egg mix and
yolk mix. In the case of albumen, the total viable
bacteria count standard is reduced to 100000 per
gram.
The pasteurization of liquid egg products, while
initially directed to reduce salmonellae to levels that
do not represent a health hazard, will also have the
same beneficial effect for other pathogens having the
same or lower thermal resistance that may be present.
The heating requirements are given in Table 7. Spray-
dried albumen shall be pasteurized at 54°C (130°F)
for 7 days, and pan-dried albumen at 52°C (125°F)
for 5 days.
Dairy Products Regulations In addition to the
general requirement as stipulated in the Act, the regu-
lations specify the compositional standards (e.g. per-
centage of butterfat in various milk categories). The
regulations reference the microbiological standards in
the Food and Drug Regulations. As the production,
processing, sale and distribution of milk and asso-
ciated products for the most part are intraprovincial,
they are also subject to regulation by each province.
Each province has specific pasteurization require-
ments with respect to time and temperatures.
Processed Products Regulations The regulations
require that a low-acid food product packed in a
hermetically sealed container be thermally processed,
until at least commercial sterility is achieved. A low-
acid food product packed in a hermetically sealed
container is exempt, if it is stored continuously under
refrigeration and if the container in which it is packed,
as well as the shipping container, is marked ‘Keep
Refrigerated’, or kept continuously frozen and the
container in which it is packed, as well as the shipping
container, is marked ‘Keep Frozen’. This duplicates
the same requirement under B.27.002 of the Food
and Drug Regulations.
 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1557

Table 6 Compliance criteria for Listeria monocytogenes in ready-to-eat foods

Food product category Action level GMP status Immediate action

RTE foods causally linked to > 0 cfu per 50 g" n/a Class I recall to retain level,
listeriosis (see Table 5) consideration of public alert
All other RTE foods supporting > 0 cfu per 25 ga n/a Class II recall to retain level, health
growth of LM with a refrigerated shelf alert consideration
life > 10 days
RTE foods supporting growth of LM 5 100 cfu gb Adequate GMP Allow sale
with a refrigerated shelf life 5 10 days
and all RTE foods not supporting
growth (see Table 5)
5 100 cfu/gb Inadequate or no GMP' Consideration of class II recall or
stop sale
> 100 cfu/gb n/a Class II recall or stop sale
~ ~~

a Enumeration by enrichment only (MFHPB-30)

Enumeration to be done by direct plating onto LPM and Oxford agar (MFLP-74)
No information on GMP is considered as no GMP and the burden of proof remains with the legal agent for the product.
In all of the above cases where L. monocytogenes is detected, the processing establishment should be inspected to determine
the source of the contamination and to ensure that corrective measures are taken.
cfu, colony forming unit; GMP, Good Manufacturing Practice: LM, Listeria monocytogenes; n/a, not applicable; RTE, ready-to-eat.

There is also a requirement that the water used to
cool the containers after thermal processing shall be of
an acceptable microbiological quality. The regulation
does not specify what is an acceptable quality. Water
used in a cooling canal system must contain a residual
amount of a bactericide at the discharge end of the
canal and records must be kept of all bactericidal
treatments.
The specific hygiene requirements detailed in the
regulations generally follow those in the Canadian
Code of Practice for the General Principles of Food
Hygiene for use by the food industry in Canada.
Meat Inspection Regulations All establishments
involved in the slaughter, preparation, manufacture,
storage, distribution and sale of meat and meat prod-
ucts in import, export and interprovincial trade must
be registered by Agriculture and Agri-Food Canada.
The regulations contain specific requirements:
governing the design, construction and main-
tenance of registered establishments and of the
equipment and facilities therein
0 prescribing the equipment and facilities to be used,
the procedures to be followed and the standards to
be maintained in registered establishments to
ensure humane treatment and slaughter of animals
and hygienic processing and handling of meat
products
0 prescribing standards for meat products that are
prepared or stored in registered establishments, for
meat products that enter into interprovincial or
international trade and for meat products in
connection with which the meat inspection legend
is applied or used.
Registered establishments have resident federal
inspectors to ensure compliance with the regulations
and to conduct product inspection sampling when
required. All processes must meet departmental
requirements. Premortem and postmortem inspec-
tions are carried out routinely in all registered slaugh-
tering plants.
The requirements for low-acid meat products pack-
aged in hermetically sealed containers duplicates the
requirements found in Section B.27.002 of the Food
and Drug Regulations The container cooling water
must be of an acceptable microbiological quality and,
in the case of water used in a cooling canal system,
contains a residual amount of a bactericide at the
discharge end of the canal, and the container be
handled in a manner that ensures that the container
remains hermetically sealed.
Fisheries and Oceans Canada
The microbial safety and quality of fish and fish prod-
ucts for export, import or interprovincial trade are
regulated under the Fish Inspection Act and the Fish
Inspection Regulations. The regulations:
prescribe grades, quality and standards
0 set the quality and specifications for containers and
the marking and inspection of containers
0 require the registration of establishments and the
licensing of persons engaged as principals or agents
in the export or import of fish or containers
0 prescribe the requirements for the equipment and
sanitary operation of establishments, of premises
operated by an importer for the purpose of import-
ing fish, and of any boats, vehicles or other equip-
ment used in connection with an establishment or
1558 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada

Table 7 Pasteurization of liquid processed egg (see Processed Eggs Regulations - Schedule, Part I)
Liquid processed egg product Minimum temperature of Minimum
the processed egg at the heating time
automatic diversion valve (mins)
"C "F

Whole egg with less than 24% milk solids 60 140 3.5
Whole egg with no less than 24% and no more than 38% egg 61 142 3.5
solids 60 140 6.2
Whole egg mix with less than 2% added salt or sweetening 61 142 3.5
agent, or both 60 140 6.2
Whole egg mix with no less than 2% and no more than 12% 61 142 3.5
added sweetening agent 60 140 6.2
Whole egg mix with no less than 2% and more than 12% added 63 146 3.5
salt 62 144 6.2
Yolk 61 142 3.5
60 140 6.2
Yolk mix with less than 2 % added salt or sweetening agent, or 61 142 3.5
both 60 140 6.2
Yolk mix with no less than 2% and no more than 12% added 63 146 3.5
sweetening agent 62 144 6.2
Yolk mix with no less than 2% and no more than 12% added 63 146 3.5
salt 62 144 6.2
Ova 63 146 3.5
62 144 6.2
Egg product with less than 24% total solids" 61 142 3.5
60 140 6.2
Egg product with no less than 24% and no more than 38% total 62 144 3.5
solids" 61 142 6.2
Egg products with more than 38% solids" 63 146 3.5
62 144 6.2
a Regardless of the total solids content, egg product must be heated to 63°C (146°F) for 3.5 min or to 62°C (144°F) for 6.2 min if
there is no less than 2% and no more than 12% added sweetening agent or salt, or both. The director, at the request of an
operator, may designate a lower minimum temperature depending on the composition of the egg product.

in connection with fishing or the import or export Table 8 Bacteriological guidelines for fish and fish products
of fish Product Microorganism Criteria"
prescribe the manner in which samples of any fish
may be taken.
The general requirement that no person shall import, Cooked or RTE Escherichia coli 5 2 4 40
export, sell for export or have in his or her possession products
for export any fish intended for human consumption All other types E. coli 5 2 4 4
that is tainted, decomposed or unwholesome has All types Staphylococcus 5 1 lo3 lo4
aureus
microbiological significance: 'decomposed' means fish All types Salmonella 5b 0 0
that has an offensive or objectionable odour, flavour, Cooked or RTE Vibrio cholerae 5b 0 0
colour, texture or contains a substance associated with products
spoilage; 'tainted' means fish that is rancid or has an a n , sample size; c, acceptance number; m, acceptable
abnormal odour or flavour; 'unwholesome' means concentration of microorganisms; M, unacceptable
fish that has in or upon it bacteria of public health concentration of microorganisms.
significance or substances toxic or aesthetically offen- The analytical unit is 25 g. Each analytical unit must be negative
sive to humans. Any of these conditions can be the for the microorganism. The analytical units may be pooled.
RTE, ready-to-eat.
result of microbial growth, i.e. spoilage.
There are a number of regulations that are specific
to the requirements for the equipment and sanitary designed to meet specific product risks (Table 8).The
operation of the fish processing establishments, for interpretation of the sampling plans and acceptance
vessels used for fishing or transporting fish for pro- criteria is the same as that described for the micro-
cessing, and for the storage of frozen fish. biological standards and guidelines under the Food
Some microbiological guidelines have been and Drug Regulations.
 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada 1559

The Canadian Shellfish Sanitation Program Canada are the basis for the classification of coastal
The Canadian Shellfish Sanitation Program (CCSP) harvesting areas for clams, oysters, mussels and whole
was developed in 1925 under the Fish Inspection Act scallops. Classifications are based on the sanitary con-
as a result of a typhoid fever outbreak in the USA in ditions of the area as defined by the shoreline survey
1924-25 involving 1500 cases and 150 deaths as a with supporting information from the microbiological
result of consuming contaminated oysters. The CSSP evaluation of the area.
is jointly administered by the Canadian Food Inspec- 1. Approved: the area is not contaminated with faecal
tion Agency (CFIA), Department of Fisheries and material, poisonous or deleterious substances or
Oceans (DFO) and Environment Canada (EC) in marine biotoxins to the extent that consumption
cooperation with Health Canada. The parameters of of the shellfish might be hazardous. In these areas
interdepartmental cooperation between EC and DFO the median or geometric mean faecal coliform level
are established by a Memorandum of Understanding must be less than 1 4 Most Probable Number
which is under revision to reflect the responsibilities (MPN) per 100ml with no more than 10% of the
of the Canadian Food Inspection Agency. samples in excess of 43 MPN per 100 ml.
Environment Canada is responsible for carrying out 2. Conditionally approved: this area must meet the
shoreline sanitary and bacteriological water quality same sanitary quality criteria as an approved area.
surveys of the shellfish growing areas according to the However, under certain conditions which are pre-
procedures, standards and protocols of the Canadian dictable and verifiable, water quality can exceed
Shellfish Sanitation Program Manual of Operations. approved area criteria. The quality can vary with:
This includes the continuing evaluation of the level of (a) the effectiveness of sewage treatment at a com-
faecal contamination in the water overlying shellfish munity, (b) rainfall or river flow, (c) seasonal
growing areas, the identification of point and non- changes in sanitary conditions (i.e. tourist or
point pollution sources that have a negative impact summer cottage activity, vessel traffic, seasonal
on these areas, and classification of these areas based industrial operation). Management plans which
on sanitary quality and general sanitary conditions. detail the criteria for opening and closing such
In order for a shellfish area to be recommended for areas, and the responsibilities of all parties are
approval, the overlying waters must be free from required for conditionally approved classifications.
hazardous concentrations of pathogenic micro- 3 . Closed: direct harvesting from this area is pro-
organisms, poisonous or deleterious substances (or hibited owing to chemical or bacteriological con-
marine biotoxins and monitored by the CFIA) as tamination. Shellfish can be used only under
outlined in the Canadian Shellfish Sanitation Program specified permit conditions for depuration, relay-
Manual of Operations. ing, experimental purposes or other approved pro-
The 1948 Canada-United States Bilateral Agree- cessing. Depending on the level of contamination,
ment on Shellfish governing trade in shellfish between harvesting may be prohibited for any purpose. The
the two countries required agreement on practices for Closed classification includes the subclassifications
sanitary control of the shellfish industry. The Can- of Restricted for Controlled Purification,
adian Manual of Operations is based on the protocols Restricted for Relaying and Prohibited Area.
and procedures of the American National Shellfish
Regional Shellfish Classification Committees, com-
Sanitation Program Manual of Operations, now
posed of DFO, EC and provincial government rep-
called the NSSP Guide. The Agreement also required
resentatives, are responsible for:
each country to facilitate inspections of each other’s
shellfish handling facilities and shellfish growing areas 0 the technical reviews of the sanitary and bac-
if requested. The United States Food and Drug Admin- teriological surveys and evaluation of growing area
istration (USFDA) has routinely audited the CSSP classification recommendations
(about every 2 years - most recently in 1996 on both 0 reviewing the policies, procedures, criteria and
Atlantic and Pacific coasts). Growing area clas- regulations affecting the implementation and appli-
sification based on water quality is the basis of the cation of shellfish growing area classification
NSSP as it is for the CSSP. According to the NSSP ‘the 0 recommendations to DFO (for closures under the
first critical control point in the sanitary control of Management of Contaminated Fisheries Regu-
shellfish is identifying harvesting areas of acceptable lations under the Fisheries Act) and CFIA (as
sanitary quality’. Water quality requirements in both required for approved areas under the Fish Inspec-
programmes are the same for shellfish aquaculture as tion Regulations under the Fish Inspection Act
they are for wild harvest. 0 implementation of the classification decisions, and
The sanitary surveys completed by Environment recommending survey priorities.

1560 NATIONAL LEGISLATION, GUIDELINES & STANDARDS GOVERNING MICROBIOLOGY/Canada
It is the policy of Environment Canada either to
use its own laboratories and personnel, or to audit
laboratories and personnel under contract, so as to
ensure impartiality of sample collection and integrity
of the data. For example, Environment Canada cur-
rently contracts out sampling and analyses under
Q N Q C (Quality AssurancelQuality Control) con-
trols to private laboratories in Quebec. In addition,
EC has a cooperative agreement with the Department
of the Environment of the Province of Prince Edward
Island to carry out sampling and analysis for its
growing areas. In all cases, the data are interpreted by
Environment Canada which then makes classification
recommendations to DFO at the regional clas-
sification committees. With reducing resources and
increasing demand from the aquaculture industry for
classified areas, EC distributed a discussion paper in
May 1997 outlining cost-sharing approaches includ-
ing stakeholder sampling and the use of third-party
laboratories.
The programme is routinely evaluated by both
internal and USFDA auditors to determine com-
pliance with CSSP and NSSP protocols. The EC
laboratories are evaluated by USFDA. In addition,
EC participates in USFDA’s Laboratory Evaluation
Officer training programme. All laboratories par-
ticipating in the CSSP must be evaluated by a Labora-
tory Evaluation Officer, have an internal QA/QC
programme and participate in a split sample
programme.
The Future
The preceding has summarized the present status of
national legislation, standards and guidelines con-
cerned with the microbiological safety and quality of
food in Canada. There are five recent initiatives, the
Canadian Food Inspection Agency Act and the Can-
adian Food Inspection System, that may well have a
profound effect on the status quo.
Under the Canadian Food Inspection Agency Act,
the Canadian Food Inspection Agency (CFIA) was
created to consolidate all federally mandated food
inspection and animal and plant health services. The
CFIA legislation sets out its responsibilities, account-
ability, regimes, powers and reporting framework.
The legislation also amended the enforcement pro-
visions and penalty structures of the federal statutes
relating to food and animal and plant health that are
enforced and/or administered by the Agency. To meet
its mandate, the Agency administers and/or enforces
the following Acts:
Canadian Agricultural Products Act
0 Consumer Packaging and Labelling Act as it relates
to food
Next Page
0 Feeds Act
0 Fertilizers Act
0 Fish Inspection Act
0 Food and Drugs Act as it relates to food
0 Health of Animals Act
Meat Inspection Act
Plant Breeders’ Rights Act
Plant Protection Act
0 Seeds Act.
The Agency is a departmental corporation, reporting
to Parliament through the Minister of Agriculture
and Agri-Food. While the setting of standards and
guidelines remains the prerogative of the mother
departments that administer the underpinning legis-
lation, these as well as the enforcement policy will
certainly be affected by the feedback information from
the inspectional services and associated laboratories.
A blueprint for the Canadian Food Inspection
System (CFIS) was prepared in 1993 by the Joint
Steering Committee of CFIS, the Federal/Provincial
Agri-Food Inspection Committee and the
Federal/Provincial/TerritorialFood Safety Committee
and revised in 1995. The goal of the system is to
integrate the activities of all food inspection depart-
ments at all levels of government where appropriate.
Priority will be placed on developing regulatory stand-
ards which will be outcome-based, and supported
by nationally accepted guidelines. Effective, ongoing
communication among stakeholders and government
agencies is a requirement of integration.
Specifically, the process of integration will include
one set of food safety standards which are nationally
recognized and a common legislative base reflective
of international developments. This will be extended
to include the establishment of common standards for
product identity, including grade, composition, net
quantity and product description.
There will be a movement from prescriptive stand-
ards to standards which are outcome- or performance-
based, where practical. Commonality will extend to
the manner and environment under which food is
produced, processed and distributed. Standard
methods for laboratory testing and reporting that are
reflective of the Canadian Food Inspection System
and international developments will be established by
the Joint Steering Committee as will an accreditation
(certification) programme for government and private
laboratories. Research into new methodologies is also
critical to the success of this process.
See also: Brucella: Characteristics. Clostridium: C/o-
stridium botulinum. Eggs: Microbiology of Egg Products.
Fish: Spoilage of Fish. Food Poisoning Outbreaks.
Listeria: Introduction; Listeria monocytogenes. Milk and
Milk Products: Microbiology of Liquid Milk. Myco-
 OENOLOGYIsee Wines 1609

Oenology see Wines: Specific Aspects of Oenology.

oils see Fermentation (Industrial): Production of Oils and Fatty Acids; Preservatives: Traditional Pre-
servatives - Oils and Spices.

Organic Acids see Fermentation (Industrial): Production of Organic Acids, e.g. Citric, Propionic;
Preservatives: Traditional Preservatives - Organic Acids

PACKAGING OF FOODS
Aaron L Brody Rubbright Brody Inc., Duluth, Georgia, USA
Copyright 0 1999 Academic Press
Packaging is intended to protect foods against
environmental invasion. Among the many external
variables that may adversely affect foods are an excess
or deficiency of moisture, oxygen, dirt, humans
(through tampering), dust, animals, insects and micro-
organisms. Packaging and processing are increasingly
becoming integrated with each other; an example is
canning, which is really a packaging and thermal
preservation operation in which the can, its product
contents, the filling temperature, air removal, closure,
heating, cooling and distribution must be an uninter-
rupted continuum or else preservation is not effected.
More traditional preservation processes such as
drying and freezing do not necessarily require close
relationships between the product, process and pack-
aging; the process and the packaging may be separate
and the preservation effect will still be achieved. In
contrast, in preservation processes such as thermal
pasteurization, modified-atmosphere packaging,
aseptic packaging, retort pouch and tray packaging,
it is necessary to integrate all the elements to ensure
the optimum preservation of the contained foods.
For example, in aseptic packaging, preservation is
achieved by sterilization of the product independently
of the package, and the packaging equipment and
assembly environment must therefore be sterile to
exclude microorganisms from the ultimately her-
metically sealed package. It is essential that the oper-
ations be connected by sterile linkages and that no
microorganisms are permitted to contaminate any
element.
For these reasons it has become increasingly
important that the packaging be incorporated into
the system if the objectives of delivering safe and high-
quality food are to be achieved.
To understand fully the role of packaging in food
preservation it is perhaps instructive to offer a few
definitions. ‘Packaging’ is a term describing the total-
ity of containment for the purpose of protecting the
food contents and includes the package material, its
structure and the equipment that marries the package
structure to the food. Package materials are the com-
PACKAGING OF FOODS 1611
ponents that constitute the structures usually known
as packages or containers. Package materials are no
longer single elements but rather composites of several
different materials. In addition, new forms of pack-
aging are increasingly replacing the traditional cans,
bottles, jars, cartons and cases.
Preservation Requirements of Common
Food Categories
Meats
Fresh Meat Most meat offered to consumers is
freshly cut, with little further processing to suppress
the normal microbiological flora present from the
contamination received during the killing and break-
ing operations required to reduce carcass meat to
edible cuts. Fresh meat is highly vulnerable to
microbiological deterioration from indigenous
microorganisms. These microorganisms can range
from benign forms such as lactic acid bacteria or
slime-formers, to proteolytic producers of undesirable
odours and pathogens such as Escherichia coli
0157:H7. The major mechanisms that retard fresh
meat spoilage are temperature reduction to (or near)
the freezing point and a reduced oxygen atmosphere
during distribution to retard microbial growth.
Reduced oxygen levels could provide conditions for
the expression of pathogenic anaerobic micro-
organisms, a situation usually obviated by the pres-
ence of competitive spoilage organisms. Reduced
oxygen levels also lead to the colour of fresh meat
being the purple of myoglobin; exposure to air con-
verts the natural meat pigment to the bright cherry-
red oxymyoglobin characteristic of most fresh meat
offered to and accepted by consumers in industrialized
societies. Reduced oxygen packaging is achieved
through the mechanical removal of air from the inter-
iors of gas-impermeable multilayer flexible material
pouches closed by heat-sealing the end after filling.
Ground Meat About 40% of fresh beef is offered in
ground or minced form to enable the preparation of
1612 PACKAGING OF FOODS
hamburger sandwiches and related foods. Ground
beef was originally a by-product, that is, the trim-
mings from reducing muscle to edible portion size.
The demand for ground beef is now so great that
some muscle cuts are specifically ground to meet the
demand. Grinding the beef further distributes the
surface and below-surface microflora and thus pro-
vides a rich substrate for microbial growth even under
refrigerated conditions. Relatively little pork is
reduced to ground fresh form; however, increasing
quantities of poultry meat are being comminuted and
offered fresh to consumers, both on its own and as a
cheaper substitute for ground beef. The major portion
of ground beef is coarsely ground at abattoir level and
packaged under reduced 0 2 levels for distribution at
refrigeration temperatures to help retard micro-
biological growth. The most common packaging tech-
nique is pressure-stuffing into chubs, which are tubes
of flexible gas-impermeable materials closed at each
end by tight-fitting metal clips. Pressure-stuffing the
pliable contents forces most of the air out of the
ground beef, and since there is no head-space within
the package, little air is present to support the growth
of aerobic spoilage microorganisms such as Lacto-
bacillus and Leuconostoc spp. At retail level the
coarsely ground beef is finely ground to restore the
desirable oxymyoglobin red colour and to provide the
consumer with the desired product.
In almost all instances, the retail cuts and portions
are placed in expanded polystyrene (EPS) trays which
are overwrapped with plasticized polyvinyl chloride
(PVC) film. The tray materials are resistant to fat and
moisture to the extent that many trays are internally
lined with absorbent pads to absorb the purge from
the meat as it ages and/or deteriorates in the retail
packages. Because of the prognosis the PVC materials
are not sealed but rather tacked so that the somewhat
water vapour-impermeable structure does not permit
loss of significant moisture during short refrigerated
distribution. Being a poor gas barrier, PVC film
permits the access of air and hence the oxymyoglobin
red colour is retained for the short duration of retail
distribution.
Case-ready Meat For many years, attempts have
been made to shift the retail cutting of beef and pork
away from the retailer’s back room and into cen-
tralized factories. This movement has been stronger
in Europe than in the USA, but some action has been
detected in the latter country in the wake of the E.
coli 0157:H7 incidents. Case-ready retail packaging
in the UK where the practice is relatively common,
involves cutting and packaging meat under extremely
hygienic conditions to reduce the probability of
microbiological contamination beyond that of the
indigenous microflora. Packaging is usually in a gas
barrier structure, typically gas/moisture barrier foam
polystyrene trays heat-sealed with polyester gas
barrier film. The internal gas composition is altered
to a high content of O2 (up to 80%) and of CO2 (up
to 3 0 % ) , with the remainder (if any) being nitrogen
as a filler gas to ensure against package collapse
arising from internal vacuum formation. The high 0 2
concentration fosters the retention of the oxy-
myoglobin red colour preferred by consumers, while
the elevated CO, level suppresses the growth of
aerobic spoilage microorganisms. Using this or similar
technologies, refrigerated microbiological shelf lives
of retail cuts may be extended from a few days to
as much as a few weeks, permitting long-distance
distribution, e.g. from a central factory to a multi-
plicity of retail establishments. One thesis favouring
the centralized packaging of ground beef is that the
probability of the presence of E . coli 0157:H7 is
reduced. On the other hand, if the pathogen is present
at the central location, the probability of its being
spread among a number of retailers is greatly
increased. Nevertheless, the use of central factories,
which would probably be under federal government
supervision in the USA, and certainly under technical
supervision, would increases the probability of the
emerging packaged meat being microbiologically safe.
Alternative packaging systems for case-ready beef
and pork include the ‘master bag’ system used widely
for freshly cut poultry (see below) in which retail cuts
are placed in conventional PVC film overwrapped
EPS trays and the trays are multipacked in gas barrier
pouches whose internal atmospheres are enhanced
with COZ to retard the growth of aerobic spoilage
microorganisms. Another popular system involves the
use of gas barrier trays with heat-seal closure using
flexible gas and moisture barrier materials. Con-
ventional non-gas-barrier trays such as EPS may be
overwrapped with gadmoisture barrier flexible films
subsequently shrunk tightly around the tray to impart
an attractive appearance. Other systems, all of which
involve removal of 0 2 , include vacuum skin pack-
aging in which a film is heated and draped over the
meat on a gas/moisture barrier tray. The film clings
to the meat so that no head-space remains, with the
result that the meat retains the purple colour of myo-
globin. In one such system the drape film is a multi-
layer whose outer gas barrier layer may be removed
by the retailer, exposing a gas-permeable film that
permits the entry of air, which reblooms the pigment
and restores the desired colour. Variations on this
double film system include packaging systems in
which the film is not multilayer but is composed
of two independent flexible layers, the outer being
impermeable to gas and moisture and the inner gas-

permeable to permit air entry to restore the red colour.
In all instances the microbiological shelf life is
extended by reduced temperature plus reduced O2
levels, which may incidentally or intentionally be
enhanced by elevated CO1 concentration.
Processed Meat Longer-term preservation of meats
may be achieved by curing, using agents such as salt,
sodium nitrite, sugar, seasonings, spices and smoke,
and by processing methods such as cooking and
drying; these treatments alter the water activity, add
antimicrobial agents, provide a more stable red
colour, and generally enhance the flavour and mouth
feel of the cured meats. Cured meats are often offered
in tubular or sausage form which means that the shape
is dictated by the traditional process and consumer
demand. Because of the added preservatives, the
refrigerated shelf life of processed meat is generally
several times longer than that of the fresh meat.
Because cured meats are not nearly so sensitive to
oxygen variations as fresh meat, the use of reduced
0 2 atmospheres to enhance the refrigerated shelf life
is quite common. The O2 reduction may be achieved
by mechanical vacuum, inert gas flushing or a com-
bination of methods. Since the conditions have been
changed to obviate the growth of anaerobic patho-
genic microorganisms, reduced oxygen conditions are
generally effective in retarding the growth of aerobic
spoilage microorganisms.
The containers for reduced O2 packaging of cured
meats are selected from a multiplicity of materials and
structures depending on the protection required and
the marketing needs: frankfurters are generally sold
in twin web vacuum packages in which the base tray
is an in-line thermoformed nylon/polyvinylidene
chloride (PVDCj web and the closure is a heat-sealed
polyester (PET)/PVDCflexible material. Sliced lunch-
eon meats and similar products are packed in ther-
moformed unplasticized PVC or polyacrylonitrile
(PAN) trays, heat-seal closed with PET/PVDC. Sliced
bacon packaging employs one of several variations of
PVDC skin packaging (in contact with the surface of
the product) to achieve the oxygen barrier. Ham may
be fresh, cured or cooked, with the cooking often
performed in the package. The oxygen barrier mater-
ials employed are usually a variation of nylon/PVDC
in pouch form.
Poultry Poultry meat is most commonly chicken,
but turkey is becoming an increasingly significant
category of protein. Further, chicken is increasingly
penetrating the cured meat market as a less expensive
but nutritionally and functionally similar substitute
for beef or pork. Since the 1970s poultry processing
in westernized societies has shifted into large-scale,
PACKAGING OF FOODS 1613
almost entirely automated killing and dressing oper-
ations. In such facilities the dressed birds are chilled
in water to near the freezing point after which they
are usually cut into retail parts and packaged in case-
ready form: expanded polystyrene trays overwrapped
with printed PVC or polyethylene film. The package
is intended to appear as if it has been prepared in the
retailer’s back room, but in reality it is only a moisture
and microorganism barrier. Individual retail pack-
ages, however, may be multipacked in gas-imper-
meable flexible materials to permit gas flush
packaging, thus extending the refrigerated shelf life
of the fresh poultry products.
Poultry is especially susceptible to infection with
Salmonella spp., which are pathogenic in large quan-
tities. Such organisms are not removed or destroyed
by the extensive washing and chemical sanitation of
current poultry processing plants, merely reduced in
numbers. Modified-atmosphere packaging has rela-
tively little effect on Salmonella and so refrigeration
during distribution is critical in the drive to avoid
increasing populations of this bacterium.
All meat products may be preserved by thermal
sterilization in metal cans or, less frequently, glass
jars. The product is filled into the container which is
hermetically sealed, usually by double-seam metal end
closure (see Fig. 2). After sealing, the cans are retorted
to destroy all microorganisms present and cooled to
arrest further cooking. The metal (or glass) serves as
a barrier to gas, moisture, microbes etc. to ensure
indefinite microbiological preservation. Cans or jars
do not, however, ensure against further biochemical
deterioration of the contents.
Fish
Fish is among the most difficult of all foods to preserve
in its fresh state because of its inherent micro-
biological population, many organisms of which are
psychrophilic, i.e. capable of growth at refrigerated
temperatures. Further, seafoods may harbour a non-
proteolytic, quasi-psychrophilic anaerobic pathogen,
Clostridium botulinum type E. The need to prolong
the refrigerated shelf life of fresh fish suggests the
application of modified-atmosphere packaging in
which reduced 0 2 levels and elevated C02 levels are
present (Table 1j. However, a reduced 0 2 atmosphere
can permit the expression of type E . botulinum, and
for this reason reduced O2 packaging for seafood is
discouraged in the USA. This is not the situation in
Europe, where gas barrier flexible and semirigid
plastic packaging similar to that described above for
case-ready fresh beef is often applied.
Packaging for fresh seafood is generally moisture-
resistant but not necessarily proof against microbial
contamination. Simple polyethylene film is employed
1614 PACKAGING OF FOODS
Table 1 Pathogens of concern in modified atmosphere-
packaged andvacuum-packagedfoods
-
Psychrotrophs growth at 3-4°C
Listeria monocytogenes
Yersinia enterocolitica
Bacillus cereus
Non-proteolytic Clostridium botulinum
-
Pseudopsychrotrophs growth at 7-8°C
Escherichia coli 0157:H7
Salmonella sp.
-
Mesophiles growth at ~ 1 0 ° C
Proteolytic Clostridium botulinum
often as liners in corrugated fibreboard cases. The
polyethylene serves not only to retain product mois-
ture but also protects the structural case against
internal moisture.
Seafood may be frozen, in which case the packaging
is usually a form of moisture-resistant material plus
structure such as polyethylene pouches or poly-
ethylene-coated paperboard cartons.
Canning of seafood is much like that of meats since
all seafoods have a p H above 4.6 and so require high-
pressure cooking or retorting to effect sterility in metal
cans (Table 2).
One variation unique to seafood is thermal pas-
teurization, in which the product is packed into plastic
cans under reasonably clean conditions, achievable
in contemporary commercial seafood factories. The
filled and hermetically sealed cans are heated to tem-
peratures of up to 80°C to effect pasteurization to
permit several weeks of refrigerated shelf life. The
system is usually effective because Clostridium bot-
ulinum type E spores are thermally sensitive and may
be destroyed by temperatures of 80°C. To ensure
against growth of other pathogens which may grow
at ambient temperatures, however, distribution at
refrigerated temperatures is dictated.
Dairy Products
Milk Milk and its derivatives are generally excellent
microbiological growth substrates and therefore
potential sources of pathogens. For these reasons,
almost all milk is thermally pasteurized as an integral
element of processing. Refrigerated distribution is
generally dictated for all products that are pasteurized
to minimize the probability of spoilage.
Milk is generally pasteurized and packaged in rela-
tively simple polyethylene-coated paperboard gable-
top cartons or extrusion blow-moulded polyethylene
bottles for refrigerated short-term (several days to 2
weeks) distribution. Such packages offer little beyond
containment and avoidance of contamination as pro-
tection benefits; they retard the loss of moisture and
resist fat intrusion. Newer forms of milk packaging
Table 2 Ranges for bacterial growth
Organism pH range
Gram-negative bacteria
Escherichia coli 4.4-9.0
Pseudomonas fluorescens 6.0-8.5
Salmonella typhimurium 5.6-8.0
Gram-positive bacteria
Bacillus subtilis 4.5-8.5
Clostridium botulinum 4.7-8.5
Lactobacillus sp. 3.8-7.2
Staphylococcus aureus 4.3-9.2
incorporate reclosure, a feature that was missing from
the traditional gable-top cartons. Further, modern
packaging environmental conditions have been
upgraded microbiologically to enhance refrigerated
shelf life by the use of pre-sterilization of the equip-
ment, shrouding and use of clean air.
An alternative, popular in Canada, employs poly-
ethylene pouches formed on vertical form/fill/seal
machines and heat-sealed after filling. This variant
has been enhanced by re-engineering into aseptic
format, a system that has not become widely accepted.
Pouch systems are generally less expensive than paper-
board and semirigid bottles, but are less convenient
for consumers. Little difference exists between the
three packaging systems from a microbiological
perspective.
In some countries, aseptic packaging is employed
to deliver fluid dairy products that are shelf-stable at
ambient temperatures. The most common processing
technology is ultra-high temperature short time
thermal treatment to sterilize the product followed by
aseptic transfer into the packaging equipment. Three
general types of aseptic packaging equipment are
employed commercially: vertical form/fill/seal in
which the paperboard composite material is sterilized
by high temperature/high concentration hydrogen
peroxide (removed by mechanics plus heat); erected
preformed paperboard composite cartons which are
sterilized by hydrogen peroxide spray (removed by
heat); and bag-in-box, in which the plastic pouch is
pre-sterilized by ionizing radiation. The former two
are generally employed for consumer sizes while the
last is applied to hotel, restaurant or institutional
sizes, largely for ice cream mixes. Fluid milk is gen-
erally pasteurized, cooled and filled into bag-in-box
pouches for refrigerated distribution.
Cheese Fresh cheeses such as cottage cheese fab-
ricated from pasteurized milk are generally packaged
in polystyrene tubs or polyethylene pouches for
refrigerated distribution. Such packages afford little
microbiological protection beyond acting as a barrier
against recontamination, i.e. they are little more than

rudimentary moisture loss and dust protectors, but
are adequate because the distribution time is so short.
Enhancement of refrigerated shelf life may be achieved
by clean filling and/or the use of a low 0 2 , high CO2
atmosphere, all of which retard the growth of lactic
acid spoilage microorganisms.
Fermented Milks Fermented milks such as yoghurts
fall into the category of fresh cheeses from a packaging
perspective, i.e. they are packaged in polystyrene or
polypropylene cups or tubs to contain and to protect
minimally against moisture loss and microbial recon-
tamination. Their closures are not hermetic and so
gas passes through both the closures and the plastic
walls, and microorganisms could enter after the
package is opened. Because the refrigerated shelf life
is short, however, few measures are taken from a
packaging standpoint to lengthen the shelf life. Clean
packaging is often used to achieve several weeks of
refrigerated shelf life. Aseptic packaging is occa-
sionally used to extend the ambient temperature shelf
life of these products. Two basic systems are
employed; one uses preformed cups, and the other
is thermoform/fill/seal. In the former, the cups are
sterilized by spraying with H20z and heating to
remove the residue prior to filling and heat-sealing a
flexible closure to the flanges of the cups, which are
impermeable to gas and water vapour. In the
thermoform/fill/seal method, a sheet of multilayer
barrier plastic sheet (usually polystyrene plus PVDC)
is immersed in HL02 to sterilize it, air-knifed to
remove the residual sterilant, heated to softening, and
formed into cups by pressure. The web containing the
connected cups is within a sterile environment under
positive pressure of sterile air. The cavities are filled
with sterile product and a flexible barrier material
web, usually an aluminium foil lamination (also ster-
ilized by H2O2immersion), is heat-sealed to the cup
flanges. Filled and sealed cups then pass through a
sterile air lock. These aseptic dairy packaging systems
may also be employed for juices and soft cheeses.
Recently, aseptic packaging of dairy products has
been complemented by ultra-clean packaging on both
preformed cup deposit/fill/seal and thermoform/
fillheal systems. In these systems, intended to offer
extended refrigerated shelf life for low-acid dairy
products, the microbicidal treatment is with hot water
to achieve a 4D kill (i.e. four times the decimal reduc-
tion time) on the package material surfaces. The same
systems may be employed to achieve ambient tem-
perature shelf stability for high-acid products such as
juices and related beverages.
Cured cheeses are subject to surface mould spoilage
as well as to further fermentation by the natural
microflora. These microbiological growths may be
PACKAGING OF FOODS 1615
retarded by packaging under reduced O2 atmospheres
which may or may not be complemented by the add-
ition of C02. To retain the internal environmental
condition, the use of gas barrier package materials
is commercial. Generally, flexible barrier materials
such as nylon plus PVDC are employed on hori-
zontal flow wrapping machines or on twin web
thermoform/vacuum/seal machines. On twin web
machines, the flat sealing web is usually a variant of
polyester plus PVDC. One problem is that some cured
cheeses continue to produce COz as a result of fer-
mentation, and so the excess gas must be able to
escape from the package or else the package might
bulge or even burst. Somewhat less gas-impermeable
materials are suggested for such cheeses.
In recent years, shredded cheeses have been popu-
larized. Shredded cheeses have increased surface areas
which increase the probability of microbiological
growth. Gas packaging under C 0 2 in gas-imper-
meable pouches is mandatory. One feature of all
shredded cheese packages today is the zipper reclosure
which does not represent an outstanding micro-
biological barrier after the package has first been
opened.
Ice Cream Ice cream and similar frozen desserts are
distributed under frozen conditions and so are not
subject to microbiological deterioration, but the
product must be pasteurized prior to freezing and
packaging. The packaging needs to be moisture resist-
ant because of the presence of liquid water prior to
freezing and sometimes during removal from refriger-
ation for consumption. Water-resistant paperboard,
polyethylene-coated paperboard and polyethylene
structures are usually sufficient for containment of
other frozen desserts.
Fruit and Vegetables
In the commercial context, fruits are generally high-
acid foods and vegetables are generally low-acid.
Major exceptions are tomatoes, which commercially
(not botanically) are regarded as vegetables, and
melons and avocados, which are low-acid.
The most popular produce form is fresh, and
increasingly fresh cut or minimally processed. Fresh
produce is a living, ‘breathing’ entity with active
enzyme systems fostering the physiological con-
sumption of O2 and production of COz and water
vapour. From a spoilage standpoint, fresh produce is
more subject to physiological than to microbiological
spoilage, and measures to extend the shelf life are
designed to retard enzyme-driven reactions and water
loss.
The simplest means of retarding fresh produce
deterioration is temperature reduction, ideally to near
1616 PACKAGING OF FOODS
freezing point but more commonly to about 4 4 ° C .
Temperature reduction also reduces the rate of micro-
biological growth, which is usually secondary to
physiological deterioration.
Since the 1960s, alteration of the atmospheric
environment in the form of modified or controlled
atmosphere preservation and packaging has been used
commercially to extend the refrigerated shelf life of
fresh produce items, such as apples, pears, straw-
berries, lettuce and now fresh cut vegetables. Con-
trolled atmosphere preservation has been largely
confined to warehouses and transportation vehicles
such as trucks and seaboard containers. In this form
of preservation, the 0 2 , CO2, ethylene and water
vapour levels are under constant control to optimize
refrigerated shelf life. For each class of produce a
separate set of environmental conditions is required
for optimum preservation effect. In modified-atmos-
phere packaging, the produce is placed in a package
structure and an initial atmosphere is introduced. The
normal produce respiration plus the permeation of
gas and water vapour through the package material
and structure drive the interior environment towards
an equilibrium gas environment that extends the
produce quality retention under refrigeration. In some
instances the initial gas may be air (passive atmos-
phere establishment). Produce respiration rapidly
consumes most of the oxygen within the package
and produces CO2 and water vapour to replace it,
generating the desired modified atmosphere.
The target internal atmosphere is to retard res-
piration rate and microbiological growth. Reduced O2
and elevated C 0 2 levels independently or in concert
retard the usual microbiological growth on fruit and
vegetable surfaces.
One major problem is that produce may enter into
respiratory anaerobiosis if the O2 concentration is
reduced to near extinction. In respiratory anaero-
biosis, the pathways produce undesirable compounds
such as alcohols, aldehydes and ketones instead of the
aerobic end products such as C 0 2 . To minimize the
production of these undesirable end products, elab-
orate packaging systems are being developed. Most
of these involve mechanisms to permit air into the
package to compensate for the oxygen consumed by
the respiring produce. High-gas-permeability plastic
films, microperforated plastic films, plastic films dis-
rupted with mineral fill, and films fabricated from
polymers with temperature-sensitive side chains have
all been proposed or used commercially.
The need for reduced temperature is emphasized in
modified-atmosphere packaging because the dis-
solution rate of CO2 in water is greater at lower
temperatures than at higher temperatures. Carbon
dioxide is one of the two major gases involved in
reducing the rate of respiration and the growth of
microorganisms.
Since the late 1980s, fresh cut vegetables, especially
lettuce, cabbage, carrots, etc., have been a major
product in both the retail trade and the hotel, res-
taurant and institutional markets. Cleaning, trimming
and size reduction lead to a greater surface area to
volume ratio and expression of fluids from the inter-
ior, increasing the respiration rate and offering a better
substrate for microbiological growth than the whole
fruit or vegetable. On the other hand, commercial
fresh cutting operations generally are far superior
to mainstream fresh produce handling in cleanliness,
speed through the operations, temperature reduction
and application of microbicides such as chlorine.
Although some would argue, on the basis of microbial
counts found in fresh cut produce in distribution
channels, that uncut produce is safer, the paucity of its
cleaning coupled with the rarity of adverse incidents
related to fresh cut produce lead to the opposite con-
clusion - that fresh cut is significantly safer micro-
biologically. Another argument is that the low O2
environment within most fresh cut produce packages
plus the risk of soil contamination lead to ideal con-
ditions for the proliferation of Clostvidium bot-
ulinum. Further, distribution temperatures are often
in excess of 1O"C, well within the range of growth
and production of spores. However, extensive testing
has demonstrated that after responsible fresh cut pro-
cessing, pathogenic spores are present in relatively
small numbers, distribution temperatures prior to
retail level are significantly lower than for uncut
produce, and times are too short for pathogenic
expression. These data indicate that while anaerobic
pathogenic problems may occur, they are significantly
less likely in fresh cut than in uncut fruit and
vegetables.
Uncut produce packaging comprises a multitude of
materials, structures and forms, ranging from trad-
itional containers such as wooden crates, to inex-
pensive ones such as injection-moulded polypropylene
baskets, to polyethylene liners within waxed, cor-
rugated fibreboard cases. Much of the packaging is
designed to help retard moisture loss from the fresh
produce or to resist the moisture evaporating or drip-
ping from the produce (or occasionally its associated
ice), to ensure the maintenance of the structure
throughout distribution. Some packaging designs rec-
ognize the issue of anaerobic respiration and incorp-
orate openings to allow passage of air into the
package, for example perforated polyethylene
pouches for apples or potatoes. Almost none of the
contemporary packaging for fresh uncut produce
encompasses any specific microbiological barriers or
countermeasures. That result is a direct extension of

the observation that uncut produce ‘processing’ is
virtually nonexistent. Packing house operations
include collection and the removal of debris and gross
dirt, and packaging is usually the least expensive
structure that will contain the contents during dis-
tribution, often at sub-optimum temperatures.
For freezing, vegetables are cleaned, trimmed, cut
and blanched, prior to freezing and then packaging
(or packaging and then freezing). Blanching and the
other processing operations reduce the numbers of
microorganisms. Fruit may be treated with sugar to
help retard enzymatic browning and other undesirable
oxidations. Produce may be individually quick frozen
(IQF) using cold air or cryogenic liquids prior to
packaging, or frozen after packaging as in folding
paperboard cartons. Frozen food packages are gen-
erally relatively simple monolayer polyethylene
pouches or polyethylene-coated paperboard to retard
moisture loss. No special effort is engineered to
obviate further microbiological contamination after
freezing, although the polyethylene pouches are gen-
erally heat-sealed.
Canning of low-acid vegetables to achieve long-
term ambient temperature microbiological stability is
the same as for other low-acid foods, with blanching
prior to placement in steel cans (today all welded side
seam tin-free steel, with some two-piece cans replacing
the traditional three-piece type), hermetic sealing by
double seaming, and retorting and cooling. Canned
fruit is generally placed into lined three-piece steel
cans using hot filling coupled with post-fill thermal
treatment. Increasingly, one end is ‘easy open’ for
consumer convenience. Newer techniques involve
placing fruit hot into multilayer gas- and moisture-
impermeable tubs and cups prior to heat-sealing with
flexible barrier materials and subsequent thermal pro-
cessing to achieve ambient temperature shelf-stability
or extended refrigerated temperature shelf life. These
plastic packages are intended to provide greater con-
venience for the consumer as well as to communicate
that the contained product is not ‘overprocessed’ like
canned food.
Tomato Products The highly popular tomato-based
sauces and pizza toppings must be treated as low-
acid foods if they contain meat, as so many do. For
marketing purposes, tomato-based products for retail
sale are commonly packed in glass jars with reclosable
metal lids. The glass jars are often retorted after filling
and hermetic sealing; major differences from the tech-
nique using metal cans include counterpressured
retorting and longer times for heating and cooling,
since the thick-walled glass is a thermal insulator.
PACKAGING OF FOODS 1617
Juices and Juice Drinks Juices and fruit beverages
may be hot-filled or aseptically packaged. Traditional
packaging has been hot-filling into steel cans and glass
bottles and jars. Aseptic packaging, described above
for paperboard composite cartons, is being applied
for polyester bottles using various chemical sterilants
to effect the sterility of the package and closure inter-
iors. Much fruit beverage is currently hot-filled into
heat-set polyester bottles capable of resisting tem-
peratures of up to 80°C without distortion. Hermetic
sealing of the bottles provides a microbiological
barrier, but the polyester is a modest oxygen barrier
and so the ambient temperature shelf life from a
biochemical perspective is somewhat limited.
Since the 1970s high-acid fluid foods such as
tomato pastes and non-meat-containing sauces have
been hot-filled into flexible pouches, usually on ver-
tical form/fill/seal machines. The hot filling generates
an internal vacuum within the pouch after cooling so
that the contents are generally shelf-stable at ambient
temperature. Package materials are usually lamin-
ations of polyester and aluminium foil with linear
low-density polyethylene (LLDPE) internal sealant;
this resists the relatively lengthy exposure to the high
heat of the contents during and immediately following
filling. The heat seal is hermetic. Some efforts have
been made to employ transparent gadwater vapour
barrier films in the structures: polyester/ethylene vinyl
alcohol laminations with the same LLDPE sealant.
Transparent flexible pouches offer the opportunity
for the consumer to see the contents, and for the hotel,
restaurant or institutional worker to identify the con-
tents without needing to read the label.
Other Products
A variety of food products that do not fall clearly into
the meat, dairy, fruit or vegetable categories may be
described as ‘prepared foods’, a rapidly increasing
segment of the industrialized society food market
during the 1990s. Prepared foods are those that
combine several different ingredient components into
dishes that are ready to eat, or simply require heating.
If the food is canned, the thermal process must be
suitable for the slowest heating component, meaning
that much of the product is overcooked to ensure
microbiological stability. If it is frozen, the com-
ponents are separate but the freezing process reduces
the eating quality. The preferred preservation tech-
nology from a quality retention or consumer pref-
erence perspective is refrigeration.
Incorporation of several ingredients from a variety
of sources correctly implies many sources for micro-
organisms - aerobic, anaerobic, spoilage, benign and
pathogenic. Where refrigeration is the sole barrier,
microbial problems are minimized by reducing the
1618 PACKAGING OF FOODS
time between preparation and consumption to less
than 1 day (under refrigeration at temperatures above
freezing) plus a nodding acknowledgment of clean-
liness during preparation. As commercial operations
attempt to prolong the quality retention periods
beyond same-day or next-day consumption, enhanced
preservation ‘hurdles’ have been introduced. These
microbiological growth retardant factors include ele-
vated salt or sugar concentrations, reduced water
activity, reduced pH to minimize the probability of
pathogenic microbiological growth, selection of ingre-
dients from reduced microbial count sources, and
modified-atmosphere packaging. The last is often sug-
gested as a potential stimulus for the growth of patho-
genic anaerobic microorganisms, since the multiple
ingredient sources can almost assure the presence of
Clostridium spores, and the reduced 0 2 low-acid con-
ditions are common to the types of products such as
potato salad, pasta dishes, etc. Further, distribution
temperatures may often be in the 5°C range or higher.
Packaging for air-packaged prepared dish products
is generally oriented thermoformed polystyrene trays
with oriented polystyrene dome closures snap-locked
into position i.e. no gas, moisture or microbiological
barriers of consequence. Refrigerated shelf life is
measured in days. When the product is intended to
be heated for consumption, the base tray packaging
may be thermoformed polypropylene or crystallized
polyester with no particular barrier closure. For modi-
fied-atmosphere packaging the tray material is a ther-
moformed, coextruded polypropylene/ethylene vinyl
alcohol with a flexible gas/moisture barrier lamination
closure heat-sealed to the tray flanges. Refrigerated
shelf life for such products may be measured in weeks.
For several years, the concept of pasteurizing the
contents, vacuum packaging and distribution under
refrigeration has been debated and commercially
developed in both the USA and Europe. The ‘sous
vide’ technique is the most publicized process of this
type. In sous vide processing the product is packaged
under vacuum and heat-sealed in an appropriate
gadwater vapour barrier flexible package structure
such as aluminium foil lamination. The packaged
product is thermally processed at less than 100°C to
destroy spoilage microorganisms and then chilled for
distribution under refrigerated or (in the USA) frozen
conditions. The US option is to ensure against the
growth of pathogenic anaerobic microorganisms. A
similar technology is cook-chill in which pumpable
products such as chili, chicken a la King and cheese
sauce are hot-filled at 80°C or more into nylon
pouches which are immediately chilled (in cold water)
to 2°C and then distributed at temperatures of 1°C.
The hot filling generates a partial vacuum within the
package to virtually eliminate the growth of any spoil-
age microorganisms that might be present.
This listing is only a sampling of the many alter-
native packaging forms offered and employed com-
mercially for foods subject to immediate micro-
biological deterioration. An entire encyclopedia
would be required to enumerate all of the known
options available to the food packaging technologist
with the advantages and issues associated with each.
Package Materials and Structures
Package Materials
In describing package materials, different conventions
are employed depending on the materials and their
origins. The commercial conventions are used with
some common indicator of quantitative meaning to
establish relative values.
Paper The most widely used package material in the
world is paper and paperboard derived from cellulose
sources such as trees. Paper is used less in packaging
because its protective properties are almost non-exist-
ent and its usefulness is almost solely as decoration
and dust cover. Paper is cellulose fibre mat in gauges
of less than 250 microns. When the gauge is 250
microns to perhaps as much as 1000 microns the
material is known as paperboard, which in various
forms can be an effective structural material to protect
contents against impact, compression and vibration.
Only when coated with plastic is paper or paperboard
any sort of protection against other environmental
variables such as moisture. For this reason, despite
their long history as packaging materials, paper and
paperboard are only infrequently used as protective
packaging against moisture, gas, odours or micro-
organisms.
Paper and paperboard may be manufactured from
trees or from recycled paper and paperboard. Virgin
paper and paperboard, derived from trees, has greater
strength than recycled materials whose fibres have
been reduced in length by multiple processing. There-
fore, increased gauges or calipers of recycled paper or
paperboard are required to achieve the same struc-
tural properties. On the other hand, because of the
short fibre lengths, the printing and coating surfaces
are smoother. Paper and paperboard are moisture-
sensitive, changing their properties significantly and
thus often requiring internal and external treatments
to ensure suitability.
Metals Two metals are commonly employed for
package materials: steel and aluminium. The former
is traditional for cans and glass bottle closures, but is

Canner’s end Canner’s end
component
,component
/
Canner’s
end seam
\
Maker’s end
(4 component (6)
Figure 1 Metal can construction. (A) Three-piece steel can.
(6)Two-piece steel or aluminium can. From Soroka (1995) with
permission.
Lining compound
Can body 7: I!
Can end resting First curl Finished
on body double seam
Figure 2 Operation of affixing or double-seaming a metal
closure to a metal can body. From Soroka (1995) with per-
mission.
subject to corrosion in the presence of air and mois-
ture and so is almost always protected by other mater-
ials. Until the 1980s, the most widely used steel
protection was tin, which also acted as a base for lead
soldering of the side seams of ‘tin’ cans. When lead
was declared toxic and removed from cans during the
1980s in the USA, tin was also found to be superfluous
and its use as a steel can liner declined. The tin in
‘tin-free’ cans was chrome and chrome oxide. The
construction and closure techniques of metal cans are
shown in Figures 1-3.
In almost every instance the coated steel is further
protected by organic coatings such as vinyls and
epoxies which are really the principal protection.
Steel is rigid, a perfect microbial, gas and water
vapour barrier, and resistant to every temperature to
whicha food may be subjected. Because steel-steel or
steel-glass interfaces are not necessarily perfect, the
metal is often complemented by resilient plastic to
compensate for the minute irregularities.
Aluminium is lighter in weight than steel and easier
to fabricate; it has therefore become the metal of
choice for beverage containers in the USA and is
favoured in other countries. As with steel, the alu-
PACKAGING OF FOODS 1619
Seaming wall radius
Bod
Seaming panel radius
Body hookradius /
Lining compound
Seaming wall
-Chuck wall
End hook
Chuck wall radius
End hook radius Body wall
Figure 3 Double-seam closure on a metal can. From Soroka
(1995) with permission.
minium must be coated with plastic to protect it from
corrosion. It is the most commonly used material for
can-making in the USA. However, aluminium cans
must have internal pressure from COz or Nz to main-
tain their structure, and so aluminium is not widely
used for food canning applications in which internal
vacuums and pressures change as a result of retorting.
Aluminium may be rolled to very thin gauges (8-
25 microns) to produce foil, a flexible material with
excellent microbial, gas and water vapour barrier
properties when it is protected by plastic film. Alu-
minium foil is generally regarded as the only ‘perfect’
barrier flexible package material. Its deficiencies
include a tendency to pinholing, especially in thinner
gauges, and to cracking when flexed.
In recent years, some applications of aluminium
foil have been replaced by vacuum metallization of
plastic films such as polyester or polypropylene.
Glass The oldest and least expensive package mater-
ial is glass, derived from sand. Furthermore, glass is
a perfect barrier material against gas, water vapour,
microorganisms, odours, etc. The transparency of
glass is often regarded by marketers and consumers
as a desirable property. Technologists may view the
transparency as less than desirable because visible
and ultraviolet radiation accelerates biochemical
(particularly oxidative) reactions.
Glass is energy-intensive to produce; it is heavy and
vulnerable to impact and vibration even though it
has excellent vertical compressive strength. For these
reasons, glass is being displaced by plastic materials
in industrialized societies.
Plastics The term ‘plastics’ describes a number of
families of polymeric materials (Table 3), each with
different properties. Most plastics are not suitable as
package materials because they are too expensive or
toxic in contact with food, or do not possess prop-
erties desired in packaging applications. The most
commonly used plastic package materials are poly-
1620 PACKAGING OF FOODS

Table 3 Packase plastic structures

Plastic Structure Oualities

~

Polyethylene (PE) H H H H Three basic types:

II I I high-density
-c - c - c - c - linear low-density
I I I I lowdensity
H H H H Moisture barrier

Polypropylene (PP) H CHS H CHJ Higher temperature than polyethylene

I I I I Low density high yield
-c-c- c- c- Very good moisture barrier
I I I I
H H H H

Ethylene vinyl alcohol (EVOH) H H Excellent O2 barrier resin

I I Moisture sensitive, poor water barrier
H O H O Used in coextrusion; expensive
I l l 1
-c-c-c-c-
I I I I
H H H H

Polyvinylidene chloride (PVDC) H CI H CI Excellent 0 2 , moisture, flavour and fat barrier

I I I I Dense
-c-c-c-c-
I I I I
H CI H CI

Polyvinyl chloride (PVC) H CI H CI Stiff, clear - without plasticizer

I I I I Soft with plasticizer
-c-c-c-c- No barrier
I l l 1
H H H H

Polyamide (PA) (Nylon) 0 O H H Temperature resistant

I1 I/ I I Very good 0 2 barrier
- C - ( C H Z )-
~ C - N - (CH2)s - N - Thermoformabie

Polyethylene terephthalate (PET) 0 0 H H High temperature after orientation

(polyester) I/ II I I
-0- c - (-J- c - 0 - c - c -
I I
H H

Polyacrylonitrile (PAN) H H H H H Very good O2 barrier

I I I I I Not processable in extrusion unless
-c-c-c-c-c- copolymer
l l l l l
H C H C H
111 111
N N

Polystyrene (PS) H H H H H Stiff, brittle, clear

1 1 1 1 1 Very little barrier
-c-c-c-c-c-
I l l l l
H c l H o H

Table 4 Properties of plastic package materials
Material Specific gravity Clarity or colour
Polyethylene
high density 0.941-0.965 Semi-opaque
medium density 0.926-0.940 Hazy to clear
low density 0.910-0.926 Hazy to clear
Polypropylene 0.900-0.91 5 Transparent
Polystyrene 1.04-1.08 Clear
Plasticized vinyl chloride 1.16-1.35 Clear to hazy
Nylon 1.13-1.1 6 Clear to translucent
aWatervapour transmission rate is measured in gm-’ for 24h at 38°C and 90% relative humidity.
bGas transmission is measured in crn3ml-’m-*for 24h at latmosphere, 30°C and 0% relative humidity.
ethylene, polypropylene, polyester, polystyrene and
nylon. Each has quite different properties (Table 4).
Plastics may be combined with each other and with
other materials to deliver the desired properties.
Polyethylene Polyethylene is the most used plastic
in the world for both packaging and non-packaging
applications. It is manufactured in a variety of dens-
ities ranging from 0.89 g cm-3 (very low density) to
0.96 g ~ m (high
- ~ density), and is lightweight, inex-
pensive, impact-resistant, relatively easily fabricated,
and forgiving. Polyethylene is not a good gas barrier
and is generally not transparent, but rather trans-
lucent. It may be extruded into film with excellent
water vapour and liquid containment properties.
Low-density polyethylene film is more commonly
used as a flexible package material. Low-density poly-
ethylene is also extrusion-coated onto other substrates
such as paper, paperboard, plastic or even metal to
impart water and water vapour resistance or heat
sealability.
Although used for flexible packaging, high-density
polyethylene is more often seen in the form of extru-
sion blow-moulded bottles with impact resistance,
good water and water vapour barrier, but poor gas
barrier properties. Any of the polyethylenes in proper
structure functions as an effective microbial barrier.
Polypropylene Like polyethylene, polypropylene is
a polyolefin, but it has better water vapour barrier
properties and greater transparency and stiffness.
Although more difficult to fabricate, polypropylene
may be extruded into films that are widely used for
making pouches particularly on vertical form/fill/seal
machines. In cast film form, polypropylene is the heat
sealant of choice on retort pouches because of its
fusion sealing properties, and because in this form it
is a good microbial barrier.
Polypropylene’s heat resistance up to about 133°C
permits it to be employed for microwave-only heating
trays. Unfortunately microwave heating alone is
insufficiently uniform to be a reliable mechanism for
PACKAGING OF FOODS 1621
Water vapour Gas Resistance to
transmission‘ transmissionb grease
~ ~~
Low High Excellent
Medium High Good
Good High Good
Good High Excellent
High High Fair to good
High to low High Good
Varies Low Excellent
reducing microbiological counts or destroying heat-
labile microbial toxins in foods.
Polyester A cyclical polymer that is relatively dif-
ficult to fabricate, polyethylene terephthalate poly-
ester is increasingly the plastic of choice as a glass
replacement in making food and beverage bottles.
Polyester plastic is a fairly good gas and moisture
barrier ;in bottle, tray or film form it is dimensionally
quite stable and strong. Its heat resistance in amorph-
ous form is sufficient to permit its use in hot-fillable
bottles. When polyester is partially crystallized the
heat resistance increases to the level of being able to
resist conventional oven heating temperatures. For
this reason crystallized polyester is employed to manu-
facture ‘dual ovenable’ trays for heat-and-eat foods
(‘dual ovenable’ means that the plastic is capable of
being heated in either conventional or microwave
ovens).
The transparency of polyester makes it highly desir-
able from a marketing standpoint for foods that are
not light sensitive.
Nylon Polyamide or nylon is a family of nitrogen-
containing polymers noted for their excellent gas
barrier properties. Moisture permeability tends to be
less than in the polyolefin polymers and nylon is
somewhat hygroscopic, meaning that the gas barrier
may be reduced in the presence of moisture. Gas and
water vapour barriers are enhanced by multilayering
with polyolefins and high-gas-barrier polymers.
Nylons are thermoformable and both soft and tough,
and so are often used for thermoformed processed
meat package structures in which the oxygen within
the package is reduced to extend the refrigerated shelf
life.
Polystyrene Polystyrene is a poor barrier to moisture
or gas. It is, however, very machinable and usually
highly transparent. Its structural strength is not good
unless the plastic is oriented or admixed with a rubber
modifier which reduces the transparency. Polystyrene

1622 PACKAGING OF FOODS
is often used as an easy and inexpensive tray material
for prepared refrigerated foods.
Polyvinyl Chloride Polyvinyl chloride is a polymer
capable of being modified by chemical additives into
plastics with a wide range of properties. The final
materials may be soft films with high gas per-
meabilities, such as used for overwrapping fresh meat
in retail stores; stiff films with only modest gas barrier
properties; readily blow-mouldable semirigid bottles;
or easily thermoformed sheet for trays. Gas and mois-
ture impermeability is fairly good but must be
enhanced to achieve ‘barrier’ status.
This material falls into a category of halogenated
polymers which are regarded by some envir-
onmentalists as less than desirable. For this reason, in
Europe and to a lesser extent in the USA, PVC has
been resisted as a package material.
Polyvinylidene Chloride Polyvinylidene chloride
(PVDC) is an excellent barrier to gas, moisture, fat
and flavours, but is so difficult to fabricate on its own
that it is almost always used as a coating on other
substrates to gain the advantages of its properties.
Ethylene Vinyl Alcohol Ethylene vinyl alcohol
(EVOH) is an outstanding gas and flavour barrier
polymer which is highly moisture sensitive and so
must be combined with polyolefin to render it an
effective package material. Often EVOH is sand-
wiched between layers of polypropylene which act as
water vapour barriers and thus protect the EVOH
from moisture.
Package Structures
Currently, rigid and semirigid forms are the most
common commercial structures used to contain foods.
Paperboard is most common, in the form of cor-
rugated fibreboard cases engineered for distribution
packaging. In corrugated fibreboard three webs of
paperboard are adhered to each other with the central
or fluted section imparting the major impact and
compression resistance to the structure. Folding
cartons constitute the second most significant struc-
ture fabricated from paperboard. Folding cartons are
generally rectangular in shape and often are lined with
flexible films to impart the desired barrier.
Metal cans have traditionally been cylindrical
(Figures 1 , 2 and 3 ) , probably because of the need to
minimize problems with heat transfer into the con-
tents during retorting. Recently, metal - and par-
ticularly aluminium - has been fabricated into tray,
tub and cup shapes for greater consumer appeal, with
consequential problems with measuring and com-
puting the thermal inputs to achieve sterilization.
Next Page
Thread Sealing surface
\ (land)
Neck ring \ /
Neck
(bead)
ring 7 \ 7 : ; i s h
parting line
I I
Bottom plate
parting line
Heel
Push-up/ ’ Base ~ 7r ~ - , ~
Figure 4 Glass bottle nomenclature. From Soroka (1995) with
permission.
During the 1990s shaped cylinders entered the market
again in efforts to increase consumer market share.
Few have been applied for cans requiring thermal
sterilization, but barrel and distorted body cans are
not rare in France for retorted low-acid foods. Analo-
gous regular-shaped cans are being used for hot filling
of high-acid beverages.
Noted for its formability, glass has traditionally
been offered in a very wide range of shapes and
sizes including narrow-neck bottles (Fig. 4) and wide-
mouth jars. Each represents its own singular problems
in terms of fabrication, closure and - when applic-
able - thermal sterilization.
Plastics are noteworthy for their ability to be
formed into the widest variety of shapes. Thin films
can be extruded for fabrication into flexible package
materials. These flexible materials may then be
employed as pouch or bag stock or as overwraps on
cartons or other structures, or as inner protective
liners in cartons, drums, cases, etc. Thicker films
(sheets) may be thermoformed into cups, tubs and
trays for containment. Plastic resins may be injection-
or extrusion-moulded into bottles or jars by melting
the thermoplastic material and forcing it, under pres-
sure, into moulds that constitute the shape of the
hollow object, e.g. the bottle or jar.
See also: Cheese: In the Market Place. Chilled Storage
of Foods: Use of Modified Atmosphere Packaging; Pack-
aging with Antimicrobial Properties. Fermented Milks:
Range of Products. Fish: Spoilage of Fish. Heat Treat-
ment of Foods: Thermal Processing Required for
Canning; Principles of Pasteurization. Ice Cream. Meat
and Poultry: Spoilage of Meat: Curing of Meat; Spoilage
 QUANTITATIVE RISK ANALYSIS 1883

1 Quality Assurance and Management see Hazard Appraisal (HACCP): The Overall Concept 1

QUANTITATIVE RISK ANALYSIS

S H W Notermans, TNO Nutrition and Food Research Institute, Zeist, The Netherlands
Copyright 0 1999 Academic Press

Introduction These were recommended following F A O N H O

Risk analysis is a structured, multidisciplinary
approach to the identification and reduction of risk.
Interest in risk analysis in the context of food-borne
pathogens, contaminants and additives has increased
due to the Sanitary and Phyto-Sanitary (SPS) Agree-
ment of the World Trade Organization (WTO). The
aim of the SPS Agreement is to endorse food safety
objectives, such as microbial standards and guide-
lines, that are based on the application of risk analysis
to sound scientific knowledge. Figure 1 illustrates the
use of risk analysis in the development of food safety
objectives (e.g. end product specifications) from the
food safety policy of the WHO/FAO Codex Ali-
mentarius Commission. Risk analysis can also be used
to determine criteria at the critical control points
in HACCP (hazard analysis critical control point)
processes.
Risk analysis involves the evaluation of risk in the
context of science, an understanding of all the activ-
ities involved and a structured approach. The process
of risk analysis consists of three essential components.
expert consultations, and later adopted by its Codex
Alimentarius Commission. They are:
1. Risk assessment: the evaluation of known or
potential adverse health effects resulting from
human exposure to food-borne hazards. The
outcome of the risk assessment is called the risk
estimate.
2. Risk management: the control of risks associated
with food-borne pathogens and contaminants, in
order to protect consumers. Risks are controlled
as effectively as possible through the selection and
implementation of appropriate measures, as
formulated by the World Health Organization
and the Food and Agriculture Organization
(FAONHO).
3 . Risk communication: an interactive process of
exchange of information and opinion between risk
assessors, risk managers and other interested
parties, such as consumers.

I-I
The concept of risk analysis as adopted by F A O N H O
Food safety policy
and several Codex Committees is primarily aimed at
: Quantitative
risk analvsis consumer protection, and involves the establishment
I
1
I
I Food safeti objectives I
I I
I
of safety objectives for foods that are based on science.
Risk analysis may also be used in selecting the most
I appropriate food processing and preservation
I I
I Food producers
Good Manut;ng Practices

Hazard analvsis critical control

k l
I
Quantitative
risk analysis
methods for compliance with the food safety object-
ives set. In addition, it is possible to use risk analysis
to set (sub)criteriaat critical control points, as defined
point process (HACCP)
Own responsibility by the hazard analysis critical control point (HACCP)
concept (see below). Thus risk analysis is used both
in compliance with the food safety objectives set by
Evaluationiverification the regulating bodies, and in meeting any additional
Figure 1 The use of risk analysis in the context of food safety. objectives set by the producers themselves.
1884 QUANTITATIVE RISK ANALYSIS

Components of Quantitative Risk used to plot dose-response curves directly applicable

Analysis to humans. Hazard characterization also involves
consideration of the characteristics of a pathogenic
Risk Assessment microorganism in relation to factors such as the
nature of the product, the processing conditions, and
Risk assessment is the evaluation of known or poten- the storage conditions. This information is necessary,
tial adverse health effects resulting from human for example, to estimate the outgrowth of the organ-
exposure to food-borne factors such as additives, con- ism in the food product of interest. There are a number
taminants and pathogenic microorganisms. Risk of uncertainties in hazard characterization, and so
assessment involves the documentation and analysis the introduction of an uncertainty factor must be
of scientific evidence, the measurement of risk and considered.
the identification of factors that influence it. This
information is used to produce the risk estimate. The Exposure Assessment This is the qualitative and/or
process of risk assessment consists of four steps: quantitative estimation of the likely intake of bio-
1. Hazard identification logical, chemical and physical agents via food. The
2. Hazard characterization ultimate goal of exposure assessment is the estimation
3. Exposure assessment of the hazardous agents in food at the time of con-
4. Risk characterization. sumption. This requires specific expertise and infor-
mation, about food consumption (e.g. from intake
There are several strategies for obtaining information surveys) and about the concentration and distribution
about the factors which contribute to risk and their of particular hazardous agents in foods. In the case
impact. One approach is a case-control study, in of food-borne microbiological hazards, the estimated
which unacceptable products are compared with concentration of microorganisms may be based on
acceptable ones. product surveillance and testing, storage conditions
Hazard Identification This is the identification of and the use of mathematical models which predict the
potential adverse health effects associated with expos- growth and death of microorganisms. There are many
ure to, inter alza, additives, contaminants and patho- sources of uncertainty involved in exposure assess-
genic microorganisms. It is a qualitative approach. ment, resulting in either underestimates or over-
For example, the microbiological hazards present in estimates. These uncertainties should be reflected in
food may be identified with reference to a list (based the risk characterization. Although it is seldom pos-
on published data) of pathogenic microorganisms able sible to provide fully quantified assessments of uncer-
to cause food-borne disease. The likelihood is deter- tainties, the introduction of a negative or a positive
mined of each listed organism being present in the bias should be made clear.
raw materials used and/or entering the food pro-
Risk Characterization This is the quantitative
cessing area. Organisms that have never been found
in either location can be deleted from the list. Any and/or qualitative estimation of the probabilities of
organisms which are completely destroyed during occurrence and severity of known or potential adverse
processing can also be deleted from the list. The pos- health effects in a given population, taking into
sibility of recontamination must then be considered. account attendant uncertainties. It is the last step in
risk assessment, and from it a risk management strat-
Any organisms which are not known to cause a food-
borne disease involving either an identical or a related egy can be formulated. Although the Codex Ali-
food product can be deleted from the list. mentarius document does not suggest that the
identification and quantification of the factors con-
Hazard Characterization This is the qualitative tributing to a risk is part of the risk characterization,
and/or quantitative consideration of the nature of the it is logical to include them.
adverse health effects associated with the biological,
Risk Management
chemical and physical agents which may be present
in food. If practicable, dose-response relationships Risk management is the process of evaluating alter-
should be assessed for all the adverse effects produced native policies in the light of the risk estimate and,
by the agents being considered, e.g. changes in organ if required, selecting and implementing appropriate
function and clinical symptoms. In the case of addi- controls, including regulation. The purpose of risk
tives and contaminants, epidemiological data are of management is the identification of acceptable risk
value in verifying the dose-response relationships levels and the development and implementation of
obtained in experimental animals. In the case of control measures within the framework of public
pathogenic microorganisms, such data may also be health policy. Risk management takes into account

the factors contributing to a risk and their quantitative
effect, and also a cost-benefit analysis of options.
The outcome of risk management is the derivation
of food safety objectives, for example banning addi-
tives or reducing their usage; setting maximum levels
for contaminants and pathogenic microorganisms;
and the obligatory use of Good Manufacturing Prac-
tices (GMP) and controls at national level. In setting
food safety objectives, risk managers should take into
account the difficulties of control, the feasibility of
monitoring, the availability of suitable methods of
analysis and the economic importance of the food.
The F A O N H O document on risk management
formulates some general principles covering a struc-
tured approach embracing risk evaluation, the assess-
ment of risk management options, decision
implementation and monitoring and review. The
document also emphasizes that the primary con-
sideration in risk management should be the pro-
tection of human health, and that the decisions and
practices associated with risk management should be
clear.
Risk Communication
Risk communication is defined as an interactive
process of exchange of information and opinion
between risk assessors, risk managers, and other inter-
ested parties. communication starts with the pro-
vision of information about food safety policy to all
parties involved in the process of risk analysis, as the
basis for the purpose and scope of risk assessment and
risk management. Clear, interactive communication is
necessary between all involved, including consumers,
and at all stages of the processes, and is likely to
assume increasing importance.
Framework for the Establishment of Food
Safety Objectives
Food safety objectives reflect the food safety policy,
which should present a general outline of what is
acceptable or not acceptable, and quantitative risk
analysis is used in the derivation process. There is
increasing consensus that food safety policy is an
international issue, and F A 0 and W H O are the inter-
national bodies responsible for setting this policy.
They have delegated this task to the F A O N H O
Codex Alimentarius Commission (CAC). This was
established in 1962 as an intergovernmental organ-
ization for developing food-related standards, guide-
lines and recommendations in order to protect the
health of consumers and facilitate international trade.
These standards are recognized by WTO, and provide
a reference point for the safety of foodstuffs traded
internationally.
QUANTITATIVE RISK ANALYSIS 1885
Risk assessment in relation to additives and con-
taminants is carried out by the Joint FAOPWHO
Expert Committee on Food Additives (JECFA). The
outcome of its risk assessment, i.e. the risk char-
acterization, is the starting point for the Codex Ali-
mentarius Committee on Food Additives and
Contaminants (CCFAC). This Committee, in which
all member states are represented, is responsible for
risk management and sets standards which are sub-
sequently adopted by CAC.
Risk assessment in the context of additives and
contaminants is a well-established activity. It involves,
firstly, the determination of dose-response rela-
tionships for additives and contaminants which can
cause an adverse health effect. This involves experi-
ments on animals and the use of a data package
obtained by testing several health parameters. From
the dose-response relationship, the so-called ‘no
observed effect’ level is estimated. This is used as
the basis for determining acceptable daily intakes for
additives, and provisional tolerable daily/weekly
intakes for contaminants, through the application of
uncertainty factors.
Risk analysis in relation to food-borne pathogens
is a newly emerging activity. Criteria and guidelines
are set by the Committee for Food Hygiene (CCFH),
and are based on results obtained from the analysis
of outbreaks of food-borne disease. At present, CCFH
lacks the assistance of a JECFA-like body for risk
assessment studies. WHO/FAO have recommended
that such a body be established, because it is unaccept-
able that a single body should carry responsibility
for both risk assessment and risk management. Both
CCFH and the JECFA equivalent should then elab-
orate the criteria and guidelines regarding the risk
assessment of food-borne pathogens. In addition,
CCFH should clarify the criteria for the selection of
pathogens for referral to the JECFA equivalent and
should clearly identify the factors to be taken into
account in its decision making, particularly in relation
to the evaluation of risk management options.
The Use of Quantitative Risk Analysis in
Food Production
The principles of quantitative risk analysis can also
be applied to food production. Internationally estab-
lished legal food safety objectives, together with safety
objectives set by individual food production com-
panies, focus on safe food production. In adhering to
these objectives, food producers make use of general
guidelines such as GMP. In addition, the use of hazard
analysis critical control points (HACCP)is mandatory
in most countries. The use of HACCP entails a sys-
tematic approach to the identification, assessment and
1886 QUANTITATIVE RISK ANALYSIS
control of hazards in a particular food operation. This
approach aims to identify problems before they occur,
and to establish measures for the control of stages in
production that are critical in terms of food safety.
The controls are thus preventive, remedial action
being taken in advance of problems developing. The
critical control points are defined as steps, points and
procedures where control can be exercised. In relation
to each point, criteria are specified such that if met,
the food produced will be safe. The traditional, largely
qualitative HACCP system can be converted into a
quantitative system using elements of quantitative risk
analysis, as indicated in Figure 2. In HACCP, a hazard
is defined as it is in risk analysis: an agent with the
potential to cause an adverse health effect. Inter-
national standards have been established for most
agents, which means that a risk assessment is not
necessary. Critical control points may be defined as
factors that contribute to the risk that a standard is
not met. The effect of such a factor should preferably
be quantified. In relation to each critical control point,
risk managers set criteria, based on this quantification.
In most cases, the actual risk is the result of a
I Hazard Analysis Critical Control Point system I
I
1 Hazard analysis I Risk assessment
I Determination of
critical control points ~
identification and quantification
of factors contributing to the risk
Risk management: setting of
criteria matching food safety objectives
Y
I System for monitoring
Corrective actions
Documentation
Figure 2 The hazard analysis critical control point (HACCP)
concept and the possible use of elements of risk analysis.
combination of several factors. A knowledge of the
effect of each individual factor enables optimization
of the process, taking into account economic factors.
This can be illustrated with a simple example
described by Notermans, Zwietering and Mead in
1994. One of the legal standards for pasteurized milk
is that Bacillus cereus must number < l o 4 organisms
per millilitre at the time of consumption. The factors
which determine the B. cereus count at the time of
consumption are the spore load of B. cereus after
pasteurization and the storage time and temperature.
The effect of each factor can be calculated, as can the
cost of control of each factor. Clearly, the wishes of the
consumer, especially in relation to storage conditions,
must be taken into consideration in reaching a final
managerial decision.
See also: Bacillus: Bacillus cereus. Good Manu-
facturing Practice. Hazard Appraisal (HACCP): The
Overall Concept; Critical Control Points; Involvement of
Regulatory Bodies; Establishment of Performance Cri-
teria. International Control of Microbiology. National
Legislation, Guidelines 81 Standards Governing
Microbiology: Canada; European Union; Japan. Pre-
dictive Microbiology 81 Food Safety. Process
Hygiene: Involvement of Regulatory Bodies
Further Reading
Codex Alimentarius Commission (1996) Terms and Defin-
itions used in Risk Analysis. Doc. CXIEXEC 9614316.
Annex 1.
FAOAVHO (1995) Applications of Risk Analysis to Food
Standavd Issues. Repovt of a Joint FAOIVIIHO Con-
sultation.
FAOAVHO (1997) Risk Management and Food Safety.
Report of a Joint FAO/WHO Consultation.
Notermans S , Zwietering MH and Mead GC (1994) The
HACCP concept: identification of potentially hazardous
micro-organisms. Food Microbiology 11: 203-214.
World Trade Organization (1994) The Results of the
Uruguay Round of Multilateval Trade Negotiations: the
Legal Texts. Agveement on the Application of Sanitary
and Phytosanitary Measures. MTN/FA 11-A1A-4.
 RAPID METHODS FOR FOOD HYGIENE INSPECTION 1887
R
RAPID METHODS FOR FOOD HYGIENE INSPECTION
Matthias Upmann, Institute of Meat Hygiene, Veterinary University of Vienna, Austria
Christine Bonaparte, Department of Dairy Research and Bacteriology, Agricultural University, Vienna, Austria
Copyright 0 1999 Academic Press
Introduction
Supplying consumers with microbiologically safe
products is a high priority with regulatory authorities
worldwide. But, since recognizing that governmental
supervision cannot assure absolute food safety, strong
emphasis is placed on the manufacturer’s respon-
sibility for the hygienic and toxicological quality of
foods, limiting the state’s task to the ‘control of the
control’. To meet these product liability demands, the
food industry increasingly relies on process control
systems and longitudinally integrated quality and
safety assurance programmes.
The underlying idea is that safety and quality of the
products are controlled best through effective man-
agement of those processing areas where hazards may
arise. After assessing the risks associated with the
food, processing steps are selected where preventive
measures will lead to the elimination of the hazard.
Establishing critical limits within these processing
steps and monitoring relevant parameters will result
in its control. But, with respect to microbial hazards
such a systematic approach known as hazard analysis
critical control point (HACCP) system suffers from
slow and cumbersome conventional methods in
microbiology which neither allow rapid evaluation
of raw materials on delivery nor ‘on-line’ control
measures during processing. Even with end-product
testing, they often permit only a retrospective assess-
ment of the food’s microbiological condition, since
many foods are highly perishable. Therefore, much
effort has been made to develop methods which
enable a more rapid estimation of the microbiological
quality of foods.
Microbiological Examination of Foods
General Considerations
To get reliable results from microbiological exam-
ination of foods many factors must be taken into
consideration. Firstly, ‘food’ is an extremely varied
matrix which contains infinite arrays of ingredients,
shows a high variability in physical composition, is
subjected to multifold processing technologies and is
stored under many different conditions. Furthermore,
its intrinsic flora may consist of high numbers of
typical quality indicating microorganisms as in the
case of fermented products. Also, they may contain
varying amounts of shelf-life limiting or even haz-
ardous microorganisms. O n the other hand there are
numerous sterilized products. In contrast to chemical
and physical contaminants, microorganisms are
mostly heterogeneously distributed in foods and their
concentration seldom remains constant. Additionally,
microbial cells may be injured sublethally due to food
manufacturing processes or food ingredients, thus
escaping detection if no preventive measures are
taken. The same problem may occur when a high
background flora prevents selective isolation of spe-
cific bacteria.
Methodological Requirements
Three main categories of analytical procedures can be
distinguished. Firstly, analysis may be directed
towards qualitative detection of specific micro-
organisms (presence/absence tests). Secondly, analysis
may be performed in order to quantify the total micro-
bial number, special indicative groups or specific
microorganisms. Thirdly, characterization of isolated
microorganisms may be desired.
Considering the broad range of analytical pro-
cedures available particular requirements were
defined which an optimum method should meet. High
sensitivity, which is defined as the lowest amount
of microorganisms detectable, should be of primary
importance. Likewise, high accuracy is essential. The
analytical result should meet the true value and repe-
titions of the analytical procedure should ideally give
the same results (Le. high precision). As explained
1888 RAPID METHODS FOR FOOD HYGIENE INSPECTION
above, rapidity is another important factor. Under
practical conditions economic considerations favour
the use of simple, inexpensive, universally applicable
and less laborious methods. Furthermore, the testing
system must operate at a high level of hygienic safety,
as for instance provided by self-contained units, espe-
cially if the user group consists of non-specialists.
Unfortunately, an optimum technique covering all
requirements does not exist. In particular, the accur-
acy of different analytical techniques is quite different,
hence validations by in-laboratory and/or inter-
laboratory comparisons against commonly agreed
standard methods are necessary. European standards
for validation and official acknowledgement of alter-
native microbiological methods are now dealt with at
the technical committee of the European Committee
for Standardization (CEN/TC) in Brussels.
Improving Methodological Rapidity
Ideally, rapid methods should enable such a quick
estimation of microbiological parameters that food
manufacturers are able to take corrective actions
immediately in the course of the manufacturing
process. However, the majority of methods char-
acterized as ‘rapid’ do not meet this demand. Never-
theless, they offer a more or less pronounced
advantage in analytical time compared to their con-
ventional equivalent by eliminating laborious and/or
subjective elements through mechanization and
automation.
Improved rapidity can be applied at each step of
the analysis, i.e. the sampling process, sample treat-
ment and detection/enumeration procedure. Although
labour-saving and automated methods speed up the
processes of sampling and sample treatment, thus
improving the laboratory’s output, the influence on
the total analysis time is usually negligible due to the
incubation time required for traditional culture-based
methods. A real shortening of the analytical time
can only be obtained if alternatives to the traditional
incubation methods are developed.
Training of Inspection Staff
New inspection techniques make great demands on
the qualifications of the inspection staff. The numer-
ous analytical options can be confusing and over-
whelming to the user. It is the user who decides
whether a microbiological test is reasonable -the fact
that it is applicable does not mean that it is necessary
or useful - and which technique should be applied.
Because new analytical procedures are based on
various technologies and designs, their performances
are highly variable. Moreover, many automated
instruments exhibit a so-called ‘black-box’ phe-
nomenon. The utmost caution is advised with such
instruments; reliable results are only feasible when
they are properly maintained and calibrated. Test
results must never be accepted in an uncritical manner.
Therefore, incorporation of accelerated methods
into the microbiological analytical repertoire must be
accompanied by training of the inspection staff. By
following the literature or attending occasional meet-
ings, one is not likely to be able to keep abreast of the
rapidly changing and developing field of inspection
techniques.
Methods with Improved Rapidity
Sampling
The sampling method depends on the material
(processing environment, solid, semisolid or liquid
foods), the surface structure (smooth/rough, hori-
zontal/vertical, fladcurved), and the expected micro-
bial contamination level. Additionally, the prac-
ticability on the spot should be considered: the use of
electrical sampling devices, for example in the pro-
cessing areas, may be a problem due to lacking plug
sockets.
With a few exceptions, such as ultrasonic sterility
testing of heat-treated milk, food samples are taken
destructively (excision, scraping) which destroys the
integrity of the food. Sampling of foods is almost
exclusively performed manually.
On the other hand, surfaces of the food processing
environment are sampled by non-destructive
methods. Mostly, contact slides or swabbing tech-
niques are employed. The former is often regarded as
‘rapid’ as there is no necessity for further sample
treatment and its simple application, although the
incubation time remains unchanged. Other methods,
such as manual or mechanical rinsing, do not have
any practical importance.
Sample Treatment
During sample treatment, the sample is comminuted,
liquefied and homogenized. Subsequently either
dilution or enrichment steps may be necessary accord-
ing to the expected level of microorganisms. Several
semi- or fully automated dilution procedures which
reduce the laboratory work have been developed
(e.g. stomacher, pipetting instruments, gravimetric
diluters).
Substituting for microbial enrichment procedures
and enabling quantitative results at low microbial
concentrations, several physical techniques for extrac-
tion and concentration of microorganisms are
employed. Techniques such as filtration,
centrifugation, ion exchange resins and the very
promising area of magnetic separations are men-
 RAPID METHODS FOR FOOD HYGIENE INSPECTION 1889

tioned. Furthermore, the polymerase chain reaction Table 1 Rapid methods for microbial detection, enumeration
(PCR), has become more applicable as a non-cultural and characterization in food microbiology: overview
means of target amplification for food analysis now- Direct methods
adays. Microcolony and single cell detection
Conventional microscopy
Microbial Detection, Enumeration and Epifluorescent techniques
Characterization Direct epifluorescent filter technique (DEFT)
Antibody direct epifluorescent filter technique (Ab-DEFT)
New time-saving detection methods utilize principles Membrane filter microcolony fluorescence technique
originally belonging to disciplines such as chemistry, (MMCF)
Flow cytometry
biochemistry, physics or immunology. This devel-
opment was rendered possible because of major tech- Indirect methods
nological advances in data-processors, which allow Methods based on growth and metabolic activity
Optical methods
rapid collection and interpretation of vast amounts of Colorimetry and fluorometry
data. Since these methods often measure parameters Turbidimetry
which are different from the traditional ones, the Pyruvate determination
correlation with traditional methods may be Thermal methods
problematic. Microcalorimetry
Electrical methods
The methods can be placed in two categories: (1) Direct conductimetry/impedimetry
direct methods based on the detection of whole cells Indirect conductimetry/impedimetry
(single cells or colonies), and (2) indirect methods Radiometry
which measure cell components, metabolites, meta- Immunological methods
bolic activities or changes caused by cell growth. Table Agglutination tests
1 gives a survey of rapid methods used for microbial lmmunodiffusion tests
detection, enumeration and characterization. Immunoassays based on labelled antibodies
Immunofluorescent assays (IF)
Direct Methods Radioimmunoassays (RIA)
Enzyme immunoassays (EIA)
Usually, colony-based techniques cannot be char- Immunomagnetic separation
acterized as rapid due to the continuing necessity Methods based on microbial cell components
for incubation, although several devices (dehydrated Luminometry
nutrient pads, spiralplater, laser or image analyser ATP-bioluminescence
etc.) can help to reduce the total laboratory work. Bacterial bioluminescence ('in-vivo bioluminescence')
Therefore, rapid direct methods are microcolony or Limulus amoebocyte lysate test
Ergosterol de termination
single-cell based.
Nucleic acid-based methods
Microcolony and Single-cell Detection DNA probe hybridization
Microscopical Techniques In order to visualize Polymerase chain reaction
Fingerprinting-like methods
objects for microscopical examination, colouring
agents are used to provide information on the total Combined methods
levels of microorganisms (e.g. methylene blue, acri- Biosensors
dine orange staining), special bacterial groups (e.g.
Gram stains) or specific types of microorganisms (e.g.
fluorescently labelled antibodies). In combination through membrane filters and are stained, most com-
with different pre-treatments (e.g. membrane monly with acridine orange which binds to nucleic
filtration, pre-incubation) and detecting principles acids. O n epifluorescence-microscopical examination,
(microscope, image analyser), microscopy has devel- aggregates of orange fluorescing cells are counted.
oped into a commonly used technique. Another technique uses tetrazolium chloride, which
Epifluorescent techniques The direct epifluorescent is reduced to purple-coloured formazan by an active
filter technique (DEFT) was originally developed for cellular respiration apparatus.
rapid assessment of bacterial numbers in raw milk. By using fluorescently labelled antibodies, specific
However, the introduction of several pre-treatment types of microorganisms can be detected. This anti-
techniques has considerably enlarged the range of body-direct epifluorescent filter technique ( Ab-DEFT)
successful applications. is especially useful in detecting pathogens. Since
Homogenized, prefiltered and subsequently pathogens usually occur in foods at low numbers and
enzyme-surfactant-treated food samples are passed microbial cell surface antigenicity has to be preserved,
1890 RAPID METHODS FOR FOOD HYGIENE INSPECTION
special product preparation steps (enrichment, im-
munocapture, pre-incubation) are necessary.
Short-term incubation of the membrane filters
before the staining process, results in the growth of
microcolonies. Hence, only viable microorganisms are
detected by this ‘membrane filter microcolony fluor-
escence (MMCF)’technique.
DEFT and related techniques have been used for
counting bacteria in milk, milk products, water,
beverages, raw meat, fish, poultry and food contact
surfaces. If large numbers of samples have to be ana-
lysed daily, an automated counting procedure linking
the microscope to an image-analysing system is advis-
able. Due to its rapidity and broad applicability DEFT
is recommended for quality control, shelf-life pre-
diction, irradiation control, as well as hygiene moni-
toring. Further details are given in Table 2.
Flow Cytometry Flow cytometry enables both quali-
tative and quantitative analysis of microbial cells in
liquids. The sample is injected in a thin, rapidly
moving carrier fluid which passes through a light
beam. The previously fluorescently labelled cells are
detected one by one with a photoelectric unit. By
using nonspecific and specific fluorochromes, different
wavelengths and measuring at different angles, it is
feasible to discriminate between bacteria in mixed
populations.
The practical use of flow cytometry is still limited
to few examples. However, since the possible appli-
cations are numerous, it should be considered as a
promising technology in the future. Some method-
ological properties are given in Table 2.
Indirect Methods
Methods Based on Growth and Metabolic Activ-
ity Several promising analytical procedures are
based on the detection of microbial growth during
incubation. Detection times ranging from a few
minutes to 30 h depend on many factors, including
inoculum density, microbial growth rate and type of
metabolic activity. Generally, detection time is related
inversely to the bacterial number: the lower the initial
bacterial content, the longer the detection time.
According to the physico-chemical properties con-
sidered, optical, thermal, electrical, and radiometric
methods are distinguished.
Optical Methods
Colorimetry and fluorometry: Specific physical or
chemical changes (pH, oxidation/reduction potential,
enzymatic transformations) associated with microbial
metabolic activity can be indicated by changes in
colour, fluorescence or colour intensity of an added
reagent dye during sample incubation. Many
chromogenic and fluorogenic dyes are used depending
on the metabolic change to be shown. A multitude of
miniaturized and computer-aided or even fully auto-
mated identification systems for pure cultures are
based on this principle.
Colorimetry and fluorometry can also be used for
quantitative purposes by measuring the required incu-
bation time in order to produce a colour reaction or
fluorochrome formation. Broadly known indicators
are litmus and bromocresol purple for detecting pH
shifts or resazurin, methylene blue, and tri-
phenyltetrazolium chloride as oxidation/reduction
indicators.
Fully automated procedures are now available
which use reflectance colorimeters or fluorometers.
These techniques are applicable for rapid estimation
of total microbial numbers or specific micro-
organisms, for product shelf-life stability, starter
culture activity, and antibiotic testing. Fur further
details see Table 2.
Turbidimetry: Increasing cell numbers lead to an
increase in optical density of liquid growth media.
Therefore, a light beam will increasingly we weakened
on transillumination when a liquid sample is incu-
bated. By varying the sample dilution, the growth
medium and the incubation temperature the result can
be narrowed to specific bacterial species or numbers.
Some methodological properties are given in Table 2.
Turbidimetry is widely applied in vitamin bioassays
and disinfectant testing. It has been used for sterility
testing in food quality control. Its application may be
limited by background turbidity of foods (fat glob-
ules, blood cells, food particles).
Pyruvate determination: Pyruvate is a key compound
in bacterial lactose metabolism and can serve as an
indicator for milk quality monitoring. Pyruvate is
measured indirectly by spectrophotometric detection
of reduced nicotinamide-adenine dinucleotide
(NADH) which is a cofactor in the enzymatic break-
down of pyruvate. Since somatic cells contribute to
the pyruvate content of milk and not all bacteria
produce pyruvate, the relation between this meta-
bolite and total microbial count is limited (see
Table 2).
Thermal Methods Bacterial growth is accompanied
by heat production, which can be used for micro-
calorimetric estimation of the bacterial content.
Highly sensitive calorimeters are necessary to detect
the heat generated. Due to multiple interfering factors,
microcalorimetry has thus far not assumed any prac-
tical importance.
Electrical Methods Measurement of electrical con-
 RAPID METHODS FOR FOOD HYGIENE INSPECTION 1891

Table 2 Methodological properties of selected rapid methods in food microbiology

Method Purpose Detection limit Rapidity Selected instruments and suppliers
(cells per ml or
qual. quant. chal:
Per 91
Direct methods
Epifluorescence microscopy
DEFT - + - ca. io4 <lh Bio-Foss (Foss Electric, Denmark),
COBRA (Biocom, France),
Autotrak (A.M. Systems, UK)
Ab-DEFT + + + ca. l o 3 clh
MMCF - + - ca. l o 3 27h
Flow cytometry + + + ca. l o 4 < 0.5 h BactoScan (Foss Electric, Denmark),
ChemFlow (Chemunex, France),
Argus Flow Cytometer (Skatron,
Norway)

Indirect methods
Methods based on growth and metabolic activity
Colorimetry, Fluorimetry + + - ca. 10’ 0.5-30 h Omnispec (Wescor, USA),
Fluoroskan (Labsystems Oy, Finland)
Turbidometry + + - ca. lo2 0.5-30 h Bioscreen analysing system
(Labsystems Oy, Finland),
AutoMicrobic System (Vitek
Systems, USA),
Cobas Bact Centrifugal Analyzer
(Roche Diagnostica, Switzerland)
Pyruvate determination (+) (+) - ca. 10’
Conductimetry/impedimetry
Direct +a + - ca. 10’ 0.5-30 h Bactometer (Bio Merieux, Germany),
BacTrac (Sy-lab, Austria),
Malthus (Malthus Instruments, UK),
RABIT (Don Whitley Scientific, UK)
Indirect + + - ca. lo-’ 0.5-30 h Malthus (Malthus Instruments, UK),
RABIT (Don Whitley Scientific, UK)
Radiometry + + - ca. lo2 1-18 h Bactec (Johnston Laboratories, USA)
Methods based on microbial cell components
ATP bioluminescence - + - ca. IO4 30 s-2 h BactoFoss (Foss Electric, Denmark),
Biocounter (LumadPerstorp
Analytical, Netherlands),
Luminometry System (Bio-Orbit Oy,
Finland),
AutoPlCOLlTE (Packard Instrument
Company, USA),
Biotrace Luminometer (Biotrace, UK)
ATP bioluminescence in - (+) - < 2 min HY-LITE (Merck, Germany),
hygiene monitoring Uni-Lite (Biotrace, UK)
Checkmate (Lumac/Perstorp
Analytical, Netherlands),
Lightning (Idexx, USA),
Systemsure (Celsis, USA)
Bacterial bioluminescence + + + ca. IO3 <lh
Nucleic acid-based methods
PCR + (+) + ca. l o o < 3 days
direct in food:
ca. i o 4
Dot Blot + (+I + ca. lo4 < 3 days
Colony hybridization + + + ca. 10’ < 3 days
qual., qualitative result (presence/absence); quant., quantitative result (enumeration); char., microbiological characterization.
+, applicable; (+), applicability restricted; -, not applicable.
alf special selective media are available.
1892 RAPID METHODS FOR FOOD HYGIENE INSPECTION
ductivity changes have been applied quite successfully
for microbial growth detection. Direct and indirect
methods have been developed depending on the
medium in which the changes are monitored. Using
electrodes, impedance, conductance and/or cap-
acitance on an alternating current, are measured
continuously.
Conductimetric methods are well established in the
dairy industry for monitoring total viable flora, indi-
cator organisms and pathogens. Application to other
foods is possible. Furthermore, conductimetry can be
useful for producing data for predictive microbiology.
Some further details are summarized in Table 2.
Direct conductimetry: Direct methods detect con-
ductivity changes in specific growth media. Nutrient
macromolecules are broken down to mostly charged
particles, thus increasing the conductivity of the
medium with incubation time.
Indirect conductimetry: Indirect conductimetry meas-
ures the conductivity reduction in a detection medium,
which is caused by absorption of COZ produced
during microbial metabolism. This method is suitable
for food products with low contamination levels,
since C 0 2 formation can be detected much earlier.
Specific growth media are not necessary.
Radiometry Radiometric measurement is also based
on the detection of COZ release during microbial
metabolism. However, in this case isotopically
labelled carbon sources in the growth medium are
converted into 14C02,the amount of which is meas-
ured in a ionization chamber. Table 2 contains more
methodological information.
The major drawback of this technique is the use
of radioactive material. Therefore, it has not been
frequently used in Europe. Alternatively, COZ pro-
duction may also be monitored by infrared spec-
trophotometry or volumetry. Both principles have
been suggested for sterility testing of liquid samples.
Immunological Methods
Today, reactions between inducible animal-derived
proteins (antibodies) and specific target molecules
(antigens) are widely applied for rapid separation,
identification, differentiation and quantification of
microorganisms and their toxins.
Agglutination and Immunodiffusion Tests Bacterial
agglutination (‘clumping’) due to the formation of
antigen-antibody complexes are well introduced in
microbiology. For example, the Salmonella dif-
ferentiation scheme of Kauffmann and White is based
on this technique. Similarly, it is used in several com-
mercial latex agglutination kits; antibody coated latex
particles show a macroscopically visible agglutination
if the antigen is present.
Immunodiffusion has also been applied for a long
time. A semisolid medium is inoculated at one spot
with the sample and at another spot with a specific
antibody. If the corresponding antigen is present, line-
shaped immunoprecipitates will develop at the place
where diffusing antibodies and antigeils meet. Several
test kits based on this technique are available.
Immunoassays Based on Labelled Antibodies In
most immunoassays the primary antigen-antibody
reaction is made detectable by means of labelling the
antibodies with marker substances. Immuno-
fluorescent (IF) assays, making use of antibodies
labelled with fluorescent reporter molecules, do not
enjoy widespread use. Equally, radioimmunoassays
(RIA) are not used frequently, due to the inherent
disadvantages of handling radioisotopes. Probably the
fastest growing and most widely used formats are
enzyme immunoassays (EIA) which employ enzyme
markers in conjunction with a colorimetric or
fluorometric substrate system. By immobilizing the
capturing antibody on a solid matrix (microtitre trays,
polystyrene or ferro-metal beads, dip-sticks) enzyme-
linked immunosorbent assays (ELISA) were created.
Other assay formats are conceivable depending on
the number of antibodies involved.
Immunoassays are promising because of their sen-
sitivity and rapidity. However, they normally require
enrichment of the target bacterium to the level of the
assay’s detection limit. Disadvantageous false-positive
or false-negative results have been significantly
reduced by advances in antibody preparation.
Immunomagnetic Separation Immunomagnetic
separation (IMS) has proved to be a very efficient
method for separating target organisms from food
materials and background flora. Antibody-coated
paramagnetic particles are mixed with the sample. By
exposure to a magnetic field, bound target cells are
separated while the sample suspension is removed. A
number of procedures may be used for subsequent
final detection, such as conventional culturing, micro-
scopy, impedance technology, ELISA, latex agglu-
tination or DNA hybridization, partly involving
amplification techniques. In addition to the short sep-
aration and concentration time, IMS technology also
overcomes the problem associated with unwanted
inhibition due to selective media components. Since
IMS can be used in conjunction with many final detec-
tion technologies, it is expected that several auto-
mated analytical procedures will make use of this
potent technique in the near future.
 RAPID METHODS FOR FOOD HYGIENE INSPECTION 1893
Methods Based on Microbial Cell Components
Luminometry
ATP bioluminescence: ATP is the universal intra-
cellular energy carrier in living somatic and microbial
cells. Although the level of ATP depends on the cel-
lular type and is influenced by several external factors
it seems to be a useful parameter in the estimation of
the active microbial population.
The amount of ATP is detected by a bio-
luminescence reaction; in the presence of ATP and
catalysed by the enzyme ‘luciferase’ the substrate ‘luci-
ferin’ is oxidized to oxyluciferine. As a ‘by-product’
of this reaction photons are emitted - one photon of
light for each molecule of ATP, provided all other
reagents are present in surplus. Therefore, photo-
metrical measurement of the light emitted gives rapid
information on the amount of metabolically active
cells. However, for microbiological food control add-
itional steps prior to the assay are required in order
to remove non-microbial (i.e. intrinsic and somatic)
ATP which often greatly exceeds the microbial ATP
content.
Nevertheless, ATP bioluminescence is a well estab-
lished technique in the industry for quality control of
raw milk, meat and fish. Further, it has been applied
to end-product testing of beer, carbonated beverages,
fruit juices, pasteurized or ultra-high temperature
milk and dairy products, testing of starter cultures, as
wellas to shelf-life prediction. Manual and automated
luminometers and standardized reagent kits are avail-
able from several suppliers.
In recent years, the application of ATP bio-
luminescence has become important for on-site
hygiene control of surfaces in industrial plants.
Various low-cost, portable luminometers are com-
mercially available (see Table 2).
Bacterial bioluminescence (‘in-vivo bio-
luminescence’): Bioluminescence also occurs naturally
in several bacterial species using flavin monon-
ucleotide as the energy source. Bacterial luciferase
mediates the oxidation of aldehydes to fatty acids.
Since the so-called lux genes encoding for bio-
luminescence are known, their bacteriophage- or
plasmid vector-mediated transfer to other bacteria has
been enabled. The resulting light emission is detected
by standard luminometers, as with ATP-bio-
luminescence (see also Table 2).
The specificity of this sensitive and rapid technique
depends on the host range of the bacteriophages used.
Wide host ranges covering several bacterial species
enable the detection of indicator organisms, whereas
species- or subspecies-specific bacteriophages are used
for the detection of pathogens. In addition, bacterial
bioluminescence enables the detection of inhibitory
substances (antibiotics, bacteriophages, antimicrobial
compounds) by making use of starter cultures con-
taining lux genes.
Limulus Amoebocyte Lysate Test The limulus am-
oebocyte lysate (LAL) test is a simple method for
the detection of viable and non-viable Gram-negative
bacteria. Certain cell-wall lipopolysaccharides (i.e.
endotoxins) of this bacterial group lead to gelation
of blood cell (amoebocytes) lysates of the Limulus
polyphemus crab. Using a dilution row and deter-
mining the limit at which no more gel formation
occurs, a semi-quantitative estimation of the Gram-
negative content is possible.
Several test-kits are available. Mostly used for
pyrogen control of pharmaceutical products the LAL-
test is applicable for predominantly Gram-negative
containing foods such as fresh meat, milk and eggs.
Another field of application may be the retrospective
assessment of the microbiological quality of heated
products.
Ergosterol Determination Ergosterol is a steroidal
component of fungal cell membranes. Therefore, the
amount of ergosterol present can be related to fungal
biomass. The chemical detection procedure includes
high-pressure liquid chromatography and detection
by ultraviolet absorption. Though yeasts also contain
ergosterol in their cell wall, the rapid ergosterol assay
seems to be a useful technique for food quality control
purposes with respect to fungal invasion.
Nucleic Acid-based Methods The application of
new methods based on nucleic acids has greatly stimu-
lated the food microbiology field during recent years.
DNAIRNA Probe Hybridization With the classical
DNA probe hybridization assay, microorganisms are
collected on a filter, cells are lysed and the liberated
DNA or RNA is immobilized in single-stranded form.
The nucleic acid is identified by hybridization with
radioactively or non-radioactively labelled DNA
probes of defined origin.
Hybridization assays for several organisms are
available commercially. However, their sensitivity
depends on the initial amount of DNA/RNA; cur-
rently most of them do not allow detection of bacterial
populations below lo5 bacteria per gram. This dis-
advantage may be overcome by pre-enriched cultures
and/or DNA amplification techniques such as PCR.
DNA hybridization with labelled DNA probes is
mainly used for identification or confirmation of pure
cultures thus providing qualitative results. It is some-
times considered as an indirect semi-quantitative
method by comparison of the signal response intensity

1894 RAPID METHODS FOR FOOD HYGIENE INSPECTION
with that at the detection limit (for example Dot Blot,
see Table 2). Quantitative results can be obtained by
colony hybridization.
Polymerase Chain Reaction (PCR) In PCR tech-
nology specific DNA sequences are detected and
multiplied in vitro. Small but specific DNA primers
are added to the sample’s liberated and denatured
target DNA. If these primers meet a complementary
nucleic acid base sequence, they will hybridize. Sub-
sequently, a thermostable DNA polymerase will
elongate the primers according to the complementary
base sequence given by the target DNA. This cyclic
process is usually repeated about 30 times, amplifying
the target DNA by 1O5-10--fold.
The primers can be designed for different purposes.
Specific primers are targeted to a known virulence
factor of a single species and allow simultaneous
detection and identification, whereas multiple primers
with a broader spectrum are suitable for several
species. Theoretically, several microorganisms in a
sample can be detected at the same time with so-called
multiplex PCR.
PCR is a sensitive, specific and rapid microbial
detection (presence/absence testing) method, which
provides qualitative results. Theoretically, a single
molecule of target DNA can be detected within one
working day. However, the PCR can be inhibited or
its sensitivity severely reduced when applied to food
samples (see Table 2). High amounts of fat and pro-
teins in complex foods as well as certain components
of selective culture media inhibit the enzymatic DNA
amplification reaction. Additionally, the small PCR
reaction volumes usually cannot be accommodated to
large sample sizes dictated by low contamina-
tion levels (e.g. 2 5 g of product for Salmonella
presence/absence testing).
Another problem in common with other rapid
methods is the fact that PCR technology cannot dis-
tinguish between genetic material from dead and
living cells. Therefore an mRNA-based modification
of PCR (NASBA, nucleic acid sequence based
amplification) has been developed which is indicative
for living cells in the sample.
Routine application of PCR technology in food
microbiology usually requires special sample pre-
treatment (e.g. purification, cell concentration, cul-
turing or selective enrichment, immunomagnetic
separation) adapted to each specific sample matrix.
Fingerpyinting-like Methods Another group of
DNA assays result in the determination of DNA pat-
terns. These fingerprinting-like methods, for example
restriction fragment length polymorphism (RFLP)
Next Page
analysis or random amplification of polymorphic
DNA (RAPD), can be used for high-resolution char-
acterization of microorganisms from pure cultures.
They may gradually replace traditional serotyping
systems. Such techniques may be useful for elucidating
the contamination routes of pathogens in the food
chain.
Combined Methods
Undoubtedly, the next major step will be the com-
bination of advances from different technologies. One
very promising example of combined methods is dis-
cussed here.
Biosensors Biosensors are a relatively new area in
the automated food microbiology. They consist of
immobilized, biologically sensitive material (e.g.
enzymes, antibodies, antigens, nucleic acids) coupled
with or in close proximity to a receiving transducer
unit which gives an electrical, optical or thermal signal
when the sensor reacts with its target. The intensity
of the signal is proportional to the concentration of
the target.
Due to problems with long-term stability, reus-
ability and sterilizability, biosensors have so far been
mostly used for detecting chemical substances. Never-
theless, their future potential is enormous, since they
can offer a very sensitive and accurate ‘on-line’ control
system for food manufacturing processes.
See also: ATP Bioluminescence: Application in Meat
Industry; Application in Dairy Industry; Application in
Hygiene Monitoring; Application in Beverage Micro-
biology. Biochemical and Modern Identification
Techniques: Introduction; Food Spoilage Flora (i.e.
Yeasts and Moulds): Food-poisoning Organisms;
Enterobacteriaceae, Coliforms and E. coli; Microfloras
of Fermented Foods. Direct (and Indirect)
ConductimetricAmpedimetric Techniques: Food-
borne Pathogens. Direct Epifluorescent Filter Tech-
niques (DEFT). Electrical Techniques: Introduction;
Food Spoilage Flora and Total Viable Count (TVC); Lactics
and other Bacteria. Enzyme Immunoassays: Overview.
Flow Cytometry. Hazard Appraisal (HACCP): The
Overall Concept; Critical Control Points; Involvement of
Regulatory Bodies; Establishment of Performance Cri-
teria. lmmunomagnetic Particle-based Techniques:
Overview. Molecular Biology - in Microbiological
Analysis. Nucleic Acid-based Assays: Overview.
PCR-based Commercial Tests for Pathogens. Pre-
dictive Microbiology & Food Safety. Total Counts:
Microscopy. Total Viable Counts: Metabolic Activity
Tests; Microscopy.
 SACCHAROMYCESIlntroduction 1907

Contents
Introduction
Saccharomyces sake
Saccharomyces cerevisiae
Saccharomyces: Brewers’ Yeast

Introduction properties seems to cause loss of sporulation ability

Yuji Oda, Department of Applied Biological Science,
Fukuyama University, Hiroshima, Japan
Kozo Ouchi, Kyowa Hakko Kogyo Co. Ltd, Tokyo,
Japan
Copyright 0 1999 Academic Press
Yeasts are classified into three groups: asco-
sporogenous yeasts, basidiosporogenous yeasts and
imperfect yeasts. Saccharomyces is the representative
of ascosporogenous yeasts and historically the most
familiar microorganism for human. This genus was
first described by Meyen when he assigned beer yeast
as S. cerevisiae in 1838, and redefined by Reess in
1870 from the observations of ascospores and their
germination. The name is derived from the Greek
words sakcharon (sugar) and mykes (fungus). The
number of species has changed according to the cri-
teria used to delimit species, and 16 species are now
accepted in the genus Saccharomyces (Table 1 j.
in these strains.
The most famous physiological characteristic of Sac-
charomyces spp. is their capacity for vigorous anaer-
obic or semianaerobic fermentation of one or more
sugars to produce ethanol and COz. These sugars
include D-glucose, D-fructose and D-mannose, except
in the case of certain mutants. Most strains of Sac-
charomyces can grow on D-galactose under aerobic or
anaerobic conditions; however, none of them utilizes
lactose, pentose, alditols and citrate as carbon sources,
assimilates nitrate as a nitrogen source or hydrolyses
exogenous urea. Among polysaccharides, starch and
pectin are exceptionally utilized by certain strains of S.
cerevisiae. They do not produce starch-like com-
pounds. Their ubiquinone is exclusively Q-6, but this
feature is common in the genera Kluyveromyces
Torulaspora and Zygosaccharomyces.
Table 1 The species accepted in the genus Saccharomyces
Species Authority
~

S. barnettii Vaughan-Martini 1995

Characteristics of the Genus S. bayanus Saccardo 1895
S. castellii Capriotti 1966
The vegetative cells of Saccharomyces species are S. cerevisiae Meyen ex E. C. Hansen 1883
round, oval or cylindrical and reproduce by multi- S. dairenensis Naganishi 1917
lateral budding. They may form pseudohyphae but S. exiguus Reess ex E. C. Hansen 1888
not septate hyphae. These yeasts are predominantly S. kluyveri Phaff, M. W. Miller and Shifrine 1956
S. kunashirensis James, Cai, Roberts and Collins 1997
diploid or occasionally of higher ploidy. Asci, which S. martiniae James, Cai, Roberts and Collins 1997
are persistent and usually transformed by direct S. paradoxus Bachinskaya 1914
change from the vegetative cells, may contain one to S. pastorianus E. C. Hansen 1904
four ascospores. The ascospores are round or slightly S. rosinii Vaughan-Martini, Barcaccia and Pollacci
oval, with smooth walls. Conjugation occurs on or 1996
S. servazzii Capriotti 1967
soon after germination of the ascospores. Some strains S. spencerorurn Vaughan-Martini 1995
of S. cerevisiae and its related species used in the S. transvaalensis van der Walt 1956
brewing and baking industries hardly form ascospores S. unisporus Jorgensen 1909
at all; continuous selection with respect to practical
1908 SACCHAROMYC€S/lntroduction

Table 2 Discriminating characteristics of species of the genus Saccharomyces

S. cerevisiae
S. bayanus
S. paradoxus
S. pastorianus
S. barnettii
S. castellii
S. dairenensis
S. exiguus
S. kunashirensis
S. martiniae
S. rosinii
S. servazzii
S. spencerorum
S. transvaalensis
S. unisporus
S. kluyveri
Reactions: +, positive; -, negative; v, variable

Identification of Saccharomyces Species be 1.OO,1.15 and 1.46, respectively. Saccharomyces

paradoxus is exclusively isolated from natural sources
Yeasts are usually classified by the characteristics of
such as tree exudates, soil and Drosophila. Cells of S.
microscopic appearance, sexual reproduction and
paradoxus are small in size and readily form asci
physiological features including (1)fermentation of
compared with the other three species. Effective sep-
certain sugars semianaerobically; (2) assimilation of
aration of the Saccharomyces sensu stricto species is
various compounds each as sole carbon or nitrogen
complicated because these species often have appar-
source; ( 3 ) growth without an exogenous supply of
ently identical morphological, physiological and sero-
certain vitamins; (4) growth in the presence of 50%
or 60% (w/w)glucose; ( 5 )growth at 37°C; (6)growth logical properties. The four species have been
in the presence of cycloheximide; ( 7 )splitting of fat, differentiated from each other by DNA reassociation
production of starch-like polysaccharides, hydrolysis study (Fig. 1). Strains with 80-100% overall
of urea; and ( 8 ) formation of acid. homology of base sequences are considered as belong-
The 16 species now accepted in the genus Sac- ing to the same species, while the strains of distantly
charomyces are divided into three groups: Sac- related taxa show homology of less than 30%. Among
charomyces sensu stricto, Saccharomyces sensu lato, the four species, S. pastorianus reveals 53 % homology
and S. kluyveri. Table 2 compares the differences of to S. cerevisiae and 72% homology to S. bayanus,
physiological properties of the Saccharomyces species.
Saccharomyces sensu stricto
Saccharomyces sensu stricto species including S. cer-
evisiae, S. bayanus, S. paradoxus and S. pastorianus
are phylogenetically closely related in the genus Sac-
charomyces. The species S. cerevisiae, S. bayanus and
S. pastorianus are specifically found in the artificial
environments of wineries and breweries. The relative Figure 1 DNA relatedness among the type strains of Sac-
genome sizes of these three species are estimated to charomyces sensu stricto species.

suggesting an intermediate position between two
unrelated species, S. cerevisiae and S. bayanus.
Since species division within Saccharomyces sensu
stricto group were clarified on the molecular level,
it became possible to determine those physiological
responses necessary for the separation of the four
taxa. Saccharomyces bayanus is the only species of
the genus that can grow in the absence of vitamins.
Maximum growth temperature immediately dis-
tinguishes S. bayanus and S. pastorianus, which never
grow at above 35"C, from S. cerevisiae and S. para-
doxus, which grow at 37°C and often at up to 40-
42°C. An active fructose transport system is present
in the group of S. bayanus and S. pastorianus, while
absent in S. cerevisiae and S. paradoxus. Sac-
charomyces cerevisiae is distinguished from s. para-
doxus with respect to assimilation of D-mannitol and
fermentation of maltose.
Saccharomyces sensu lato
Saccharomyces servazzii and S. unisporus are unusual
members of the genus as judged from narrow fer-
mentative profiles and ability to grow in the presence
of 0.1 % cycloheximide. Assimilation of ethylamine,
cadaverine and lysine can differentiate S. unisporus
from S. servazzii.
Saccharomyces exiguus together with S. servazzii
and S. unisporus is characterized by much lower G+C
values (34.7-36.6%) than other Saccharomyces
species (39.3-41.9%0), but do not grow in the presence
of 0.1 Y
' O cycloheximide. Saccharomyces barnettii and
S. spencerorum have been separated from S. exiguus
and its anamorph, Candida holmii, on the basis of
DNA relatedness. Separation of these three taxa is
confirmed by conventional taxonomic tests. Sac-
charomyces spencerorum obviously differs from S.
exiguus in the assimilation of glycerol as a sole carbon
source and ethylamine, cadaverine and lysine as sole
nitrogen sources.
Saccharomyces castellii, S. dairenensis and S. rosinii
have not been distinguished by conventional physio-
logical criteria although they represent separate taxa
based on DNA relatedness. Physiological profiles of
S. dairenensis and S. transvaalensis are similar, but S.
transvaalensis has characteristics of larger vegetative
cells and large refringent ascospores. The latter species
shows intermediate levels of DNA homology to the
type strains of S. dairenensis and S. castellii. Com-
bination of physiological patterns and electrophoretic
karyotypes permits with some difficulty the separation
of most of these four species. Saccharomyces dai-
renensis is still a heterogenous taxon including at least
two unrelated groups since three strains show less
than 32% homology to the type strain of S. dai-
renensis.
SACCHAROMYC€S/lntroduction 1909
Saccharomyces kunashirensis and S. martiniae are
recently defined from the analysis of 18s ribosomal
RNA (rRNA) gene sequences.
Saccharomyces kluyveri
Saccharomyces kluyveri is easily distinguishable from
other species of the genus since it is characterized by
a wide assimilative and fermentative profile including
the ability to utilize ethylamine-HC1, cadaverine and
lysine as sole nitrogen sources for growth as well as
the ability to both assimilate and ferment melibiose.
The distinct character was already anticipated by
molecular taxonomic studies which showed no nucle-
otide homology between S. k l u y e r i with either Sac-
charomyces sensu stricto or sensu lato strains, where
DNA homology values were never above 22%.
Molecular Methods to Differentiate
Species
The conventional taxonomic tests to assess physio-
logical features are fundamental for identification, but
the results have shown to be insufficient for species
delimitation and discrimination of interstrain vari-
ability. The genetic basis behind many of these char-
acteristics is often either poorly understood or
unknown. In the past DNA reassociation study has
significantly contributed to molecular taxonomy of
the genus Saccharomyces: this method was used to
reestablish several species names, reduce other names
to synonyms, describe new species, and raise the like-
lihood of the existence of additional species. However,
the equipment used to measure DNA association is
highly specialized and expensive and the amount of
data obtainable in an average week's work is relatively
small. Discrimination of the closely related species
has been confirmed by other molecular techniques,
such as whole-cell protein patterns, multilocus
enzyme electrophoresis, fructose transport system,
mitochondrial DNA restriction analysis, electro-
phoretic karyotypes, random amplified polymorphic
DNA polymerase chain reaction (RAPD-PCR) and
restriction fragment length polymorphism (RFLP)
patterns, and rRNA gene sequencing. These methods
are valid additions to the conventional taxonomic
tests, and those applicable to the food industry are
described below.
Electrophoretic Karyotypes
Chromosomal patterns resolved by pulse field gel elec-
trophoresis are called electrophoretic karyotypes. By
comparing the results from this method and DNA
reassociation, it has been demonstrated that elec-
trophoretic karyotypes of the two strains are identical
when DNA sequence homology is over 85%, while
1910 SACCHAROMYC€S/Introduction
Figure 2 Electrophoretic karyotypes of Saccharomyces
species. Chromosomal DNA of type strains was separated in
0.8% agarose gel in 0.5 x TBE (45 mmol I-' Tris-borate, pH 7.3,
1 mol I-' EDTA) at 125 V and 14°C. Pulse times were 3 min for 24
h followed by 5 min for 16 h. Lanes: 1, S. cerevisiae; 2, S. bayanus;
3, S. paradoxus; 4, S. pastorianus; 5, S. barnettii; 6, S. castellii;
7, S. dairenensis; 8, S. exiguus; 9, S. kunashirensis; 10, S. mar-
tiniae; l l , s. rosinii; 12, s. servazzii; 13, s. spencerorum; 14, s.
transvaalensis; 15, S. unisporus; 16, S. kluyveri.
low DNA relatedness corresponds to completely dif-
ferent chromosomal patterns. Since similar but not
identical karyotypes are not interpreted as either dif-
ferent species or polymorphisms in the same species,
karyotyping is not so reliable as DNA base sequence
comparisons, but is undoubtedly an important
adjunct. Electrophoretic karyotypes can serve as a
rapid, inexpensive and relatively easy first approach
for the evaluation of a group of physiologically similar
strains.
The general feature for ascosporogenous yeasts is
the presence of one to five bands of chromosomal
DNA larger than 1000 kb as in S. kluyveri (Fig. 2),
whereas in most Saccharomyces species, chromo-
somes smaller than 1000 kb are observed. Chromo-
somes of Saccharomyces sensu stricto species were
resolved into 12 to 16 bands in the range 200 kb to
2200 kb. None of the other species contains chromo-
somes smaller than 300kb. The patterns of Sac-
charomyces sensu stricto species are similar, and are
distinguishable from the other species at a glance. A
multivariate analysis of the polymorphisms in the
numbers and molecular weights of chromosomes has
revealed that the Saccharomyces sensu stricto strains
could be separated into four clusters that correspond
to the four species.
-
RAPD PCR
Analysis by RAPD-PCR involves the use of small
random primers and low stringency primer annealing
conditions to amplify arbitrary fragments of template
DNA. The single primer will anneal at any point on
the genome where a near-complementary sequence
exists, and if two priming sites are sufficiently close,
then PCR amplifies the fragment between them. A
number of fragments of various sizes may be pro-
duced; formed patterns are specific for the particular
DNA template used. This technique is suitable for
typing and identification of microorganisms, but
several problems are present. First, the whole patterns
of electrophoresis are not always the same in inde-
pendent experiments, and only the reproducible bands
should be scored. Second, the results are affected by
the nucleotide sequence of the primer used.
After the PCR products have been resolved, genetic
distance is calculated manually as the number of dif-
ferent bands between two patterns divided by the sum
of all bands in the same patterns. A value of 0 indicates
that the two strains had identical patterns, and a value
of 1 indicates that the two strains had completely
different patterns. The dice matrix obtained from
these data is used to construct an unrooted den-
drogram.
Ribosomal RNA Gene Analysis
The analysis of rRNA genes, which can elicit exact
data without pairing two samples, is one of the prom-
ising methods among these tools applied to the phylo-
genetic study of yeast. In S. cerevisiae, the co-
transcribed genes for small (17S-l8S), 5.8.5, and large
(25s-28s) rRNA and 5s rRNA genes occur as tan-
demly repeated units on chromosome XII. Sequence
comparisons of the rRNA genes have shown a rela-
tively high degree of evolutionary conservation and
have been used as bases for inferring phylogenetic
relationships. Almost the complete 18s rRNA gene
sequence of Saccharomyces species has been
sequenced and their relationships investigated in
detail, but the entire region of 18s rRNA is not simply
and rapidly determined by anyone.
The region spanning the internal transcribed
spacers (ITSs) and the 5.8s rRNA gene entire is
amplified by PCR using PITS1 (5'-
TCCGTAGGTGAACCTGCGG-3') and pITS4 (5'-
TCCTCCGCTTATTGATATG-3 '), which are derived
from conserved regions of the 1 8 s and 28s rRNA
genes, respectively. The size is over 800bp for
Saccharomyces sensu stricto species and S. trans-
vaalensis, and less than 800bp for the other Sac-
charomyces species (Fig. 3). Furthermore, restriction
analysis of ITS region allows the separation of S.
cerevisiae, S. bayanus and S. pastorianus. These may
be useful methods to identify Saccharomyces isolates.
 SACCHAROMYC€S/lntroduction 1911

Figure 3 Size of ITS regions amplified from the type strains of Saccharomyces species. Each lane number corresponds to thatof
the strain in Figure 2.

Detection, Isolation and Cultivation laid on the agar plate and incubated for 2-3 h a t 30"C,
the colour of the colony changes to pink or red.
Composition of media for yeasts is shown in Table 3. Selective isolation of yeasts and estimation of viable
The presence of yeasts in food is usually investigated cell number require special techniques to repress the
using nutrient media such as YM agar which is prin- growth of bacteria and fungi. The use of acidified
cipally composed of yeast extract and malt extract. agar (< pH 5.0) is adequate for isolation of yeasts from
Potato-dextrose agar is suitable for storage of cul- samples containing bacteria. When acidified medium
tures, but not satisfactory for detection because each alone is inadequate to eliminate bacterial growth,
species develops a less characteristic colony on this chloramphenicol dissolved in ethanol (20 mg m1-I) is
medium. Colonies of Saccharomyces and some species added at a final concentration of 50 pgml-l. Unlike
of Hansenula and Pichia, which ferment glucose vig- other antibiotics that depress bacterial growth, chlor-
orously, are simply discriminated on the agar plate: amphenicol can be added to the medium before auto-
when medium containing 0.5% glucose, 0.05% 2,3,5- claving. Addition of either 0.2% sodium propionate
triphenyltetrazolium chloride and 1.5% agar is over- or 2-3 YOethanol to an acidic medium has only limited
effect on prolonging the growth of fungi. However, it
Table 3 Composition of culture media
is possible to isolate yeasts selectively by the difference
in growth rates in the presence of sodium propionate.
Medium Contents Percentage Saccharomyces sensu stricto species frequently
WIV
appear after enrichment in YM broth in which glucose
YM Yeast extract 0.3 was added at 10-20%. The addition of a suitable
Malt extract 0.3 indicator such as bromphenol blue permits the moni-
Peptone 0.5
Glucose 1
toring of changes in p H and periodic adjustments if
Agar (if required) 2 desired. Glucose and other components should be
YPD Yeast extract 1 autoclaved separately to avoid browning. Less than
Peptone 2 0.1 g of sample is added to 10 ml of the modified
Glucose 2 YM broth containing chloramphenicol and sodium
Agar (if required) 2
Potato-dextrose agar Potato extracta 23 (volume)
propionate in a test tube. After static incubation for
Glucose 2 the desired period, about 0.1 ml obtained from near
Agar 2 the bottom is transferred to another medium. The
YNB glucose Yeast nitrogen base 0.67 fermentation rate can be followed by recording the
without amino acidsb weight loss of the medium. This procedure is repeated
Glucoseb 2
Agar (if required) 2
several times to enrich yeasts fermenting glucose vig-
Fowell's acetate agar Sodium acetate 0.5 orously. The culture broth is successively diluted and
Agar 2 spread on a plate of acidified YM agar.
McClary's acetate agar Glucose 0.1 For cultivation, Saccharomyces yeasts are generally
Potassium chloride 0.18 grown on YPD medium (see Table 3) rather than YM
Yeast extract 0.25
Sodium acetate 0.82
medium, at 25-30°C. The most common system for
Agar I .5 aerobic growth uses liquid medium in an Ehlenmeyer
Gorodkowa agar Glucose 0.1 flask on an orbital shaker. Shaking at 150-200 r.p.m.
(modified) Peptone 1 provides efficient aeration for 100 ml volume of
Sodium chloride 0.5 medium in a 300ml flask. The growth rate varies
Agar 2
Malt extract agar Malt extract 5
depending on strain, medium and incubation tem-
Agar 3 perature, typically within the range of 90 min to 3 h
per doubling time. The cell concentration can be
a The filtrate autoclaved for 1 h at 120°C after washed, peeled

and finely grated potato (100 g) is soaked Tn 300 ml tap water

determined using an electronic particle counter or
for several hours in a refrigerator and filtered through cloth. a haemocytometer and microscope. Alternatively, a
Tenfold concentrated solution is filter-sterilized and added. spectrophotometer can be used to measure turbidity

1912 SACCHAROMYCESllntroduction
at 600 nm. Typically, a yeast culture in a logarithmic
phase of growth of 0.1 will contain about 2 x l o 6
cells.
Ascospores are induced on the sporulation media,
most of which have been developed for Sac-
charomyces species. Young cells grown on YM agar
for 2-3 days are spread on Fowell’s or McClary’s agar
based on sodium acetate, Gorodkowa agar or malt
extract agar, and incubated at least 4-6 weeks. Freshly
isolated cells sporulate on the isolation medium and
ascospores can be observed after cultivation for about
1 month, while the cells cultured on the nutrient
medium many times require certain sporulation media
to convert asci.
Importance to the Food Industry
The genus Saccharomyces is the most extensively util-
ized group of yeasts for the benefit of humans. Sac-
charomyces cerevisiae and related species are
employed in three main processes of the food industry
(Table 4). The first is the production of industrial
alcohol and alcoholic beverages, including wine, beer,
saki. and potable spirits. Saccharomyces pastorianus
including S. carlsbergensis was initially recognized as
a lager strain. Saccharomyces bayanus has been
mostly associated with the wine industry. Second is
the baking industry; originally, spent yeasts from the
brewing and distilling industries were used for baking,
but became insufficient as the baking industry
expanded. Yeasts for dough leavening are now propa-
gated to meet these growing needs. The third process
includes the production of biomass, extracts, auto-
lysates and flavouring compounds. The yeast used in
such processes can be either purpose-grown or a by-
product.
Saccharomyces exiguus and its anamorph, Candida
holmii, are the predominant yeasts responsible for the
leavening of sourdough which is usually prepared by
adding a commercially produced culture containing
lactic acid bacteria.
No other species of Saccharomyces is of commercial
importance, although some strains of Torulaspora
and Zygosaccharomyces spp., formerly accepted in
the genus Saccharomyces, are used for baking and
production of miso and shoyu, respectively.
Saccharomyces species are found in many foods
and sometimes cause spoilage. Wild strains which
contaminate the pure culture reduce the fermentation
Table 4 Application of Saccharomyces species in the food
industry
1. Industrial alcohol and alcohol beverages
2. Bakery products
3. Biomass, extracts, autolysates and flavouring compounds
Next Page
rate and diminish the quality of final products in
the brewing process. Killer wild yeasts will dominate
within a short period when inoculated strains are
killer-sensitive. In saki. brewing, killer sake strains
were constructed by the methods of back-crossing
and cytoduction to overcome these problems.
Most species of the genus Saccharomyces have ‘gen-
erally recognized as safe’ (GRAS) status from the
fact that many strains have been applied to the food
industry. There is no report of disease in healthy
humans and other warm-blooded animals caused by
Saccharomyces sensu stricto species.
See color Plates 28 and 30.
See also: Saccharomyces: Saccharomyces sake: Sac-
charomyces cerevisiae; Saccharomyces carlsbergensis
(Brewer’s Yeast).
Further Reading
Barnett JA (1992) The taxonomy of the genus Sac-
charomyces Meyen ex Reess: a short review for non-
taxonomists. Yeast 8: 1-23.
Barnett JA, Payne RW and Yarrow D (1990) Yeasts: Char-
acterization and Identification, 2nd edn. Cambridge:
Cambridge University Press.
Benitez T, Gasent-Ramirez JM, Castrejon F and Codon AC
(1996)Development of new strains for the food industry.
Biotechnol. Prog. 12: 149-163.
Evans IH (1996) Yeast Protocols. Methods in Molecular
Biology 52. Ottawa: Humana Press.
James SA, Cai J, Roberts IN and Collins MD (1997) A
phylogenetic analysis of the genus Saccharomyces based
on 18s rRNA gene sequences: description of Sac-
charomyces kunashirensis sp. nov. and Saccharomyces
martiniae sp. nov. International Journal of Systematic
Bacteriology 47: 453-460.
Kurtzman CP (1994) Molecular taxonomy of the yeasts.
Yeast 10: 1727-1740.
Kurtzman CP and Fell J W (1997) The Yeast - a Taxonomic
Study, 4th edn. Amsterdam: Elsevier.
Panchal CJ (1990) Yeast Strain Selection. New York: Marcel
Dekker.
Reed G and Nagodawithana T W (1991) Yeast Technology,
2nd edn. New York: Van Nostrand Reinhold.
Rose AH and Harrison JS (1987) The Yeast, 2nd edn. Vol.
1, Biology of Yeasts. London: Academic Press.
Rose AH and Harrison JS (1993) The Yeasts, 2nd edn. Vol.
5, Yeast Technology. London: Academic Press.
Russell I, Jones R and Stewart GG (1987) Yeast - the
primary industrial microorganism. In: Stewart GG,
Russell I, Klein RD and Hiebsch RR (eds) Biological
Research on Industrial Yeasts. Vol. 1 , p. 1. Boca Raton:
CRC Press.
Spencer JFT and Spencer D M (1990) Yeast Technology.
Berlin: Springer.
Spencer JFT and Spencer D M (1997) Yeast - in Natural
and Artificial Habitats. Berlin: Springer.
Vaughan-Martini A and Barcaccia S (1996) A recon-

THERMUS AQUATICUS
C K K Nair, Radiation Biology Division, Bhabha Atomic Research Centre, Mumbai, India
Copyright 0 1999 Academic Press
The bacteria belonging to the genus Thermus are
aerobic, thermophilic, Gram-negative and rod-
shaped, and have attracted considerable attention
because of the biotechnological potential of their ther-
mostable enzymes, particularly their DNA poly-
merases which constitute the most important
component of the polymerase chain reaction. These
bacteria are isolated from neutral to alkaline hot
springs of various geographical locations in the world.
Thermus aquaticus was first discovered by Thomas
D. Brock in the 1960s during a long-term study of
microbial life in hot springs of Yellowstone National
Park in Wyoming, USA, as a pigmented rod-shaped
Gram-negative bacteria capable of growing at tem-
peratures as high as 79°C. After the discovery of this
extremophile, search for microbes in hot springs and
in environments around deep sea hydrothermal vents
(called smokers, which are natural undersea chimneys
through which super-heated mineral-rich fluid as hot
as 350°C escapes) led to the isolation of a number of
species belonging to the genus Thermus. Some of these
hyperthermophiles are listed in Table I.The cell walls
of the genus Thermus has a characteristic peptide-
glycan composition.
Some of the species have distinct features such as
halotolerance (e.g. T.thermophilus),non-pigment for-
mation (e.g. T. scotoductus), and filamentous nature
Table 1 Different species of hyperthermophiles belonging to
the genus Therrnus and their site of isolation
/solate Site of isolation
Thermus aquaticus Yellowstone National Park, USA
T: brockianus Yellowstone National Park, USA
T filiformis Rotorea, New Zealand; Takaanu, New
Zealand
T: thermophilus Mine, Japan: Izu-Atagawa, Japan; Hruni,
Iceland; Savusavu beach, Fiji; Stream
Is S. Miguel, Azores
T: scotoductus Selfoss, Iceland: Bloomington, USA;
London, UK: Calasde Vizele, Portugal
T: oshimai S. Pedrodosal, Portugal: Hveresarli
Hengill, Iceland
THERMUS AQUATICUS 2139
(T. filiformis). However, the classification of the
various species of the genus Thermus has been pri-
marily based on whole genome DNA : DNA hybrid-
ization, as most biochemical and taxonomic
parameters were found unsuitable. Recently, molecu-
lar biology techniques involving characterization of
genomic macro-restriction fragment length poly-
morphism (RFLP) using pulsed-field gel electro-
phoresis (PFGE)and characterization of rDNA genes,
ribotyping by restriction fragment length poly-
morphism, have been used in taxonomic studies and
classification. Among the various species, Thermus
aquaticus is the most widely studied and well
characterized.
Characterization of Thermus aquaticus
A number of strains of T. aquaticus have been isolated
from different thermal springs. This organism has also
been isolated from artificial environments such as hot
tap water in geographical locations quite distant from
thermal springs.
T aquaticus is a Gram-negative, non-sporulating,
and non-motile rod of 0.5-0.8 pm diameter and 5.0-
10.0 pm length. It forms long filaments of 20.0 pm
or greater in stationary phase or at supra-optimal
temperatures. The rods occur singly or in aggregates,
by individual rods joining end to end or side to side.
Large spheres of 10-20pm in diameter are often
formed in old cultures. Deoxyribonucleic acid base
composition of T. aquaticus is 65.4-67.4 mol% G+C.
The growth of this bacteria is inhibited by fairly low
concentrations of cycloserine, streptomycin, peni-
cillin, novobiocin, tetracycline and chloramphenicol.
The growth of this organism is also inhibited by
100 pg ml-' sodium lauryl sulphate, 500 pg ml-'
sodium azide, 200 pg ml-' oxgall or 2 % NaCI. Nutri-
tional studies show that T aquaticus does not require
vitamins or amino acids although the growth is con-
siderably faster in enriched medium than in synthetic
basal salts medium (Table 2). With 0.1-0.33% tryp-
tone and yeast extract in basal medium, the organism
21 40 THERMUS AQUATICUS
Table 2 Media used for the isolation of Thermus aquaticus
Basal salts medium Nitrilotriacetic acid
Micronutrient solution
FeCI, solution
CaS04.2H20
MgS04.7H20
NaCl
KN03
NaN0,
Na2HP04
Dissolve in 1 I distilled water, adjust to pH 8.2 with NaOH.
Micronutrient solution H2S0,, conc.
MnS04.H,O
ZnS04.7H20
H803
CuS04.5H20
Na2Mo04.2H20
CoCI2.6HPO
Dissolve in 1 I distilled water
FeCI, solution FeCI,
Dissolve in 1 I distilled water
grows well but at 1% tryptone and yeast extract there
was inhibition of growth. The bacterial growth was
good in 1%vitamin-free casein hydrolysate and 0.5%
glutamic acid, as sole source of nitrogen and carbon.
Ammonium as source of nitrogen and with acetate,
succinate, sucrose, glucose or fructose as source of
carbon supports the growth of this bacterium. T.
aquaticus is an obligate aerobe; it has a p H optimum
of 7.5-7.8 and does not grow below p H 6.0 and above
pH9.5. The optimum temperature for growth is
70"C, with a maximum at 79°C and minimum at
40°C. The generation time of this organism under
optimum conditions is about 50 min. At temperatures
75°C and above the organism exhibits filamentous
growth. Filamentous forms are also observed in sta-
tionary cultures after growth at 65-70°C. In older
cultures large spherical bodies are often formed due
to extrusion of the protoplasts or long rods or fila-
ments from one of the poles, and also filaments with
swollen ends can be seen frequently in these cultures.
The rod-shaped forms have a tendency to aggregate
either as linear arrays or as rosettes. The aggregation
phenomenon is due to the presence of a slime on the
surface of the organism.
Isolation and Enrichment
Thermus aquaticus can be isolated from soil or water
from hyperthermal environments. Basal salts medium
(Table 2) with 0.1% tryptone and 0.1% yeast extract
in cap tubes is inoculated with the samples and incu-
bated unshaken at 75°C for 1-2 days.
The growth of the bacterium is indicated by visible
turbidity. When the turbidity is heavy the colour of
the bacterial mass turns yellow or orange. Under the
0.1 g
0.5 ml
1.O ml
0.06 g
0.1 g
0.008 g
0.103g
0.689 g
0.1 11 g
0.5 ml
2.28 g
0.5 g
0.5 g
0.025 g
0.025 g
0.0459
0.2905 g
microscope the organism appears as rods, large fila-
ments and/or large spheres. As this organism is a non-
spore-former, cultures with spores are discarded. Pure
cultures can be isolated by streaking on enrichment
plates containing the same medium solidified with 3%
agar. The plates are sealed with Saran Wraps and
incubated for 1-2 days at 70°C to obtain yellow-
orange colonies. These colonies are re-streaked and
stock cultures are prepared as agar slants from the
isolated colonies of the second plate. For longer
storage the isolates are preserved by freeze-drying.
Biochemical Tests
Acid without gas is produced when T. aquaticus is
grown on rich media with glucose, fructose, mannose,
sucrose or maltose. Xylose is not utilized by the organ-
ism. T. aquaticus coagulates Brom Cresol Purple
(BCP)-containing milk with slight acid formation and
liquifies gelatin. Potassium nitrate is weakly reduced
to nitrite and formation of ammonia is weakly
positive.
Biotechnological Value of Thermus
aquaticus
In the past 15-20 years the considerable value of
enzymes produced by thermophilic organisms includ-
ing T. aquaticus has been realized. Although their
ability to survive and moreover flourish in envir-
onments that can exceed 95°C suggests a robust set
of enzymes, not all of them are thermostable. Likewise
not all enzymes recovered from mesophiles are ther-
molabile. The intrinsic properties that make a given
enzyme thermostable are not fully understood. Efforts

to classify enzymes based on structural motifs as well
as other features including numbers of salt bridges,
surface-to-volume ratio and disulphide linkages have
had only limited success. Given the evolutionary
diversity between enzymes isolated from thermophiles
and mesophiles, it is difficult to correlate any par-
ticular amino acid difference to thermostability.
Nevertheless, in general, thermophilic organisms tend
to be a valuable source of thermostable enzymes. T.
aquaticus is perhaps one of the best studied ther-
mophiles or extremophiles and has been the source of
a number of valuable enzymes. Thermostable enzymes
are useful in a number of biotechnological processes
since they typically have faster catalytic rates, they
can be operated at high temperatures where con-
tamination is suppressed and they have a longer half-
life which makes them less expensive to use. In other
applications, thermostable enzymes are a dis-
advantage as they cannot be inactivated and they can
continue to affect the food product well after the
optimal product composition is reached.
One of the most cited examples of a process
whereby the inclusion of a thermostable enzyme dra-
matically improved the process is the use of the T.
aquaticus DNA polymerase in the polymerase chain
reaction (PCR).Although the original versions of PCR
were accomplished using thermolabile Escherichia
coli DNA polymerase, the process was compromised
by the need to add enzyme after each step and the
build up of inactive enzyme. The T. aquaticus DNA
polymerase eliminated the need to add enzyme after
each step. PCR is described in detail elsewhere and its
application for the detection of food-borne pathogens
holds the promise of revolutionizing food safety
testing.
In addition to the T. aquaticus DNA polymerase,
other enzymes from this organism have significantly
improved a host of other biotechnological appli-
cations. For example, T. aquaticus DNA ligase is used
in the ligase chain reaction (LCR). LCR is an amp-
lification-based process in which a pair of adjacent
oligonucleotides in a set of diametrically opposed
oligonucleotide primer pairs are joined in a series of
sequential ligation steps. The adjacent primers are
only joined when the nucleotide at the junction
matches the targeted 3' end of the upstream primer.
LCR is therefore diagnostic for that nucleotide and
has been used to format assays to discriminate
between two targets that differ only in a single nucle-
otide. Another T aquaticus enzyme of value is TaqI,
a restriction endonuclease that shares thermostable
properties along with other DNA modifying enzymes
from this organism. Aqualysin is a thermostable serine
protease isolated from T. aquaticus and may have
applications in food processing where its high heat
Next Page
THERMUS AQUATICUS 2141
stability is valuable. Other thermostable catalytic
enzymes of potential value from T. aquaticus are
superoxide dismutase and fumarase. Finally, a P-gal-
actosidase has been isolated which may have value to
hydrolyse lactose as well as being used for synthetic
reactions to create oligosaccharides.
Regulation
In addition to hot springs, this bacterium has also
been found in hot water taps and other thermal niches.
Thus this organism may be considered as a predictor
for thermal pollution. There have been no reports of
illness due to T. aquaticus and no toxic material or
poison has so far been isolated from this organism.
See also: PCR-based Commercial Tests for Patho-
gens.
Further Reading
Bej AK, Mahbubani M H and Atlas R M (1991) Amp-
lification of nucleic acids by polymerase chain reaction
(PCRj and other methods and their applications. Critical
Reviews in Biochemistry and Molecular Biology 26:
301-334.
Brock TD (1967) Life at high temperature. Science 197:
1012-101 9.
Brock TD and Freeze H (1969) Thermus aquaticus gen.
n. and sp. n, a non-sporulating extreme thermophile.
Journal of Bacteriology 98: 289-297.
da Costa MS, Duarte JC and Williams RAD (edsj (1989)
Microbiology of the Extreme Environments and Its
Potential for Biotechnology. London: Elsevier Applied
Sciences.
Madigan M T and Marrs BL (1997) Extremophiles. Sci-
entific American April 66-71.
Moreira LM, da Costa MS and Correia IS (1997) Com-
parative genomic analysis of isolates belonging to the
six species of the genus Thermus using pulse field gel
electrophoresis and ribotyping. Archives of Micro-
biology 168: 92-101.
Saiki T, Kimura R and Arima K (1972) Isolation and char-
acterization of extremely thermophilic bacteria from hot
springs. Agricultural and Biological Chemistry 36:
235 7-23 6 6.
Sato S, Hutchinson 111, CA and Harris JI (1997) A thermo-
stable sequence specific endonuclease from Thermus
aquaticus. Proceedings of the National Academy of Sci-
ences USA 74: 542-546.
Williams RAD and da Costa M S (1992) The Genus
Thermus and Related Microorganisms. In: Balows A,
Truper HG, Dworkin M, Harder W and Schleifer K H
(eds) The Prokaryotes, 2nd edn. P. 3745. New York:
Springer.
Williams RAD, Smith KE, Welch SG and Micallef J (1996)
Thermus oshimai sp. nov., isolated from hot springs in
 ULTRASONIC IMAGING/Non-destructive Methods to Detect Sterility of Aseptic Packages 2195
I UHT Treatments see Heat Treatment of Foods: Ultra-high Temperature (UHT) Treatments.
ULTRASONIC IMAGING
Non-destructive Methods to Detect Sterility of
Aseptic Packages
Laura Raaska and Tiina Mattila-Sandholm, VTT Biotechnology and Food Research, Finland
Copyright 0 1999 Academic Press
Introduction
The safety criteria for aseptic foods are very important
because or the long shelf life and unrestricted storage
conditions of these foods. Microorganisms in asep-
tically processed food cause quality problems either
by spoiling the product or by increasing the possible
health risk. Aseptic products must be absolutely free
of microorganisms, including their spores. When mar-
keting ultra-high temperature (UHT) treated prod-
ucts, to ensure an acceptably low percentage of
unsterile units it is necessary to check an appropriate
number of packed samples for sterility from every lot.
A sampling rate of about 1% is generally recom-
mended when samples are evaluated for their micro-
biological safety and sensory quality. However, the
system of destructive sterility testing by sampling cur-
rently in use by foodstuff producers does not guar-
antee consumer safety and causes large losses of
ready-to-use food. At the commissioning stage of a
new production line, tens of thousands of samples are
tested by destructive microbiological methods, which
are both uneconomical and a burden on the envir-
onment. Checking every single food container and its
product content in a non-destructive way is expected
to increase consumer safety and avoid losses of food-
stuffs and packaging materials.
Traditionally, microbial quality control methods
have focused on assessing specific food-borne patho-
gens. A wide range of kits and instruments are now
commercially available for the detection of specific
pathogens such as Clostridium botulinum, Sal-
monella, Listeria and Escherichia coli. However, in
order to check the safety of commercial sterile prod-
I
ucts, methods that detect the growth of any micro-
organism are needed. To date, there are only two
commercial non-invasive methods on the market. The
Electester (TuomoHalonen Oy, Toijala, Finland)
assesses viscosity changes in the product by first oscil-
lating the product and then measuring the pattern of
the subsequent damping of the induced motion in the
fluid. Tap Tone (Benthos Inc., North Falmouth, MA,
USA) uses an electric field to create a tone, by inducing
vibrations in the aluminium foil of the package; the
amplitude of the tone changes if, for example, gas is
produced in a package without a head space. These
indirect methods are often appropriate for detecting
a number of different microorganisms, but as micro-
organisms produce different effects, the methods must
be checked using a wide variety of microorganisms in
order to establish the extent of their applicability.
Increasing consumer demand has accentuated the
need for rapid, non-destructive on-line measurements
of food quality. The ideal method, in addition to being
non-destructive, should be nonspecific, to ensure that
all types of microbial growth are detected. Preferably
the method should measure factors that can be dir-
ectly attributed to any organic growth in the product.
A method with high sensitivity means that the neces-
sary incubation time can be significantly less than that
needed for standard microbial cultivation. Fur-
thermore, in order to permit extensive testing the
method should be rapid. A non-destructive sterility
test method that shortens the incubation time, is more
reliable than the standard methods used today and
requires little labour input has great economic import-
ance for companies producing aseptic food products.
2196 ULTRASONIC IMAGING/Non-destructive Methods to Detect Sterility of Aseptic Packages
Ultrasound methods
Absorption 60-210 kHz 26 mm
Absorption 1-6 MHz 32 mm
Second harmonic 50 mm
Scattering 7-12 MHz 90 mm
Absorption 7-12 MHz 90 mm
Scattering 3-12 MHz 25 mm
Scattering 7-12 MHz 8 mm
Second harmonic 100 mm
Absorption 3-12 MHz 25 mm
Absorption 7-12 MHz 32 mm
Absorption 7-12 MHz 8 mm
Shearwave 0 5 MHz
Velocity burst 125 mm
Velocity spike 22 mm
Velocity spike 125 mm
0 2 4 6 8 1 0 1 2
Figure 1 The sensitivity of different ultrasound methods to detect contamination. The shaded bars show the number undetected
out of 12 samples; open bars are false positives. The ultrasound method, frequency and transducer distance shown are listed at the
left of the graphs. (A) Inoculum level < 700 cfu ml-‘ (B) Inoculum level < 10 cfu ml-’.
Potential Non-destructive Sterility Test
Methods
Contact Ultrasound Method
One interesting method for non-destructive testing is
ultrasound technology. Ultrasonics can be used in
food engineering for many purposes, for example to
measure the different physical properties of food-
stuffs. Its applicability to quality control of both fresh
and processed foodstuffs has been studied. The sta-
bility of reconstituted orange juice, the skin texture
of oranges, cracks in tomatoes and defects in husked
sweetcorn have been investigated using ultrasonics. It
is also possible to determine the presence of foreign
materials such as metal and bone particles in food
products with this method. Ultrasonic energy can be
used for non-destructive measurement of the thickness
of eggshells. It has also been shown that ultrasound
imaging can be used for non-destructive testing of the
microbiological quality of aseptic milk products.
The investigation was conducted with the primary
idea that any change in properties compared with
those of the controls is an indicator of a abnormality.
The ability of some contact ultrasound techniques to
detect contamination in UHT milk is shown in Figure
I . The sensitivity of the ultrasound methods was
greater with a high inoculum level than with a low
level. In addition, there were more false alarms when
a low inoculum level was used. The most sensitive
and accurate ultrasound techniques were second har-
monic generation, absorption at 60-210 kHz, absorp-
tion at 1-6MHz, and scattering at different
Ultrasound methods
Second harmonic 50 mm
B
Absorption 60-210 kHz 26 mm
Scattering 3-12 MHz 25 mm
Absorption 1-6 MHz 32 mm
Absorption 3 -1 2 MHz 25 mm
Scattering 7-12 MHz 90 mm
Absorption 7-12 MHz 90 mm
Scattering 7-12 MHz 90 mm
Second harmonic 100 mm
Absorption 7-12 MHz 32 mm
Absorption 7-12 MHz 8 mm
Velocity burst 125 mm
Shearwave 0 5 MHz
Velocity spike 125 mm
Velocity spike 22 mm
I , I ,
0 2 4 6 8 10 2
frequencies and transducer distances. The meas-
urements were clearly dependent on transducer dis-
tance and sound intensity. However, laser fluorescence
and shearwave could not distinguish between con-
taminated products and controls, and in the case of
velocity measurements, the number of undetected
samples was high both with low and high inoculum
levels. Repeatable and reliable ultrasound results were
also shown to be dependent on the spreading and
distribution of microbes in the package; shaking prior
to measurement was significant, especially in the case
of highly contaminated samples and more viscous
products like Nantua fish sauce.
The potential of second harmonic generation and
absorption was studied more thoroughly in an inves-
tigation using Tetrapak packages provided with ‘win-
dows’, and water as a contact medium. The contact
ultrasound measurement system was a semiautomatic
system where the sample was manually changed (Fig.
2). The measurement vessel housed the transducers
for the second harmonic measurement in addition to
the ones used for the absorption measurement. An
optical switch indicated when the package was pos-
itioned correctly. Small air bubbles which accu-
mulated on the ultrasound window were cleaned off
with brushes. Using this method, it was possible to
record both the absorption and the second harmonic
values at the same time. If only two ‘windows’ were
used it was necessary to turn the package through
180”. One package was measured at a time; the
change in absorption (1.1-5.6MHz) and the gen-
eration of second harmonics (1MHz -+2 MHz,
 ULTRASONIC IMAGING/Non-destructive Methods to Detect Sterility of Aseptic Packages 2197
Contact
medium
(bo)
Mat: PVC 20 mm
Bubble brush
Transducer
Sample SIDE VIEW
Alignment Optical switch
aroove
.. .
....
FRONT VIEW
Absorption Second
and width harmonic
Figure 2 The measurement vessel used for assessing ultra-
sound techniques.
3 MHz -+ 6 MHz) were measured; a powerful math-
ematical transform (partial least squares regression
analysis) was used to extract the characteristic fea-
tures from the data vector of the sample; and the
characteristic vectors used for statistical inference
and the samples differing from the controls were
identified.
Absorption and second harmonic measurements
were used to detect contaminated UHT milk pack-
ages. Measurements were performed through the
‘windows’ without breaking the package. All the con-
taminants could be detected after 24 h of incubation.
During that time the microbial counts in UHT milk
varied between 10’ colony forming units (cfu) per
millilitre and l o 6cfu ml-’. The detection threshold for
second harmonic generation was 5% and for absorp-
tion 1.5%. The difference between ultrasound meas-
urements of control and contaminated samples was
greatest in the case of E . coli and smallest in the case
of Pseudomonas fluorescens. Absorption appeared
to be a more stable but less sensitive measurement
technique than second harmonic generation.
Although second harmonic ( 3 MHz + 6 MHz) and
- 350r
.
E
- 3.30 - n
a
- 3 10 L
““I
m
2.70
2 50
2 30
2 10
1 2 3 4 5 6 7 8 9 101112131415
E col~ L plantarum Controls
Figure 3 Detection ability of second harmonic (3 MHz) of Esch-
erichia col/ (samples 1-5) and Lactobacillus plantarum (samples
6-1 0) contaminated UHT milk packages from controls (samples
11-1 5) after 24 h. The measurement values have been corrected
for the width of the package. The microbial levels were E. coli
6.9 log cfu ml-’, L. planfarum 6.5 log cfu ml-’.
absorption could distinguish between control and
contaminated packages, differences in their overall
response could be detected. The ability of second
harmonic ( 3 MHz) measurement to distinguish UHT
milk packages contaminated with E. coli or Lacto-
bacillus plantarum from control ones is shown in
Figure 3.
The results of using second harmonic and absorp-
tion methods to detect contaminated UHT milk and
Pursoup vegetable soup packages are presented in
Table 1 and Table 2. The packages were inoculated
with several important contaminants and changes in
ultrasound measurements compared to the controls
were followed for 4 days. During the first day the
measurements were performed 5-7 h after the inocu-
lation, when the microbial counts were lower than
10 cfuml-*. From these results, absorption seems to
be the most promising ultrasound measurement tech-
nique in detecting contamination in UHT milk. The
best discrimination was obtained after 72 h of incu-
bation when the microbial counts were lo5-
108cfuml-l. However, E. coli, L. plantarum and P.
fluorescens were detected 5-7 h after the inoculation.
Candida kefyr, Bacillus subtilis and Clostridium spo-
rogenes could be detected after 24 h of incubation
owing to their slow growth rate. Second harmonic
generation seems to be slightly better than absorption
for detecting contamination in Pursoup vegetable
soup. During the first incubation day 80% of the
contaminated Pursoup vegetable soup packages
would be detected by second harmonic generation. In
the case of Nantua fish sauce, the second harmonic
could detect only 20-40% and absorption 10% of
the contaminated packages.
Variation between replicated measurements was
slight but between samples was significant (see Fig.
3 ) . The reliability of ultrasound measurements was
2198 ULTRASONIC IMAGING/Non-destructive Methods to Detect Sterility of Aseptic Packages

Table 1 The probability (%) of second harmonic generation further improved by taking into consideration the
and absorption detecting contaminated UHT milk samples. The effects of parameters such as temperature, air bubbles,
measurement values have been corrected for the width of the
package. The detection threshold level for second harmonic was
amount of inoculum, changes in package width
5% and for absorption 1.5% during incubation and differences in package weight.
In particular, the effect of air bubbles in the package
2nd Harmonic 3+6 MHz
lncubation time windows and changes in package width after inocu-
lation and during incubation were shown to be
Microorganism 1 day 2 days 3 days 4 days
important error factors. The use of brushes removed
Escherichia coli 20 20 40 nd the air bubbles and also reduced the variation between
Lactobacillus plantarum 20 20 50 nd replicate measurements and samples. The width was
Pseudomonas fluorescens 40 40 40 nd
shown to vary between UHT milk packages and width
Candida kefyr nd 40 40 80
Bacillus subtilis nd 40 40 40 also changed after the inoculation because of the
Clostridium sporogenes nd 100 100 100 needlestick. The possibility of measuring the width
of the package using the same transducers used for
Probability (day) 30 40 50 70
interlocating the sample was investigated, and found
to be feasible with the transducer pair used in absorp-
tion measurement for UHT milk and Pursoup vege-
Absorption
lncubation time table soup. Nantua fish sauce, however, attenuates
Microorganism 1 day 2 days 3 days 4 days the signal too much for the pulse echo measurement
to be successful.
E. coli 40 80 90 nd
L. plantarum 60 40 100 nd Non-contact Ultrasound Method
i? fluorescens 80 80 80 nd
C. kefyr nd 100 60 100 The non-contact ultrasound method is based on the
5. subtilis nd 60 80 100 emission and reception of ultrasound by piezoelectric
C. sporogenes nd 100 100 100
transducers. This includes three steps:
Probability (day) 60 80 90 100
generation of ultrasound with pulsed lasers
nd, no data. detection of ultrasound with an interferometric
probe
data acquisition and signal processing.
Table 2 The probability (%) of second harmonic generation Generation of ultrasound with pulsed lasers is ach-
and absorption detecting contaminated Pursoup vegetable soup
samples. The measurement values have been corrected for the
ieved by electromagnetic radiation from the laser,
width of the package. The detection threshold level for second which is absorbed in the surface region of the sample,
harmonic was 5% and for absorption 1.5% causing heating; thermal energy then propagates into
2nd Harmonic 3-6 MHz
the specimen as thermal waves, the heated region
lncubation time undergoes thermal expansion, and thermoelastic
stresses generate elastic waves (ultrasound) which
Microorganism 1 day 2 days 3 days 4 days
propagate deep within the sample. The ultrasound
Escherichia coli nd nd nd nd waves are detected with an interferometric probe
Lactobacillus plantarum 80 80 60 80 which is designed as a high-resolution optical spec-
Pseudomonas fluorescens 100 80 100 100
Candida kefyr 100 40 100 100
trometer to detect changes in frequency of the scat-
Bacillus subtilis 60 100 100 80 tered or reflected light. This method, which is being
Clostridium sporogenes 80 100 100 80 developed in France by SFIM-ODS and Technogram
Probability (day) 80 80 90 90 in cooperation with Danone, has shown some prom-
Absorption ising results in detecting contamination in aseptic food
lncubation time products.
Microorganism 1 day 2 days 3 days 4 days
Calorimetric and Volumetric Methods
E. coli 60 60 60 60
L. plantarum 70 60 70 60 The calorimetric and volumetric methods were devel-
i? fluorescens 100 100 100 100 oped in the Netherlands at the Delft University of
C. kefyr 80 60 80 100
Technology, in cooperation with the Unilever
B. subtilis 20 40 20 40
C. sporogenes 60 100 80 20 Research Laboratorium. Metabolically active and
growing microorganisms consume energy and in turn
Probability (day) 70 60 70 60
generate small amounts of heat. This phenomenon is
nd, no data. used in the calorimetric method which detects small
 ULTRASONIC IMAGING/Non-destructive Methods to Detect Sterility of Aseptic Packages 21 99
Time (min)
Figure 4 Thermograms of different bacteria in UHT milk at
30°C. Squares, Escherichia coli; triangles, Salmonella arizonae;
circles, Bacillus cereus; crosses, S. cremoris H414.
temperature increases of a product caused by growing
microorganisms. The method uses a specially designed
calorimeter, smart temperature sensors and data pro-
cessing equipment. The calorimeter contains 100 cav-
ities for 1 litre packages. Each cavity is equipped
with a smart sensor. The system is able to follow
temperature changes of 100 packs simultaneously for
an indefinite period. The system is microprocessor-
controlled and is built in such a way that the influences
of environmental temperature changes are minimized.
The temperature changes in the products tested in this
system are sensed using ‘smart’ sensor chips that are
in direct contact with the package material of the
product. With current technology, the heat pro-
duction of certain organisms can be detected in the
same time period as that needed for the destructive
test method based on ATP bioluminescence. However,
not all microorganisms produce enough heat during
their growth cycle to be detected. In Figure 4, ther-
mograms of some bacteria growing in UHT milk are
shown.
In practice the implementation of the calorimetric
and the volumetric methods is very similar, and by
combining the two, the advantages of both non-
specificity and sensitivity can be achieved. A prototype
has been developed for simultaneous volume-tem-
perature monitoring of two 1 litre Tetrapaks. Relative
temperature changes of less than 10 mK and relative
volume changes of less than 0.3 ml (0.03%) can reli-
ably be distinguished. These results are achieved by
ensuring a good thermal insulation and by applying
smart sensor interfacing and smart data processing.
This method has been shown to be very attractive
for automated, non-destructive sterility testing of a
number of food packs under laboratory conditions.
It is not applicable for intensive, 100% sterility testing
of the production lot, because the continuous testing
procedure could last from a few hours up to a few
days.
lmpedimetric Method
For intensive, non-destructive sterility testing of
100% of the production lot a new impedance method
has been studied in the Netherlands. The main
problem to be solved in measuring the impedance of
aseptically packaged food is how to pass an electric
signal through the package. Most food containers are
designed to prevent any contact between the food and
the surrounding environment to ensure the highest
food quality for the longest time possible. An inter-
mediate aluminium foil layer acts as a Faraday’s cage
and does not allow electromagnetic fields to penetrate
it. The small change in packaging technology required
to apply the impedance method is already feasible and
prototypes of new packages with one small electrode,
fixed on the inside surface of the packaging material
and reachable from outside, are being tested. The
inside electrode can be in galvanic contact with the
food or it can be isolated from the food by a thin
thermoplastic layer. As a second electrode, the alu-
minium foil itself is used. In the impedance method,
the changes in conductance and capacitance of the
food are measured. The changes in impedance depend
on the number of ions moving in the liquid - cations
moving to the negatively charged electrode and anions
to the positively charged electrode. The increase in
conductance and capacitance caused by the metabolic
activity of the microbes leads to a decrease in the
impedance.
It appears that non-invasive sterility testing of the
whole production lot guarantees high quality of the
food immediately after it is produced, but not a few
months or a year later, when it may actually be con-
sumed. For such a guarantee, intensive quality check-
ing of the package itself is also needed. For aseptically
packed foods, this check principally focuses on the
inner thermoplastic layer of the packaging laminate,
since any possible leakage in this layer may result in
the contents reaching the barrier layer (aluminium
foil) or the fibre layer, at which time the other barrier
properties of the laminate are lost, even if no actual
liquid leakage occurs through the laminate. This pro-
cedure is destructive, time-consuming and makes sig-
nificant demands on reliability and costs. At the same
time, it does not ensure individual consumer safety as
only a very small part of the production lot is tested.
The impedance method is easily applicable for sim-
ultaneous sterility testing and leakage detection. For
this purpose, the small-surface electrode has to be in
galvanic contact with the food. ‘Internal’ leakage has
been simulated by making a hole in the thermoplastic
layer, ensuring direct contact between the foil and the
liquid. What is observed in case of leakage is an
increase of the impedance components Rz and Cx that
2200 ULTRASONIC IMAGING/Non-destructive Methods to Detect Sterility of Aseptic Packages

100000~
and rapidity (permits extensive on-line measurement)
(Table 3). Changes in physical parameters are meas-
10000
R ured by ultrasound imaging, using Doppler tech-
niques as well as contact and non-contact ultrasound
methods. The impedance method detects changes in
the conductance and capacitance of the food. The
drawback of these non-destructive methods is that
the presence of microorganisms is not detected dir-
ectly but rather by a secondary parameter which
changes with the presence and growth of the micro-
organism. These can be viscosity changes in the
product or gas production by the microorganism.
(4 Frequency (kHz) However, there are indications that these methods
are also sensitive to physical parameters other than

look
120

*OM
viscosity changes or gas production. Nevertheless, the
effects of different microorganisms on the properties
of liquid food products vary considerably and little
is known about how different microbes change the
physical properties of liquid food products; research
in this area is certainly needed.
The smart temperature sensor method, which meas-
ures the minute temperature increases produced by
growing microbes, is the only method that directly
measures microbial growth. However, the meas-
“7 r 0 r r N m m d
0 0 0 0 0 0 0 0 0
urements must be carried out before and during the
4 : ; ; : ; ; y ;
I I + +
W
+ + + + +
exponential growth phase of the microbe, which
(6) Frequency (kHz) makes the assessment time long and uncertain. In
Figure 5 The increase of R, (A) and C, (B)results in decrease
addition, not all microbes produce detectable
of the phase shift of the measured impedance w = arcfg amounts of heat during their exponential growth
(llwCR,Cr) at low frequency, which also can be used as an phase.
indication of leakage. R,, resistance; C,, capacitance. Delft Uni- Several promising non-destructive sterility test
versity of Technology, Netherlands. methods have been developed and studied at the
laboratory scale. Most of these methods presuppose
is easily detectable at frequencies below 10 kHz (Fig. some modifications in packaging technology.
5). Research has shown, however, the potential for new
industrial scale test methods. Optimization and on-
Conclusions line measurement tests at industrial scale are needed to
verify the potential and applicability of these methods.
The marketing study demonstrated the need to
develop new non-invasive methods for detecting the
Acknowledgements
growth of microorganisms and the spoilage of prod-
ucts; although new methods have been investigated Non-destructive sterility testing methods have been
and prototypes developed, none of these methods has investigated in an international European project
yet succeeded in meeting the three ‘ideal’ criteria of called Endtest ‘Development of non-destructive ster-
nonspecificity (detects growth of any microorganism), ility testing equipment for aseptic products’. The
high sensitivity (needs only a short incubation time) project involved research institutes as well as com-

Table 3 Methods for non-invasive sterility contol in aseptically packaged foods

Method Type of changes registered Nonspecificity Sensitivity Rapidity
Ultrasonic imaging Physical structures ++ ++ ++
Ultrasonic Doppler Viscosity, physical structures ++ ++ ++
Contact ultrasound Physical structures ++ ++ +++
Smart temperature sensors Temperature +++ +++ +
Impedance Electrical impedance +++ ++ +++
Key: +, low; ++, medium; +++, high.
 ULTRASONIC IMAGING/Non-destructive Methods to Detect Sterility of Aseptic Packages 2201

panies from the Netherlands (Delft University of Tech- (1994)Methods for noninvasive sterility control in asep-
nology, Unilever Research Laboratorium, Laboratory tically packaged foods. Trends in Food Science and Tech-
of Celsis-Lumac), Finland (Process Flow Ltd, VTT nology 5 : 379-383.
Biotechnology and Food ~ ~ university
~ of Haeggstrom
~ ~ E (1997)
~ Ultrasound
~ detection
h of microbe
, con-
Helsinki), Sweden (Tetra pak Research & Devel- tamination in premade food. Acta Polytechnica Scan-
dinavica, Applied Physics Series 214: 115.
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Haus HA and Melcher JR (1989) Electromagnetic Fields
Danone, INRA, CRSA). and EnerRy. P. 260. Prentice Hall: New -jersey.
.
See also: ATP Bioluminescence: Application in Dairy Javanaud C-i1988) Applications of ultrasound to food
systems. Ultrasonics 26: 117-123.
Industry. Heat Treatment of Foods: Ultra-high Tem-
Margulies TS and Schwarz W H (1994) A multiphase con-
perature (UHT) Treatments. Packaging of Foods: Pack-
tinuum theory for sound wave propagation through
aging of Solids and Liquids. dilute suspensions of particles. Journal o f the Acoustic
Society of America 96: 319-331.
Meijer GC, Kerkvliet H M M and Toth FN (1994) Non-
Further Reading invasive detection of micro-organisms using smart tem-
perature sensors. Sensors Actuators B Chemical 18: 276-
Ahvenainen R, Mattila T and Wirtanen G (1989) Ultra-
281.
sound penetration through different packaging mater-
Nihtianov SN (1996) Method for measuring the con-
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ductivity of fluids. Patent Application 96201096.3-
packaged UHT milk. Lebensm. Wissensch. Technol. 22:
2204, April 24 1996.
26 8-2 72.
Nihtianov SN and Meijer GC (1995) Non-invasive impe-
Ahvenainen R, Wirtanen G and Manninen M (1989) Ultra-
sound imaging - a non-destructive method for moni- dimetric sterility testing of aseptically packed food prod-
toring the microbiological quality of aseptically-packed ucts. Proceedings o f the Anniversary Scientific
milk products. Lebensm. Wissensch. Technol. 22: 382- Conference: ‘Fifty Years Technical University - Sofia’,
386. Fourth Edition of the National Scientific Conference
Ahvenainen R, Wirtanen G and Mattila-Sandholm T (1991) ‘Electronic Engineering’, Sozopol 1995. Vol. 1, p. 52.
Ultrasound imaging - a nondestructive method for moni- Nihtianov SN, Meijer GCM, Kerkvliet H and Demeijer E
toring changes caused by microbial enzymes in asep- (1996) New methods for non-destructive sterility testing
tically-packed milk and soft ice-cream base material. of aseptically packed food products. Proceedings of the
Lebensm. Wissensch. Technol. 24: 397-403. 1996 National Sensor Conference. 20-21 March 1996,
Dubois M, Enguehard F and Bertrand L (1994) Analytical Delft, the Netherlands. P. 139.
one dimensional model to study the ultrasonic precursor Nihtianov SN, Kerkvliet H M M and Meijer GCM (1996)
generated by a laser. Physical Review E 50: 1548-1551. Non-invasive sterility-testing device of aseptically
Gastagnede B, Deschamps M, Mottay E and Mourad A packed food products by simultaneous volume-tem-
(1994) Laser impact generation of ultrasound in com- perature monitoring. Fifth Edition of the National Sci-
posite materials. Acta Acoustica 2: 83-93. entific and Applied-Science Conference, Electronics ET
Gestrelius H (1994) Ultrasonic Doppler: a possible method ’96, Sozopol, Bulgaria, 27-29 September 1996.
for noninvasive sterility control. Food Control 5: 103- Pless P, Futschik K and Schopf E (1994) Rapid detection
105. of salmonellae by means of a new impedance-splitting
Gestrelius H (1996) Aseptic packaging of food - non- method. Journal of Food Protection 5715):369-376.
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International Conference ASEPT - Food Safety ’96, 4- parameter in liquid media using focused ultrasound.
6 June 1996, Lava], France. P. 321. Journal of the Acoustic Society of America 93: 162-172.
Gestrelius H, Hertz TG, Nuamu M, Persson H W and Lind- Wirtanen G, Ahvenainen R and Mattila-Sandholm T (1992)
strom K (1993)A nondestructive ultrasound method for Nondestructive detection of spoilage of aseptically-
microbial quality control of aseptically packaged milk. packed milk products: effect of frequency and imaging
Lebensm. Wissensch. Technol. 26: 334-339. parameters on the sensitivity of ultrasound imaging.
Gestrelius H, Mattila-Sandholm T and Ahvenainen R Lebensm. Wissensch. Technol. 25: 126-132.

VAGOCOCCUS
Lucia Martins Teixeira and Maria da Gloria S Carvalho, Departamento de Microbiologia Medica, lnstituto de
Microbiologia, Universidade Federal do Rio de Janeiro, Brazil
Richard R Facklam, Streptococcus Laboratory, Respiratory Diseases Branch, Division of Bacterial and Mycotic
Diseases, Centers for Disease Control and Prevention, Atlanta, USA
Copyright 0 1999 Academic Press
Introduction
The genus Vagococcus was established as a separate
genus in 1989, to accommodate the motile cocci
resembling lactococci, which were earlier referred to
as motile lactic streptococci and were shown to be
diverse from all known lactococci. Results of 16s
rRNA sequencing studies demonstrated that such
strains formed a separate line of descent within the
lactic acid bacteria and represented a new species,
which was named V. fluvialis. A subsequent molecular
taxonomic investigation resulted in the description of
a second species, in 1990, named b! salmoninarum.
Although distinct, the genus Vagococcus has a close
phylogenetic relationship with the genera Entero-
coccus and Lactococcus, and some species are difficult
to differentiate solely on the basis of phenotypic char-
acteristics. The significance of vagococci as agents of
infections and their presence in food products of
animal origin is still unclear. The scarcity of reports
may be due, at least in part, to their recent recognition
as a separate taxon.
Description of the Genus
The members of genus Vagococcus (wandering
coccus) are facultatively anaerobic, catalase-negative
Gram-positive cocci. Cells occur singly or arranged
in pairs or as short chains. The colony morphology
resembles that of enterococcal and streptococcal
strains. Colonies are raised and grey-white and they
are a- or non-haemolytic on agar media containing
sheep blood. They have fermentative metabolism,
with L-lactic acid being the predominant end product
of glucose fermentation. They can react with Lance-
field streptococcal groups D and N antisera. The cell
wall peptidoglycan type is LYS-D-ASP. The DNA G+C
content ranges from 33.6 to 36.5 mol%. Two species
of Vagococcus have been described: V. fluvialis and V.
VAGOCOCCUS 2215
salmoninarum. The type strain is V. fluvialis ATCC
49515 (NCFB 2497). The type strain for I? sal-
moninarum is ATCC 51200 (NCFB 2777).
The physiological tests for differentiating the vag-
ococci and other catalase-negative Gram-positive
cocci are listed in Table I.The presumptive iden-
tification of a Vagococcus can be accomplished by
demonstrating that the strain hydrolyses esculin in the
presence of bile (bile-aesculin-test-positive), produces
leucine aminopeptidase and pyrrolidonyl arylamidase
(LAP and PYR test-positive), and is susceptible to
vancomycin. Growth at 10°C, 45"C, and growth in
broth containing 6.5% NaCl can be variable. Motility
can also vary (only V. fluvialis strains are usually
motile). The vagococci are non-pigmented, and are
negative for production of gas, and arginine and hip-
purate hydrolysis. Delayed or weak reactions with
anti-streptococcal group D serum as well as reaction
with group N antiserum can be observed with some
vagococcal strains when Lancefield hot-acid cell
extracts are tested by the capillary precipitation
method.
The difficulty in distinguishing these micro-
organisms,.~especially V. fluvialis, from other lactic
I
acid bacteria by phenotypic criteria is widely recog-
nized. O n the basis of phenotypic characteristics, most
isolates are initially classified as unidentified or atyp-
ical enterococci, because they resembled some of the
less common atypical arginine-negative enterococcal
species. Results of motility and arginine hydrolysis
tests can be helpful in the differentiation from the
lactococci, which usually give negative and positive
reactions, respectively. All Vagococcus strains tested
to date reacted with the AccuProbe Enterococcus
culture confirmation test manufactured by Gen-Probe
(San Diego, CA, US). This probe is based on a DNA
oligomer having a structure complementary to a
segment of enterococcal rRNA. Although testing with
221 6 VAGOCOCCUS

Table 1 Phenotypic characteristics of facultatively anaerobic, catalase-negative, Gram-positive coccus genera

Phenotypic characteristic
Growth at:
Genus VAN GAS PYR LAP BE NaCl 70°C 45°C MOT HEM
Chains
Vagococcus S + aln
Enterococcus SC + alpln
Lactococcus S + aln
Leuconostoc R aln
Weissella R aln
Abiotrophia S + a
Streptococcus S -d alpln
Globicatella S + a
Clusters and tetrads
Pediococcus a
Tetragenococcus CY
Aerococcus a
Gemella aln
Helcococcus n
Alloiococcus' n
Dolosigranulum n
Facklamia n
lgnavigranum + a
VAN, susceptibility to vancomycin (30 pg disc); GAS, production of gas from glucose in Lactobacillus Mann, Rogosa, Sharpe (MRS)
broth; PYR, production of pyrrolidonyl arylamidase; LAP, production of leucine aminopeptidase; BE, reaction on bile-aesculin medium;
NaCI, growth in broth containing 6.5% NaCI; MOT, motility: HEM, haemolysis on blood agar containing 5% sheep blood; a , a-
haemolysis; p, p-haemolysis; n, no haemolysis; s, susceptible; R, resistant; -, 295% of strains with negative results; +, b95% of
strains with positive results; V, variable reactions (some strains positive, some negative).
astrainsare generally positive after long incubation (5 days or more).
bSome strains grow slowly at 45°C.
"Some enterococcal strains are vancomycin-resistant but still show a small inhibition around the disc; other strains grow right up
to the disc and are vancomycin-resistant under the defined screening test criteria.
dGroupA streptococci are PYR-positive and all other streptococci are PYR-negative.
'Streptococcus bovis and S. equinus are bile-esculin-positive as well as 5-1 0% of the viridans streptococci.
'Alloiococcus strains grow anaerobically only under defined conditions.

the enterococcal genetic probe does not allow dif-
ferentiation between the enterococci and the vag-
ococci, it can also be used as a tool to separate the
vagococci from the lactococci, which are negative.
Characterization of the Species
Confirmation that a strain is a Vagococcus requires
complete identification the species level, It is gen-
erally accomplished by using a series of additional
conventional physiological tests. In addition to the
tests included in Table 1, the following tests are
usually included: hydrolysis of arginine, hydrolysis of
hippurate, pigment production, pyruvate utilization,
tellurite tolerance, the Voges-Proskauer reaction, and
acid production from L-arabinose, methyl-a-D-gluco-
pyranoside, glycerol, inulin, lactose, maltose, D-man-
nitol, melibiose, raffinose, ribose, D-sorbitol, sorbose,
sucrose and trehalose.
As already pointed out, even using extensive testing,
the differentiation of vagococcal strains from entero-
cocci is sometimes problematic. Tests for production
of acid from L-arabinose and raffinose may be useful,
since V% fluvialis strains are negative and the motile
Enterococcus species, E. gallinarum and E . cas-
seliflavus, are positive. The arginine test may also be
a clue for such differentiation as most strains belong-
ing to these enterococcal species are positive.
However, uncommon arginine-negative variants of
the Physiological group 11enterococcal species and E .
columbae have biochemical characteristics which are
very similar to those of the vagOCOCCi, especially lf#
fluvialis. Table 2 lists some of the tests that can be
used to differentiate between them.
Both vagococcal species V. fluvialis and sal-
moninarum share several phenotypic characteristics.
They usually produce acids from maltose, ribose and
trehalose, and are negative for the following tests:
arginine, arabinose, inulin, lactose, melibiose and
sorbose. Variable results are obtained for pyruvate
utilization, tellurite tolerance and Voges-Proskauer
tests. They can be distinguished on the basis of a
few phenotypic characteristics, such as motility, and
production of acids from mannitol, raffinose and
sorbitol (Table 2). Tests for production of acids from

Table 2 Phenotypic characteristics used to differentiate between arginine-negative variants of physiological group II enterococcal
species, Enterococcus columbae and species of Vagococcus
Phenotypic characteristic
Species MAN SOR ARG ARA SBL RAF
E. faecalis + - - - + -
E. casseliflavus + - - + V +
E. gallinarum + - - + - +
E. columbae + - - + + +
V: fluvialis + - - - + -
!L salmoninarum - - - - - +
MAN, mannitol; SOR, sorbose; ARG, arginine; ARA, arabinose; SBL, sorbitol; RAF, raffinose; TEL, tellurite; MOT, motility; PIG,
pigment; SUC, sucrose: PYU, pyruvate; MGP, methyl-a-o-glucopyranoside; GLY, glycerol; - , 3 95% of strains with negative
results; +, 3 95% of strains with positive results; V, variable reactions (some strains positive, some negative).
"Occasional exceDtions occur.
sucrose and glycerol, as well as pyruvate utilization
and tellurite tolerance are also of some help. In the
original description of V. fluvialis this microorganism
was reported to be LAP-negative, PYR-variable and
negative for growth in 6.5% NaCl broth, but all
strains tested at the Centers for Disease Control have
been both LAP- and PYR-positive and have dem-
onstrated growth in 6.5% NaCl broth.
In addition to the determination of physiological
characteristics, analysis of electrophoretic whole-cell
protein profiles was shown to be a reliable method
for the identification of vagococcal strains. V. fluvialis
isolates correspond to a unique protein profile that is
distinct from the protein profile characteristics of V.
salmoninarum strains, enterococcal and lactococcal
species. Genus- and species-specific oligonucleotide
probes derived from 1 6 s rRNA were also shown to
facilitate the precise identification of vagococcal
isolates.
Data on the antimicrobial susceptibility char-
acteristics are only available for a few V. fluvialis
isolates. Results of minimum inhibitory concentration
determinations indicated that V. fluvialis strains are
susceptible to ampicillin, cefotaxime, trimethoprim-
sulphamethoxazole and vancomycin, and are resistant
to clindamycin, lomefloxacin and ofloxacin. Strain-
to-strain variation was observed in relation to 17
other antimicrobial agents tested, including cefaclor,
cefazolin, cefixime, ceftriaxone, cefuroxime, chlor-
amphenicol, ciprofloxacin, clarithromycin, erythro-
mycin, gentamicin, meropenem, oxacillin, penicillin,
piperacillin-tazobactam, rifampin, tetracycline and
tobramycin.
Molecular characterization of V. fluvialis strains,
using analysis of chromosomal DNA restriction pat-
terns by pulsed-field gel electrophoresis (PFGE) after
digestion with SmaI, resulted in distinctive PFGE pat-
terns, suggesting the nonclonal nature of V. fluvialis,
and indicating the potential ability of this typing tech-
nique to discriminate between Vagococcus isolates.
VAGOCOCCUS 2217
TEL MOT PIG SUC PYU MGP GLY
+ - - +a + - +
V +a +a + V + V
- += - + - + -a
- - - + + - -
-a + - +a v + +
- - - + + + -
~ ~~~~
Importance of the Genus in Animal and
Human Diseases: Importance for the
Food Industry and Potential Hazard for
the Consumer
As the members of the genus Vagococcus were only
recently distinguished from other related bacteria, the
involvement of the vagococci as infectious agents has
been possibly hindered by difficulties in their precise
identification, as they may have been misidentified or
overlooked in diagnostic laboratories. Also, the role
of Vagococcus in food spoilage, decreasing its shelf
life, as well as the production of metabolic compounds
important for the food industry, is still unclear.
The pathogenic role of these microorganisms was
first recognized when they were identified among the
agents associated with a complex of similar fish dis-
eases known under the general denomination of strep-
tococcosis, caused by different taxons of Gram-
positive cocci. Such complex of diseases has long been
considered a serious problem in cultured marine fish
in the Far East. Nowadays, with the development of
intensive aquaculture, fish infections caused by Gram-
positive cocci, including vagococcosis, have become a
major problem in various parts of the world, affecting
different fish species destined for human con-
sumption. Heavy economic losses caused by these
infections are in some cases estimated to encompass
45% of total fish production. On the other hand,
except for reports of the isolation of V. salmoninarum
from diseased salmonid fish (brown trout, rainbow
trout and Atlantic salmon) and of V. fluvialis from
domestic animals (cats, horses and pigs), information
on the significance of the vagococci as pathogens
followed by proper identification of the micro-
organism is still very limited.
More recently, a report from our laboratories pro-
vided the first evidence of the possible connection of
a vagococcal species as a cause of infections in human
beings. Very little clinical information was submitted
2218 VAGOCOCCUS
with the cultures identified. One strain was isolated
from the peritoneal fluid of a nephrology patient, and
another from an infected bite wound in a person who
was bitten by a lamb. Two other human isolates were
recovered from blood cultures, and no additional
information on the clinical condition or associated
illness was available. Two additional strains have been
received for identification. One was isolated from a
finger wound of a patient in Canada, and the other
was recovered from the cerebrospinal fluid of a patient
with meningitis in Argentina. All vagococcal strains
isolated from humans until now are V fluvialis. No
human infection associated with V salmoninarum has
been documented.
Apart from the economic impact, concerns have
been generated about the potential health hazard that
handling or consumption of colonized or infected
fish, as well as other animals or their products can
represent for humans. The presence of lactic acid
bacteria, including vagococci, in foodstuffs, is not
usually considered to be a major concern, because
they are widely distributed in nature. Therefore, they
are not considered to be significant pathogens of
humans, as they are probably only associated with
opportunistic infections. However, recent evidence of
acquisition of serious infections by people who had
skin injuries during handling of Streptococcus iniae
(another agent of fish streptococcosis that was not
considered pathogenic for humans) -colonized or dis-
eased fish grown by aquaculture, raises the question
on the potential health hazard. During the inves-
tigation of S. iniae transmission from farm-cultured
fish (Tilapia)to humans, many V fluvialis strains were
isolated from the surface of the fish. It is not known
whether these isolates had any effect on the fish or
whether they could be transmitted to humans. One
additional example suggesting the possibility of
animals or their food products as sources of trans-
mission of infections, due to Gram-positive cocci, to
humans, is given by Lactococcus garvieae, another
agent of fish streptococcosis that has also been shown
to cause disease in cattle and in humans.
In the light of the above-mentioned, the vagococci
should be considered to be among the emerging zoo-
notic pathogens that have been isolated from various
species of fish, as well as mammals, and finally
humans. Diagnostic laboratories as well as those
devoted to the analysis of food products of fish and
other animal origin, especially fresh produces, must
be aware of the methods for the precise detection of
this microorganisms, as they may serve as vehicles
for transmission of infections caused by this newly
recognized pathogen. As more attention and accurate
procedures are incorporated into the identification
schemes to detect and characterize vagococcal strains
in the diagnostic setting, more information will
become available to help in answering the many ques-
tions raised about the significance of these micro-
organisms.
Suggested Laboratory Procedures for
Isolation and Identification
Recommendations for Isolation and Preliminary
Testing
Enriched infusion agar and broth, such as trypticase
soy, heart infusion, Todd-Hewitt, Lactobacillus
Mann, Rogosa, and Sharpe (MRS) or brain-heart
infusion, support the growth of vagococci and 5%
sheep blood agar plates are recommended to verify
haemolytic activity. If the specimen to be processed
for primary isolation is likely to contain other bac-
teria, such as food samples, bile-aesculin medium may
be an option as a primary isolation-selective medium.
Vagococci growth is better when incubated for 18-
2 4 h in 3-10% COz atmosphere. Special attention
must be paid to the temperature requirements for
optimal growth of each vagococcal species: V. fluvialis
cultures should be incubated at 35-37°C and V sal-
moninarum cultures at 25°C. The most consistent
Gram stains can be prepared from growth in thio-
glycolate broth. The catalase test should be performed
by flooding the growth of the bacteria on a blood-
free medium with 3 % hydrogen peroxide and observ-
ing for bubbling (positive reaction) or not (negative
reaction).
Bile-aesculin Test The bile-aesculin medium can be
used in agar slants or agar plates. Inoculate the bile-
esculin medium with one to three colonies and incu-
bate it at normal atmosphere for up to 7 days. A
positive reaction is recorded when a black colour
forms over one-half or more of the slant, or when any
blackening occurs on the agar plate. N o change in the
colour of the agar indicates a negative reaction.
NaCl Tolerance Test Growth in broth containing
6.5% NaCl is determined in heart infusion broth base
with an addition of 6% NaCl (heart infusion base
contains 0.5% NaCl), 0.5% glucose and bromcresol
purple indicator. When a frank growth occurs, the
glucose is fermented and the broth colour changes
from purple to yellow, but it is not necessary for a
positive result: an obvious increase in turbidity
without a change in colour is also considered to be a
positive test. One or two colonies or a drop of an
overnight broth culture is inoculated into the broth
containing 6.5% NaC1. The inoculated broth is incu-
bated up to 7 days.
 VAGOCOCCUS 221 9

LAP and PYR Tests The LAP and PYR disc tests are carbohydrate is determined in heart infusion broth
performed in the same manner, and the discs are containing the specific carbohydrate (1% ) and brom-
available from several commercial suppliers. The cresol purple indicator (0.0016%).The carbohydrate
strains to be tested are grown overnight on blood agar broth is inoculated with a drop or a loopful of an
plates. The discs are placed on the blood agar plate in overnight broth culture or with several colonies taken
an area of little or no growth. The discs are inoculated from a blood agar plate. The inoculum should be
heavily; two or more loopfuls of inoculum are neces- from a fresh culture. The carbohydrate broth is incu-
sary for satisfactory results. The plates with the discs bated for up to 7 days. A positive reaction is recorded
are incubated at room temperature for 10 min, the when the broth turns yellow.
detection reagent is added, and the reactions are read
after 3 min. The development of a red colour is posi- Gas Production Production of gas from glucose is
tive; no change in colour or a yellow colour is nega- determined in Lactobacillus MRS broth. Two or more
tive; and the development of a pink colour indicates colonies from a blood agar plate or a drop of broth
a weak positive reaction. The test should be discarded culture are used to inoculate the broth. The inoculated
after 10 min if still negative. tube is overlaid with melted petroleum jelly and incu-
bated for up to 7 days. Gas production is indicated
Tests for Growth at 10 and 45°C Growth at 10 when the wax plug is completely separated from the
and 45°C is determined in heart infusion broth base broth. Small bubbles that may accumulate over the
medium containing 0.1% dextrose and bromcresol incubation period are not read as positive.
purple indicator. The tests are performed by inocu-
lating the broths with a single colony or a drop of an Arginine Deamination The deamination of arginine
overnight broth culture and incubating at the respect- is determined in Moeller decarboxylase medium con-
ive temperatures for up to 7 days. The time between taining 1% L-arginine. The medium is inoculated with
inoculation and placement at the proper temperature a fresh culture and is then overlaid with sterile mineral
should not be longer than 10min. When the test oil (1-2ml per 5 m l of arginine medium) and incu-
cultures are inspected for growth during the incu- bated for up to 7 days. A positive reaction is recorded
bation period, the tubes should be returned to the when the broth turns deep purple, indicating an alka-
proper temperature without being allowed to warm line reaction. A yellow colour or no colour change of
or cool. A positive result is indicated by frank growth, the broth indicates a negative reaction.
which may be accompanied by a colour change in
the indicator. A colour change is not necessary to Motility Test Motility is determined in modified
determine a positive reaction; an increase in turbidity Difco motility medium. The medium is prepared by
indicates growth and a positive test. Be sure to rotate adding 16 g of motility test medium (Difco), 4 g of
the tube vigorously after the incubation period. Some nutrient broth powder (Difco), and 1g of NaCl to 11
bacterial strains have a tendency to settle to the of distilled water. The medium is inoculated with a
bottom of the tube, and turbidity will not be apparent single stab (with an inoculating needle, not a loop)
until the contents of the tubes are mixed. about 1in (2.54 cm) into the centre of the medium in
the tube. The inoculated tube is placed in a 25-30°C
Vancomycin Susceptibility Identification Test To incubator. Motility is indicated by the spread of
determine susceptibility to vancomycin, several col- growth to the bottom and sides of the tube. Growth
onies of the strain are transferred to one-half of a along and slightly away from the stab indicates nega-
trypticase soy agar plate containing 5 % sheep blood tive motility.
and spread with a loop or cotton swab to achieve
confluent growth. The vancomycin susceptibility Pigment Production Test Production of pigment is
testing disc (30 pg) is placed in the heavy part of the determined by examining a cotton swab that has been
streak. The inoculated plate is incubated at 5% C 0 2 smeared across growth on a trypticase soy-S% sheep
atmosphere for 18 h. Strains with any zone of growth blood agar plate that has been incubated for 24 h in
inhibition are considered susceptible, and strains that a 5% C 0 2 atmosphere. Production of pigment is
exhibit growth up to the disc are considered resistant. indicated by a yellow colour of the growth. A cream,
white or grey colour does not indicate pigmentation.
Carbohydrate Fermentation Test The carbo-
hydrates that may be tested are L-arabinose, methyl- Pyruvate Utilization Test A fresh culture is used
a-D-glucopyranoside, glycerol, inulin, lactose, to inoculate a tube of pyruvate broth. The broth is
maltose, D-mannitol, melibiose, raffinose, ribose, incubated for up to 7 days. A positive reaction is
sorbitol, sorbose, sucrose and trehalose. Acid from indicated by the development of a yellow colour. If
2220 VAGOCOCCUS
the broth remains green or greenish yellow the test Gram-positive cocci, excluding the streptococci and
result is negative. A yellow colour with only a hint of enterococci. Clinical Microbiology Reviews 8 : 479-495.
green is interpreted as positive. Facklam RR
Tellurite Tolerance Test Tolerance to tellurite is
determined on agar medium containing 0.04% potas-
sium tellurite. The agar may be contained in a tube
or plate. The medium is inoculated with a fresh culture
of the strain to be tested and incubated up to 7 days.
Tolerance (positive result) is indicated whenever black
colonies form on the surface.
Voges-Proskauer Test The Colbentz modification of
the Voges-Proskauer test is used to determine the
production of acetylmethyl carbinol. The strains are
grown on blood agar plates overnight. With a loop,
all growth on the plate is transferred to 2 ml of Voges-
Proskauer broth. The broth is incubated for 6 h or
overnight and then tested. Ten drops of solution (A
(a-naphthol) and 10 drops of solution B (sodium
hydroxide and creatine) are added to 0.5ml of the
culture in the Voges-Proskauer broth. The tube is
shaken vigorously, and a positive reaction is indicated
when a red colour develops within 30 min. A pink or
rust colour is interpreted as a weak positive reaction.
See also: Enterococcus. Lactococcus: Introduction.
Streptococcus: Introduction.
Further Reading
Carvalho MGS, Teixeira LM and Facklam RR (1998) Use
of tests for acidification of methyl-a-D-ghcopyranoside
and susceptibility to efrotomycin for differentiation of
strains of Enterococcus and some related genera. Journal
of Clinical Microbiology 36: 1584-1587.
Collins MD, Ash C, Farrow JAE, Wallbanks S and Williams
M (1989) 16s ribosomal ribonucleic acid sequence
analyses of lactococci and related taxa. Description of
Vagococcus fluvialis gen. nov., sp. nov. Journal of
Applied Bacteriology 67: 453-460.
Facklam RR and Elliott JA (1995) Identification, clas-
sification, and clinical relevance of catalase-negative,
I Vegetable oils see Preservatives: Traditional Preservatives - Vegetable Oils.
and Teixeira L M (1997) Enterococcus. In:
Collier L, Balows A and Sussman M (eds) Topley &
Wilson’s Microbiology and Microbial Infections, 9th
edn. Vol2, p. 669. London: Edward Arnold.
Pot B, Devriese LA, Hommez J et a1 (1994) Characterization
and identification of Vagococcus fluvialis strains isolated
from domestic animals. Journal of Applied Bacteriology
77: 362-369.
Schliefer KH and Kilpper-Balz R (1987) Molecular and
chemotaxonomic approaches to the classification of
streptococci, enterococci and lactococci: a review. Sys-
tematic Applied Microbiology 10: 1-19.
Schliefer KH, Kraus J, Dvorak C, Kilpper-Balz R, Collins
M D and Fischer W (1985) Transfer of Streptococcus
lactis and related streptococci to the genus Lactococcus
gen. nov. Systematic Applied Microbiology 6: 183-195.
Schmidtke LM and Carson J (1994) Characteristics of Vag-
ococcus salmoninarum isolated from diseased salmonid
fish. Journal of Applied Bacteriology 77: 229-236.
Teixeira LM, Merquior VLC, Vianni MCE et a1 (1996)
Phenotypic and genotypic characterization of atypical
Lactococcus garvieae strains isolated from water buf-
falos with subclinical mastitis and confirmation of L.
gurvieae as a senior subjective synonym of Enterococcus
seriolicida. International Journal of Systematic Bac-
teriology 46: 664-668.
Teixeira LM, Carvalho MGS, Merquior VLC, Steigerwalt
AG, Brenner DJ and Facklam RR (1997) Phenotypic
and genotypic characterization of Vagococcus fluvialis,
including strains isolated from human sources. Journal
of Clinical Microbiology 35: 2778-2781.
Wallbanks S, Martinez-Murcia AJ, Fryer JL, Phillips BA
and Collins M D (1990) 1 6 s rRNA sequence deter-
mination for members of the genus Carnobacterium and
related lactic acid bacteria and description of Vagococcus
salmoninarum sp. nov. International Journal of Sys-
tematic Bacteriology 40: 224-230.
Weinstein MR, Litt M, Kertesz DA et a1 (1997) Invasive
infections due to a fish pathogen, Streptococcus iniae.
N e w England Journal of Medicine 337: 589-594.
Williams AM and Collins M D (1992) Genus- and species-
specific oligonucleotide probes derived from 1 6 s rRNA
for the identification of vagococci. Letters in Applied
Microbiology 14: 17-21.
WATER QUALlTYASSESSMENT/RoutineTechniquesfor Monitoring Bacterialand Viral Contaminants 2281
WATER QUALITY ASSESSMENT
Contents
Routine Techniques for Monitoring Bacterial and Viral Contaminants
Modern Microbiological Techniques
Routine Techniques for
Monitoring Bacterial and Viral
Contaminants
John Watkins, CREH Analytical, Leeds, UK
Liz Straszynski,Alcontrol Laboratories, Bradford, UK
David Sartory, Severn Trent Water, Shrewsbury, UK
Peter Wyn-Jones, Sunderland University, UK
Copyright 0 1999 Academic Press
Water is a basic requirement of living organisms.
Throughout the world waterborne and water-asso-
ciated diseases affect millions of people. In developed
countries, clean water is readily available. It will have
been subjected to treatment and disinfection, and will
have been monitored for both chemical and micro-
biological qualities. In many areas of the world,
although water is available and does not have to be
paid for, the quality of the water is poor. Where there
is no treatment or organized distribution of water, the
incidence of gastroenteritis is high, particularly in
young children. As well as being directly consumed,
water is also used in the manufacture and preparation
of food and drink, and is an important medium for
recreation. Water quality standards must take account
of all these uses, and the test procedures used must
identify deterioration quickly and accurately.
Contamination of water with animal or human
faecal material can introduce large numbers of patho-
genic microorganisms, many of which have a low
infective dose. Distribution of these organisms in
drinking water can result in large numbers of people
becoming infected in a short space of time, resulting
in an outbreak of waterborne disease. Contamination
may occur in the water before treatment, treatment
may be ineffective or contamination may occur in the
distribution system after effectively treated water has
been distributed. Water and distribution systems may
also contain relatively large numbers of saprophytic
microorganisms. Increases in temperature during the
summer months. or increases in temDerature while
water is stored and distributed in buildings, together
with reduced amounts of disinfectants may result in
growth of potentially hazardous species. These con-
ditions may permit the growth of pseudomonas aeyu-
ginosa or Legionella spp., and here testing for faecal
contamination is of little value.
Rationale of Water Testing
Regular sampling and analysis provide data on the
quality of raw water, the efficiency of water treatments
and the integrity of distribution systems. The range of
pathogenic microorganisms is extensive and therefore
water is examined for microbiological indicators of
contamination. The use of indicator organisms is
based on the assumption that if they are present, the
pathogens may also be present, but that if they are
absent then the water is suitable for consumption or
use in manufacturing. To be useful, indicators must
always be present when contamination has occurred
and must be present in higher numbers than patho-
gens.
The principal bacterial indicators are the coliforms,
faecal coliforms, enterococci, Clostridium per-
fringens, the sulphite-reducing clostridia and total
aerobic bacteria. The coliform group encompasses a
wide range of genera of the family Enterobacteriaceae.
Many of the group are common in soil and natural
waters and can grow in water. Their presence in
drinking water is undesirable, but does not necessarily
indicate any health hazard. The faecal coliforms are
a group of thermotolerant species which may indicate
faecal contamination. The key species of the faecal
coliform group is Escherichia coli, commonly found
in the faeces of humans and animals and is thus a
definitive indicator of faecal contamination. Other
species of faecal coliforms (e.g. Klebsiella pneumoniae
2282 WATER QUALlTYASSESSMENT/RoutineTechniques for Monitoring Bacterial and Viral Contaminants
and Enterobacter cloacae), although associated with
faeces, also occur naturally in the environment, on
vegetation and in soils. The enterococci and the clo-
stridia are able to survive in water for substantially
longer periods and are therefore better indicators than
the coliforms of remote pollution and serious
contamination of raw water, when treatment and
disinfection have removed coliforms and faecal
coliforms. They are also used for determining the
hygienic operation of new installations such as treat-
ment works or boreholes and the proper laying, dis-
infection and repair of water mains. The total viable
bacterial count (plate count) is a measure of the
hygienic quality of the water and not necessarily a
measure of its suitability for consumption. Counts in
treated water are usually low, but they may increase
dramatically if water becomes stagnant and the tem-
perature increases. There have been many arguments
about indicators reliably establishing the presence of
pathogens, particularly those pathogens which are
resistant to disinfection. Certainly, there have been a
number of waterborne outbreaks of disease where
indicator analysis has been satisfactory.
Current Legislation Governing Water
Quality
The European Community Directive relating to the
quality of potable water and water intended for food
production (known as the Drinking Water Directive)
was implemented in July 1980. It lists microbiological
criteria, providing guide (G) values and maximum
admissible concentrations (MACs), and details ana-
lytical methods and sampling frequencies. It does not
require direct monitoring for specific pathogens but
does state that water should be free from such organ-
isms. The United Kingdom adopted these standards in
the Water Supply (Water Quality) Regulations 1989,
(amended 1990 and 1991). Private water supplies
were similarly covered in the Private Water Supply
(Water Quality) Regulations, 1991. In the USA,
drinking water quality is covered by the National
Primary Drinking Water Standards (1994) and in
Canada by the Guidelines for Canadian Drinking
Water Quality (1996).
Current Standards
The World Health Organization (WHO) Guidelines
for Drinking Water Quality recommend that all
100 ml water samples intended for drinking must not
contain Escherichia coli or thermotolerant coliforms.
Where water is treated it should not contain coliforms
on entry to the distribution system, and they should
not be detected in 95% of samples from a distribution
system. Under European legislation, treated waters
should contain (per 100ml) no coliforms, no faecal
coliformslE. coli and no enterococci. The current
value for sulphite-reducing clostridia is not more than
one in 20 ml. These are MAC values. For the aerobic
plate counts there are guide values per millilitre of
less than ten for colonies at 37°C and less than 100
for colonies at 22°C. There is an additional provision
for the plate count that there should be no significant
increase above those levels normally seen during
routine analysis. If plate count levels normally exceed
the guide values, this is acceptable providing that
levels do not increase significantly. For distribution
systems and at point of use, 95% of samples must be
free from coliforms but faecal coliforms must be
absent at all times. Standards for the United States are
based on the monitoring of coliforms in distribution,
where 95% of samples must be coliform free (or no
more than one sample per month may be positive if
less than 40 per month are taken). Isolation of coli-
forms requires that there be additional sampling and
testing for E . coli. There are no standards for the
other indicator organisms. In Canada the MAC for
coliforms is stated to be zero, but there is compliance
if no sample contains more than 10 coliforms and
no faecal coliforms per 100ml and no consecutive
samples contain coliforms. In both the USA and
Canada, aerobic plate counts are monitored, pri-
marily as an adjunct to the coliform test as high
numbers of heterotrophic bacteria (500-1000 m1-l)
can interfere with standard methods of coliform
enumeration.
Frequencies of sampling and analysis for each indi-
cator are set by each country, and are typically based
on the sizes of the populations supplied.
Methods of Analysis
Early methods developed for water analysis were
based on most probable number (MPN) or multiple
tube techniques using liquid culture media. There
was always the requirement for confirmation of any
positive tests and therefore first results were always
presumptive. Although relatively simple to perform,
such test procedures were time consuming and of long
duration, with up to four days required to obtain
confirmed results. In the UK, a chemically defined
medium, Minerals Modified Glutamate Medium, was
introduced in 1969. This is still used by some labora-
tories. The introduction of enzyme-specific chromo-
genic and fluorogenic substrates has made the MPN
test procedure popular again, and removed the need
for confirmations. Results can now be available in
18 hours (Table I).
Membrane filtration was introduced in the 1950s
WATER QUALlTYASSESSMENT/RoutineTechniques for Monitoring Bacterial and Viral Contaminants 2283
Table 1 Isolation media for the detection of faecal indicators
lndicator organism lsolation medium
Coliforms and faecal coliforms Minerals modified glutamate
(UK) medium
UK Membrane lauryl sulphate
broth
UK and USA Colilert (Idexx)
UK Membrane lactose
glucuronide agar
USA m-END0 Broth
USA m-FC Medium
Enterococci (UK) Slanetz and Bartley agar
Clostridia Tryptose sulphite cycloserine
agar, OPSP agar
Total viable bacterial count Yeast extract agar, plate count
(UK, USA) agar, R2A Agar
and is currently the basis of the standard methods
used in the UK for coliforms, faecal coliforms,
enterococci and clostridia. The standard medium for
coliforms and faecal coliforms is membrane lauryl
sulphate broth. Coliforms are incubated at 37°C and
faecal coliforms at 44"C, for 14 h, with both having
a pre-incubation period of 4 h at 30°C. In the USA m-
Endo medium with incubation at 35°C is employed
for total coliforms and mFC agar with incubation at
44°C for faecal coliforms. More recently, methods
based on defined substrate formulations have been
widely used. Enterococci are enumerated on Slanetz
and Bartley medium incubated at 37°C for 48 h for
non-contaminated waters or at 44°C for 48 h for
contaminated waters. Kanamycin aesculin azide
medium may be used as an alternative. Clostridia
present a more complex problem. Sample pas-
teurization may be required before incubation on
tryptose sulphite cycloserine (TSC) agar or ole-
andomycin polymixin sulphadiazine (OPSP) agar.
Chromogenic and/or fluorogenic substrates may be
added to the above media to make confirmation
unnecessary. Yeast extract agar is the medium of
choice for plate counts in the UK, whereas in the USA
plate count agar is used. Comparative trials suggest
the two media give similar results. Many bacteria of
water origin have simple nutritional requirements and
the use of a nutritionally weak medium (for example
R2A agar) together with a lower incubation tem-
perature and longer incubation period can recover
more bacteria from water samples than yeast extract
or plate count agars.
incubation time and Confirmation
temperature
37°C 48 h Oxidase, lactose peptone and
tryptone water
30°C 4 h, 37°C 14 h or 44°C Oxidase, lactose peptone and
14h tryptone water
37°C 18 h None required
30°C 4 h, 37°C 14 h None required
35°C 20-22 h Lauryl tryptose broth, brilliant
green bile broth
44.5"C 24 h None required
37°C 48 h Bile aesculin azide or
kanamycin aesculin azide
agar
37°C 48 h Litmus milk
37°C 24 h, 22°C 72 h, 20°C None required
7 days
Confirmation of Isolates
The media used for the isolation of faecal indicators
may also isolate other bacteria which can give a
typical positive reaction. Confirmation of positive
MPN tubes or colonies on membranes is usually
required. Coliform bacteria by definition ferment
lactose at 37°C with the production of acid and do
not possess the enzyme cytochrome oxidase. Faecal
coliforms are coliforms that grow and can produce
acid from lactose at 44°C. Some coliforms which are
not faecal in origin may also grow at 44°C. For this
reason, in the UK, Escherichia coli is considered to be
the only true faecal coliform. It also produces indole
from tryptophan at 44°C. Acid and indole production
at 44°C can be found in coliforms other than E . coli
and the test results should be viewed with caution.
Experience has shown that coliforms other than E.
coli can be isolated by some food microbiology
methods. Biochemical test kits are commercially avail-
able for the identification of Enterobacteriaceae, but
these can give widely differing results between kits
and between single strains even when a single strain
is tested in duplicate. The introduction of chromo-
genic and fluorogenic substrates has removed much
of the ambiguity of confirmations although iden-
tification of coliforms can still be uncertain.
Coliforms are confirmed by subculture of typical
colonies to nutrient agar and after growth, testing for
oxidase and inoculation of lactose peptone water for
the production of acid at 37°C within 48 h. Faecal
coliforms are confirmed as oxidase negative and by
the production of acid in lactose peptone water at
44°C and indole in tryptone water at 44°C. Entero-
cocci are confirmed by their ability to hydrolyse
2284 WATER QUALITYASSESSMENT/RoutineTechniquesfor Monitoring Bacterial and Viral Contaminants
aesculin when grown on bile aesculin azide agar or
kanamycin aesculin azide agar. Sulphite-reducing clo-
stridia can be confirmed as Clostridium perfringens
by inoculation into litmus milk and incubation at
37°C for up to 5 days. Fermentation of the lactose
causes acid production and clotting of the milk.
Quality Assurance
Legislation on water quality assessment in the water
industry has included the requirement for laboratories
to have internal quality control and participate in
external quality assurance schemes. Because of the
difficulties of preparing and keeping suspensions of
bacteria for quality control, manufacturers have now
developed dried suspensions which can be stored and
rehydrated to give consistent suspensions for day to
day quality assurance. These suspensions can also be
used in some of the validation procedures for new
methods and for the assessment of new analysts.
Interpretation of Results
Coliforms in water can derive from a number of
sources of which faecal contamination is only one. In
the absence of other indicators, they may be derived
from vegetation, soil or surface water. In addition
there is ample evidence that coliforms can grow in
distribution systems in association with biofilms.
Although their presence in water is undesirable, the
water may be perfectly safe for drinking, but its use for
other purposes may be unsatisfactory. The presence of
faecal coliforms, especially Escherichia coli, and other
indicators means that water is probably contaminated
with faecal material and immediate remedial action
must be undertaken. Aerobic plate counts above the
guide values in the EC Directive do not mean that
water is unfit for human consumption. High counts
at 37°C are suspicious and may require further inves-
tigation. High counts at 22°C suggest stagnation and
warming of water with the concomitant growth of
normal water bacteria. Cleaning and disinfection are
required in some circumstances, e.g. hospitals, where
counts in excess of 1000ml-I at 22°C may require a
water distribution system to be disinfected. Excep-
tional counts may be used as indications for the
growth of other microorganisms in such as cooling
towers and swimming pools. Pseudomonads, in par-
ticular Pseudomonas aeruginosa, aeromonads and
Legionella spp. can colonize water systems where
conditions are suitable, and high aerobic counts may
signal the need to look for pathogens.
Other Pathogenic Microorganisms
There is a wide range of pathogens that can
contaminate water, including bacteria, viruses and
eukaryotic parasites. Analysis for bacteria such as
Campylobacter spp., Salmonella spp., Pseudomonas
spp. and Aeromonas spp. follow conventionally pub-
lished methods. Immunological techniques such as
enzyme-linked immunosorbent assays (ELISA) can
also be adapted for water analysis. Methods for the
isolation of Legionella spp. are currently detailed in
a draft British Standard BSI 6068 (1994).The proto-
zoan Cryptosporidium parvum is an important water-
borne parasite that has been implicated in a number of
waterborne disease outbreaks. Methods for detection
have been published in the UK and USA and both
methods are currently under revision. The significance
and occurrence of this organism in water has led to
the rapid development of faster and more efficient
techniques for its detection, including the use of small
volume processing, immunomagnetic separation and
flow cytometry.
Escherichia coli 0157:H7 has received much atten-
tion and can be transmitted by water. Evidence
suggests that it is removed by conventional water
treatment and is readily killed by conventional dis-
infectants. However, there have been several out-
breaks where water was the vehicle of infection. In
one instance, four people died. One of the main prob-
lems with such pathogens occurring at low levels in
water is the opportunities for contamination of foods,
where they may be able to multiply, or the con-
tamination of food wash waters where they can be
recycled.
Other Indicators
A wide range of other microorganisms have been
described as useful indicators of faecal contamination.
Coliphages are bacterial viruses which are specific to
Escherichia coli and other coliforms. They can be
divided into two groups. The somatic bacteriophages
gain access to the cell through the cell wall and the F
specific bacteriophages attach to the F or sex pilus.
The latter have been described as useful indicators of
the presence of enteric viruses in water.
Bacteriophages of Bacteroides fragilis have also
been suggested as useful indicators, as have the bac-
teria themselves. Both are specific to the faeces of
humans and farm animals. Bifidobacterium spp. can
also be found in humans and some animals. Sorbitol-
fermenting strains have been specifically linked to
human faeces. Both groups of bacteria are strictly
anaerobic. Rhodococcus caprophilus is a Nocardia-
like actinomycete found in farm animals (cattle, sheep,
WATER QUALINASSESSMENT/Routine Techniques for Monitoring Bacterial and Viral Contaminants 2285
pigs and horses) and may be a useful indicator of
contamination with animal faeces.
Routine Techniques for Monitoring Viral
Contamination
Viruses in Water
The aquatic environment (which includes fresh and
marine water, raw and treated sewage, sludge and
sediments) often provides conditions in which patho-
genic viruses released from the body may retain infect-
ivity, and so may cause disease on entry into a new
host. Human enteric viruses enter the water cycle
when faecal waste is discharged into the sewerage
system; they may end up in water which, after treat-
ment, is used for drinking. The purpose of sewage
treatment is to remove microorganisms and other
organic matter from sewage so that the resulting
effluent can be discharged to receiving waters with
minimal adverse effects. It also renders any receiving
water fit for use as a raw water source for drinking
water abstraction, as up to 90% of viruses are
removed during sewage treatment. Viruses may rarely
also gain access to raw water sources by percolation
through the soil into groundwater used as a spring or
a borehole supply. The treatment process to produce
wholesome drinking water involves the removal of
organic material by flocculation and sedimentation
followed by disinfection with chlorine. Most viruses
will be removed by flocculation and the remainder
will be inactivated by disinfection. It is thus important
to be able to monitor both raw and finished waters
for viral contamination where there is likely to be a
risk that these are present.
Human Enteric Viruses
Enteric viruses, i.e. those inhabiting the gastro-
intestinal tract, may be divided into two groups
according to their effects on the gut lining. The entero-
viruses cause little damage to the gut epithelium and
thus do not usually cause gastroenteritis. Although
they multiply in the gut and therefore may be patho-
genic, in the majority of infections they do not migrate
to other tissues. This group includes poliovirus,
coxsackieviruses and echoviruses. They cause a
variety of symptoms, mainly in children. They are
shed into the sewage in large numbers and include
poliovirus vaccine strains, which will be present all
year round. The viral pathogens which do cause
gastroenteritis include the rotaviruses, small round-
structured viruses (SRSVs, Norwalk viruses) and
astroviruses. It is important to distinguish between
the enteroviruses, which is a taxonomic group, and
enteric viruses, which is a term describing the habitat
of all viruses in the gastrointestinal tract, and includes
the enteroviruses.
Coincident with their pathogenesis is the ability of
enteric viruses to grow in cell culture. Thus, most of
the enteroviruses can be grown in BGM cells, a
monkey kidney cell line used for many years in the
detection of waterborne viruses. Enteroviruses are
easily detected in this cell system after concentration.
Because of this, and because enteroviruses are resist-
ant to the p H changes carried out during the filtration
process (see below), enteroviruses have been the group
of waterborne viruses most investigated over the past
20 years. Conversely, the viruses of gastroenteritis
cannot be grown in cell culture and are thus much
more difficult to detect, as non-culture based systems
are required.
The burden of disease imposed by enteric viruses
varies, and in many cases is difficult to estimate, since
many infections are subclinical or cause only minor
symptoms. The enteroviruses cause relatively mild
disease in the vast majority of infections, whereas
rotaviruses and SRSVs cause moderate to severe diar-
rhoea with or without vomiting. The proportion of
infections associated with waterborne spread is also
difficult to gauge but will be very small compared with
person to person spread. Most reports of waterborne
disease are anecdotal. SRSVs have caused well-docu-
mented outbreaks of viral gastroenteritis when fin-
ished drinking water has been polluted by sewage,
including contamination of a borehole in a tourist
resort, water tanks at a mobile home park, well water
used to make ice, and cross connections of drinking
water supplies with water supplies used for irrigation.
Concentration and Detection of Enteric Viruses
Concentration All viruses are obligate intracellular
parasites and so will not multiply outside the body.
This, coupled with the diluting effects of water, means
that direct detection of these agents in any kind of
water (with the exception of raw sewage) requires
the sample to be concentrated before virus can be
detected. The degree of concentration will reflect the
nature of the water and the potential quality of virus
it contains. Thus raw waters will require less con-
centration than finished waters. Typically, volumes of
about 101 are taken from river waters, whereas it is
necessary to take samples of 100-1000 1 to detect any
viruses in finished waters.
The most common concentration technique is
adsorption/elution, where virus is adsorbed to an
insoluble matrix then eluted in a smaller volume of
fluid. Concentration from water samples usually
involves passage of the sample through a filter which
adsorbs the virus. Filters may be membranes or cart-
ridges. In the UK, membranes are most popular, but

2286 WATER QUALlTYASSESSMENT/RoutineTechniques for Monitoring Bacterial and Viral Contaminants
in the US, where more potable water supplies are
monitored, cartridge filters are used more frequently
since they can handle greater volumes without clog-
ging. Membrane materials include cellulose nitrate
(commonest), cellulose acetate and glass fibre/epoxy
resin, and are either 142 mm or 293 mm in diameter.
Since retention of virus is by adsorption, not entrap-
ment, the pore size used is determined by the turbidity
of the sample. For membranes, the smallest pore size
used is 0.45 pm and the largest, used for turbid waters,
is 5 pm. A glass-fibre prefilter may be used for river
waters but is not necessary for other types.
Viruses are negatively charged at neutral pH. Most
filters are also negatively charged and it is necessary
to precondition the water sample to increase the posi-
tive ion concentration on the virus particles. This is
done by lowering the pH to about 3.5 and sometimes
adding aluminium ions (as aluminium chloride) to the
sample. Viruses will then adsorb efficiently to the
filter.
Following filtration, adsorbed virus is eluted by
passing through the filter 100-400ml of a protein
solution, either beef extract or skimmed milk at p H 9,
which displaces the virus into the eluant. It is then
further concentrated (secondary concentration) by
lowering the p H to the isoelectric point of the protein,
which results in the formation of a floc to which virus
is adsorbed. This floc can be deposited by low-speed
centrifugation, resuspended in a small volume (about
10 ml) of phosphate buffer and frozen until assayed.
This is the method most popular in the UK and rec-
ommended by the Standing Committee of Analysts
(SCA).
There are several variations on adsorption/elution.
Some laboratories use glass fibre tube filters, which
are more economic, but clog faster. Many EU and US
laboratories use positively charged filters which avoid
the need for preconditioning the water sample, but
these are considerably more expensive. Novel tech-
niques being evaluated include adsorption to posi-
tively charged nylon membranes, adsorption to glass
powder and glass wool, and to immunomagnetic
beads. The glass wool method has been used very
successfully in France for analysis of drinking water
samples for enteroviruses.
Entrapment techniques can be used to concentrate
viruses from water; the most effective of these is
ultrafiltration, which has been used in Italy and the US
for analysis of drinking water supplies. Ultrafiltration
can be effected either through hollow fibre systems or
tangential flow membranes using a 100 000 M,cut-
off level. The advantages of this system are that a
wide range of viruses may be concentrated, usually
no secondary concentration is needed, and there
is no need to condition the water before filtration.
Next Page
However, the capital costs are high and filtration of
turbid waters can be a difficult and lengthy process.
Detection Detection of viruses in concentrates is best
done by cell culture, which is the only way to detect
infectious virus. As indicated above, of the viruses
likely to be present in water, only the enteroviruses
will grow well in culture. They are detected quan-
titatively by plaque assay under agar or by a liquid
culture assay such as the MPN technique. Plaque
assays are statistically more reliable in terms of the
number of cultures usually used, but liquid culture
assay will detect a wider range of viruses. Plaque assay
may be done with BGM cells in a monolayer or with
cells suspended in the agar. The latter approach is
between three and 1 0 times more sensitive than the
monolayer assay due to the larger number of cells
available for virus infection, and is the SCA rec-
ommended method. The US Environmental Pro-
tection Agency (1984) recommends both methods.
Plaques usually develop after 3-5 days.
To demonstrate the presence of infectious virus of
the types which grow less well in culture, more
complex procedures must be used. Rotaviruses and
astroviruses will undergo limited replication in
different cell lines and may be detected by immuno-
fluorescence. Hepatitis A virus grows very slowly in
a limited range of cells and may be detected by radio-
immunoassay. At present there is no cell culture
system for SRSVs.
Molecular biology methods (principally reverse-
transcriptase-polymerase chain reaction, RT-PCR)
offer an alternative approach to the detection of non-
culturable viruses. Methods for the RT-PCR detection
of several enteric virus types from water and related
materials are available, and have proved as sensitive
as culture where the techniques have been tested in
parallel. The main drawback to RT-PCR is that it
does not specifically detect infectious virus, but a
positive result in such an analysis will nevertheless
indicate viral pollution of the water. Routine analysis
for these fastidious viruses is not practical at present.
Significance of Waterborne Viruses
The detection of viruses in water indicates faecal pol-
lution. If animal viruses, which may gain access to
water through soil run-off, are excluded, then it must
be inferred that the water is polluted with sewage,
since in the absence of virus multiplication in the
environment, faeces from an infected individual are
the only source of the virus. In this sense enteroviruses
act as another indicator of pollution. Viruses are more
resistant than many bacterial indicators and their
infectious dose is lower than that for most bacterial
infections. In the food context therefore, waterborne

I Xanthan Gum see Fermentation (Industrial): Production of Xanthan Gum.
XANTHOMONAS
Arun Sharma Food Technology Division, Bhabha Atomic Research Centre, Mumbai, India
Copyright 0 1999 Academic Press
The genus was created by Dowson in 1939 following
a proposal by Burkholder in 1930, for a group of
plant pathogens until then assigned to the genus Phy-
tomonas. Bradbury in 1984 widened the definition of
the genus to include characteristics such as the absence
of denitrification, the nature of xanthomonadin
pigment, and many more biochemical and physio-
logical properties. The word Xanthomonas (Xan.
tho’mo. nas or Xan. tho’. monas) is composed of the
Greek adjective Xanthus meaning yellow and femi-
nine noun monas meaning unit. In modern Latin it
literally translates to ‘yellow monad’. The bacteria
belonging to the genus Xanthomonas are commonly
associated with the diseases of plants. Xanthomonas
species are seen as yellow-pigmented colonies on a
nutrient agar plate and as Gram-negative, short and
usually straight rods under a microscope. However,
Pseudomonas maltophila has recently been included
in the genus, which is neither a plant pathogen nor
does it produce the characteristic yellow pigment.
Characteristics of the Genus
Cells of bacteria belonging to this genus are short,
straight rods, but not vibrioid. The size is in the range
0.4-1.0 x 1.2-3.0 pm. The cells are monotrichous
with a polar flagellum. The bacteria of the genus
do not form spores, sheaths, appendages or buds.
Xanthomonads are chemoorganotrophic, use low-
molecular-weight compounds and some are able to
depolymerize natural polysaccharides and proteins.
The cells carry out aerobic respiratory metabolism
and are non-fermentative. The members of the genus
show oxidase negative (or weakly positive) and cat-
alase positive reactions.
The major means of glucose catabolism is the
Entner-Duodoroff pathway (Fig. 1). Acid is produced
from mono- and disaccharides. In a weakly buffered
medium acid is produced from many carbohydrates
but not from rhamnose, inulin, adonitol, dulcitol,
inositol or salicin, and rarely from sorbitol. Acetate,
citrate, malate, propionate, and succinate are utilized
XANTHOMONAS 2323
I
Glucose
/- Koeinase
Glucose-6-phosphate
Glucose-6-phosphate dehydrogenase
Y
6-Phosphogluconic acid
,
’ Dehydrase
V
2-Keto-3-deoxygluconic acid 6-phosphate
(KDPG)
KDGP aldolase
V
I V
Pyruvate + Glyceraldehyde 3-phosphate
Y
Glycolysis
t
Pyruvate
Figure 1 Entner-Doudoroff pathway.
but generally benzoate, oxalate and tartarate are not
used. The tests for indole production from tryp-
tophan, and acetoin production (Voges-Proskauer
test) are negative. The growth on nutrient agar is
inhibited by 0.1-0.02% triphenyltetrazolium chlor-
ide. Hydrogen sulphide is produced from cysteine and
by most species from thiosulphate and peptone due
to desulphurase activity. Proteins are usually readily
digested and milk becomes alkaline with the growth
of Xanthomonas. Asparagine is not sufficient as the
only source of carbon and nitrogen. Most species
hydrolyse starch and Tween 80 rapidly. Nitrates are
not reduced. Some species hydrolyse cellulose and
pectin. G+C content of the members of the genus is
in the range 63-71 mol%. A majority of the species
are plant pathogens.
The typical colonies on an LB agar plate (Fig. 2)
are about 2-5 mm diam., mucoid or buttery, with a
raised centre and entire margins. Minimum growth
requirements are complex and include a need for
2324 XANTHOMONAS

Br

O
‘H

Br
OCH3
Figure 3 Xanthamonadin: yellow pigment of Xanthornonas.

New Approaches to the Classification of

Xanthomonas
Phenotypic properties are generally inadequate for
differentiating xanthomonads because of overlapping
characters among the strains. This has led to the
development of the pathovar system of nomenclature
Figure 2 Typical colonies of Xanthornonas on LB agar plate. for Xanthomonas. This system relies on the sus-
ceptibility of the host as the means of identifying the
methionine, glutamic acid and nicotinic acid in pathogen. However, many Xanthomonas pathovars
various combinations. Optimum temperature for have a wide host range. Therefore, new approaches to
growth is in the range 25-27°C. No growth is classification of xanthomonads have been developed.
observed above 40°C and below 5°C.
Xanthomonadin Production The yellow pigment
produced by most xanthomonads is a group of mem-
Classification of Xanthomonas brane bound, non-water-soluble, brominated aryl-
polyenes called xanthomonadins (Fig. 3 ), which are
Xanthomonas is one of the important genera of the
used as chemotaxonomic and diagnostic markers of
family Pseudomonadaceae in the order Pseudo-
the genus.
monadales. The genus was originally described as
Phytomonas in the first edition of BergeyS Manual. Nutritional Screening Carbon substrate utilization
The present description of Xanthomonas in relation patterns have been found to be sufficiently uniform
to other genera of the family Pseudomonoadaceae as among the various genomic groups within xantho-
shown in Bergey’s Manual of Determinative Bac- monads to allow their differentiation.
teriology (7th edition) is:
Division: Protophyta Fatty Acid Composition The qualitative and quan-
Class: Schizomycetes titative differences in the whole-cell fatty acid methyl
Order: Pseudomonadales ester patterns have been used to determine the rela-
Class: Pseudomonadaceae tionships between species and strains.
Genus: Xanthomonas
Species: campestris Phage typing Taxonomic classification based on
bacteriophage sensitivity of the different strains of
The general identification of Xanthomonas to Xanthomonas has also been attempted.
species level is based on the ability of the organism
to: Serological Typing A number of serological methods
have been successfully used to identify and diagnose
hydrolyse gelatin and starch;
different species and strains of xanthomonads. Both
produce nitrites and ammonia from nitrates;
polyclonal and monoclonal antibodies, produced to
0 form yellow non-water-soluble pigment in nutrient
whole cells or flagellar extracts of the organism have
agar;
been employed for the identification of xanthomonads
form brown pigment in beef extract agar.
and groupings of strains of different species. Tech-
Another key for the classification of Xanthomonas niques, such as immunofluorescence microscopy,
is based on the plant host that the bacterium attacks. enzyme immunoassays and dot-blot immunoassays,
The members of the genus are known to attack both have been used for carrying out specificity tests. Based
monocotyledons and dicotyledons. A detailed clas- on studies with polyclonal antibodies three serovars
sification of the genus is given in Bergey’s Manual of of X. albilineans have been identified. Monoclonal
Determinative Bacteriology. It is possible to classify a antibodies have been used for the identification of
given species to subspecies level based on the pathogen xanthomonads and groupings of strains of X . cam-
race and host-cultivar relationship. pestris and X . albilineans. Serological studies using

monoclonal antibodies have shown that Pseudo-
monas gardneri is also a xanthomonad.
Sodium Dodecyl Sulphate-Polyacrylamide Gel Elec-
trophoresis (SDS-PAGE) Development of rapid
high-resolution fingerprinting techniques such as
protein electrophoresis in combination with the com-
puter assisted processing of SDS-PAGE pattern of
proteins make it possible to characterize and compare
a large number of strains at infra-subspecies levels.
The electrophoretic groupings corresponded in some,
but not in all cases, to the existing pathovars. Numer-
ical analysis on SDS-PAGE protein patterns of
Xanthomonas strains has shown a good agreement
between groupings obtained by gas chromatographic
analysis of cellular fatty acids, and DNA-DNA
hybridization.
DNA Based Techniques These techniques include
DNA-DNA hybridization, DNA-RNA hybridization
using 16s and 23s rRNA, restriction fragment length
polymorphism (RFLP)analysis, plasmid profile analy-
sis, and polymerase chain reaction (PCR) based detec-
tion using random or specific primers. Based on DNA-
DNA hybridization experiments strain clusters have
been identified that deserve independent status as
species. As a result of such studies Pseudomonas mal-
tophila, a member of the rRNA similarity group V
of Pseudomonas together with P. geniculata and P.
gardeneri, has been allocated to the genus Xan-
thomonas and is now known as X . maltophila. The
organism is not a phytopathogen and it does not
produce the xanthomonadin pigment. It is also
reported to be an animal pathogen. On the other hand
RFLP analysis has shown striking similarities among
different X . campestris pathovars.
Conventional methods for detection and iden-
tification of Xanthomonas rely on pure culture isol-
ation on selective media, followed by morphological
and biochemical characterization of the isolates.
Pathogenicity is ascertained by testing for Koch's pos-
tulates. A pathogenic microbe is further screened on
a number of cultivars to identify the presence of race
differences. Traditionally these methods have given
reliable results. However, with the discovery of so
many new strains it has been increasingly difficult to
establish their relationship with each other and to
classify them. Moreover, the traditional methods are
cumbersome when quick results are required, for
example in seed certification and quarantine pro-
cedures. Methods based on protein profiling and fatty
acid analysis are also time consuming and need pure
culture of each isolate for assessment. Polyclonal anti-
sera often cross-react and fail to identify pathovars or
races of the organism. Even monoclonal antisera may
XANTHOMONAS 2325
react with some or all the strains of a pathovar. There-
fore, strain identification has become a difficult task.
Development of probes based on more specific gene
sequences such as those of hrp genes may provide
a useful tool for the detection and identification of
phytopathogenic xanthomonads.
Internal Subdivisions of Xanthomonas
In Bergey's Manual of Determinative Bacteriology
five species of Xanthomonas were recognized. These
species could be distinguished on the basis of bio-
chemical reactions as shown in Table i.X. campestris
can grow at 35"C, hydrolyse aesculin (6,7-dihydroxy-
coumarin 6-glucoside), exhibits mucoid growth, and
can liquefy gelatin. It causes proteolysis of milk and
H2S production from peptone. Salt tolerance of X.
campestris is high (2-5%). It can utilize arabinose,
glucose, mannose and cellobiose to produce acid. X .
fragarzae can not cause proteolysis of milk and does
not produce H2S.It can use glucose and mannose to
produce acid. Its tolerance to salt is low (0.5-1.00/,).
X . albilineans does not show gelatin hydrolysis and
mucoid growth, otherwise it is similar to X . fragariue.
It has the least salt tolerance (< 0.50/). X . axonopodis
differs slightly from X . albilineans in that it produces
H2S and can not use mannose to produce acid. X.
ampelina differs from X . albilineans in that it has
urease activity and use arabinose to produce acid.
Two more species, X . populi and X . maltophila, have
been recently added to the genus.
X . fragariae, a pathogen of strawberries;
X . albilineans, a pathogen of monocots;
X . axonopodis, a pathogens of monocots;
X . ampelina, a pathogen of grapes;
X . populi, a pathogen of poplars;
0 X . maltophila, recently transferred from the genus
Pseudomonas;
0 X . campestrzs, a pathogen of dicots.
Xanthomonas campestris
The determination of the genus Xanthomonas and its
species is relatively easy, however, the characterization
of X . campestris pathovars poses problems. An unam-
biguous identification of the pathovars of X . cam-
pestris can be of great use in plant pathology. The X.
campestris pathovars that are defined by the host or
disease symptoms are difficult to identify by other
phenotypic characteristics. X . campestris group is the
largest of all and causes diseases in many plant species.
It is, therefore, classified into pathovars differentiated
by the host reaction. However, application of the
newer techniques of classification has been useful. A
relationship of nutritional properties, host specificity
2326 XANTHOMONAS
Table 1 Biochemical characters of different Xanthomonas species
Characteristic X. campestris X. fragariae
Growth at 35°C + +
Aesculin hydrolysis + +
Mucoid growth + +
Gelatin liquefaction + +
Milk proteolysis + -
H2Sfrom peptone + -
Urease - -
NaCl tolerance (%) 2-5 0.5-1 .O
Acid production from
Arabinose + -
Glucose + +
Mannose + +
Cellobiose + -
and DNA homology groups has been observed.
Genetic diversity among the strains of different patho-
vars of X. campestris has also been studied for a
number of pathovars. It is believed that the variability
could be more pronounced in the regions where the
host plant originated. Ribosomal RNA and DNA
probes could be useful tools for the epidemiological
studies and in following the genetic evolution of the
strains.
Methods of Detection and Enumeration
in Foods
Starch hydrolysis is a major distinguishing feature of
xanthomonads from other pseudomonads. Soluble
starch has been used in several agar media for isol-
ation for X. campestris.
Typical solid media for the general enumeration of
xanthomonads may contain (per litre):
Potato starch, 10.0 g; K2HP04.3H20, 3.0 g;
KHlP04, 1.5 g; (NH4)2S04, 2.0 g; L-methionine,
0.5 g; nicotinic acid, 0.25 g; L-glutamic acid, 0.25 g;
pH 6.8-7.0; with 15 g agar;
Potato starch, l o g ; yeast extract, 5.0g;
(NH4)H2P04, 0.5 g; K2HP04, 0.5 g; MgS04.7H20,
0.2 g; NaCl, 5.0 g, pH 7.4 with 15 g agar.
After autoclaving and cooling to 50°C the medium
is fortified with one or more i f the antibiotics such
as cephalexin 20 pg ml-', kasugamycin, 20 pg ml-l,
chlorothalomine 15 pg ml-', gentamycin 2 pg mlb',
and dyes such as brilliant cresyl blue 1pg ml-', methyl
green 1pgml-' and methyl violet 1 pgml-'. Xantho-
monads are generally resistant to these antibiotics.
After spread plating a given sample on the agar
plates the typical yellow colonies of Xanthomonas
are seen after incubating the plates at 2622°C for
48 h. The starch hydrolysis is visualized as the zone
of clearance around the colonies. In the absence of
dyes, iodine solution (1Yo) is spread on the plate to
visualize the zone of hydrolysis by starch.
X. albilineans X. axonopodis X. ampelina
Immunoassay for the Detection of X. campestris
Both polyclonal and monoclonal antibodies are
employed for detection using radioimmunoassay or
enzyme immunoassay. Production of antibodies, both
monoclonal and polyclonal, has been described. These
antibodies have been used for the detection of Xan-
thomonas campestris pv. campestrzs in crucifer seeds.
For assay the isolate is streaked on a solid medium.
The plates are incubated at 26e2"C for 48 h. The
putative Xanthomonas colonies could be directly
tested by enzyme linked immunosorbent assay
(ELISA).
A protocol for an indirect double antibody ELISA
for the detection of X. campestris strains is as follows:
Coat polystyrene micotitre plates with poly-L-lysine
by adding 0.1 mg ml-' solution in carbonate buffer
(pH 9.6) and incubating for 30 min at 21°C;
Wash microtitre plates three times with phosphate-
buffered saline containing 0.01% Tween 20
(PBST);
Add antigen (untreated or boiled bacterial cells lo'
cells mi-') or bacterial protein extract (1-10 pg);
Add PBST containing 0.5% BSA and incubate for
30 min at 37°C;
Add antibody and incubate for 3 h at 4°C and wash
the plates three times with PBST;
Add goat anti-mouse alkaline phosphatase con-
jugate (1: 1000 dilution in PBST containing 0.1%
albumin) and incubate 2 h at 37°C;
Wash three times with PBST and incubate with
the enzyme substrate (0.75 mg ml-l p-nitrophenyl
phosphate in 10% diethanolamine buffer v/v,
pH 9.8) for 0.5-1 h at 37°C;
Read absorbance at 405 nm.
Phytopathogenic Potential of
Xanthomonas
Among the bacterial diseases of plants, the most wide-
spread and destructive losses are caused by the Gram-

Table 2 Some common plant diseases caused by
Xanthomonas campestris
Plant Disease Causative agent
Cotton Leaf spot X.C. pv. malvacearum
Rice Leaf blight X. c. pv. oryzae
Cereals Bacterial blight X.C. pv. translucens
Walnut Bacterial blight X.C. pv. juglandis
Soybean Bacterial pustule X.C. pv. glycines
Sugarcane Gumming disease X.C. pv. vascularum
negative bacteria of the genus, Erwinia, Pseudomonas
and Xanthomonas. The genus Xanthomonas is of
great economic importance because of its broad host
range. More than 145 species of plants are known to
be infected, mainly by biotrophic pathogens, belong-
ing to this genus. These pathogens cause many types
of disease symptoms including spots, blights, cankers
and vascular wilts. The type of physiological function
that is affected first depends on the cells and tissues
of the host plant that become infected. Thus the infec-
tion of xylem vessels interferes with the translocation
of water leading to vascular wilts and cankers,
whereas infection of foliage interferes with the
photosynthetic process as in leaf spots, blights, and
pustules. Some common plant diseases caused by
Xanthomonas are listed in Table 2.
Angular leaf spot disease of cotton is caused by X.C.
pv. malvacearum. The disease is present wherever
cotton is grown. The bacterium attacks the leaves as
well as young cotton bolls. In rice, X.C. pv. oryzae
causes leaf blight disease. Bacterial blight or stripe of
several cereals and streak of sorghum and maize is
caused by X.C. pv. translucens. X.C. pv. juglandis
causes blight of walnuts. In the case of soybean, bac-
terial pustule disease caused by X.C. pv. glycines is
known to inflict considerable losses in yield.
Gumming disease of sugarcane affecting yields of
sugar is caused by X.C. pv. vascularum.
Bacterial pathogen-plant interactions involve an
interplay of the various virulence factors, the hyper-
sensitivity response and pathogenicity ( h r p ) and avi-
rulence (avr) genes of the pathogen and the disease
resistance genes in plants. The virulence factors com-
prise agents such as the hydrolytic enzymes, toxins,
polysaccharides, and plant growth regulators secreted
by the pathogen that damage or alter plant cells and
provide optimal environment for the growth of the
pathogen. On the other hand avirulence factors or the
products of avirulence genes of the pathogen invoke
hypersensitive response and death of the surrounding
cells in the resistant host. This restricts the spread of
the pathogen and in turn restricts its host range. H r p
genes in the pathogen regulate both the avr-induced
hypersensitivity reaction as well as pathogenicity.
XANTHOMONAS 2327
Table 3 Pre-harvest disease of fruits and vegetables caused
by Xanthomonas campestris
Fruithegetable Name of disease Causative agent
Lima beans Bacterial blight X.C. pv. phaseoli
Tomato Bacterial spot X.C. pv. tomato
Pepper Bacterial spot X.C. pv. vesicatoria
Cabbage Black rot X.C. pv. campestris
Citrus Canker X.C. pv. citri
Food Spoilage Potential of Xanthomonas
Pre-harvest Spoilage
Xanthomonads are the cause of a number of pre-
harvest diseases of fruits and vegetables. They cause
blights, spots, cankers and rots of different fruits and
vegetables (Table 3).
Lima bean pods can be spoiled by the common
blight caused by X.C. pv. phaseoli. Bacterial spots of
tomato and pepper caused by X.C. pv. tomato and
X.C. pv. vesicatoria reduce quality and marketability
of these important vegetables. One of the most feared
diseases of citrus fruits, citrus canker, is caused by
X.C. pv. citri. The commodity loses its aesthetic
appeal, quality and marketability, thereby causing
severe economic losses.
Post-harvest Spoilage
Being a part of the natural microflora xanthomonads
are invariably associated with plants and plant prod-
ucts. The role of xanthomonads in the post-harvest
spoilage of plant foods and food products has been
poorly investigated. However, the association of
various hydrolytic enzymes with the growth of xan-
thomonads suggests their high food spoilage poten-
tial. Many xanthomonads are known to possess
proteolytic, amylolytic, cellulolytic, pectolytic and
lipolytic activities. As fruits and vegetables provide
ample substrate for these enzymes the food spoilage
potential of xanthomonads is high. Xanthomonads
may also bring about yellow discoloration of foods
due to their pigment-producing potential. Xantho-
monads also produce xanthan gum, which may cause
undesirable gumminess in certain foods and fruit
juices. Products formed from the infected plants and
plant products are also likely to undergo spoilage by
the action of hydrolytic enzymes.
Production of Xanthan Gum by
Xanthomonas
Xanthan gum is a high-molecular-weight poly-
saccharide gum. It is produced extracellularly by
strains of Xanthomonas campestris in a medium con-
taining carbohydrate and protein. Industrially it is
 Next Page

2328 XANTHOMONAS

HO
d...;
M 3 Na, K, ’/&a

Figure 4 Structure of xanthan gum.

produced by a pure culture fermentation of a carbo- Table 4 Applications of xanthan gum in the food industry
hydrate-containing medium with the strains of X. Product Function
campestvis developed for this purpose. Xanthan gum Stabilizer
Ice-cream
is a substituted cellulose. It is composed of p(1+4)- Sauces and gravies Thickener
linked glucose backbone. Every alternate glucose Dressings Stabilizing and suspending agent
residue in the backbone is connected to a mannose Non-alcoholic beverages Stabilizer
through an a(1+3) bond, which is connected to a Cake mixes and batters Suspending agent
Relishes Thickener
glucoronic acid residue through an a(1+2) linkage.
Processedcheese Stabilizer
The glucoronic acid residue is in turn connected to Milk-based desserts Thickener
another mannose unit through a p(1 4 4 ) linkage (Fig. Syrups and toppings Thickener
4). The repeating five-sugar unit thus comprises two Noodles Stabilizer
glucose and two mannose molecules and one glu- Spring roll pastry Stabilizer
curonic acid molecule. The mannose units have acetyl
and pyruvate groups. The contents of acetate and
food uses. The thickening, stabilizing, jelling and
pyruvate are in the ranges 4-5% and 2-6%, respect-
ively, in xanthan gum. The polymer has a molecular emulsifying properties of this polysaccharide make it
weight of 1-10 x l o 6kDa. useful in food industry. It imparts good flavour release
v
characteristics and sensory qualities to food. It can
Xanthan gum has the following useful properties:
be pumped, sprayed and spread easily. Some of the
It gives high viscosities at low concentrations; applications of xanthan gum in the food industry are
It has remarkable emulsion stabilizing and sus- given in Table 4. Xanthan gum is used as a stabilizer
pending ability; in ice-creams and other milk-based products, in con-
Its viscosity is stable to changes in pH, salts and fectionery products and noodles, in salad dressings,
temperature; and non-alcoholic beverages. It is used as thickener
Xanthan gum solutions show pseudoplastic behav- in soups, sauces, gravies, shakes, syrups, relishes and
iour, i.e. with an increase in shear rate the viscosity toppings. It is also used as a suspending agent in a
of a xanthan solution decreases and vice versa. number of foods including dressings, cake mixes and
batter. In its non-food uses, xanthan gum finds appli-
Application of Xanthan Gum in Food cation in the preparation of polishes, paints, adhe-
sives, ceramics, and explosives. In the petroleum
Industry
industry xanthan gum is used for enhanced oil
Because of the above properties of xanthan gum it recovery.
serves as an excellent stabilizing, thickening and emul-
sifying agent. Xanthan gum has a wide range of appli- See also: Enzyme Immunoassays: Overview. Fer-
cations. It has several pharmaceutical, food and non- mentation (Industrial): Production of Xanthan Gum.
 YEASTS: PRODUCTION AND COMMERCIAL USES 2335
YEASTS: PRODUCTION AND COMMERCIAL USES
Richard Joseph, Central Food Technological Research Institute, Mysore, India
Copyright 0 1999 Academic Press
Introduction
Commercial yeast production worldwide exceeds 1.8
million t per annum. The yeasts are used mostly by
the baking industry, but also by the brewing and
distilling industries. Yeast is also a commercial source
of natural flavourings, flavour potentiators and the
dietary supplements.
Yeasts are unicellular eukaryotes, and in several
ways are akin biochemically to higher organisms.
They have been shown to be suitable for the expres-
sion of valuable mammalian and plant proteins, and
have therefore emerged as an important bio-
technological asset in recent years.
The emphasis of this article is on the practical
aspects of the production and preservation of bakers’
yeast.
History
The practice of yeast husbandry can be dated to the
Neolithic, i.e. long before scientific knowledge about
microorganisms was available. More authentic evi-
dence dates from 4-5 millennium BC when the arts
of leavening, brewing and wine-making were well
known. The excavation of Thebes in Egypt has
revealed models of baking and brewing dating from
the 11th dynasty (about 2000 BC).
The use of yeasts therapeutically is revealed in the
Ebes Papyrus, one of the earliest known medical docu-
ments, dating from the 16th century BC. Hippocrates
(4-5th century BC), the well-known Greek physician,
also used yeasts therapeutically.
The first yeasts used for baking were obtained from
the mashes produced in the manufacture of beer. The
first compressed yeasts used for baking and brewing
were made in England in about 1792, and by 1800
they were available throughout northern Europe. The
large-scale commercial production of bread in the US
was facilitated by the introduction of an improved
strain of compressed yeast in 1868, by Charles
Fleischmann. The vigorous research and development
effort that ensued yielded yeast strains suited to each
type of fermentation and leavening. For example,
some of these strains were able to tolerate high sugar
or salt concentrations, or the high temperatures used
in fermentation and the proving of dough.
Anton van Leeuwenhoek (1632-1723) of Holland
was possibly the first human to set eyes on a yeast,
when he observed a droplet of fermenting beer with
the aid of one of the first microscopes, capable of
250-270-fold magnification. Interest in yeasts, and in
microorganisms in general, then lay almost dormant
until Louis Pasteur (1822-1 895) carried out extensive
systematic studies which revealed the nature of yeasts
and their extraordinary biochemical capabilities.
Classification
Yeasts are unicellular fungi reproducing asexually by
budding or fission and sexually by spore formation.
Emil Christian Hansen’s studies, over a span of 30
years, provided insight into the biological features of
yeasts and facilitated their differentiation and their
characterization as species.
Currently more than 500 species of yeasts, belong-
ing to around 50 genera, are known. Yeasts belong
to the division Eumycota, within the subdivisions
Ascomycotina, Basidiomycotina, and Deutero-
mycotina.
Bakers’ yeast and the yeasts used in brewing, wine-
making and distilling are strains of Saccharomyces
cerevisiae, belonging to the family Saccharo-
mycetaceae in Ascomycotina.
Saccharomyces cerevisiae
The genus Saccharomyces (translation ‘sugar fungus’)
derives its name from its common occurrence in
sugary substrates such as nectar and fruits. Strains of
S. cerevisiae have been isolated from diverse sources,
including breweries, wine, berries, cheese, pear juice
and must, honey, eucalyptus leaves, kefir, Drosophila,
2336 YEASTS: PRODUCTION AND COMMERCIAL USES

soil and human skin, sputum and leg ulcers. S. cer- Commercial Production of Bakers’ Yeast
evisiae has around 87 synonyms worldwide.
Morphology
Sources
After 3 days’ growth in malt extract at 25”C, S.
cerevisiae cells are either globose in shape (5.0- Industries requiring yeast cultures can either obtain
10.0)x (5-12.0) pm or ellipsoidal or cylindrical, them from culture collection centres or isolate and
measuring 3.0-9.5 pm x 4.5-21.0 ym. The cells may develop their own cultures. In either case, the propa-
occur singly or in pairs, short chains or clusters. gation and maintenance of cultures for long-term use
Streak cultures on malt agar are butyrous and ensures consistency of performance and quality.
cream to brownish. They are either smooth and However, basic facilities and expertise within the
slightly raised with shallow striations, or raised, industry are required.
folded and (often) subdivided. They can be either Saccharomyces can be isolated from natural
glossy or dull. sources, and maintained in pure culture by con-
ventional microbiological techniques. The source
Reproduction material (fermenting sugary materials, fruit juices or
Asexual reproduction usually occurs by budding. The soil) is usually serially diluted and plated onto potato-
buds arise on the ‘shoulders’ and at either pole of the dextrose agar (PDA) or yeast extract-peptone-dex-
cell. The vegetative cells are diploid or polyploid, and trose agar (YEPDA). The growth of yeasts in pref-
this phase predominates in the life cycle of the yeast. erence to bacteria is achieved by the p H of the medium
Sexual reproduction involves the production of being below neutral (usually 4-6) and the incorp-
asci, within which ascospores develop directly fol- oration of antibacterial antibiotics.
lowing meiosis of the diploid nucleus. The sporulation Enrichment culture is a technique by which strains
of S. cerevisiae is encouraged by media containing with characteristics required by industry (e.g. tol-
acetate, such as acetate agar. Sporulation also occurs erance to high temperatures) can be isolated from
on potato-dextrose agar. True sexual reproduction is natural habitats. The required strains are selected
found in some strains, which exhibit heterothallism, either by gradually increasing exposure to the factors
of the bipolar physiological type. Compatibility is to which tolerance is required, or by cultivation with
determined at one mating type locus, which may very high levels of the factors over a long period of
contain either of two alleles. Conjugation occurs time.
either by the fusion of two ascospores or by the
fusion of two haploid somatic cells of germinating
Maintenance of Cultures
ascospores. Haploid vegetative clones can be raised
by the germination of isolated spores. If yeasts are used regularly, for example in a batch
process with a constant periodicity, the simplest
Hybrid Strains
method of maintaining stock cultures is to use agar
Cells of opposite mating types can be fused to produce slopes or broth. For semi-continuous and continuous
hybrid yeast strains. This technique can be used to processes, fresh yeast cultures must be available
combine industrially desirable traits such as high because the overgrowth of less efficient and low-per-
growth rates, high yields, resistance to drying and COz forming variants of the yeasts in continuous processes
production. A range of hybrids has been developed, is a common problem.
suited to different needs. These include rapidly fer- Normally, slope and broth cultures are subcultured
menting strains which produce high volumes of COz, once every 2 months. After allowing for adequate
for automated bakeries; strains with intermediate growth at ambient temperature, they are kept at 4-
activity, for traditional bakeries; and strains which 8°C until use. The drawback of this simple technique
ferment more slowly, for in-store bakeries. Strains is the risk of contamination and the development of
have also been developed which have improved resist- genetic variants which are less efficient than the yeasts
ance to drying, osmotolerance and tolerance to freez- originally selected. These undesirable effects can be
ing. prevented by the incorporation of a ‘selection pres-
If sexual mating is difficult to achieve owing to very sure’ to ensure the retention of strains which perform
low yields of viable spores, modern techniques for well in preference to any variants which perform less
improving strains can be used. These include proto- well. Examples include the incorporation of a high
plast fusion and the construction of recombinant sugar or salt concentration in the maintenance
DNA, which can be achieved with relative ease using medium, to retain yeasts which will be tolerant to
the tools and techniques of molecular genetics. high concentrations in the fermentation.
 YEASTS: PRODUCTION AND COMMERCIAL USES 2337

Preservation of Cultures Table 1 Composition of molasses

Yeasts that are sensitive to dehydration in slope or Percentage of total

stab cultures may be maintained by overlaying with weight of molasses
mineral oil. It is generally observed that microbes in Component Range Average
soil culture retain their original characteristics over a Water 17-25 20
relatively long period of time. This is a simple and Sucrose 30-40 35
inexpensive method, in which sterilized garden soil Glucose 4-9 7
Fructose 5-12 9
containing 60% moisture is inoculated with the Other reducing substances 1-5 3
culture and, after allowing for growth at ambient Other carbohydrates 2-5 4
temperature for a week, is kept at 4 4 ° C . All micro- Ash 7-15 12
organisms except non-sporulating bacteria sporulate Nitrogenous compounds 2-6 4.5
in soil, and in this form remain viable and functional Non-nitrogenous acids 2-8 5
Waxes, sterols and phospholipids 0.1-1 0.4
for up to 2 years. Vitamins Trace Trace
Cultures can also be frozen, keeping them func-
tionally intact for long periods. The inclusion of gly-
cerol (5-20%) in the suspension medium and storage many countries is subject to excise. Alternative sub-
at -20°C are recommended. Well-equipped culture strates for the production of bakers' yeast have there-
collection centres have facilities for the extended fore had to be considered. Starchy substrates, derived
storage of cultures in liquid Nz at -196°C or by from low-grade grains and tubers, are the logical
lyophilization. It is recommended that lyophilized cul- alternative to molasses, but suitable manufacturing
tures be stored at 4-8"C, but they can withstand the processes need to be developed. Sugar-cane juice and
ambient temperatures imposed during transit by post. sugar-beet extract can also be used for the production
Frozen cultures stored in liquid N2 and lyophilized of bakers' yeast, if the process is economically viable.
cultures maintain their genetic constitution and func- Nitrogen
tional characteristics for long periods of time.
S. cerevisiae can utilize inorganic nitrogenous com-
pounds such as ammonium sulphate, ammonium
Growth Requirements
chloride or even ammonia. Urea can be used to
S. cerevisiae is a heterotroph, i.e. it requires preformed provide Nz in the commercial production of yeasts.
organic compounds for growth. It is also a mesophile,
Minerals
growing best in the temperature range 25-40°C. In
common with other living organisms, bakers' yeast The major minerals required for growth by S. cer-
has basic nutritional requirements for carbon and evisiae are phosphorus (an important component of
nitrogen sources, minerals and vitamins. nucleic acids), potassium, calcium, sodium, mag-
nesium and sulphur. Iron, zinc, copper, manganese
Carbon and cobalt are required as trace elements. These
A limited range of sugars is utilized as a carbon source requirements are largely met by the molasses, with
by S. cerevisiae. Glucose and fructose are readily util- the exceptions of phosphorus and magnesium.
ized, and of the disaccharides, sucrose and maltose Phosphorus is supplied in the commercial pro-
are preferred. Other malto oligosaccharides can also duction of yeasts as phosphoric acid or as phosphate
be utilized, but less readily. Notably, S. cerevisiae of sodium, potassium or ammonium. Magnesium is
cannot utilize pentoses, other hexoses, the disac- supplied as magnesium sulphate in the growth
charides lactose or cellobiose or the polysaccharides. medium.
In industry, the preferred carbon sources are cane
Vitamins
or sugar-beet molasses, which have a fermentable
sugar concentration of 50-55?0 and around 80% S. cerevisiae requires biotin, pantothenic acid, inositol
total soluble solids. The composition of typical molas- and thiamin for growth. These, except for thiamin,
ses is shown in Table 1. However, it should be noted are usually available from molasses in adequate quan-
that being a by-product of the sugar industry, molasses tities. Sugar-beet molasses, however, is deficient in
is considerably variable in composition. biotin and hence requires supplementation with syn-
However, improvements in the processes for the thetic biotin. The biotin requirement of yeast is
recovery of sugar have resulted in the production of reported to be increased when urea is used as the Nz
molasses containing a lower concentration of fer- source in the growth medium. A mixture of L-aspar-
mentable sugars. In addition, molasses is also the tate and oleic acid was found to completely eliminate
substrate for the production of ethanol, and so in the requirement for biotin.
2338 YEASTS: PRODUCTION AND COMMERCIAL USES
Molasses must be supplemented with thiamin to
enable maximum growth of the yeast.
Oxygen
S. cerevisiae possesses a remarkable ability to adapt
to thrive in varying levels of available 0 2 . In very low
levels of 02,its metabolism responds by shutting
off the respiratory enzymes. The yeast then leads a
fermentative life, in which sugar is partially and non-
oxidatively utilized for energy and the ‘waste’ product
is ethanol. In contrast, when adequate 0 2 is available,
sugar is converted by the respiratory enzymes to CO2
and H20, as well as to intermediates needed for the
cell biomass.
Therefore in microaerophilic conditions (often
erroneously termed ‘anaerobic’ conditions), the yeast
grows significantly more slowly than in aerobic con-
ditions. The maximum theoretical yields of yeast
solids under microaerophilic (anaerobic) and aerobic
conditions have been calculated as 7.5 kg and 54.0 kg
respectively per 100 kg of sugar utilized. In the case
of S. cerevisiae, the concentration of the substrate
(sugar) influences the availability of 02,with con-
sequent effects on the growing culture. If the medium
contains > 5% glucose, glucose utilization via the
TCA (tricarboxylic acid) cycle is almost completely
blocked. Even if the culture is aerated, glucose can
only be fermented, this phenomenon being known as
the ‘glucose effect’ or the ‘Crabtree effect’. If the
glucose level is then lowered to 0.1% and aeration
continued, the metabolism of the yeast shifts from
fermentation to respiration.
For aerobic growth, therefore, the sugar has to be
supplied incrementally so that the rate of growth of
the yeast (p)does not exceed 0.2 and the respiratory
quotient (RQ) is maintained at the value of 1. The
production of bakers’ yeast should therefore take
place in aerobic growth conditions, facilitating the
efficient oxidation of glucose to C 0 2 and HzO and the
concomitant formation of ATP, required for cellular
metabolism and the build-up of biomass. In aerobic
conditions, as much as a third of the available sugar is
metabolized via the hexose monophosphate pathway,
generating NADPH, which is mainly utilized in syn-
thetic reactions. Thus, under aerobic growth con-
ditions the yeast’s metabolism is elegantly balanced,
generating chemical energy for cellular metabolism
and precursor molecules for cell growth and pro-
liferation. The TCA cycle is particularly important,
being involved in the production of both the biomass
precursor molecules and the chemical energy. S. cer-
evisiae also possesses the enzymes involved in the
‘glyoxylate shunt’. This replenishes the TCA inter-
mediates taken up for biomass formation.
Value pH
S. cerevisiae grows optimally at p H 4.5-5.0, although
it can tolerate a pH range of 3.6-6.0. At higher pH
values, the yeast’s metabolism shifts, producing gly-
cerol instead of ethanol in microaerophilic conditions.
In the production of bakers’ yeast an initial pH at
the lower end of the range inhibits the growth of
bacterial contaminants. As the process progresses
towards harvesting, the p H is raised slightly so that
any colouring matter taken up by the yeast from the
molasses is desorbed.
Temperature
S. cerevisiae has one of the shortest generation times
amongst yeasts, 2.0-2.2 h at 30°C. Bakers’ yeast pro-
duction is optimal when the temperature of the cul-
tivation medium is maintained at 28-30°C. However,
productivity, in terms of grams of yeast solids pro-
duced per litre of cultivation medium per hour,
depends on the feed rate in fed-batch fermentations.
Despite growth at diminished rates being non-expo-
nential, high productivity is still achieved.
Manufacturing Processes used for
Bakers’ Yeast
The overall process is summarized in Figure 1,
Preparation of Medium
The molasses (containing around 50% fermentable
sugars and 80% soluble solids) is usually diluted with
an equal weight of water, and the pH is adjusted to
4.5-5.0 with sulphuric acid. The diluted molasses is
clarified using a desludger centrifuge. Clarification by
filtration is recommended for beet molasses, but is not
necessary for cane molasses. The clarified molasses is
then sterilized by the high temperature short time
(HTST) process. Other sterilization methods, which
involve prolonged heating at low temperatures, cause
caramelization and hence a decrease in the fer-
mentable sugar content.
Medium supplements are added, these typically
being: a nitrogen source (e.g. urea, 2.5gl-I); potas-
sium orthophosphate (KH2PO,), 0.96 g I-’; hydrated
magnesium sulphate (epsomite, MgS04.7H20),
0.1 g1-l; and a defoamer (silicone, fatty acid deriva-
tives or edible oil), 0.01 g I-’.
The molasses is then transferred to the fermenter,
filling it to about two-thirds of its total volume, and
pitching yeast is added. More clarified molasses is
added incrementally in the fed-batch cultivation.
A substrate of starch (from corn, sorghum or
tubers), hydrolysed by acids or enzymes, or of sugar
cane juice, does not require elaborate clarification.
Supplementation with a nitrogen source, minerals and
 YEASTS: PRODUCTION AND COMMERCIAL USES 2339

Diluted
molasses

Filter

Air

Water removal Extruder Dryer Packaging Cold storage

Active dry yeast

Figure 1 Production of bakers' yeast

vitamins is, however, necessary. With suitable sup-

plements, these raw materials are well-suited for the 'Gassed liquid' level
production of bakers' yeast, although economic con- 1 0 3 c 0 0 0 n0 0 00 0 0 0 0 0 (

siderations may dictate otherwise. ................366 cm ................

-
Cultivation
In industry, bakers' yeast is produced in fermentation
j
tanks with a capacity of 200 m3 or more. Tanks, and D
7
all connecting tubes, should preferably be made of
stainless steel. E,
W
The design and operation of fermenters for the m
W

production of bakers' yeast must be carefully con-

sidered given the interrelationship between aeration,
the specific growth rate of yeast and the substrate
concentration. The facility for bubbling compressed
air (as a source of 0 2 ) into the medium, to ensure
effective aeration, is therefore particularly important.
In practice, 0 2 transfer in a fermentation system can
be manipulated by adjusting the bubble size and by
the dispersion of air in the cultivation medium, by
using mechanical agitation close to the point of entry
of air into the medium. The level of dissolved O2
during fermentation can be determined by using O2
electrodes.
The features of a typical cultivation tank used for
the production of bakers' yeast, an airlift fermenter,
17 cm
Figure 2 Airlift fermenter.
are shown in Figure 2. It consists of a cylindrical
vessel, provided with a sparger (aerater) at the bottom
for producing air bubbles in the medium. Aerobic
growth results in the generation of almost 14 650 J of
heat per gram of yeast solids, and because cultivation
has to be carried out at 28-30°C, cooling is necessary.
This is achieved by cooling coils, either within or
2340 YEASTS: PRODUCTION AND COMMERCIAL USES
outside the vessel. Directional flow of the cultivation
medium is achieved by pumping air in at the bottom
of a ‘draft tube’. The ratio between the depth of the
cultivation medium and the diameter of the vessel has
been shown to influence 0 2 transfer. If the depth of
the broth exceeds 3 m, the use of compressed air, to
overcome the hydrostatic pressure of the liquid, is
recommended. The design of the air sparger is also
important for effective O2transfer. The inclusion of a
motor-driven impeller further increases the effect-
iveness of 0 2 transfer, but the additional investment
and energy consumption involved may undermine
their cost-effectiveness.
The manufacture of bakers’ yeast begins with a
number of stages which build up production, and
involve the inoculation of the medium with pitching
yeast. This development process is usually divided
into eight stages, in which the yeast solids are grad-
ually built up from the slope or flask culture of yeast.
Over the eight stages, 0.2 kg of yeast solids give a final
yield of about 100000kg of yeast. In the first two
stages, sterilized medium and pure yeast cultures are
employed in pressurized tanks, but the subsequent
stages are operated in open tanks. The entire process
is known to involve 24 generations of the yeast. Cul-
tivation may be terminated before the normal final
stage, in which case a yeast cream is obtained by
centrifugation and used for pitching as required.
Maturation
At the end of the final stage of yeast cultivation, the
feed rate is greatly reduced. This allows the yeast cells
to mature and results in a low proportion of budding
cells, which confers higher stability on compressed
yeast in storage.
Finishing Stages
Yeast Cream The culture broth can be centrifuged
in a continuous centrifuge (with a vertical nozzle) at
4000-5000 g, leading to almost complete recovery of
the yeast cells. In the first run, around two-thirds of
the fluid can be removed and in subsequent runs
further concentration of the cells is achieved, pro-
ducing a slurry called ‘yeast cream’, which contains
about 20% yeast solids. Yeast cream can be stored at
4°C for a number of days, with good retention of
viability.
Compressed Yeast This is prepared from yeast
cream by filtration or by pressing in a filter press.
Rotary continuous vacuum filters can also be used.
The pressed cake thus obtained is mixed with 0.1-
0.2% of emulsifiers such as monoglycerides, digly-
cerides, sorbitan esters and lecithin, and then extruded
through nozzles. The extruded material, in the form of
thick strands, is cut into suitable lengths and packaged
(usually in packs of about SOOg) in wax paper or
polythene sheet. The compressed yeast must be
rapidly cooled, and stored at 5 4 ° C .
Active Dry Yeast Active dry yeast is useful in situ-
ations (e.g. homes) where storage at low temperatures
is not possible. It is prepared by spreading out the
pressed yeast cake to produce thin strands or small
particles, which are then dried. Generally, a tunnel
drier is used, taking 2-4h with the air inlet tem-
perature maintained at 28-42°C. However, more
modern equipment, achieving either continuous
drying or fluidized-bed drying (airlift drying), is also
available. Emulsifiers such as sucrose esters or sor-
bitan esters (0.5-2.00/) are mixed with the dried yeast
to facilitate rehydration. Antioxidants, such as butyl
hydroxyanisole at 0.1%, are also added, to prevent
undesirable oxidative changes. Active dry yeast has a
moisture content of 4.0-8.5%.
Yeast Products and Uses
Nutritional Yeast
Yeast which has been heat-killed and dried is a source
of protein and the B vitamins, and useful for sup-
plementing foods and animal feeds. Currently the
yeast used for these purposes is derived from brewing,
wine-making or distilling, but the cultivation of yeast
exclusively for food/feed supplementation is a pos-
sible future development. The official definitions and
standards for such products are laid down by the
International Union of Pure and Applied Chemistry
(IUPAC), the National Formulary (NF XII) of the
American Pharmaceutical Association and the Food
and Drug Administration (FDA) of the US.
Lysine-enriched Yeast
Strains of bakers’ yeast which can convert precursor
molecules such as 5-formyl-2-oxovalerate or 2-oxo-
adipate to lysine, with high efficiency, have been
reported. The use of such strains for the fortification
of lysine-deficient cereals has potential.
Vitamin-enriched Yeast
The addition of thiazole and pyrimidine to the cul-
tivation medium has been shown to cause bakers’
yeast to synthesize high levels of thiamin (around
600 pg g-’).
The irradiation of bakers’ yeast with ultraviolet
light has been shown to convert ergosterol to cal-
ciferol (vitamin D2),with a vitamin potency reaching
as much as 180 000 USP units per gram of yeast. Such
strains will be useful for the fortification of food, feed
and pharmaceuticals.
 YEASTS: PRODUCTION AND COMMERCIAL USES 2341
Yeast Lysates and Yeast Extract
Yeast autolysates are prepared by imposing conditions
which trigger the yeast’s native hydrolytic enzymes.
These digest the yeast proteins and nucleic acids,
converting them into soluble substances with an
acceptable flavour and taste. The process involves the
addition of ethyl acetate and NaCl (l-3%) to a yeast
slurry containing 14-16’70 yeast solids. The mixture
is maintained at 45°C for about 20 h, and then the
whole autolysate is concentrated to a paste or dried
to a powder. The soluble fraction can be separated
out by centrifugation, and then concentrated.
Yeast hydrolysates can also be obtained, by adding
hydrochloric acid to a yeast slurry containing 65-
80% yeast solids and subjecting the mixture to reflux
for about 12 h. It is then neutralized with sodium
hydroxide solution, filtered, decolorized, con-
centrated and dried. Concentrates of hydrolysates,
containing 42% solids, 18% NaCl and 3 % Nz can be
obtained.
Biochemicals
S. cerevisiae is a good and economical source of
several biochemicals which are in great demand by
biochemists and molecular biologists. S. cerevisiae is
a source of the pyridine nucleotides NAD and NADP
and their reduced forms and of several ribonucleotides
and deoxyribonucleotides, including AMP, ADP and
ATP.
Heterologous Gene Expression in S. cerevisiae
With the advent of the recombinant DNA technology
a major application has been to extract a desired plant
or mammalian gene and introduce it into a suitable
microorganism with the aid of a vector for expression.
As microorganisms can be grown to huge numbers in
a short period of time in fermenters, copious amounts
of the ‘foreign’ gene product can be produced. The
Human Genome Project makes great use of S. cer-
evisiae and its vectors.
Future Developments
Yeast has come to occupy a unique place in science
and technology: being a unicellular microorganism
with a short life span, it is readily amenable to cul-
tivation and to manipulation to reflect process needs.
It is also amenable to traditional and modern methods
of genetic engineering, using its natural recombination
processes as well as in vitro techniques. Yeasts are
eukaryotes and their biochemistry has much in
common with that of higher organisms, including
glycosylation and cell sorting. Yeasts are therefore
poised to be major players in biotechnology in the
future.
See also: Fermentation (Industrial): Basic Con-
siderations; Media for Industrial Fermentations. Genetic
Engineering: Modification of Yeast and Moulds.
Saccharomyces: Saccharomyces cerevisiae; Sac-
charomyces carlsbergensis (Brewer’s Yeast). Single Cell
Protein: Yeasts and Bacteria. Wines: Microbiology of
Wine-making.
Further Reading
Chapman JW (1991) Trends in Food Science and Tech-
nology. P. 176. Elsevier Sci. Pub. Ltd (UK).
Collar C (1996) Food Sci. Technol. Int. 2 ( 6 ) :349-367.
Doran PM (1995) Bioprocess Engineering Principles.
London: Academic Press.
Edelmann K, Stelwagen P and Oura E (1980) In: Stewart G
and Russell I (eds) Current Develop. Yeast Research. P.
51. Toronto: Pergamen Press.
Oura E, Soumalainen H and Viskari R (1982) In: Rose AH
(ed) Economic Microbiology. P. 87. New York: Aca-
demic Press.
Peppler HJ (1979) In: Peppler HJ and Perlman D (eds)
Microbial Technology, 2nd edn., vol 1. I? 157. New
York: Academic Press Inc.
Reed G (1982)In: Reed G (ed)Prescott & Dunn’s Industrial
Microbiology, 4th edn. P. 593. Westport: AVI Publishing.
Reed G and Peppler HJ (1973) Yeast Technology. Westpor:
AVI Publishing.
Rose AH and Harrison JS (eds) (1993) The Yeast, 2nd edn.,
vol 5. Yeast Technology. London: Academic Press.
Sat0 T (1966) Bakers’ Yeast. Tokyo: Korin-Shoin Pub.
Trivedi NB and Jacobson G (1986) In: Adams M R (ed)
Progress in Industrial Microbiology. I? 45. Amsterdam:
Elsevier.
White J (1954) Yeast Technology. London: Chapman &
Hall.
 ZYGOSACCHAROMYCES 2359

I Zygomycetes see Fungi: Classification of the Zygomycetes.

ZYGOSACCHAROMYCES
John P Erickson and Denise N McKenna Bestfoods Technical Center, Somerset, New Jersey, USA
Copyright 0 1999 Academic Press

The yeast genus Zygosaccharomyces contains eight Macroscopic and microscopic morphology obser-
recognized species, of which three pose serious eco- vations cannot differentiate Zygosaccharomyces from
nomic spoilage risks to processed food manufacturers. other yeast or individual species within the genus.
They are Z . bailii, Z . rouxii and Z . bisporus. Z . O n various mycological agars, colonies are smooth,
bailii is particularly troublesome in mayonnaise, salad round, convex and cream-coloured. Microscopic
dressings, pickled vegetables, teas and various fruit observation shows large ovoid elongated cells and
drinks. In contrast, Z. vouxii spoilage is more closely multilateral budding. All eight species vigorously
associated with high sugar or salt-based ingredients ferment glucose and produce asci with one to four
and finished products like fruit concentrates, syrups, globose or ellipsoidal ascospores inside. The three
candied fruit pieces and confectioneries such as mar- food and beverage spoilage species can be dis-
zipan, chocolate candy fillings, etc. The Z. bisporus tinguished by sucrose fermentation properties,
spoilage profile is similar to Z . rouxii, but occurs presence/absence of growth at 37°C and acetic acid
less frequently. The types of food ingredients and resistance (Table 1). However, the incubation time
processed foods spoiled by Zygosaccharomyces are and laborious media preparation required to conduct
generally considered shelf-stable in that they readily these tests make them unsuitable for routine quality
inactivate a broad spectrum of food-associated micro- control applications. From a practical perspective, the
organisms ranging from bacteria to moulds. Hence, physiochemical attributes of the ingredient or finished
Z. bailii, Z. rouxii and, to a lesser extent, Z. bisporus product can furnish useful information regarding
possess several intrinsic physiological resistance species identification. For example, if yeast was reco-
factors which allow them to survive and thrive in vered from a highly acidified food with a 3 0.90 water
normally hostile environments. The most prominent activity (a,$,),there is a strong likelihood Z. bailii was
resistance factors are acetic acid (vinegar) tolerance,
chemical antimycotic preservative resistance and
growth under high osmotic pressure. Effective con- Table 1 Key taxonomic, biochemical and physiological tests
required to differentiate three Zygosaccharomyces - food
tamination control is dependent upon ingredient spoilage species
ecology knowledge, stringent/consistent sanitation
standards, tight formulation specifications and Results
focused quality assurance strategies. Equally import- Tests Z. bailii Z. rouxii Z. bisporus
ant, finished product microbiological stability streng- Glucose fermentation Positive Positive Positive
ths and weaknesses must be well documented and (slow)
fully understood. Sucrose fermentation Variable Variable Negative
(slow)
Growth at 37°C Variable Variable Negative
Taxonomic and Ecological Growth in presence of Positive Negative Positive
1% acetic acid
Characteristics Water activity (a,) 0.80-0.85 < 0.80 < 0.80
The general taxonomic properties of Zygo- tolerance
saccharomyces are identical to ubiquitous foods yeast Adapted from Kreger-Van Rij NJW (1984) The Yeasts, A
genera such as Saccharomyces, Candida and Pichia. Taxonomic Study, 3rd edition. Amsterdam: Elsevier Science.
2360 ZYGOSACCHAROMYCES
detected. Conversely, a low acid food with high sugar
or salt content is more likely to be contaminated and
spoiled by Z . rouxii.
Zygosaccharomyces isolates from alcoholic bev-
erages are more difficult to discern since Z . bailii and
Z . rouxii exhibit comparable ethanol and low pH
tolerance, and the a,v of most wines is 30.95. Also,
with the exception of wines that contain sulphur
dioxide, very few wines and fruit cordials are pre-
served with antimycotic acids such as acetic, benzoic
and sorbic.
The ecological distribution of Z . bailii, Z . rouxii
and Z . bisporus is not well defined. The most fre-
quently cited natural habitats are mummified fruits,
tree exudate, and at various stages of raw sugar refin-
ing and commercial syrup production. It appears that
Zygosaccharomyces prefers specialized, selective eco-
logical niches. But technical limitations have strongly
influenced ecological investigations in two ways: first,
the lack of reliable and simple-to-use selective recov-
ery media prevented detection in highly competitive
and dynamic environments, and second, slow,
complex identification methods deterred screening
large numbers of yeast isolates. Recent advances in
selective plating media combined with powerful new
genetic assays such as polymerase chain reaction
(PCR)have greatly enhanced the ability to conduct in-
depth and long-term ecological studies in both natural
and food-processing plant environments.
Physiological and Preservative
Resistance Characteristics
Z. bailii
Among the three Zygosaccharomyces food/beverage
spoilage species, Z . bailii possess the most pro-
nounced and diversified antimicrobial resistance
attributes. Extensive research conducted over four
decades has repeatedly documented resistance to high
concentrations of monocarboxylic acids, chemical
antimycotic preservatives and ethanol. Z . bailii also
grows over wide p H and a,< ranges - 2.0-7.0 and
0.85-0.99, respectively. Even more significant for the
food industry, the yeast can survive and defeat potent
broad-spectrum synergistic preservative com-
binations (hurdles) that impart microbiological shelf-
stability protection to acidified processed foods. Spe-
cifically, Z . bailii has been shown to be innately resist-
ant to commonly used food and beverage
preservatives, including acetic acid, lactic acid, pro-
pionic acid, benzoic acid, sorbic acid, sulphur dioxide
and ethanol. Optimum preservative resistance is medi-
ated by glucose levels, with 10-20% sugar con-
centrations producing maximum resistance responses.
Depending upon accompanying formulation factors
such as pH, it is not atypical to encounter Z . bailii
strains spoiling fruit drinks and pourable salad dress-
ings preserved with > 1000 p.p.m. of potassium
sorbate. Intrinsic preservative resistance mechanisms
are extremely adaptable and robust. Their func-
tionality and effectiveness are unaffected or mar-
ginally suppressed by physiochemical environmental
conditions such as low pH, low a , high osmotic
pressure and sparse nutrients. Interestingly, there is
strong evidence that Z. bailii preservative resistance
is stimulated by the presence of multiple antimicrobial
constituents. Cellular acetic acid uptake was inhibited
when sorbic acid, benzoic acid or ethanol was incorp-
orated into yeast culture medium. Similarly, ethanol
levels up to 10% did not adversely alter Z . bailii
intrinsic sorbic acid and benzoic acid resistance at
p H 4.0-5.0. Sugar substrate investigations also
revealed negligible effects on preservative resistance.
Comparable sorbic and benzoic acid resistance was
observed regardless of whether Z. bailii cells were
grown in culture medium containing glucose, fructose
or sucrose as the fermentable substrate. Conversely,
synergistic interaction between salt and acetic acid
generated antagonistic effects against Z . bailii. As salt
levels increased, the yeast was inactivated by lower
amounts of acetic acid.
Sugar fermentation within the Zygosaccharomyces
genus is unique. Unlike the vast majority of yeast
genera, fructose is metabolized more rapidly than
glucose. For species like Z. bailii and Z . rouxii this
produces a phenomenon known as fructophily, in
which yeast growth rates are greatly accelerated when
a food’s fructose level approaches and exceeds 1% of
product composition. The slow and delayed fer-
mentation of sucrose is directly linked to fructose
metabolism: sucrose, a disaccharide composed of
glucose and fructose, is hydrolysed by food acids, i.e.
low p H conditions. Hence, in acidic processed foods
and beverages there is a steady accumulation of
glucose and fructose during storage. If sucrose is the
primary carbohydrate ingredient and Z . bailii con-
tamination is present, cell growth can be impeded for
several weeks until sufficient quantities of fructose
and glucose are available to support reproduction and
proliferation. This is usually preceded by a 2-4-week
lag before visible spoilage defects are noticeable. In
toto, overt product quality deterioration does not
surface until 2-3 months after manufacturing. Z .
bailii’s key preservative, physiological and metabolic
resistance properties are summarized in Table 2.
2. rouxii and 2.bisporus
These two species differ from Z . bailii in their inferior
resistance to acetic acid and chemical antimycotic

Table 2 Key preservative and physiological resistance factors
of Zygosaccharomyces bailii
factor Resistance properties
Acetic acid (pH 4.0) 33%
Benzoic acid (pH 4.0) 31000 p.p.m.
Sorbic acid (pH 4.0) 31000 p.p.m.
Sulphur dioxide 3500 p . p m
Ethanol 2 10-1 5%
Sodium chloride <IO%
Sugar (glucose) 2 60%
PH 12.0
Water activity (a,) 3 0.80-0.85
Temperature range 3 8-37°C
Atmosphere 3 Facultative (aerobic-stimulatory)
preservatives. In contrast, both yeasts grow at lower
a,vthan Z . bailii. Z . rouxii is capable of spoiling honey
and related high-sugar foods at a, as low as 0.62,
whereas Z. bailii growth potential ceases in the 0.80-
0.85 range. Comparative salt tolerance further illus-
trates exceptional capacity of Z . rouxii to survive high
osmotic stresses. It can grow in liquid sugars at > 75"
Brix and salt concentrations close to 20%, cor-
responding to 0.75 and 0.85 a,\,respectively. With the
exception of a few rare strains, the growth of Z .
rouxii and Z . bisporus is completely inhibited by
3 500 p.p.m. of sorbic and benzoic acids within a 4.0-
4.5pH range. They are especially sensitive to acetic
acid, rapidly dying off at 2 1% levels.
Physiochemical environmental effects on sugar and
salt-tolerant yeast have not been studied as com-
prehensively as Z . bailii. This is most likely due to the
latter's broader economic impact regarding food and
beverage categories vulnerable to spoilage. Com-
parative studies of sugar- and salt-tolerant 2. rouxii
strains indicated that distinct physiological differences
existed. The sugar-tolerant strains grew over a 1.8-8.0
pH range in high sugar media, whereas salt-tolerant
strains were more sensitive to pH conditions. At 1mol
sodium chloride concentration, growth was detected
over a 3.0-6.6 pH range. When sodium chloride mol-
arity was doubled, growth was restricted to a narrow
4.0-5.0 range. Z. rouxii optimum growth tem-
perature increased as a,v decreased. Surprisingly, the
optimum temperature reached 35°C at 60.96 a,
levels, which is more typical of mesophilic bacteria
incubation requirements.
Zygosaccharomyces Heat Resistance
The heat resistance profiles of Z . bailii, Z . rouxii
and Z . bisporus are comparable to other ascospore-
forming yeasts. Z . bailii asci were significantly more
heat-resistant than Z . rouxii at 0.963 and 0.858 a, in
liquid medium adjusted to pH4.5. Z . bailii vegetative
cells were also more heat-resistant than Z . rouxii.
ZYGOSACCHAROMYCES 2361
As expected, asci and vegetative cell heat resistance
increased as a,v decreased. Six log reductions (D64sC)
were calculated as 1.2mid0.963 a,,, and
5.4 min/0.858 a,,,. Commercially processed fruit
drinks and sugar syrups are normally pasteurized at
75-85°C. This provides a large quality assurance
margin with respect to Zygosaccharomyces thermal
destruction efficacy.
Zygosaccharomyces Preservative and
Environmental Resistance Mechanisms
Due to the pioneering and elegant research efforts of
Dr A.D. Warth the Z . bailti preservative resistance
mechanism has been elucidated and thoroughly
understood. The organism utilizes an inducible, active
transport pump to counteract the toxic effects pro-
duced by undissociated preservative molecule build-
up inside individual cells. The pump provides two
levels of protection. First, it physically expels pre-
servative molecules from the cell, which assists in
maintaining low, non-injurious preservative levels
within the cell. Second, the rapid and efficient purging
of undissociated molecules prevents deleterious cyto-
plasm pH changes that could disrupt or shut down
critical metabolic pathways. Because the pump
requires energy to function optimally, high sugar
levels enhance Z. bailii preservative resistance. It is
equally effective in excreting monocarboxylic organic
acids and lipophilic straight chain fatty acid pre-
servatives such as sorbic acid and benzoic acid. Add-
itionally, the active transport successfully operates
across a wide p H and a , range, and broad nutritional
conditions.
The resistance to osmotic stress of Z. rouxii is
primarily associated with internal synthesis of polyols,
mainly glycerol and arabitol, which raises intra-
cellular pressure, bringing it in balance with the exter-
nal osmotic gradient. Cell membrane and wall
composition as well as ATPase enzyme activity may
be important in augmenting poly01 formation, thus
regulating osmotolerance.
Zygosaccharomyces Spoilage in Food and
Beverages
Z . bailii is the most troublesome and persistent spoil-
age yeast confronting acidified food and beverage
manufacturers. Early reports of inexplicable fer-
mentation spoilage in mayonnaise and salad dressing
date back to the 1920s. Several incidents described
violent fermentation coupled with the recovery of a
few yeasts. More detailed investigations in the 1940s
and 1950s confirmed Z. bailii spoilage in cucumber
pickles, sundry pickled vegetable mixes, acidified
2362 ZYGOSACCHAROMYCES
Table 3 Food and Drug Administration yeast fermentation -
spoilage recalls in dressings and related acidified processed
foods (1978-1 996)
Year Product
-
1978 Imitation mayonnaise
1981 Mayonnaise (single serving pouch)
1984 Low-calorie Roquefort dressing
1985 Homestyle salad dressing, real mayonnaise
1986 Real mayonnaise
1987 Carbonated beverages (soda and fruit concentrate)
1988 Ketchup
1990 Reduced-calorie mayonnaise, fat-free French
dressing
1991 Light mayonnaise
1992 Lite Caesar dressing, lite creamy Parmesan dressing,
olive oil vinaigrette dressing
1993 Salad dressing, tartar sauce, coleslaw dressing,
burger tartar sauce, thousand island dressing,
Parmesan pepper dressing, lite Parmesan pepper
dressing
1995 White salad dressing
1996 Salad dressing
sauces, mayonnaise and salad dressings. Spoilage
invariably occurred in acidic shelf-stable foods which
relied upon acetic acid (vinegar) to negate micro-
biological growth risks. Around the same time, spor-
adic gaseous fermentation spoilage incidents suddenly
appeared in high-acid/sugar fruit syrups and bev-
erages preserved with moderate benzoic acid levels
of 400-500 p.p.m. Again, Z . bailii was indisputably
identified as the spoilage culprit. The near sim-
ultaneous emergence of Z . bailii spoilage in two diver-
gent processed food categories was probably due to
improved laboratory and field evaluation techniques,
better communication channels, movement towards
consolidated, mass-production manufacturing facil-
ities and large-scale complex distribution networks.
Remarkably, 50 years later, Z. bailii spoilage risks
and root causes closely parallel the processed food
industry’s situation in the late 1940s to early 1950s.
Despite quantum leaps in formulation control, food
process equipment design and construction, and sani-
tation technologies (automated clean-in-place, sani-
tary transfer valves, etc.), Z. bailii remains
problematic in mayonnaise, salad dressings, tomato
ketchup, pickled/brined vegetables, low to moderate
Brix fruit concentrates, and various non-carbonated
fruit drinks. This is mute testimony to the organism’s
adaptability, resilience and overall hardiness. Fur-
thermore, Z . bailii spoilage is expanding into new
food categories. Two recent examples are spoilage
incidents in prepared mustards and fruit-flavoured
carbonated soft drinks containing citrus, apple and
grape juice concentrates. The specialized but per-
sistent nature of Z . bailii spoilage problems is exem-
plified in Table 3,which summarizes the US Food and
Drug Administration yeast fermentation - spoilage
recalls in high acetic acid and/or chemically preserved
foods and beverages covering an 18-year period
(1978-1996). Most likely, Table 3 represents a small
fraction of economic losses produced by Z . bailii
contamination. Obviously, company rejections, pro-
longed holding times and spoilage risks caught before
finished production lots reached retail distribution
channels are not accounted for.
In most cases it is difficult to pinpoint the specific
cause of spoilage. This is because the problem does
not show up until 2-4 months after production and
the finished product conformed to applicable for-
mulation, processing and microbiological spe-
cifications at the time of manufacture. Z . bailii
spoilage manifestations are readily recognized by both
customers and consumers. Overt physical and organ-
oleptic decomposition signs include product oozing
from jars or bottles, emission of pungent yeast and
alcoholic odours, occasional emulsion breakage
(dressings), sediment formation (beverages) and
brown surface film development on product surfaces.
Z . bailii and Z . rouxii spoilage must never be taken
lightly. Under extreme circumstances, internal COz
pressure increases inside glass jars or bottles to the
level where on-the-shelf explosions may take place.
Although the possibility is remote, personal injury
could result from flying debris. Plastic containers and
polyfilm pouches burst open rather than explode. If
slip and fall injuries were caused by spilled product
residue, the company is exposed to contentious liabil-
ity issues. A plausible scenario is that individuals
become angry after being soiled by high-velocity
product expelled from jars or bottles immediately
upon opening. More often than not, these types of
incidents result in unwanted government involve-
ment, or costly consumer complaint investigations.
Z. rouxii and Z. bisporus exclusively spoil high-
sugar/low a, foods and food ingredients. The most
prevalent types are sugar syrups, fruit syrups, molas-
ses, honey, fruit concentrates, sweetened wines and
cordials and confectionaries such as marzipan and
candy fillings. These yeasts ferment the food product,
leading to effervescence, alcohol odour or taste, and
turbidity which is more easily noted in clear sugar
syrups. However, Z. rouxii and Z . bisporus ferment
slower and less aggressively than Z. bailii. Thus, the
potentially serious ramifications generated by vig-
orous Z. bailii fermentative metabolism are not a
concern with osmotolerant yeast.
As mentioned previously, it is generally impossible
to ascertain the exact reason why Z. bailii or Z . rouxii
contamination occurred in processed food lots made
2-3 months earlier. Circumstantial and inferential evi-
dence is readily attainable from additional finished

product and environmental sample testing, but accur-
ate and concrete information gathering is highly
suspect when the investigation focuses on recon-
structing and interpreting events that happened weeks
ago. However, trend analysis of past spoilage inci-
dents suggests that certain commonalties exist among
diverse Zygosaccharomyces contamination failures.
They include undetected introduction of the offending
Z. bailii or Z. rouxii strain into the plant environment
from a low-level heterogeneous contaminated ingre-
dient lot. This is followed by yeast build-ups inside
key processing equipment because of inadequate or
poorly executed sanitation procedures. In tandem,
routine quality assurance/quality control (QA/QC)
monitoring protocols missed or overlooked con-
tamination risks. Eventually, finished product cross
contamination occurred during production runs, and
QA/QC standard operating procedures (SOPS)lacked
appropriate detection sensitivity, discrimination abil-
ities and sampling discipline/focus to discover the
problem. These shortcomings and deficiencies are
compounded in product formulations which are
excessively sensitive to yeast growth. Certain fruit
beverages can be spoiled by Z. bailii contamination
as low as one viable cell in 3 10 1 of finished product.
No sanitation or microbiological QA/QC programme
can cope with this degree of risk. The only viable
alternatives would be reformulation to increase sta-
bility and/or application of high-lethality thermal-
processing parameters.
-
Zygosaccharomyces Pract i caI
Food/Beverage Industry Aspects
Recovery and Enumeration - Q N Q C Microbiology
Several media were developed for the selective detec-
tion and quantification of preservative-resistant and
sugadsalt-tolerant yeast in susceptible foods and bev-
erages. As regards the former yeast group, standard
non-differential yeast and mould plating media (malt
extract or potato dextrose agars) were supplemented
with 20.5% glacial acetic acid and/or 0.05% sodium
benzoate. Recommended incubation times ranged
from 5 to 14 days. Common incubation conditions
were 20-25"C, aerobic atmosphere and acidifying
media to 4.0-4.5 p H range to ensure proper select-
ivity. The addition of 0.5% glacial acetic acid was As discussed earlier, Zygosaccharomyces colonies on
usually sufficient to produce a final p H of 4.0-4.5.
Specialized yeast enrichment broths were co-devel-
oped to increase recovery sensitivity below the typical
b 10 per gram or millilitre detection threshold of direct to identify Z. bailii and Z . rouxii do not lend them-
microbiological plating methods. Up to 1000-fold selves to routine microbiological Q C applications.
sensitivity increases were reported. The enrichment Yeast identification kits have been available for over
broths operate on three principles:
ZYGOSACCHAROMYCES 2363
0 Rich nutrient content and absence of acidulants
concomitantly resuscitate debilitated cells and
maximize growth rates.
0 Use of antibiotics to eliminate bacterial growth.
0 Plating or streaking on selective media to separate
spoilage from innocuous yeast strains.
The detection and enumeration of sugar- and salt-
tolerant yeast ( Z . rouxiij rely upon high solute con-
centrations in plating media and enrichment broths.
Typically, 40-60% glucose is added to the basal
medium to lower a , and establish a high osmotic
pressure environment, The most popular basal plating
medium is unacidified total plate count agar, which
is often supplemented with antibiotics that obviate
bacterial growth. Incubation conditions and length
are identical to preservative-resistant yeast. One con-
troversial aspect is whether plating diluent must
contain 40-60% glucose in order to prevent osmotic
shock (transfer from low to high a , environment) that
dramatically decreases yeast recovery rates. Recent
observations suggest that 30°C is the best incubation
temperature for yeast detection. Faster growth rates
and larger surface colonies occurred at 30°C vs 25°C
incubation for 3-5 days.
It is also widely accepted that all yeast and mould
plating assays should employ surface plating tech-
niques which simplify counting procedures, improve
discrimination between food particles and yeast col-
onies, and enhance recovery rates due to high oxygen
tension. Surface plating effectiveness is further opti-
mized when the sample aliquot is analysed by mem-
brane filtration methods (for example, hydrophobic
grid membrane filtration j. Membrane filtration phys-
ically separates viable yeast cells from food and bev-
erage ingredients that may inhibit or slow yeast
growth. The filtration step is superior to manual
spreading and streaking regarding depositing and dis-
tributing individual yeast cells over the entire agar
surface, which increases enumeration accuracy and
improves analytical reliability and simplicity. Rec-
ommended preservative-resistant and sugar-tolerant
Zygosaccharomyces microbiological Q C plating and
enrichment media are summarized in Table 4.
Zygosaccharomyces Identification
non-selective and semi-selective media are mor-
phologically similar to many common yeasts, and
traditional biochemical and physiological tests used
10 years. However, they have limited Q C benefits
 Next Page

2364 ZYGOSACCHAROMYCES

Table 4 Recommended enumeration and recovery media for preservative-resistant and sugarhalt-tolerant Zygosaccharomyces
Medium
Acidified tryptone glucose
yeast extract agar
(ATGYE)
Z. bailii selective agar
medium (ZBM)"
Dichloran-18% glycerol
agar (DGl8)
Malt-yeast extractdo%
glucose agar (MY50G)
Plate count 30% sugar
agar (PCAS)
Composition (per litre) Preparation lncubation Special comments
timeltempera ture
100 g Glucose 1. Mix and boil dry 5 days 1. 0.5% glacial acetic
monohydrate ingredients 25-30°C acid in final medium:
5 g Tryptone 2. Autoclave at 121"C for pH ca. 3.9
5 g Yeast extract 15minat 15Ibsteam 2. Best total recovery of
15 g Agar 3. Cool to 45-52°C preservative-
5 ml Glacial acetic acid 4. Add 5 ml glacial acetic resistant yeast
acid and mix thoroughly 3. Not selective for Z.
5. Pour plates, let solidify. bailii
Store at 4-6°C for up to 2
weeks
30 g Sabouraud's 1. Same as ATGY E except 3-5 days 1. 0.5% glacial acetic
dextrose broth autoclave 121"C for 5 min 30°C acid in final medium:
30 g Fructose at 15 Ib steam pH 3.7-4.0
25 g Sodium chloride 2. Add potassium sorbate 2. Filter sterilize 10%
15 g Agar and glacial acetic acid potassium sorbate
5 g Tryptone after cooling to 45-52°C solution before use:
2.5 g yeast extract 100 p.p.m. in final
0.1 g Trypan blue dye medium
(optional) 3. Highly specific for Z.
5 ml Glacial acetic acid bailii
1 ml Potassium sorbate 4. Targeted to pickled
10% solution vegetables, fruit
concentrates and
salad dressings
5. Requires validation in
alcoholic and acidified
beverages
Commercially available As directed by 5 days 1. Recovered Z. bailii
manufacturer 25°C and Z.rouxii
2. Best yeast recovery
when mixed with
xerophilic mould
500 g Glucose 1. Mix and boil dry 5 days 1. Does not recover Z.
10 g Malt extract ingredients 25°C bailii
10 g Agar 2. Autoclave at 121"C for 2. Colonies small at 5
2.5 g Yeast extract 15 min at 15 Ib steam days
3. Cool to 45-52°C
4. Pour plates, let solidify.
Store at 4-6°C for up to 2
weeks
200 g Glucose 1. Mix and boil dry 3 days 1. Used with
100 g Fructose ingredients 30°C hydrophobic
23 g Plate count agar 2. Cool to 45-50°C membrane filtration
0.1 g Trypan blue dye 3. Add 20 ml antibiotic method
(optional) solution and mix 2. Leverages
20 ml Sterile antibiotic thoroughly fructophily response
solution 4. Pour plates, let solidify. of Z. rouxii to speed up
Store at 4-6°C for up to 2 growth rate
weeks
~~

aModified from Erickson JP (1993) Hydrophobic membrane filtration method for the selective recovery and differentiation of
Zygosaccharomyces bailii in acidified ingredients. J. Food Prot. 56: 234-238.

because of labour requirements, complexity and
length of testing, which can take 37 days for a final
result. A trained laboratory staff is needed to reduce
human error. These kits are useful plant investigation,
trouble-shooting and problem-solving tools.
Various genetic DNA probe techniques have been
successfully applied to identify spoilage yeast, includ-
ing Z.bailii and Z.rouxii. Three technologies men-
tioned were random amplified polymorphic DNA
(RAPD), RAPD-polymerase chain reaction, and
microsatellite polymerase chain reaction assays. All
have been described as extremely precise and faster
than traditional cultural identification methods.
Whether there is enough industry demand and volume
 APPENDIX I: BACTERIA AND FUNGI

The genera listed here are those associated with food, agricultural products and environments in which food is
prepared or handled.

Abiotrophia Chromobacterium Kocuria

Acinetobacter Chryseobacterium Kytococcus
Actinobacillus Chryseomonas Lactobacillus
Actinomyces Citrobacter Lactococcus
Aerococcus Clostridium Leclercia
Aeromonas Comamonas Leptotrichia
Agrobacterium Corynebacterium Leuconostoc
Alcaligenes Cryptococcus Listeria
Alloiococcus Debaryomyces Malassezia
Anaerobiospirillum Dermabacter Methylobacterium
Arcanobacterium Dermacoccus Microbacterium
Arcobacter Dietzia Micrococcus
Arthrobacter Ed wardsiella Mobiluncus
Aureobacterium Eikenella Moellerella
Bacillus Empedobacter Moraxella
Bacteroides Enterobacter Morganella
Bergeyella Enterococcus Myroides
Bifidobacterium Erwinia Neisseria
Blastoschizomyces Erysipelothrix Nocardia
Bordetella Escherichia Ochrobactrum
Branhamella Eubacterium Oerskovia
Brevibacillus Ewingella Oligella
Brevibacterium Flavimonas Paenibacillus
Brevundimonas Flavobacterium Pantoea
Brochothrix Fusobacterium Pasteurella
Brucella Gardnerella Pediococcus
Budvicia Gemella Peptococcus
Burkholderia Geotrichum Peptostreptococcus
Buttiauxella Gordona Photobacterium
Campylobacter Haemophilus Pichia
Candida Hafnia Plesiomonas
Capnocytophaga Hansenula Porphyromonas
Cardiobacterium Helicobacter Prevotela
Carnobacterium King ella Propionibacterium
CDC Klebsiella Proteus
Cedecea Kloeckera Prototheca
Cellulomonas Kluyvera Providencia
Aii APPENDIX I: BACTERIA AND FUNGI

Pseudomonas Shigella Trichosporon

Psychrobacter Sphingobacterium Turicella
Rahnella Sphingomonas Veillonella
Rals tonia Sporobolomyces Vibrio
Rhodococcus Staphylococcus Weeksella
Rhodotorula Stenotrophomonas Weissella
Rothia Stomatococcus Xanthomonas
Saccharomyces Streptococcus Yarrowia
Salmonella Suttonella Yersinia
Serratia Tatumella Yokenella
Shewanella Tetragenococcus Zygosaccharomyces
 APPENDIX II: LIST OF SUPPLIERS

The suppliers below are mentioned in the text as main sources of specialist equipment, culture media or diagnostic
materials. This list is not intended to be comprehensive.

3 M Microbiology Products Becton Dickinson Microbiology

3M Center Systems
Building 260-6B-01 7 Loveton Circle
St Paul Sparks
MN 55144-1 000 M D 21 152-0999
USA USA

bio resources
ABC Research Corporation
9304 Canterbury
3437 SW 24th Avenue
Leawood
Gainesville
KS 66206
FL 32607
USA
USA
BioControl Systems
Adgen Ltd 19805 North Creek Parkway
Nellies Gate Bothwell
Auchincruive WA 98011
Ayr KA6 5HW USA
UK
BioControl Systems, Inc
Agi-Diagnostics Associates 12822 SE
Cinnaminson 32nd Street
New Jersey Bellevue
USA WA 98005
USA

ANI Biotech OY Bioenterprises Pty Ltd

Temppelikatu 3-5, 00100 28 Barcoo Street
Helsinki PO Box 20 Roseville
Finland NSW 2069
Australia
Applied Biosystems
The Perkin-Elmer Corporation Biolog, Inc
12855 Flushing Meadow Drive 3938 Trust way
St Louis Hayward
MO 63131 1824 CA 94545
USA USA
Aiv APPENDIX II: LIST OF SUPPLIERS

Bioman Products, Inc Celsis-Lumac Ltd

400 Matheson Blvd Cambridge Science Park
Unit 4 Milton Road
Mississauga Cambridge
Ontario CB4 4FX
LAZ 1N8 UK
Canada
Charm Sciences Inc
bioMerieux 36 Franklin Street
Chemin de I'Orme Malden
69280 Marcy L'Etoile MA02148 3141
France USA

bioMerieux (UK) Chemunex Corporation

Grafton House St John's Innovation Centre
Grafton Way Cowley Road
Basingsto ke Cambridge
Hants RG22 6HY CB4 4WS
UK UK

bioMerieux Vitek, Inc Crescent Chemical Co, Inc

595 Anglum Drive 1324 Motor Parkway
Hazelwood Hauppauge
MO 63042 2320 NY 11788
USA USA

diAgnostix, Inc
Bioscience International
11607 Mcgruder Lane 1238 Anthony Road
RockviI le Burlington
MD 20852 4365 NC 27215
USA
USA
DIFCO
Biosynth AG
PO Box 331058
PO Box 125
Detroit
9422 Staad
MI 48232
Switzerland
USA
Biotecon
Diffchamb (UK)
Hermannswerder haus 17
1 Unit 12 Block 2/3
14473 Potsdam
Old Mill Trading Estate
Germany
Mansfield Woodhouse
Biotrace Nottingham NG19 9BG
666 Plainsboro Road UK
Suite 1116
Plainsboro Diffchamb SA
NJ 08536 8 Rue St Jean de Dieu
USA 69007 Lyons
France
Celsis
2948 Old Britain Circle Digen Ltd
Chattanooga 65 High Street
TN 37421 Wheatley
USA Oxford OX33 1UL
UK
Celsis International plc
Cambridge Science Park DiverseyLever
Milton Road Weston Favell Centre
Cambridge Northampton
CB4 4FX NN3 8PD
UK UK
 APPENDIX II: LIST OF SUPPLIERS Av

Diversy Ltd Envirotrace (BioProbe)

Technical Lane 675 Potomac River Road
Greenhill Lane McLean
Riddings VA 22100
DE55 4BA USA
UK
Foss Electric (UK)
Parkway House
Don Whitley Scientific Ltd
Station Road
14 Otley Road
Didcot
Shipley
Oxon OX11 7NN
West Yorkshire
UK
BD17 7SE
UK
Fluorochem Ltd
Wesley Street
DuPonVQualicon Old Glossop
E35711001A Derbyshire
Rouote 141 & Henry Clay Road SK13 9RY
PO Box 80357 UK
Wilmington
DE 19880 0357
USA Foss Electric AIS
69 Slangerupgade
PO Box 260
Dynal DK-3400 Hillerod
PO Box 158 Skoyen Denmark
0212 Oslo
Norway
GENE-TRAK Systems
94 South Street
Dynal (UK) Ltd Hopkinton
Station House MA 01748
26 Grove Street USA
New Ferry
Wirral
Gist-Brocades Australia
Merseyside L62 5AZ
PO Box 83
UK
Mooreban k
NSW 2170
Australia
Dynal (USA)
5 Delaware Drive
Lake Success Gist-Brocades BV
NY 11042 PO Box 1345
USA 2600 M A Delft
The Netherlands

Dynatech Laboratories Inc

14340 Sulleyfield Circle I.U.L.
Chantilly 1670 Dolwick Drive
VA 22021 Suite 8
USA Erlanger
KY 41 01 8

Ecolab Ltd
David Murray John Building IDEXX Laboratories, Inc
Swindon One IDEXX Drive
Wiltshire Westbrook
SNl 1NH ME 04092
UK USA
Avi APPENDIX II: LIST OF SUPPLIERS

Industrial Municipal Equipment Malthus Instruments Ltd

Inc (ime, Inc) Topley House
1430 Progress Way 52 Wash Lane
Suite 105 Bury
Ridersburg Lancashire
MD 21784 BL9 6AU
USA UK

Merck (UK) Ltd

Merck House
Innovative Diagnostic Systems
2797 Peterson Place Poole
Dorset BH15 1TD
Norcross
UK
GA 30071
USA
Meridian Diagnostics Inc
3741 River Hills Drive
Cincinnati
International BioProducts Tecra OH 45244
Diagnostics USA
14780 NE 95th Street
Redmond
MicroBioLogics
WA 98052
217 Osseo Ave N
USA
St Cloud
MN 56303 4455
USA
Lab M Ltd
Topley House Microbiology International
52 Wash Lane 10242 Little Rock Lane
Bury Fredrick
Lancashire MD 21702
BL9 6AU USA
UK
Microgen Bioproducts
1 Admiralty Way
Launch Diagnostics Ltd Camberley
Ash House Surrey GU15 3DT
Ash Road UK
New Ash Green
Longfield MicroSys, Inc
Kent DA3 8JD 2210 Brockman
UK Ann Arbor
MI 48104
USA
Lionheart Diagnostics Bio-lek
Instruments, Inc Minitek-BBL
Highland Park BD Microbiology Systems
Box 998 7 Loveton Circle
Winooski Sparks
VT 05404 0998 MD 21152
USA USA

Mitsubishi Gas Chemical

M. 1. Biol America, Inc
BioPharma Technology Ltd 520 Madison Avenue
BioPharma House 25th Floor
Winnall Valley Road New York
Winchester SO23 OLD NY 10022
UK USA
 APPENDIX II: LIST OF SUPPLIERS Avii

M-Tech Diagnostics Pharmacia Biotech

49 Barley Road 800 Centennial Avenue
Thelwall PO Box 1327
Warrington Piscataway
Cheshire WA4 2EZ NJ 08855 1327
UK USA

National Food Processors Assoc Prolab Diagnostics

1401 New York Unit 7 Westwood Court
NW Clayhill Industrial Estate
Washington Neston
DC 20005 Cheshire L64 3UJ
USA UK

Neogen Corporation QA Life Sciences Inc

620 Lesher Place 6645 Nancy Ridge Drive
Lansing San Diego
MI 48912 CA 92121
USA USA

New Horizons Diagnostic Corp Radiometer Ltd

91 10 Red Branch Road Manor Court
Suite B Manor Royal
Columbis Crawley
MD 21045 2014 West Sussex
USA RHlO 2PY
UK
Olympus Precision Instruments
Division R-Biopharm GmbH
10551 Barkley Dolivostr 10
Suite 140 D-64293
Overland Park Darmstadt
KS 66212 Germany
USA
RCR Scientific Inc
Organon Teknika AKZO NOBEL
206 West Lincoln
100 Akzo Avenue
PO Box 340
Durham
Goshen
NC 27712
IN 46526 0340
USA
USA
Oxoid, Inc
Remel
21 7 Colonnade Road
12076 Santa Fe Drive
Nepean
Lenexa
Ontario
KS 66215
K2E 7K3
USA
Canada

Oxyrase Inc Rhone-Poulenc Diagnostics Ltd

PO Box 1345 3.06 Kelvin Campus
Mansfield West of Scotland Science Park
OH 44901 Maryhill Road
USA Glasgow G20 OSP
UK
Perkin Elmer Corporation
50 Tanbury Road SciLog, Inc
Mail Station 251 14 Ellis Potter Ct
Wilton Madison
CT 06897 0251 WI 5371 1-2478
USA USA
Aviii APPENDIX II: LIST OF SUPPLIERS

Silliker Laboratory Inc Unipath, Oxoid Division

1304 Halstead Street Wade Road
Chicago Heights Basingstoke
IL 6041 1 Hampshire
USA RG24 8PW
UK
Spiral Biotech
7830 Old Georgetown Road Unipath, Oxoid Division (USA)
Bethesda 800 Proctor Avenue
MD 20814 Ogdensburg
USA NY 13669
USA
Tecra Diagnostics
28 Barcoo Street Vicam
PO Box 20 29 Mystic Avenue
Roseville Somerville
NSW MA 02145
Australia USA

Tecra Diagnostics (UK) Wescor, Inc

Batley Business Centre 1220 E
Technology Drive 1220 N
Batley Logan
W Yorkshire WF17 6ER UT 84321
UK USA
 GUIDE TO USE OF THE ENCYCLOPEDIA

Structure of the Encyclopedia

The material in the Encyclopedia is arranged as a series of entries in alphabetical order. Some entries comprise
a single article, whilst entries on more diverse subjects consist of several articles that deal with various aspects
of the topic. In the latter case the articles are arranged in a logical sequence within an entry.
To help you realize the full potential of the material in the Encyclopedia we have provided three features to
help you find the topic of your choice.

1. Contents Lists
Your first point of reference will probably be the contents list. The complete contents list appearing in each
volume will provide you with both the volume number and the page number of the entry. On the opening
page of an entry a contents list is provided so that the full details of the articles within the entry are immediately
available.
Alternatively you may choose to browse through a volume using the alphabetical order of the entries as
your guide. To assist you in identifying your location within the Encyclopedia a running headline indicates
the current entry and the current article within that entry.
You will find ‘dummy entries’ where obvious synonyms exist for entries or where we have grouped together
related topics. Dummy entries appear in both the contents list and the body of the text. For example, a dummy
entry appears for Butter which directs you to Milk and Milk Products: Microbiology of Cream and Butter,
where the material is located.

Example

If you were attempting to locate material on Dairy Products via the contents list.

DAIRY PRODUCTS see BRUCELLA: Problems with Dairy Products; CHEESE: In the Market Place; Microbiology of
Cheese-making and Maturation; Mould-ripened Varieties; Role of Specific Groups of Bacteria; Microflora of White-
brined Cheeses; FERMENTED MILKS: Yoghurt; Products from Northern Europe; Products of Eastern Europe and
Asia; PROBIOTIC BACTERIA: Detection and Estimation in Fermented and Non-fermented Dairy Products

At the appropriate location in the contents list, the page numbers for articles under Brucella, etc. are given.

If you were trying to locate the material by browsing through the text and you looked up Dairy Products then
the following information would be provided.
xiv Guide to use of the Encyclopedia

DAIRY PRODUCTS see BRUCELLA: Problems with Dairy Products; CHEESE: In the Market Place; Micro-
biology of Cheese-making and Maturation; Mould-ripened Varieties; Role of Specific Groups of Bacteria;
Microflora of White-brined Cheeses; FERMENTED MILKS: Yoghurt; Products from Northern Europe; Products
of Eastern Europe and Asia; PROBIOTIC BACTERIA: Detection and Estimation in Fermented and Non-
fermented Dairv Products.

Alternatively, if you were looking up Brucella the following information would be provided.

BRUCELLA
Contents
Characteristics
Problems with Dairy Products

2. Cross References
All of the articles in the Encyclopedia have an extensive list of cross references which appear a t the end of
each article, e.g.:

ATP BlOLUMlNESCENCElApplication in Dairy Industry.

See also: Acetobacter. ATP Bioluminescence: Application in Meat Industry; Application in Hygiene
Monitoring; Application in Beverage Microbiology. Bacteriophage-based Techniques for Detection of
Food-borne Pathogens. Biophysical Techniques for Enhancing Microbiological Analysis: Future
Developments. Electrical Techniques: Food Spoilage Flora and Total Viable Count (TVC). Immuno-
magnetic Particle-based Techniques: Overview. Rapid Methods for Food Hygiene Inspection. Total
Viable Counts: Pour Plate Technique: Spread Plate Technique: Specific Techniques; MPN; Metabolic
Activity Tests; Microscopy. Ultrasonic Imaging: Non-destructive Methods to Detect Sterility of Aseptic
Packages. Ultrasonic Standing Waves.

3. Index
The index will provide you with the volume number and page number of where the material is to be located,
and the index entries differentiate between material that is a whole article, is part of a n article or is data
presented in a table. O n the opening page of the index, detailed notes are provided.

4. Colour Plates
The colour figures for each volume have been grouped together in a plate section. The location of this section
is cited both in the contents list and before the See also list of the pertinent articles.

5. Contributors
A full list of contributors appears at the beginning of each volume.
 CONTRIBUTORS

Lahsen Ababouch lmad Ali Ahmed

Department of Food Microbiology and Quality Control Central Food Control Laboratory, Ajman Municipality
lnstitut Agronomique et Veterinaire Hassan II PO Box 3717
Rabat Ajman
Morocco UAE

D Abramson Peter Ahnert

Agriculture & Agri-Food Canada Department of Biochemistry
Cereal Research Centre Ohio State University
195 Dafoe Road Columbus
Winnipeg OH 4321 0
Manitoba USA
R3T 2M9
Canada William R Aimutis
Land 0' Lakes Inc.
Ann M Adams PO Box 674101
Seafood Products Research Center St Paul
US Food and Drug Administration Minnesota
PO Box 3012 55164-01 01
22201 23rdDrive SE USA
Bothell
WA 98041-301 2
J H AI-Jedah
USA
Central Laboratories
Ministry of Public Health
Martin R Adams
Qatar
School of Biological Sciences
University of Surrey
Guildford Cameron Alexander
GU2 5XH Macromolecular Science Department
UK Institute of Food Research,
Reading Laboratory
G E Age Earley Gate
PO Box 553 Whiteknights Road
Wageningen Reading
The Netherlands RG6 6BZ
UK
M Ahmed
Food Control Laboratory Marcos Alguacil
PO Box 7463 Departmento de Genetica
Dubai Facultad de Ciencias, Universidad de Malaga
United Arab Emirates Spain
xvi Contributors

M Z Ali Les Baillie

Central Laboratories Biomedical Sciences
Ministry of Public Health DERA
Qatar CBD Porton Down
Salisbury
W i Its hire
M D Alur UK
Food Technology Division
Bhabha Atomic Research Centre
Mumbai 400085 Gustavo V Barbosa-Canovas
Biological Systems Engineering
India
Washington State University
Pullman
R Miguel Amaguatia Washington 99164-6120
US Food and Drug Administration USA
Washington, DC
USA J Baranyi
Institute for Food Research
Vilma Moratade de Ambrosini Reading
Centro de Referencia para Lactobacilos and Universidad UK
Nacional de Tucuman
Casilla de Correo 21 1 Eduardo Barzana
(4000)-Tuc uman Departamento de Alimentos y Biotecnologia
Argentina Facuitad de Quimica
Universidad Nacional Autonoma de MBxico
Mexico City 0451 0
Wallace H Andrews
US Food and Drug Administration Mexico
Washington, DC 20204
USA Carl A Batt
Department of Food Science
Cornell University
Dilip K Arora
lthaca
Department of Botany
NY 14853
Banaras Hindu University
USA
Varanasi 221 005
India
Derrick A Bautista
Saskatchewan Food Product Innovation Program
B Austin
Department of Applied Microbiology and Food Science
Department of Biological Sciences
University of Saskatchewan
Heriot-Watt University
Canada
Riccarton
Edinburgh EH14 4AS
Scotland, UK S H Beattie
Hannah Research Institute
Ayr KA6 5HL
Aslan Azizi
UK
Iranian Agricultural Engineering Research Institute
Agricultural Research Organization
Evin Tehran H Beck
Iran Department for Health Service
South Bavaria
Veterinarstrasse 2
S De Baets 85764 Oberschleissheim
Laboratory of Industrial Microbiology and Biocatalysis
Germany
Department of Biochemical and Microbial Technology
Faculty of Agricultural and Applied Biological Sciences
University of Gent Reginald Bennett
Coupure links 653 FDA
B-9000 Center for Food Safety and Applied Nutrition
Gent Washington, DC
Belgium USA
 Contributors xvii

Marjon H J Bennik Deepak Bhatnagar

Agrotechnological Research Institution (ATO-DLO) Southern Regional Research Center
Bornsesteeg 59 Agricultural Research Service
6709 PD US Department of Agriculture
Wagen i ngen LA
The Netherlands USA

Merlin S Bergdoll (dec)

J R Bickert
Food Research Institute
Halosource Corporation
University of Wisconsin-Madison
First Avenue South
Madison, WI
Seattle
USA
WA 98104
R G Berger USA
Food Chemistry
University of Hannover Hanno Biebl
Germany GBF - National Research Centre for Biotechnology
Braunschweig
K Berghof Germany
BioteCon Gesellschaft fur Biotechnologische
Entwicklung und Consulting
Hermannswerder Haus 17 Clive de W Blackburn
14473 Potsdam Microbiology Unit
Germany Unilever Research Colworth
Colworth House
P A Bertram-Drogatz Sharnbrook
Mediport VC Management GmbH Bedford
Wiesenweg 10 UK
12247 Berlin
Germany
I S Blair
Gail D Betts Food Studies Research Unit
Campden and Chorleywood Food Research Association University of Ulster at Jordanstown
Chipping Campden Shore Road
Gloucestershire Newtownabbey
GL55 6LD Go. Antrim
UK Northern Ireland
BT37 9QB
R R Beumer
Wageningen Agricultural University
G Blank
Laboratory of Food Microbiology
Department of Food Science
Bomenweg 2 University of Manitoba
NL 6703 HD Wageningen
Winnipeg
The Netherlands
MB
Canada
Rijkelt R Beumer
Wageningen University and Research Centre
Department of Food Technology and Nutritional Sciences Hans P Blaschek
Bomenweg 2 Department of Food Science and Human Nutrition
NL 6703 HD Wageningen University of Illinois
The Netherlands 488 Animal Science Lab
1207 West Gregory Drive
Saumya Bhaduri Urbana
Microbial Food Safety Research Unit IL 61801
Eastern Regional Research Center USA
Agricultural Research Service
US Department of Agriculture
600 East Mermaid Lane D Blivet
Wyndmoor AFSSA
PA 19038 Ploufragan
USA France
xviii Contributors

R G Board Astrid Brandis-Heep

South Lodge Philipps Universitat
Northleigh Fachbereich Biologie
Bradford-on-Avon Laboratorium fur Mikrobiologie
Wiltshire D-35032 Marburg
UK Germany

Enne de Boer Susan Brewer

Inspectorate for Health Protection Department of Food Science and Human Nutrition
PO Box 9012 University of Illinois
7200 GN Zutphen Urbana
The Netherlands Illinois
USA
Christine Bonaparte
Department of Dairy Research and Bacteriology Aaron L Brody
Agricultural University Rubbright Brody Inc.
Gregor Mendel-Str. 33 PO Box 9561 87
A-1 180 Vienna Duluth
Austria Georgia
30095-9504
Kathryn J Boor USA
Department of Food Science
Cornell University Bruce E Brown
lthaca B. E. Brown Associates
NY 14853 328 Stone Quarry Priv.
USA Ottawa
Ontario
A Botha K1K 3Y2
Department of Microbiology Canada
University of Stellenbosch
Stellenbosch 7600 G Bruggeman
South Africa Laboratory of Industrial Microbiology and Biocatalysis
Department of Biochemical and Microbial Technology
W Richard Bowen Faculty of Agricultural and Applied Biological Sciences
Biochemical Engineering Group University of Gent
Centre for Complex Fluids Processing Coupure links 653
University of Wales Swansea 8-9000
Singleton Park Gent
Swansea Belgium
SA2 8PP
UK Andreas Bubert
Department for Microbiology
Catherine Bowles Theodor-Boveri Institute for Biosciences
Leatherhead Food Research Association University of Wurzburg
Leatherhead Am Hubland
Surrey 97074 Wurzburg
UK Germany

Patrick Boyaval Ken Buckle

INRA Department of Food Science and Technology
Laboratoire de Recherches de Technologie Laitiere The University of New South Wales
65 rue de Saint-Brieuc Sydney
35042 Australia
Rennes Cedex
France Lloyd B Bullerman
Department of Food Science and Technology
F Bozoglu University of Nebraska
Department of Food Engineering PO Box 830919
Middle East Technical University Lincoln
Ankara NE 68583-0919
Turkey USA
 Contributors xix

Justin0 Burgos Maria da Gloria S Carvalho

Food Technology Section Departamento de Microbiologia Medica
Department of Animal Production and Food Science lnstituto de Microbiologia
University of Zaragoza Universidade Federal do Rio de Janeiro
SDain Rio de Janeiro 21941
Brazil
Frank F Busta
0 Cerf
Department of Food Science and Nutrition
Alfort Veterinary School
University of Minnesota
7 Avenue du General de Gaulle
St Paul
F-94704
Minnesota 55108
Maisons-Alfort Cedex
USA
France

Daniel Cabral Lourdes Perez Chabela

Departmento de Ciencias Biologicas Universidad Autonoma Metropolitana-lztapalapa
Facultad de Ciencias Exactas y Naturales Mexico
Pabellon II 4to piso - Ciudad Universitaria Apartado Postal 55-535
1428 Buenos Aires CP 09340 Mexico DF
Argentina Mexico
Perng-Kuang Chang
Maria Luisa Calderon-Miranda Southern Regional Research Center
Biological Systems Engineering Agricultural Research Service
Washington State University US Department of Agriculture
Pullman LA
Washington 99164-61 20 USA
USA E A Charter
Canadian lnovatech Inc.
Geoffrey Campbell-Platt 31212 Peardonville Road
Gyosei Liaison Office Abbotsford
Gyosei College BC V2T 6K8
London Road Canada
Reading
Parimal Chattopadhyay
Berks RGl 5AQ
Department of Food Technology and Biochemical
UK
Engineering
Jadavpur University
lain Campbell Calcutta-700 032
International Centre for Brewing and Distilling India
Heriot-Watt University
Yusuf Chisti
Edinburgh
-EHl4 4AS Department of Chemical Engineering
University of Almeria
Scotland
E-04071 Almeria
Spain
Frederic Carlin
lnstitut National de la Recherche Agronomique Thomas E Cleveland
Unite de Technologie des Produits Vegetaux Southern Regional Research Center
Site Agroparc Agricultural Research Service
84914 US Department of Agriculture
Avignon LA
Cedex 9 USA
France Dean 0 Cliver
University of California, Davis, School of Veterinary
Brigitte Carpentier Medicine
National Veterinary and Food Research Centre Department of Population Health and Reproduction
22 rue Pierre Curie One Shields Avenue
F-94709 Davis
Maisons-Alfort Cedex California 95616-8743
France USA
xx Contributors

T E Cloete N D Cowell
Department of Microbiology and Plant Pathology Elstead
Faculty of Biological and Agricultural Sciences Godalming
University of Pretoria Surrey
Pretoria 0002 GU8 6HT
South Africa UK

Roland Cocker Julian Cox

Cocker Consulting Department of Food Science and Technology
Bergeendlaan 16 The University of New South Wales
1343 AR Almere Sydney
The Netherlands Australia

Timothy M Cogan C Gerald Crawford

Dairy Products Research Centre US Department of Agriculture
Teagasc Agricultural Research Service
Fermoy Eastern Regional Research Center
Ireland 600 E. Mermaid Lane
Wyndmoor
David Collins-Thompson PA 19038
Nestle Research and Development Center USA
210 Housatonic Avenue
New Milford Theresa L Cromeans
Connecticut Department of Environmental Sciences and Engineering
USA School of Public Health
University of North Carolina
Janet E L Corry North Carolina
Division of Food Animal Science USA
Department of Clinical Veterinary Science
University of Bristol Kofitsyo S Cudjoe
Langford
Department of Pharmacology
Bristol
Microbiology and Food Hygiene
BS40 5DT
Norwegian College of Veterinary Medicine
UK
PO Box 8146 Dep
0033 Oslo
Aldo Corsetti Norway
Institute of Dairy Microbiology
Faculty of Agriculture of Perugia
David Cunliffe
06126 S. Costanzo
Perugia Macromolecular Science Department
Institute of Food Research
Italy
Reading Laboratory
Earley Gate, Whiteknights Road
Polly D Courtney Reading
Department of Food Science and Technology RG6 6BZ
Ohio State University UK
2121 Fyffe Road
Columbus
OH 43210 Ladislav Curda
USA Department of Dairy and Fat Technology
Prague Institute of Chemical Technology
Czech Republic
M A Cousin
Department of Food Science
Purdue University G J Curie1
West Lafayette Unilever Research Vlaardingen
Indiana PO Box 114
47907-1 160 3130 AC Vlaardingen
USA The Netherlands
 Contributors xxi

G D W Curtis Stephen P Denyer

Bacteriology Department Department of Pharmacy
John Radcliffe Hospital The University of Brighton
Oxford Coc kcroft BuiIding
UK Moulescoomb
Brighton
BN2 4GJ
Michael K Dah1 UK
Department of Microbiology
University of Erlangen P M Desmarchelier
Staudtstrasse 5 Food Safety and Quality
91058 Erlangen Food Science Australia
Germany PO Box 3312
Tingalpo DC
Queensland 41 73
Crispin R Dass Australia
The Heart Research Institute Ltd
145 Missenden Road Janice Dewar
Camperdown CSlR Food Science and Technology
Sydney PO Box 395
NSW 2050 Pretoria 001
Australia South Africa

Vinod K Dhir
E Alison Davies
Biotec Laboratories Ltd
Technical Services & Research Department
32 Anson Road
Aplin & Barrett Ltd (Cultor Food Science)
Martlesham Heath
15 North Street
lpswich
Beaminster
Suffolk
Dorset
IP5 3RD
DT8 3DZ
UK
UK

M W Dick
Brian P F Day Department of Botany
Campden and Chorleywood Food Research Association University of Reading
Chipping Campden Reading
Gloucestershire RG6 6AU
GL55 6LD UK
UK
Vivian M Dillon
Department of Biology and Biochemistry
J M Debevere University of Bath
Laboratory of Food Microbiology and Food Preservation Bath
Faculty of Agricultural and Applied Biological Sciences UK
University of Ghent
Coupure Links 654 Eleftherios H Drosinos
9000 Ghent Department of Food Science and Technology
Belgium Laboratory of Microbiology and Biotechnology of Foods
Agricultural University of Athens
lera Odos 75
Joss Delves-Broughton Athens
Technical Services and Research Department Greece
Aplin & Barrett Ltd (Cultor Food Science)
15 North Street F M Dugan
Beaminster USDA-ARS Western Regional Plant Introduction Station
Dorset Washington State University
DT8 3DZ Washington
UK USA
xxii Contributors

B Egan Nana Y Farkye

Marine Biological and Chemical Consultants Ltd Dairy Products Technology Center
Bangor Dairy Science Department
UK California Polytechnic State University
San Luis Obispo
CA 93407
H M J van Elijk USA
Unilever Research Vlaardingen
PO Box 114
Manuel Fidalgo
3130 AC Vlaardingen
Departmento de Genetica
The Netherlands
Facultad de Ciencias, Universidad de Malaga
Spain
Hartmut Eisgruber
Institute for Hygiene and Technology of Foods of Animal
Christopher W Fisher
Origin, Veterinary Faculty
Department of Food Science and Human Nutrition
Ludwig-Maximilians University
University of Illinois
80539 Munich
Urbana
Germany
IL 61801
USA
Phyllis Entis
QA Life Sciences, Inc. G H Fleet
6645 Nancy Ridge Drive CRC for Food Industry Innovation
San Diego Department of Food Science and Technology
CA 92121 The University of New South Wales
USA Sydney
New South Wales 2052
John P Erickson Australia
Microbiology - Research and Development
Bestfoods Technical Center Harry J Flint
Somerset Rowett Research Institute
New Jersey Greenburn Road
USA Bucksburn
Aberdeen
Douglas E Eveleigh UK
Department of Microbiology
Rutgers University Samuel Formal
Cook College Department of Microbiology and Immunology
76 Lipman Drive Uniformed Services University of the Health Sciences
New Brunswick F Edward Hebert School of Medicine
NJ 08901-8525 4301 Jones Bridge Road
USA Bethesda
MD 20814
USA
Richard R Facklam
Streptococcus Laboratory Pina M Fratamico
Respiratory Diseases Branch US Department of Agriculture
Division of Bacterial and Mycotic Diseases Agricultural Research Service
Centres for Disease Control and Prevention Eastern Regional Research Center
Mail Stop CO-2 600 E. Mermaid Lane
Atlanta Wyndmoor
GA 30333 PA 19038
USA USA

M Fandke Colin Fricker

BioteCon Gesellschaft fur Biotechnologische Thames Water Utilities
Entwicklung und Consulting Manor Farm Road
Hermannswerder Haus 17 Reading
14473 Potsdam RG2 OJN
Germany UK
 Contributors xxiii

Daniel Y C Fung N P Ghildyal

Department of Animal Sciences and Industry Fermentation Technology and Bioengineering
Kansas State University Department
Manhattan Central Food Technological Research Institute
Kansas66506 Mysore 570013
USA India

H Ray Gamble
M Gibert
United States Department of Agriculture
lnstitut Pasteur
Agricultural Research Service
Unite Interactions Bacteries Cellules
Parasite Biology and Epidemiology Laboratory
28 rue du Dr Roux
Building 1040, Room 103, BARC-East
75724 Paris
Beltsville
Cedex 15
MD 20705
France
USA

lndrawati Gandjar Glenn R Gibson

Department of Biology Microbiology Department
Faculty of Science and Mathematics Institute of Food Research
University of Indonesia Reading
Jakarta UK
Indonesia
M C te Giffel
Mariano Garcia-Garibay
Wageningen Agricultural University
Departamento de Biotechnolog/a
Laboratory of Food Microbiology
Universidad Autonoma Metropolitana
Bomenweg 2
Iztapalapa, Apartado Postal 55-535
NL 6703 HD Wageningen
Mexico City 09340
The Netherlands
Mexico

Maria-Luisa Garcia-Lopez A Gilmour

Department of Food Hygiene and Food Technology Food Science Division (Food Microbiology)
University of Leon Department of Agriculture for Northern Ireland
24071-Leon Agriculture and Food Science Centre
Spain Newforge Lane
Belfast
S K Garg BT9 5PX
Department of Microbiology Northern Ireland, UK
Dr Ram Manohar Lohia Avadh University
Faizabad 224 001
Giorgio Giraffa
India
lstituto Sperimentale Lattiero Caseario
A Gasch Via A. Lombard0
BioteCon Gesellschaft fur Biotechnologische 11 - 26900 Lodi
Entwicklung und Consulting Italy
Hermannswerder Haus 17
14473 Potsdam R W A Girdwood
Germany Scottish Parasite Diagnostic Laboratory
Stobhill Hospital
Michel Gautier Glasgow
Ecole Nationale Superieure d’Agronomie GL21 3UW
lnstitut National de la Recherche Agronomique UK
65 rue de SrBrieuc
35042
Andrew D Goater
Rennescedex
Institute of Molecular and Biomolecular Electronics
France
University of Wales
Gerd Gellissen Dean St
Rhein Biotech GmbH Bangor
EichsFelder Str. 11 Gwynedd
40595 Dusseldorf LL57 1UT
Germany UK
xxiv Contributors

Marco Gobbetti M K Gowthaman

lnstituto di Produzioni e Preparazioni Alimentari Fermentation Technology and Bioengineering
Facolta di Agraria di Foggia Department
Via Napoli 25 Mysore 57001 3
71 100 Foggia India
Italy
Lone Gram
Millicent C Goldschmidt
Danish Institute for Fisheries Research
Department of Basic Sciences
Department of Seafood Research
Dental Branch
Technical University of Denmark Bldg 221
The University of Texas Health Center at Houston
DK-2800 Lyngby
6516 John Freeman Avenue
Denmark
Houston
Texas 77030
USA AGE Griff ioen
Stichting EFFl
Lorena Gomez-Ruiz PO Box 553 Wageningen
Departamento de Biotechnologia The Netherlands
Universidad Autonoma Metropolitana
Iztapalapa, Apartado Postal 55-535
Mexico City 09340 Mansel W Griffiths
Mexico Department of Food Science
University of Guelph
Katsuya Gomi Guelph
Division of Life Science Ontario
Graduate School of Agricultural Science N1G 2W1
Tohoku University Canada
Japan
C Gronewald
M Marcela Gongora-Nieto BioteCon Geseilschaft fur Biotechnologische
Biological Systems Engineering Entwicklung und Consulting
Washington State University Hermannswerder Haus 17
Pullman 14473 Potsdam
Washington 99164-61 20
Germany
USA

S Gonzalez Isabel Guerrero

Universidad Nacional de Tucuman, Argentina Universidad Autonoma Metropolitana-lztapalapa
Cerela-Conicet Mexico
San Miguel de Tucuman Apartado Postal 55-535
Argentina CP 09340 Mexico DF
Mexico
Silvia N Gonzaler
Centro de Referencia para Lactobacilos (Cerela) and
Universidad Nacional de Tucuman G C Giirakan
Chacabuco 145 (4000) Middle East Technical University
Tu c uman Ankara
Argentina Turkey

Leon G M Gorris Carlos Horacio Gusils

Unit Microbiology and Preservation Centro de Referencia para Lactobacilos and Universidad
Unilever Research Vlaardingen Nacional de Tucuman
PO Box 114 Casilia de Correo 21 1
3130 AC Vlaardingen (4000)-Tucuman
The Netherlands Argentina
Grahame W Gould
17 Dove Road Thomas S Hammack
Bedford US Food and Drug Administration
MK41 7AA Washington, DC 20204
UK USA
 Contributors xxv

S A S Hanna, A D Hitchins
48 Kensington Street Center for Food Safety and Applied Nutrition
Newton Food and Drug Administration
MA 02460 Washington, DC
USA USA

Karen M J Hansen Jill E Hobbs

Saskatchewan Food Product Innovation Program George Morris Centre
University of Saskatchewan 345, 21 16 27th Avenue NE
Saskatoon Calgary
SK Alberta
S7N 5A8 T2E 7A6
Canada Canada

J Harvey
Food Science Division (Food Microbiology) Ailsa D Hocking,
Department of Agriculture for Northern Ireland CSlRO Food Science Australia
Agriculture and Food Science Centre Riverside Corporate Park
Newforge Lane North Ryde
Belfast New South Wales 21 13
BT9 5PX Australia
Northern Ireland, UK
Cornelis P Hollenberg
Wilma C Hazeleger lnstitut fur Microbiology
Wageningen University and Research Centre Heinrich-Heine-Universitat Dusseldorf
Department of Food Technology and Nutritional Sciences 40225 Dusseldorf
Bomenweg 2 Germany
NL 6703 HD Wageningen
The Netherlands
Richard A Holley
G M Heard Department of Food Science
University of Manitoba
CRC for Food Industry Innovation
Department of Food Science and Technology Winnipeg
University of New South Wales Manitoba
R3T 2N2
Sydney
Canada
New South Wales 2052
Australia
Wilhelm H Holzapfel
Nidal Hila1 Institute of Hygiene and Toxicology
Biochemical Engineering Group Federal Research Centre for Nutrition
Centre for Complex Fluids Processing Bundesforschungsanstalt
Department of Chemical and Biological Process Haid-und-Neu-Str. 9
Engineering D-7613 Karlsruhe
University of Wales Swansea Germany
Singleton Park
Swansea SA2 8PP
Rolf K Hommel
UK
Cell Technologie Leipzig
Fontanestr. 21
G Hildebrandt Leipzig
Institute for Food Hygiene D-04289
Free University of Berlin
Germany
Germany

Colin Hill Dallas G Hoover

Department of Microbiology and National Food Department of Animal and Food Sciences
Biotechnology Centre University of Delaware
University College Newark
Cork DE 19717-1 303
Ireland USA
xxvi Contributors

Thomas W Huber B Jarvis

Medical Microbiology and Immunology Ross Biosciences Ltd
Texas A&M College of Medicine Daubies Farm
Temple Upton Bishop
Texas Ross-on-Wye
USA Herefordshire
HR9 7UR
UK
Robert Hutkins
Department of Food Science and Technology Ian Jenson
University of Nebraska Gist-brocades Australia Pty, Ltd
338 FIC Moorebank
Lincoln NSW
NE 68583-0919 Australia
USA
Juan Jimenez
Departmento de Genetica
Cheng-An Hwang
Facultad de Ciencias, Universidad de Malaga
Nestle Research and Development Center
Spain
210 Housatonic Avenue
New Milford
Karen C Jinneman
Connecticut
Department of Veterinary Science and Microbiology
USA
University of Arizona
Tucson
John J landolo AZ 85721
Department of Microbiology and Immunology USA
University of Oklahoma Health Sciences Center
Oklahoma City Juan Jofre
OK 73190 Department of Microbiology
USA University of Barcelona
Spain

Y limura Eric Johansen

Department of Applied Chemistry and Biotechnology Department of Genetics and Microbiology
Yamanashi University Chr. Hansen N S
Kofu 10-1 2 B0ge Alle
Japan DK-2970
H~rsholm
Denmark
Charlotte Nexrnann Jacobsen
Department of Dairy and Food Research Nick Johns
Royal Veterinary and Agricultural University Independent Research Consultant
Rolighedsvej 3,O 15 Collingwood Close
1958 Frederiksberg C Steepletower
Denmark Hethersett
Norwich NR9 3QE
Mogens Jakobsen UK
Department of Dairy and Food Research
Royal Veterinary and Agricultural University Eric A Johnson
Rolighedsvej 3,O Department of Food Microbiology
1958 Frederiksberg C Food Research Institute, University of Wisconsin
Denmark Madison
WI
USA
Dieter Jahn
Institute for Organic Chemistry and Biochemistry Clifford H Johnson
Albert Ludwigs University Freiburg US Environmental Protection Agency
Albertstr. 21 Cincinatti
79104 Freiburg Ohio
Germany USA
 Contributors xxvii

Rafael Jordan0 Embit Kartadarma

Department of Food Science and Technology Department of Food Science and Technology
Campus Rabanales, University of Cordoba The University of New South Wales
E-I4071 Cordoba Sydney
Spain Australia

Richard Joseph K L Kauppi

Department of Food Microbiology University of Minnesota
Central Food Technological Research Institute Department of Food Science and Nutrition
Mysore St Paul
570 013 USA
India

C A Kaysner
Vinod K Joshi
US Food and Drug Administration
Department of Post-harvest Technology
22201 23rd Drive SE
Dr YSP University of Horticulture and Foresty
Bothell
Nauni
Washington 98021
Solan-173 230
USA
India

Vijay K Juneja William A Kerr

United States Department of Agriculture Department of Economics
Eastern Regional Research Center University of Calgary
600 East Mermaid Lane 2500 University Drive NW
Wyndmoor Calgary
Pennsylvania Alberta
USA T2N I N 4
Canada
G Kalantzopoulos
Department of Food Science and Technology Tajalli Keshavarz
Agricultural University of Athens Department of Biotechnology
Greece University of Westminster
115 New Cavendish Street
Chitkala Kalidas London
Field of Microbiology W I M 8JS
Department of Food Science UK
Cornell University
lthaca NY 14853
USA George G Khachatourians
Department of Applied Microbiology and Food Science
University of Saskatchewan
A Kambamanoli-Dimou
Saskatoon
Department of Animal Production
Canada
Technological Education Institute
Larissa
Greece W Kim
Department of Microbiology
Peter Kampfer University of Georgia
lnstitut fur Angewandte Mikrobiologie Athens
Justus-Liebig-Universitat Giessen Georgia
Senckenbergstr. 3 USA
D-35390 Giessen
Germany
P M Kirk
N G Karanth CAB1 Bioscience UK Centre (Egham)
Fermentation Technology and Bioengineering Bakeham Lane
Department Egham
Mysore 570013 Surrey
India TW20 9TY
xxviii Contributors

Todd R Klaenhammer Pushpa R Kulkarni

Departments of Food Science and Microbiology University Department of Chemical Technology
Southeast Dairy Foods Research Center University of Mumbai
Box 7624 Matunga
North Carolina State University Mumbai 400 019
Raleigh India
NC 27695-7624
USA Madhu Kulshreshtha
Division of Plant Pathology
Indian Agricultural Research Institute
Hans-Peter Kleber
New Delhi 11012
lnstitut fur Biochemie
India
Fakultot fur Biowissenschaften
Pharmazie und Psychologie Susumu Kumagai
Universitat Leipzig Department of Biomedical Food Research
Talstr. 33 National Institute of Infectious Diseases
Leipzig Toyama 1-23-1
D-04103 Shinjuku-ku
Germany Tokyo 162-8640
Japan
Thomas J Klem
Department of Food Science G Lagarde
Cornell University lnovatech Europe B.V.
USA Landbouwweg
The Netherlands
Wolfgang Kneifel Keith A Lampel
Department of Dairy Research and Bacteriology US Food and Drug Administration
Agricultural University Center for Food Safety and Applied Nutrition HFS-327
Gregor Mendel-Str. 33 200 c St sw
A-1 180 Vienna Washington
Austria DC 20204
USA
Barb Kohn
VICAM LP S Leaper
313 Pleasant Street Campden and Chorleywood Food Research Association
Watertown Chipping Campden
MA 02172 Gloucestershire
USA GL55 6LD
UK

C Koob J David Legan

BioteCon Gesellschaft fur Biotechnologische Microbiology Department
Entwicklung und Consulting Nabisco Research
Hermannswerder Haus 17 PO Box 1944
14473 Potsdam DeForest Avenue
Germany East Hanover
NJ 017871
P Kotzekidou USA
Department of Food Science and Technology
J J Leisner
Faculty of Agriculture
Department of Veterinary Microbiology
Aristotle University of Thessaloniki
Royal Veterinary and Agricultural University
PB 250
Stigb~jlen4
GR 540 06
DK-1870 Frederiksberg C
Thessaloniki
Denmark
Greece
H L M Lelieveld
K Krist Unilever Research Vlaardingen
Meat and Livestock Australia PO Box 114
Sydney 3130 AC Vlaardingen
Australia The Netherlands
 Contributors xxix

D F Lewis R H Madden
Food Systems Division Food Microbiology
SAC Food Science Department
Auchincruive Department of Agriculture for Northern Ireland and
Ayr KA6 5HW Queen’s University of Belfast
Scotland Newforge Lane
UK Belfast
BT9 5PX
Northern Ireland
M J Lewis
Department of Food Science and Technology
University of Reading
UK T Mahmutoglu
TATKO TAS
E Litopoulou-Tzanetaki Gayrettepe
Department of Food Science, Faculty of Agriculture Istanbul
Aristotle University of Thessaloniki Turkey
54006
Thessaloniki
Greece K A Malik
Chairman
Aline Lonvaud-Funel Pakistan Agricultural Research Council
Faculty of Enology Islamabad
University Victor Segalen Bordeaux 2 Pakistan
351, Cours de la Liberation
33405 Talence Cedex
France Miguel Prieto Maradona
Department of Food Hygiene and Food Technology
S E Lopez University of Leon
Departamento de Ciencias Biologicas 24071-Leon
Facultad de Ciencias Exactas y Naturales Spain
Pabellon I1 4to piso - Ciudad Universitaria
1428 Buenos Aires
Argentina
Scott E Martin
Department of Food Science and Human Nutrition
G Love University of Illinois
Centre for Electron Optical Studies 486 Animal Sciences Laboratory
University of Bath 1207 West Gregory Drive
Claverton Down Urbana
Bath IL 61 801
BA2 7AY USA
UK

Robert W Lovitt
Biochemical Engineering Group L Martinkova
Laboratory of Biotransformation
Centre for Complex Fluids Processing
Institute of Microbiology
Department of Chemical and Biological Process
Academy of Sciences of the Czech Republic
Engineering
Prague
University of Wales Swansea
Singleton Park Czech Republic
Swansea
SA2 8PP
UK Tina Mattila-Sandholm
VTT Biotechnology and Food Research
Majella Maher Tietotie 2
National Diagnostics Centre Espoo
National University of Ireland PO Box 1501
Galway FIN-02044 v1T
Ireland Finland
xxx Contributors

D A McDowell Vikram V Mistry

Food Studies Research Unit Dairy Science Department
University of Ulster at Jordanstown South Dakota State University
Shore Road Brookings
Newtownabbey South Dakota 57007
Co. Antrim USA
Northern Ireland
BT37 9QB D R Modi
Department of Microbiology
Denise N McKenna Dr Ram Manohar Lohia Avadh University
Microbiology - Research and Development Faizabad 224 001
Bestfoods Technical Center India
Somerset
New Jersey Richard J Mole
USA Biotec Laboratories Ltd.
32 Anson Road
M A S McMahon Martlesham Heath
Food Studies Research Unit lpswich
University of Ulster at Jordanstown Suffolk
Shore Road IP5 3RD
Newtownabbey UK
Co. Antrim
Northern Ireland M C Monte1
BT37 9QB Station de Recherches sur la Viande
INRA
T A McMeekin 63122 Saint Genes Champanelle
School of Agricultural Science France
University of Tasmania
Hobart M Moresi
Australia lstituto di Tecnologie Agroalimentari
Universita della Tuscia
Via S C de Lellis
Luis M Medina 01 100 Viterbo
Department of Food Science and Technology Italy
Campus Rabanales
University of Cordoba Andre Morin
E-14071 Cordoba Imperial Tobacco Limited
Spain 3810 rue St-Antoine
Montreal
Aubrey F Mendonca Quebec H4C 1B5
Iowa State University Canada
Department of Food Science and Human Nutrition
Ames Maurice 0 Moss
Iowa School of Biological Sciences, University of Surrey
USA Guildford
GU2 5XH
UK
James W Messer
US Environmental Protection Agency M A Mostert
Cincinnati Unilever Research Vlaardingen
Ohio PO Box 114
USA 3130 AC Vlaardingen
The Netherlands
M C Misra
Fermentation Technology and Bioengineering Donald Muir
Department Hannah Research Institute
Central Food Technological Research Institute AYr
Mysore 570013 KA6 5HL
India Scotland, UK
 Contributors xxxi

Maite Muniesa M de Nijs

Department of Microbiology TNO Nutrition and Food Research Institute
University of Barcelona Division of Microbiology and Quality Management
Spain PO Box 360
3700 AJ Zeist
E A Murano The Netherlands
Center for Food Safety and Department of Animal
Science S H W Notermans
Texas A&M University TNO Nutrition and Food Research Institute
Texas PO Box 360
USA 3700 AJ Zeist
The Netherlands
M J Murphy
Martha Nufiez
CBD Porton Down
Centro de Referencia par Lactobacilos (Cerela)
Salisbury Chacabuco 145 (4000)
SP4 OJQ
Tucuman
UK
Argentina

K Darwin Murrell George-John E Nychas

Agricultural Research Service Department of Food Science and Technology
US Department of Agriculture Laboratory of Microbiology and Biotechnology of Foods
Beltsville Agricultural University of Athens
Maryland 20705 lera Odos 75
USA Athens
11855
C K K Nair Greece
Radiation Biology Division
Bhabha Atomic Research Centre R E O’Connor-Shaw
Mumbai 400 085 Food Microbiology Consultant
India Birkdale
Queensland
Australia
Motoi Nakao
Horiba Ltd Louise O’Connor
Miyanohigashimachi National Diagnostics Centre
Kisshoin National University of Ireland
Minami-ku Galway
Kyoto Ireland
Japan
601-8510 Triona O’Keeffe
Department of Microbiology and National Food
A W Nichol Biotechnology Centre
Charles Sturt University University College
NSW Cork
Australia Ireland

Rachel M Oakley
D S Nichols United Biscuits (UK Ltd)
School of Agricultural Science High Wycombe
University of Tasmania Buckinghamshire
Hobart HP12 4JX
Australia UK

Poonam Nigam Yuji Oda

Biotechnology Research Group Department of Applied Biological Science
School of Applied Biological and Chemical Sciences Fukuyama University
University of Ulster Fukuyama
Coleraine BT52 1SA Hiroshima 729-0292
UK Japan
xxxii Contributors

Lucy J Ogbadu Photis Papademas

Department of Biological Sciences Department of Food Science and Technology
Benue State University University of Reading
Makurdi Whiteknights
Nigeria Reading
Berkshire
Guillermo Oliver RG6 6AP
Centro Referencia para Lactobacilos and Universidad UK
Nacional de Tucuman
Casilla de Correo 21 1
A Pardigol
(4000)-Tucuman
BioteCon Gesellschaft fur Biotechnologische
Argentina
Entwicklung und Consulting
Hermannswerder Haus 17
Ynes R Ortega
14473 Potsdam
Seafood Products Research Center
Germany
US Food and Drug Administration
PO Box 3012
22201 23rdDrive SE E Parente
Bothell Dipartimento di Biologia, Difesa e Biotecnologie Agro-
WA 98041-301 2 Forestali
USA Universita della Basilicata
Via N Sauro 85
Andres Otero 85100 Potenza
Department of Food Hygiene and Food Technology Italy
University of Leon
24071-Leon
Spain Zahida Parveen
University of Huddersfield
Kozo Ouchi Department of Chemical and Biological Sciences
Kyowa Hakko Kogyo Co. Ltd Queensgate
1-6-1 Ohtemachi Huddersfield
Chiyoda-ku HD1 3DH
Tokyo 100-81 85 UK
Japan
P Patakova
Barbaros H Ozer
Department of Food Science and Technology Faculty of Food and Biochemical Technology
Institute of Chemical Technology
Faculty of Agriculture
Prague
University of Harran
Czech Republic
63040
Qanliurfa
Turkey Pradip Patel
Science and Technology Group
Dilek drer Leatherhead Food Research Association
GAP Regional Development Administration Randalls Road
Sanliurfa Leatherhead
Turkey Surrey
KT22 7RY
J Palacios UK
Universidad Nacional de Tucuman, Argentina
Cerela-Conicet
San Miguel de Tucuman Margaret Patterson
Argentina Food Science Division
Department of Agriculture for Northern Ireland and The
Ashok Pandey Queen's University of Belfast
Laboratorio de Processos Biotecnologicos Agriculture and Food Science Centre
Universidade Federal do Parana Newforge Lane
Departmento de Engenharia Quimica Belfast
CEP 81531-970 Curitiba-PR BT9 5PX
Brazil UK
 Contributors xxxiii
~~~ ~~ ~

P A Pawar J I Pitt
Fermentation Technology and Bioengineering CSlRO Food Science Australia
Department Riverside Corporate Park
Central Food Technological Research Institute North Ryde
Mysore 570013 New South Wales 21 13
India Australia

Janet B Payeur M R Popoff

National Veterinary Services Laboratories lnstitut Pasteur
Veterinary Services Unite Interactions Bacteries Cellules
Animal and Plant Health Inspection Service 28 rue du Dr Roux
Department of Agriculture 75724 Paris
1800 Dayton Road Cedex 15
Ames France
IA 50010
USA U J P,otter
Centre for Electron Optical Studies
Gary A Payne University of Bath
Department of Plant Pathology Claverton Down
North Carolina State University Bath
Raleigh BA2 7AY
North Carolina UK
USA
B Pourkomailian
Ron Pethig Department of Food Safety and Preservation
Institute of Molecular and Biomolecular Electronics Leatherhead Food RA
University of Wales Randalls Road
Dean St Surrey
Bangor UK
Gwynedd
LL57 1UT K Prabhakar
UK Department of Meat Science and Technology
College of Veterinary Science
L Petit
Tirupati 517 502
Unite Interactions Bacteries Cellules
India
lnstitut Pasteur
28 rue du Dr Roux W Praphailong
75724 Paris National Center for Genetic Engineering and
Cedex 15 Biotechnology
France Rajdhevee
William A Petri Jr Bangkok
Department of Medicine, Division of Infectious Diseases Thailand
University of Virginia Health Sciences Center
MR4, Room 21 15,300 Park Place
M S Prasad
FermentationTechnology and Bioengineering
Charlottesville
Department
VA 22908
Mysore 570013
USA
India
M R A Pillai
Isotope Division J. C du Preez
Bhabha Atomic Rsearch Centre Department of Microbiology and Biochemistry
Mumbai 400 085 University of the Orange Free State
India PO Box 339
Bloemfontein 9300
D W Pimbley South Africa
Leatherhead Food Research Association
Randalls Road Barry H Pyle
Leatherhead Montana State University
Surrey Bozeman
KT22 7RY Montana
UK USA
xxxiv Contributors

Laura Raaska E W Rice

VTT Biotechnology and Food Research US Environmental Protection Agency
PO Box 1501 Cincinnati
FIN-02044 VTT Ohio 45268
Finland USA

Moshe Raccach
Jouko Ridell
Food Science Program
Department of Food and Environmental Hygiene, Faculty
School of Agribusiness and Resource Management
of Veterinary Medicine
Arizona State University East
University of Helsinki
Mesa
Finland
Arizona 85206-01 80
USA
R K Robinson
Fatemeh Rafii, Department of Food Science
Division of Microbiology University of Reading
National Center for Toxicological Research, US FDA Whiteknights
Jefferson Reading
AR Berkshire RG6 6AP
USA UK
M I Rajoka,
National Institute for Biotechnology and Genetic Hubert Roginski
Engineering (NIBGE) Gilbert Chandler College
PO Box 577 The University of Melbourne
Faisalabad Sneydes Road
Pakistan Werri bee
Victoria
Javier Raso
Biological Systems Engineering 3030
Washington State University Australia
Pullman
Washington 99164-61 20 Alexandra Rompf
USA Institute for Organic Chemistry and Biochemistry
K S Reddy Albert Ludwigs University Freiburg
Department of Meat Science and Technology Albertstr. 21
College of Veterinary Science 79104 Freiburg
Tirupati 517 502 Germany
India
S M Reddy T Ross
Department of Botany School of Agricultural Science
Kakatiya University University of Tasmania
Warangal Hobart
506 009 Australia
India
Wim Reybroeck T Roukas
Department for Animal Product Quality and Department of Food Science and Technology
Transformation Technology Aristotle University of Thessaloniki
Agricultural Research Centre CLO-Ghent Greece
Melle
Belgium
M T Rowe
U G Reyes Food Microbiology
Food Science Australia Food Science Department
Private Bag 16 Department of Agriculture for Northern Ireland and
Sneydes Road Queen’s University of Belfast
Werri bee Newforge Lane
Victoria Belfast
VIC 3030 BT9 5PX
Australia Northern Ireland
 Contributors xxxv

W Michael Russell Barbara Schalch

Departments of Food Science and Microbiology Institute of Hygiene and Technology of Food of Animal
Southeast Dairy Foods Research Center Origin
Box 7624 Ludwig-Maximilians-University Munich
North Carolina State University Veterinary Faculty
Raleigh Veterinarstr. 13
NC 27695-7624 81369 Munich
USA Germany

P Scheu
G Salvat BioteCon Gesellschaft fur Biotechnologische
AFSSA Entwicklung und Consulting
Ploufragan Hermannswerder Haus 17
France 14473 Potsdam
Germany

R Sandhir Bernard W Senior

Department of Biochemistry Department of Medical Microbiology
Dr Ram Manohar Lohia Avadh University University of Dundee Medical School
Faizabad 224 001 Ninewells Hospital
India Dundee
DDl 9SY
UK
Robi C Sandlin
Department of Microbiology and Immunology Gilbert Shama
Uniformed Services University of the Health Sciences Department of Chemical Engineering
F Edward Hebert School of Medicine Loughborough University
4301 Jones Bridge Road UK
Bethesda
MD 20814 Arun Sharma
USA Food Technology Division
Bhabha Atomic Research Centre
Mumbai 400 085
Jesus-Angel Santos
India
Department of Food Hygiene and Food Technology
University of Leon
M Shin
24071-Leon
Faculty of Pharmaceutical Sciences
Spain
Kobe Gakuin University
Kobe
A K Sarbhoy Japan
Division of Plant Pathology
Indian Agricultural Research Institute J Silva
New Delhi 110012 Universidad Nacional de Tucuman, Argentina
India Cerela-Conicet
San Miguel de Tucuman
Argentina
David Sartory
Severn Trent Water Dale1 Singh
Shrewsbury Microbiology Department
UK CCS Haryana Agricultural University
Hisar
125 004
Joanna M Schaenman India
Department of Medicine
Division of Infectious Diseases Rekha S Singhal
University of Virginia Health Sciences Center University Department of Chemical Technology
MR4, Room 21 15,300 Park Place University of Mumbai
Charlottesville Matunga
VA 22908 Mumbai 400 01 9
USA India
xxxvi Contributors

Emanuele Smacchi N H C Sparks

Institute of lndustrie Agranie (Microbiologia) Department of Biochemistry and Nutrition
Faculty of Agriculture of Perugia 06126 S. Constanzo Scottish Agricultural College
Perguia Auchincruive
Italy AYr
Scotland
Christopher A Smart
Macromolecular Science Department
Institute of Food Research M Van Speybroeck
Reading Laboratory Laboratory of Industrial Microbiology and Biocatalysis
Earley Gate Department of Biochemical and Microbial Technology
Whiteknights Road Faculty of Agricultural and Applied Biological Sciences
Reading R66 6BZ University of Gent
UK Coupure links 653
8-9000
Gent
H V Smith
Belgium
Scottish Parasite Diagnostic Laboratory
Stobhill Hospital
Glasgow D J Squirrel1
G21 3UW CBD Porton Down
Scotland. UK Salisbury
SP4 OJQ
0 Peter Snyder UK
Hospitality Institute of Technology and Management
670 Transfer Road
Suite 21A E Stackebrandt
St Paul DSMZ - German Collection of Microorganisms and Cell
MN 55114 Cultures
USA Brunswick
Gerrnany
Mark D Sobsey Deutsche Sammlung von Mikroorganisem und
Department of Environmental Sciences and Engineering Mascheroder
School of Public Health Weg 1 B
University of North Carolina 38124, Braunschweig
North Carolina Germany
USA
Jacques Stark
Carlos R Soccol
Gist-brocades Food Specialties
Laboratorio de Processos Biotecnologicos
R&D
Departamento de Engenharia Quimica
Delft
Universidade Federal do Parana
The Netherlands
CEP 81531-970
Curitiba-PR
Brazil Colin S Stewart
Rowett Research Institute
M El Soda Greenburn Road
Department of Dairy Science and Technology Bucksburn
Faculty of Agriculture Aberdeen
Alexandria University UK
Alexandria
Egypt
G G Stewart
R A Somenrille International Centre for Brewing and Distilling
Neuropathogenesis Unit Heriot-Watt University
Institute for Animal Health Riccarton
West Mains Road Edinburgh
Edinburgh Scotland
EH9 3JF EH14 4AS
UK UK
 Contributors xxxvii

Gordon S A B Stewart (dec) Jyoti Prakash Tamang

Department of PharmaceuticalSciences Microbiology Research Laboratory
The University of Nottingham Department of Botany
University Park Sikkim Government College
Nottingham Gangtok
NG7 2RD Sikkim 737 102
UK India

Duncan E S Stewart-Tu11 A Y Tamime

University of Glasgow Scottish Agricultural College
Glasgow Auchincruive
G12 8QQ AYr
UK UK

A Stolle J S Tang
Institute of Hygiene and Technology of Food of Animal American Type Culture Collection
Origin 10801 University Blvd
Ludwig-Maximilians-UniversityMunich Manassas
Veterinary Faculty VA 201 10-2209
Veterinarstr. 13 USA
81369
Munich
Chrysoula C Tassou
Germany
National Agricultural Research Foundation
Institute of Technology of Agricultural Products
Liz Straszynski S. Venizelou 1
Alcontrol Laboratories Lycovrisi 14123
Bradford Athens
UK Greece

M Stratford
S R Tatini
Microbiology Section
University of Minnesota
Unilever Research
Department of Food Science and Nutrition
Colworth House
1334 Eckles Ave
Sharnbrook
St Paul
Bedfordshire
MN 55108
MK44 1LQ
USA
UK

M Surekha D M Taylor
Department of Botany Neuropathogenesis Unit
Kakatiya University Institute for Animal Health
Warangal West Mains Road
506 009 Edinburgh
India EH9 3JF
UK
B C Sutton
Apple Tree Cottage John R N Taylor
Blackheath Cereal Foods Research Unit
Wenhaston Department of Food Science
Suffolk University of Pretoria
IP19 9HD Pretoria 0002
UK South Africa

Barry G Swanson Lucia Martins Teixeira

Food Science and Human Nutrition Departamento de Microbiologia Medica
Washington State University lnstituto de Microbiologia
Pullman Universidade Federal do Rio de Janeiro
Washington 99164-6376 Rio de Janeiro 21941
USA Brazil
xxxviii Contributors

Paula C M Teixeira D R Twiddy

Escola Superior de Biotecnologia Consultant Microbiologist
Rua Dr Antonio Benardino de Almeida 27 Guildford Road
4200 Port0 Horsham
Portugal West Sussex
RH12 1LU
J Theron UK
Department of Microbiology and Plant Pathology
Faculty of Biological and Agricultural Sciences N Tzanetakis
University of Pretoria Department of Food Science
Pretoria 0002 Faculty of Agriculture
South Africa Aristotle University of Thessaloniki
54006
Linda V Thomas Thessaloniki
Aplin & Barrett Ltd Greece
15 North Street
Beaminster C Umezawa
Dorset Faculty of Pharmaceutical Sciences
DT8 3DZ Kobe Gakuin University
UK Kobe
Japan
Angus Thompson
Technical Centre F Untermann
Scottish Courage Brewing Ltd Institute for Food Safety and Hygiene
Sugarhouse Close University of Zurich
160 Canongate Switzerland
Edinburgh
EH8 8DD Matthias Upmann,
UK Institute of Meat Hygiene
Meat Technology and Food Science
Ulf Thrane Veterinary University of Vienna
c/o Eastern Cereal and Oilseed Research Centre Veterinhrplatz 1
K.W. Neatby Building, FM 1006, A-1 210 Vienna
Agriculture and Agri-Food Canada Austria
Ottowa
Ontario K1A OC6
Turner Uraz
Canada
Ankara University
Faculty of Agriculture
Mary Lou Tortorello
Department of Dairy Technology
National Center for Food Safety and Technology
Ankara
US Food and Drug Administration
Turkey
6502 South Archer Road
Summit-Argo
M R Uyttendaele
Illinois 60501
Laboratory of Food Microbiology and Food Preservation
USA
Faculty of Agricultural and Applied Biological Sciences
University of Ghent
Hau-Yang Tsen
Coupure Links 654
Department of Food Science
9000 Ghent
National Chung Hsing University
Belgium
Taichung
Taiwan
Republic of China E J Vandamme
Laboratory of Industrial Microbiology and Biocatalysis
Nezihe Tunail Department of Biochemical and Microbial Technology
Department of Food Engineering Faculty of Agricultural and Applied Biological Sciences
Faculty of Agriculture University of Gent
University of Ankara Coupure links 653
Diskapi 8-9000
Ankara Gent
Turkey Belgium
 Contributors xxxix

P T Vanhooren Philip A Voysey

Laboratory of Industrial Microbiology and Biocatalysis Microbiology Department
Department of Biochemical and Microbial Technology Campden and Chorleywood Food Research Association
Faculty of Agricultural and Applied Biological Sciences Chipping Campden
University of Gent Gloucestershire
Coupure links 653 GL55 6LD
8-9000 UK
Gent
Belgium
Martin Wagner
Institute for Milk Hygiene
L Le Vay Milk Technology and Food Science
School of Ocean Sciences University for Veterinary Medicine
University of Wales Veterinarplatz 1
Bangor 1210 Vienna
UK Austria

P H In’t Veld Graeme M Walker

National Institute of Public Health and the Environment Reader of Biotechnology
Microbiological Laboratory for Health Protection Division of Biological Sciences
PO Box 1 School of Science and Engineering
3720 BA Bilthoven University of Abertay Dundee
The Netherlands Dundee
DDI I H G
Scotland
Kasthuri Venkateswaran
Jet Propulsion Laboratory
National Aeronautics and Space Administration P Wareing
Planetary Protection and Exobiology, M/S 89-2, 4800 Natural Resources Institute
Oakgrove Dr. Chatham Maritime
Pasadena Kent
CA 91 109 ME4 4TB
USA UK

V Venugopal John Watkins

Food Technology Division CREH Analytical
Bhabha Atomic Research Centre Leeds
Mumbai 400 085 UK
India
Ian A Watson
Christine Vernozy-Rozand University of Glasgow
Food Research Unit National Veterinary School Glasgow
Lyon G I 2 8QQ
France Ecole Nationale Vetenaire de Lyon UK
France
Bart Weimer
B C Viljoen Center for Microbe Detection and Physiology
Department of Microbiology and Biochemistry Utah State University
University of the Orange Free State Nutrition and Food Sciences
Bloemfontein Logan
South Africa UT 84322-8700
USA

Birte Fonnesbech Vogel

Danish Institute for Fisheries Research Irene V Wesley
Department of Seafood Research Enteric Diseases and Food Safety Research
Technical University of Denmark Bldg 221 USDA, ARS, National Animal Disease Center
DK-2800 Lyngby Ames IA 50010
Denmark USA
XI Contributors

W B Whitman Caroline L Willis

Department of Microbiology Public Health Laboratory Service
University of Georgia Southampton,
Athens UK
Georgia
USA F Y K Wong
Food Science Australia
Martin Wiedmann Cannon Hill
Department of Food Science Queensland
Cornell University Australia
lthaca
NY 14853
Brian J B Wood
USA
Reader in Applied Microbiology
R C Wigley Dept. of Bioscience and Biotechnology
Boghall House University of Strathclyde
Linlithgow Royal College Building
West Lothian George Street
EH49 7LR Glasgow
Scotland G1 1XW
Scotland
R Andrew Wilbey
Department of Food Science S D Worley
University of Reading Department of Chemistry
Whiteknights Auburn University
Reading Auburn
UK AL 36849
us
F Wilborn
BioteCon Gesellschaft fiir Biotechnologische Atte von Wright
Entwicklung und Consulting Department of Biochemistry and Biotechnology
Hermannswerder Haus 17 University of Kuopio
14473 Potsdam PO Box 1627
Germany FIN-70211 Kuopio
Finland
A G Williams
Hannah Research Institute
AYr Chris J Wright
KA6 5HL Biochemical Engineering Group
UK Centre for Complex Fluids Processing
Department of Chemical and Biological Process
Alan Williams Engineering
Campden and Chorleywood Food Research Association University of Wales Swansea
Chipping Campden Singleton Park
Gloucestershire GL55 6LD Swansea
UK SA2 8PP
UK
J F Williams
Department of Microbiology Peter Wyn-Jones
Michigan State University Sunderland University
East Lansing UK
MI 48824
USA Hideshi Yanase
Department of Biotechnology
Michael G Williams Faculty of Engineering
3M Center Tottori University
260-68-0 1 4-1 01 Koyama-cho-minami
St Paul Tottori
MN55144-1000 Tottori 680-0945
USA Japan
 Contributors xli

Yeehn Yeeh Gerald Zirnstein

Institute of Basic Science Centers for Disease Control
lnje University GA
Obang-dong USA
Kimhae 621-749
South Korea

Seyhun Yurdugul
Middle East Technical University Cynthia Zook
Department of Biochemistry Department of Food Science and
Ankara University of Minnesota
Turkey St Paul
MN 55108
Klaus-Jurgen Zaadhof USA
Institute for Hygiene and Technology of Foods of Animal
Origin
Veterinary Faculty
Ludwig-Maximilians University
80539 Munich
Germany
 INDEX

NOTE

Page numbers in bold refer to major discussions. Page numbers suffixed by T refer to Tables; page numbers
suffixed by F refer to Figures. vs denotes comparisons.

This index is in letter-by-letter order, whereby hyphens and spaces within index headings are ignored in the
alphabetization. Terms in parentheses are excluded from the initial alphabetization.

Cross-reference terms in italics are general cross-references, or refer to subentry terms within the same main
entry (the main entry is not repeated to save space).

Readers are also advised to refer to the end of each article for additional cross-references - not all of these
cross-references have been included in the index cross-references.

A AccuProbe system (coiztinued)

14-3-3 protein 287
AAL toxin 1518
ABC-STD 105 1979
ABC-transporters, Bacillus subtilis
producing 138
AB milk products 780-781
abortion
brucellosis 320-321
listeriosis 1198
abrasives, cell disruption by 698
Absidia 861
characteristic appearance on cultural
media 857
accreditation of HACCP 1831
accreditation of laboratories 1128
advantagesidisadvantages 1129-1 130
assessment preparations 1132-1 133
auditing 1132, 1133
documentation 1132, 1133
future prospects 1133-1134
implementation of schemes 1131-1133
maintenance of schemes 1133-1134
organizations 1128-1129, 1130
quality manager 113 1
Quality Manual 1131
standards 1128-1130
trace-ability 1128
AccuProbe system 1238-1244
advantages/limitations 1241-1242
comparison with other methods 12437
Listeria monocytogeizes detection 1238-
1239
protocol 1239-1241, 1239T, 1240T
microorganisms detection 1 2 4 2 1
principle 1238-1239
results 1242-1243, 1243T, 1243T
RNA extraction from cultures 1242F
sampling points and application 1241F
acetaldehyde 184
accumulation in yoghurt manufacture
787
production
lactic acid bacteria 184, 2105
Streptococcus thermophdus 213 1
acetate
carbon dioxide reduction to 1338
formation
cheese 384-385
lactic acid bacteria 2104-2105
metabolism in gut 1354
acetic acid 184, 705, 706T, 1292, 2258,
2263
applications 562
chemical properties 1731F
in cider 426
effectiveness for microbial control 562
effect on cytoplasmic pH 1733
effect on food 1730
formation 1-2
butanol-acetone fermentation 447-
448
quinoproteins 5-6
as preservative 1715, 1730
spectrum of activity 1711
production 2258
Brettanomyces 303
acetic acid (cotztinued)
Brevibacterium 312
lactic acid bacteria 184
see also acetic acid bacteria
recovery in industrial fermentations 691
in sausages 1267
in sourdough bread 301
structure and chemical properties 1732T
in vinegar 2259
acetic acid bacteria 1, 2260
classification 1
cocoa fermentation 468
ethanol oxidation 6F
isolation 4
tolerance to acid 1735
vinegar production 2259
wine-making 2306
see also Acetobacter
acetification 2260
composition (GK) value 2260
exothermic reaction 2261
microorganisms uses 2262
processes 2260-2262
quick vinegar process 2261-2262, 2261F
reactions 2260
submerged culture method 2262
surface culture technique 2261
see also vinegar
Acetobactev A1
characteristics of genus 1-3
classification 181-182
culture media for 4T
dendrogram of 16s rDNA 181, 181F
I ii INDEX
Acetobacter (continued)
detection 3-5
differentiation between species 3
differentiation from other genera 958,
958T
enrichment cultures 3
enzymes 2
ethanol oxidation 2260.2261F
on grapes 960
growth and requirements for 1
habitats 3
identification
features 4
phenotypic 4-5, 5T
importance to food industry 5-7
food processes 5-6
food spoiling see below
inhibition by ethanol 2
isolation 3
metabolism 1, 4
nomenclature 1
phenotypic features 4-5, 5T, 958T
pH range tolerance 1
plasmids 2
species 1
importance 5
spoilage of foods 6-7
fruit spoilage 3
sugar metabolism 2
vinegar production 2260
see also acetic acid bacteria
Acetobacteraceae 955
16s rDNA signature nucleotides 182T
phenotypic identification 4
Acetobacter aceti 2
Acetobacter carinus 2
Acetobacter diazotrophicus 2
Acetobacter eiwopaeus, acetification process
2262
Acetobacter hansenii 6
Acetobacter melanogenum 2
Acetobacter methanoliczis 2
Acetobacter pasteurianus 2, 7
Acetobacter peroxidans 2
Acetobacter rancens 2
Acetobacter xylinum 2, 6, 7
acetoin 1271
a-acetolactate synthase 920
acetone
aqueous, aflatoxin extraction 1528,
1527T
precipitation of metabolites in
fermentations 693
acetone-butanol-ethanol (ABE)
fermentation 431
Clostridium acetobutylicum 429
see also butanol-acetone fermentation
acetyl-coA (acetyl coenzyme A) 718, 719
b-oxidation of fatty acids 1306
pyruvate oxidation to 1276
acetyl-coA carboxylase 1308
acetyl-coA dehydrogenase 1306, 1307F
acetyl-coA synthetase 1306, 1307F
3-acet);l-DON, oral toxicity 1546T
15-acetyl-DON, oral toxicity 1546T
Achromobacter 1875
Achromobacter piechaudii 38
acidis) 1729
antimicrobial actions 1729
dissociation 1730-1731
in fermented meat products 749-750
organic see organic acids
pK 562
production, Cellulomonas 369
taste in foods 1731, 1733F
weak 2263, 1731, 1733, 1733F
diffusion through membrane 1735
from wood smoke 1739, 1740T
see also specific acids
acid cleaners 1810
acid-fast bacteria 1500, 1504
cell wall structure 163, 176
acid-fast stains 1384, 1385T, 1504 Acinetobacter (continued)
acidic foods isolation 10-1 1
microbial growth control 561-562 enrichment procedure 10
spoilage microorganisms and 1009 media 11
acidification 1730-1731, 1731 National/International Regulations
acidulants power 1732F procedures 1 3
effect on Candida 359 nomenclature 8-9
of media 1731-1732 optimum growth temperature 11
milk 382 pathogenicity 9
see also acidulants selective growth 10-1 1
acidified tryptone glucose yeast extract agar species 8-9, 8-9
(ATGYE) 2364T spoilage of foods 9
acidocin B 188. 1153 fish 1489
Acidomonas, classification 181-182 meat 1258,2052
acidophiles 174 taxonomy 1487,1876
acidophilus milk 779, 2086 transformation assay 11
acidophilus yogburt 781 virulence of strains 15
acid phosphatase stain method, whole cell hybridization 10F
Saccharomyces sake 1915 Acinetobacter baumanii 9
acid-resistant bacteria 1735 in intensive care units 15
acid shock proteins 561 Acinetobacter calcoaceticus-A. baumanii
acid tolerance, fermentation 1571 complex 1 3
acid tolerance response (ATR), by Acinetobacter johnsonii 7
adaptation 1736, 1736F distribution in food 9
acid-tolerant bacteria 1735 Acinetobacter lwoffii 7, 8-9
acidulants 1730, 1757 distribution in food 9
acidification power 1732F acoustic wave transducers 274
amount added 1730 Acremonium 863-864, 865, 868-870, 894
bread-making 291T acridine orange 1388, 1387T
buffering ranges 1732F Botrytis detection 281
chemical properties 1730-1731, 1732T Lactobacillus bulgaricns staining 1137F,
effect on cytoplasmic pH 1732-1734 1137F
effect on foods 1731 acridine orange direct count (AODC) 527
food 705,706T actagardine 192
function 1731-1732 Actrnomucor 861
interactions and factors affecting 1736- Actinomvcetales 211
1737 Actinomicetes 211
minimum inhibitory concentrations cellular differentiation 167, 167F
1733F metabolism 1298
taste in foods 1731, 1733F actinomycetomas 2137
temperature effect 1737 actinomycin 1334
acid washes 1810 actinorhodin 1334
Acinetobacter 7-8, 1875 active dry yeast (ADY)291-292, 291F, 2340
antibiotic resistance 15 active packaging see antimicrobial
biotechnological applications 15-16 packaging; modified-atmosphere
biotransformation 16 packaging (hfAP)
characteristics of genus 8 active transport 1273, 1273, 1280, 1290,
classification 8 1291F
culture media, acidification 11 acyl carrier protein 1308F
detection adaptation, microbial
in foods 14F to acids 1735-1736. 1736F
methods 9-13 to environmental stress, biofilm bacteria
differentiating characteristics 1876T 255-256
ecology 9 by fungi, to redox potential and pH 559-
epidemiological typing 13 561.559F
extracellular polymer production 16 to pH changes 559-561
genomic species 8-9, 9T to redox potential and pH changes 559-
distribution 9 561,559F
growth in food 13 to stress, hurdle technology 1075
habitats 9 yeasts, to acids 1736, 1736F
identification added value, microorganisms to industry
biochemical tests 11 485
chemical analyses of cellular additives see food additives
components 12 additive tree methods 179
commercial systems 11-12 ADE2 gene 911
at genomic species level 11 adenine auxotrophic mutants, sake yeasts
at genus level and metabolic characters 1917
11 adenosine diphosphate (ADP) 17
molecular methods 12-13 bioluminescent assays, adenylate kinase
PCR 12-13 assay us 24T
physiologic tests 11-12, 12T preparations 18
ribotyping 1 3 adenosine monophosphate (AMP),
importance of genus in food industry 13- adenylate kinase role 17. 17
15 adenosine iriphosphate (L4TP)’80,88, 94,
dairy products 15 220,2170
eggs 15 in adenylate kinase assay 17, 18, 19
meats and poultry 13-14 analysis, rationale for 95
seafood 14-15 assay 2170
importance of genus in other applications 2170
environments 15 limitations 2170
clinical 15 schemes 88-89
water and soil 15 technique 2170
 INDEX I iii

adenosine triphosphate (ATP) (continued) adherence (continued) Aeromonas (continued)

assay (continued)
see also ATP bioluminescence
background levels 81, 82
bioluminescence see ATP
bioluminescence
biomass measurement 80
brewing yeast vitality analysis 103
content of microorganisms 81
energy conversion pathways 1272-1279
see also metabolic pathways
formation 97
anaerobic 556
electrochemical p H gradient 556
Embden-Meyerhof-Parnas pathway
1274-1275,1274F
fermentation 556-557, 1284
Zymomonas 2367
see also specific metabolic pathways
free 89
kits for monitoring 220
malolactic fermentation 2312
microbial (bacterial) 89
microorganism number 80, 88
in milk and dairy products 89
quantification 80
reasons for measuring 88
recycling reaction 97
somatic 89
sources 95
structure 80F, 88F
synthesis, protons and energy-transducing
systems 1286-1287,1288F
adenylate kinase (AK) 16-24, 97, 107
advantages 18
amplification reaction 17
ATP bioluminescence sensitivity increase
by 97
beverage industry 107
commercial systems available 107
concerns over dead cell detection 107
mechanism 107F
bioluminescent assay 17-18
applications 21
assav cauabilitv 19-20
ATP-baied assay us 17-18, 18F, 19F,
20F, 24T
detergent effects 18-19, 1 8 7
extraction step 18-19
factors affecting correlation with
colony forming units 19
hygiene monitoring 21, 2 1
incubation time effect 19, 19F
materials and methods 18-19
matrix effects 20
sensitivities 21, 21T, 21T
typical procedure 21
as cell marker 17
chemicals affecting 20
distribution/sources 1 7
in immunoassays 21-22, 22F
microbial 17-18
molecular mass 1 7
molecule number in bacteria 1 7
nonmicrobial, detection 20, 20T
reaction catalysed 1 7
specific assays using 21-22, 22F
bacteriophage-mediated 22-23,22F,
23F
magnetic beads-mediated 21-22, 22F,
22F
for Staphylococcus aureus 23,23F, 23T
adenyl cyclase, edema factor (EF) as 130
adenylic acid, biosynthetic pathway 1297F
adherence
Brettanomyces to insects 307
Escherichia coli O157:Hi 647
lactobacilli 1370
phage to bacteria 1471
probiotics 1370
Salmonella enterrtidrs 1938
Staphylococcus aureus 2066
see also bacterial adhesion growthisurvival at low temperature 27,
adhesins 164 27-28,27T
adhesion atmosphere 29
bacteria see bacterial adhesion background microflora effect 29
biofilm bacteria 253-254, 255 factors affecting 28-29
strength 256 salt and pH affecting 28-29
adipic acid, as preservative 1730 growth temperatures 25, 27-28
adjunct cultures haemolytic and proteolytic activities 28
cheese-making 382-383 hybridization groups 3 1
Lactobacillus bulgaricus 1140 identification 3 5
meat curing and 1261, 1261T importance to food industry 27-30, 3 7
adjuvants 625 isolation from food, stages 31, 31F
adsorbents meat spoilage 1266
commercial 691T 0-antigen LPS from smooth strains 28
properties 691 pathogenicity 3 1
adsorption, metabolite recovery in industrial pathogenic serotypes 29
fermentations 691-692 phenons 2 7
column operation 691-692, 692F phenospecies 31
concentrations of solutes 692F Plesiomonas differences 2 5 1
adsorption chromatography 691 prevalence in foods 27-28, 27T
adsorption curve 532 psychrotrophic nature 27, 27-28, 2 7 1
adsorption effect 539 in shellfish 2003-2004
adsorptionielution methods, viruses 2284- significance in foods 37
2285 S-layer 28
adsorption isotherm 692 speciation 35
adulterants, detection by enzyme methods 26-27
immunoassays 632 species 25-26, 31
adverse food reactions 1004 biochemical characteristics 26, 26T
Advisory Committee on Dangerous new 26
Pathogens (ACDP)2221 pathogenicity 29-30
aeration virulence factors 28
solid-state fermentation 671 Aeromonas caviae 26
submerged fermentation 666 Pathogenicity 29, 30T
aerobes
~~~~~ ~ prevalence in foods 2 7 , 2 7 1
facultative 173 Aeromonas hydrophila 26, 30
on meatimeat products 1255, 1255T capsular layer 28
microaerophilic 173 prevalence in foods 27, 27T
obligate 173 psychrotrophic nature 27, 27-28, 27T
strict 556. 1284 spices and essential oils effect 1721
transport systems 1273T Aeromonas (Ryans)agar 31
types 173, 1 7 4 1 Aeromonas salmonicida
aerobic metabolism, ATP formation 1272- detection 3 5
1279 rapid diagnostic test 36
see also metabolic pathways Aeromonas sobria 26
aerobic plate count (APC)method 2160, prevalence in foods 27T
2161 Aeromonas veronti biotype sobvia 28
aerobiosis 557F aerosols, microorganisms in 1816
Aerococcus, characteristics 1 1 6 5 1 aesculin, hydrolysis 618, 620
aerolysin, Aeromonas expression 28 Cellulomonas 368
Aeromonas 25-30,30-37 affinity sensor 274
biochemical tests 26, 26T aflatoxin(s) 1325T, 1512, 1514
capsular layer 28 adverse effects 1540
carrier rates 29 in butter and cream 1454
chemo-organotrophic facultative carcinogenicity 1514, 1519, 1533, 1540
anaerobes 25 concerns 1512
classification 25, 26F detection 75
clinical laboratories 26 using biosensors 270
detection 30-37 disease associated 1325
rapid methods 36 effect on animal health 1519
detection by culturing 31-35 in fermented foods 1326
common media 31-32 meat products 747-748
differential media 3 5 funei oroducine 1514. 1540
enrichment techniques 32 .&pergillus ?0-71,72, 1512
from fish 35 Aspergillus flavus 71, 72, 77-78, 74F
from food 34-35 Aspernillus parasiticus 72, 74F
media 3 3 1 dekccon 70-71
methods 32-33 fungal characteristics 77T
most probable number method 33-34 genes involved 71
nonselective approach 32 in koji moulds 71
selection isolation techniques 34-35 mechanisms 78
from water 35 reduction methods 78
detection using modern methods 35-37 levels in seeds 77
molecular methods 3 7 maximum level allowed 77
serology 35-37 occurrence 1520
distribution 30 in foods 1540
endotoxin 28 food types and geographic distribution
exotoxins 28 1523T
extracellular protein expression 28 production inhibition by Lactobacillus
fermentation and 34 2100
fimbriae and adhesins 28 regulations affecting 77
general characteristics 25-27, 25T solvents for extraction 1527T
I iv INDEX
aflatoxinis) (continued)
solvents for separation 1529T
structures 74F, 1515F
synthesis and functions 77-78
tblerance limits 1533T
toxicity 1514, 1519, 1540
types 1454,1514, 1515F, 1533, 1540
see also mycotoxins
aflatoxin B,63, 1540
actions 77
Aspergillus flavus producing 7 2 , 7 7
Aspergillus producing 6 3
as carcinogen 1514
hepatocellular carcinoma 77
mass spectrum 1531F
radioactive labelling 1536, 1536F, l536F
species producing 77T
structure 74F, 1541F
toxicity 77T, 1540
aflatoxin Bz
Aspergillus flavus producing 72
species producing and toxic effects 77T
structure 74F
aflatoxin G,
Aspergillus parasrticus producing 72-73
mass spectrum 1531F
species producing and toxic effects 77T
structure 74F
aflatoxin Gz
Aspergillus parasiticus producing 72-73
species producing and toxic effects 77T
structure 74F
aflatrem 78, 76F
XFNOR, PCR commercial test validation
1638-1639
Africa
fermented fish products 757
fermented foods 736-737
agar
standard methods, formulation 2 1 5 5 1
see also specific types
agar-based kits
food-poisoning organisms 238-239
miniaturized techniques 222-223
Agaricus bisporus 909, 1401F
agglutination tests 627
Brucella 323
latex see latex agglutination
rapid detection of microbes 1892
air
compressed, laboratory supply 1123
contamination risks from 1793, 1804
microorganism concentration 1816, 1817
microorganism transport 1816-1 817
milk contamination from 1437
particle removal 1820-1821, 1820F
particles 1680
sizesiclasses 1820F
quality 1680, 1820
UV treatment 2212
air-blast coolers 407
airborne contamination 1816-1822
controlireduction 1818-1819
heat inactivation 1819-1821
need for 181 6
particle removal 1820-1821, 1820F
measurement methods 1817-1818
Andersen perforated disc sampler
1817-18 18, 1818F
centrifugation 1818, 1818F
filtration 1818, 1818F
impinger 1818, 1818F
slit sampler 1817, 1818F
see also air filtration
milk 1437
recontamination prevention 1821
risks 1793, 1804, 1817
risks from 1793, 1804
sources 1816-1817
validationiassurance and maintenance
1821-1822
air conditioning 1121, 1804
air conditioning (continued) alcoholic beverages (continued)
airborne contamination 1816 Candida role 357
air filters 1680-1681 fermentation 2098
\.alidationiassurance and maintenance nisin application 197
1821-1822 Saccharomyces carlsbergensis role 1926
air filtration 1680-1681, 1820-1821, 1820F Saccharomyces cerevisiae role 1921-
airlift fermenters 2339-2340, 2339F 1922,1925, 1926
air sampler Saccharomyces role 1925
Andersen perforated disc 1817-1818, Schizosaccharomyces significance 1987
1818F smoke-processed 1738
centrifugal 1818, 1818F from sorghum see sorghum beer
slit sampler 1817, 1818F spoilage
alanine 1292 Gluconobacter 960
b-alanine method, Saccharomyces sake Saccharomyces cerevisiae 1922
detection 1915 Zymomonas fermentation 2369-2370
‘alarm water content’ 534, 535T see also alcohol fermentation; beer;
albicidins, detoxification by Pantoea fermented beverages; wine; specific
agglomerans 1628 beverages
Albuginaceae 880 alcohol oxidase
albumen AOXl uromoter 1688
p H effect on Salmonella growth 565, production in Pichia expression system
566F 1687-1688
see also eggs alcohols
Alcaligenes 38-42 assimilation and transformation by
antibiotic resistance 4 1 Rhodotorula 1904-1905
biosensor for heavy metals 40 flavour in fermented meat products 751T
biotechnological applications 38, 38-40 secondary, in cheeses 1660T
biodegradable plastics 38-39 in wine 2308
bioremediation 39-40 alcohol vapour, antimicrobial packaging 419
Bordetella relationshipidifferentiation 4 1, aldehydes
41T flavour in fermented meat products 7 5 l T
catabolism of PCBs 39 from wood smoke 1739
classification 38, 39 ale, production 1 9 2 7
curdlan synthesis 40 Alexandrium 1672
detection methods 4 1 algae
enzymatic production of amino acids 40 biomass 2024,2026
food microbiology 40 blue-green, toxigenic 1674
heavy metal removal 39-40 see also cyanobacteria
heavy metal toxicity resistance 39-40 classification 2021
location/distriburion 38 cultivation 2021-2024
psychrotrophic nature 40 closed systems 2024
relevance to food industry 38, 40 contamination avoidance 2023
species 38 harvesting 2024
xenobiotic-metabolizing isolates 39 mass-culture open systems 2023-2024
Alcaligenes defragrans 38 photobioreactors 2024
Alcaligenes eutrophus productivities 2023
glucose-utilising mutant 39 substrate requirements 2022-2023
polyhydroxbutyrate production 38-39 water types and quality 2026
Alcaligenes latus, polyhydroxbutyrate doubling time 2035T
production 38-39 as food 2021
alcohol dehydrogenase 787 types 2022T
alcohol (ethanol) see also single-cell protein (SCP)
Acetobacter inhibition 2 heavy metals 2026
as biocide 1799, 1801 macroalgae, cultivation 2021-2022
cellulose as source 2371 nucleic acid consumption from 2026
formationiproduction phycotoxin production 1672
Cellulomonas 3 70-3 71 single-cell protein see single-cell protein
from lactose 2371 (SCP)
from starch in bread fermentation 293, toxins see phycotoxins
294F unicellular
in wine 2307 cultivation 2022
see also alcohol fermentation as food 2021
measurement by biosensors 686 see also dinoflagellates
metabolite from kefir fermentation 802 yellow-brown, toxigenic 1673
oxidation by acetic acid bacteria 6F algal blooms 1672
Acetobacter 2260, 2261F Alicyclobacillus acidoterrestris 149, 150
precipitation of metabolites in detection 153, 1 5 4
fermentations 693 alimentary toxic aleukia (ATA) 1542
Saccharomyces cereuisiae tolerance 1919 aliphatic esters 733-734
Saccharomyces sake tolerance 1914,1917 aliphatics, flal-ours 734
sake yeasts tolerance 1917 alkaline cleaners 1810, 1811
utilization, Breuibacterium 31 1, 31 1 T alkaline peptone water (XPW)31
yeast single-cell protein production 2030 Vzbrro cholerae enrichment 2244
Zymomonas tolerance to high levels 2368 alkaline phosphatase
alcohol fermentation 1285 calf, in enzyme immunoassays 629
beet molasses 2371 milk pasteurization indicator 1035
sorghum beer 763- alkalinophiles 174
vinegar production 2259-2260 alkanes
Zymomonas 2367 flavour in fermented meat products 751T
see also alcoholic beverages; oxidation and assimilation 721, 723F
Saccharomyces cereuisiae 4-alkanolides 734
alcoholic beverages 5-alkanolides 734
 INDEX I v

alkenes, flavour in fermented meat products Alternaria solani 47, 4 5 1 amino acid auxotrophs, Bacillus subtilis 136
751T Alternaria sonchi 45T amino acid complexes, hazardous 1743
allergic skin test (AST) 326, 326 Alternaria tenuis 43 D-amino acid oxidase, Rhodotorula 1901
allergy Alternaria tenuissima 44, 4 5 1 g-aminobutyric acid, production, by
sulphur dioxide 1753 Alternaria triticina 45T Lactobacillus brevis 1150
treatment, Lactobacillus acidothilus role Alternaria zinniae 4 5 1 aminoglycosides 2138
1364 alternariol, Alternaria producing 49T synthesis 1330
allicin, antimicrobial compound 1577 alternariol monomethyl ether (AWE), 6-aminopenicillanic acid, structure 1320F
allozymes 229 Alternaria producing 49T aminopeptidase, Brevibacterium linens 313
all purpose Tween 80 (APT) medium 1145, Alteromonas, in fish 807-808 aminopterin 626
1145T Alteromonas putrefaciens 2008 ammonia
ALTA 187 cells as biosensor 270 formation in gut 1354
altenuene, Alternaria producing 49T altertoxin I (ATX-I),Alternaria producing neoplastic growth in colon 1354
Alternaria 42-50, 862, 868-869, 894 49T productionisynthesis 677
appearance on cultural media 857 altertoxin I1 (ATX-II),Alternarra producing Brevibacteriurn linens 396
characteristics 42-46 49T cheese maturation 391
conidia 45T altertoxin 111 (ATX-1111, Alternaria ammonium ions, conversion to organic
detection in foods 48-49 producing 49T nitrogen 1296, 1296F
culture methods 48-49 aluminium cans 1619 ammonium salts, utilization by fungi 1297-
direct methods 48 Amadori compounds 1742 1298
endophytic 48 ambrox 734,734F ammonium sulphate, salting-out of proteins
phyiloplane detection 48-49 America 693
platingidiluting methods 49 fermented foods 737 amnesic shellfish poisoning 2000-2001,
seeds 48 see also USA 1673
ecological aspects 46-47 American National Standards Institute 1841 amoebiasis 2292
enzymes 46 American Organization of Analytical pathogenesis 2293
on fresh foods 46 Chemists (AOAC), HGMF methods treatment 2294
grouping by macroscopic appearance 45T 1079-1081 see also Entamoeba hrstolytica
growthidispersal, environmental factors amines amoebic colitis 2293
affecting 46 biogenic see biogenic amines amoebic liver abscesses 2293-2294
growth requirementsiconditions 46T in colon 1354 amoebic ulcers 2293
metabolites 47-48 in fermented meat products 748-749 amoebopores 2293
mycotoxins 46,47-48, 47-48,49T, 1512 N-nitrosopyrrolidine formation 1767 amperometric biosensors 277
optimal temperature and water activity production 748-749 amperometric transducers 272-273
46T enterococci 1369 organic conductors 273
as pathogen 47 Pediococcus 1646 ampicillin-dextrin agar (ADA) 31
physiological aspects 46 amino acidis) amplified fragment length polymorphism
phytotoxins 47-48 aromatic 1296 (AFLP), Clostridium beijerinckii 43 1
host-specific 47-48,48T synthesis 1295 AmpliSensor system, polymerase chain
nonspecific 47 aromatic compounds from, in fermented reaction 1604F
as saprophyte 42, 46 meat products 750 amylase
species 42-46, 45T aspartate, synthesis 1296, 1296F Debaryomyces occidentalis 519
identification 44 deamination fungal, synthesis by recombinant DNA
important 44, 4 5 1 Brevibacterium 31 1 technology 915
spores 43 Clostridium 1292 production, Aspergillus 907
on harvested crops 47 Proteus 1857-1 85 8 a-amylase
sporulation 47, 46T see also deamination; specific amino Aspergillus oryzae 68
patterns 45 acids egg pasteurization indicator 1036
taxonomy 4 2 , 4 2 , 4 3 decarboxylation Flavobacterium 825
conidia number 43 Brevibacterium 311 genes 68
toxins 42, 857, 1518 in cheese maturation 391 production, by moulds 21 11
in foodstuffs 1518 degradationicata holism recovery from fermented broth 693, 693F
see also phytotoxins (above) bacteria in fish 815 test, egg yolk 571
type species 43-44 Brevibacterium 308 vinegar production and 2259
see also Alternaria alternata fermented meat products 750 Anabaena spp. 1674
Alternaria alternata 42 energy release (aerobic) 1277-1278 anaerobes 173, 1284
characteristics 43-44 essential, mycoprotein products 2042T aerotolerant 173
colonization 46 fermentation 1291 facultative 200, 557
ecological aspects 46-47 in fermented meat products 750 on meatimeat products 1255T
infection appearance 43F in fermented milks 783 phosphotransferase system 1290
metabolites 47-48 free
.~ growth, yields 560
mycotoxins 47-48 cheeses 1660T growth rates 559
as saprophyte 46 fish 807 heat-treated fish product spoilage 820
structure and conidia 44F Droteolvsis of cheese 385-386 normal gut flora 198
type species 42 grdups 1235 obligate (strict) 173, 556
Alternaria brassicae 44, 45T metabolic pathways 1291 methanogens 1334
Alternaria brassicicola 44, 45T metabolism, Lactococcus lactis role sulphate-reducers 520
Alternaria cheiranthi 45T 1168-1169 transoort svstems 12737
Alternaria citri 44, 4 5 1 mycoprotein products 20421 types'173, i 7 4 1
Alternaria cucumerina 4 5 1 as nitrogen source 1289 anaerobic bioreactor 1336-1337
Alternaria dauci 45T Pediococcus requirement 1643 anaerobic conditions, E . coli regulatory
Alternaria gaisen 45T single-cell Drotein content systems 560
Alternaria helianthi 45T algal 20'24,2025T anaerobic digestion 1336
Alternaria helianthinfciens 45T yeast 2030T anaerobic metabolism
Alternaria infectoria 45T svnthesisiproduction 1295 carbohydrate catabolism 1281-1282
Alternaria longipes 48, 4 5 1 . Alcaligenes, enzymatic 40 catabolic pathways 1281-1286
Alternaria longissima 45T Brevibacterium 311 carbohydrate breakdown 1281-1282
Akernaria peponicola 45T pathways 1295-1296 Embden-Meyerhof 1282
A!ternaria petroselini 45T transamination, Brevibacterium 3 11 Entner-Doudoroff pathway 1282-
Alternaria porri 45T transport 1290 1283
Alternaria radicina 4 5 1 uptake and accumulation 1290 fermentation 1283-1286
Alternaria raphani 45T utilization, Brevibacterium 31 1 monophosphate shunt 1282
I vi INDEX
anaerobic metabolism (continued)
carbohydrate catabolism (continued)
see also fermentation; specific
pathways
energy release 1279-1288
sites 1286-1287
substrates utilized 1280
substrate uptake mechanisms 1280-1281
anaerobic respiration 1284
Shewanella putrefaciens 2010, 2010F
anaerobiosis 557F, 1189
analytes 625, 627
anamorphic state 62, 861, 887
anatase 2213
anatoxin a 1674
anatoxin ajs) 1674
Andersen perforated disc sampler 1817-
1818,1818F
aneuploidy, in fungi 928-929
ang-khak 2115
animal bioassays
botulinum toxin see botulinum toxin
(BoNT)
emetic toxin from Bacillus cereus 120-
121
rabbit ileal loop assay 120
animal feeds
antibiotic bans 1836
bovine spongiform encephalopathy
association 284
ochratoxin A in 1541
single-cell proteins 2035
nucleic acid content 2032
animals
anthrax 129
carcasses, anthrax 130, 134, 134
doublinev time 2035T
methanogenesis in gastrointestinal tract
133 7-1 3 3 8
monitorin# for contamination 837
mycotoxins effect on health 1518-1519
Trichinella prevalence 2182-2183
see also specific animalslinfections
anion removal 562
Anisakiasis. symptoms of human disease
1054
Anisakis simplex, life cycle 1054
Anoxyphotobacteria 177
anthocyanins 732
anthraquinone 73 1
anthrax 118, 129, 129-130
animals 129
cutaneous 130, 134, 130F
gastrointestinal 130, 142
intestinal and oropharyngeal types 130
pulmonary 130
see also Bacillus anthracis
anthrax toxin 130
antibacterial effects, fermented milks 798
antibacterial substances
detection in food, by electrical techniques
581-582
see also antibiotic(s); antimicrobial
compounds
antibiotic(s) 1320
in animal feeds 1836
bacteria producing 1320, 1330, 1332,
1331F
beta-lactam 1320F
Bifidobacterium selection 216-217
biosynthesis
gene control 1334
induction 1332
inhibition of enzymes 1332
recombinant DNA technology 1333-
1334
Brevibacterium linens sensitivity 309
definition 1330, 1711
depletion of intestinal lactobacilli 1362
diarrhoea associated 1363
drug residue hazards 1003
effect on colonic microflora 1353
antibioticis) (contiizued) antigenis) (continued)
enterococci sensitivity 1371 antibody reaction see antibody-antigen
Enterococcus cultivationiderection 619- reaction
620 as biosensors 269
fungal synthesis 1320-1324, 1320T coupling with enzymes 629
kefir microflora 802 food spoilage fungi detection 231
Lactobacillus brevis sensitivity 1145T Geotrichum candidum 945
Lactobacillus bulgaricus sensitivity 1141, polyclonal antibody production 626
1142T radioimmunoassay (RIA) 628
markers for Bacillus subtilis 137, 137T antigenic variation, Giardia trophozoites
in media 954
Botrytzs detection 280, 280 antimicrobial actions, acids 1729
see also specific media types antimicrobial compounds 1577
in milk, effect on Lactobacillus bulgaricus bacteriocins 1573-1574
1141 see also bacteriocins; nisin
mode of action 2138 biofilm resistance 256-257, 257F, 257T
novel 1334, 1902 ecology of natural systems 1570-1572
Pedzococctcs sensitivity 1643. 1643T electroporation treatment medium 1461
polypeptide, synthesis' 1330 enzymes 1575
as preservatives 1711, 1712T future developments 1575-1576
uses 1715-1716 GRAS status 417
production, in fermented meat products lactic acid bacteria 1574
1326 lactoferrin 1589-1590
resistance see antibiotic resistance below lysozyme 1582-1587
Rhodotorula sensitivitv 1902 milk Droteins 1587-1591
virev of action 1330
~ ~~ ~ ~~~.
~ ~~~~ in mddified atmosphere packaging 412
Staphylococcus sensitivity 2073T natural s)stems 1570, 1572-1575
Streptomyces 21 37-21 3 8, 21 37F, 21 38T nisin 1573-1574
mode of action 2138 see also nisin
susceptibility testing, using flow plant-derived 190, 1718T
cytometry 832 essential oils 1718-1720
types 1320, 1320, 1321T sources 1576-1582,1574
Yersinia susceptibility 2347 see also essential oils; spices
Zymomonas mobilis sensitivityiresistance production, recombinant DNA
2366T technology 939
antibiotic properties, Monascus pigments spices as 1717
1485 storage and natural systems 1570
antibiotic resistance transferrins 1575
Acinetobacter 15 antimicrobial effects, low pH 562
Alcaligenes xylosoxidans subsp. antimicrobial herbal extracts 420
xylosoxzdans 41 antimicrobial packaging 416-420
Bacteroides 198 applications 419-420
Enterobacter 603 edible films/coatings 418-419, 4 1 8 1
enterococci 624 materials 417-41 8
gene transfer, Acinetobacter 15 new developments 417-418
Gram-negative bacteria 163 Microban 417, 1692
Klebsiella 1107-1108, 1112 polymers/films 41 7
Lactobacillus brevis 1144 principles 417
Rhodotorula 1902 regulations and control 420
Salmonella 1948 sachet technology 419
Salmonella typhi 1945-1946 antimicrobial soaps 1799
Staphylococcus 2073T contamination 1799, 1799F
Zymomonas mobilis 2366T antimutagenic activity, of fermented milks
see also multidrug resistance 797
antibodies 625, 625-626 antioxidants
binding to antigens 627 acids 1730
characterization 627 importance of fermented foods and 738
conjugate fluorescent 1388, 1387T sulphur dioxide 1752
immobilization aoules 782
blocking and 1090 API 20E system 128, 2 2 5 2 239
for immunomagnetic separation see advantages and disadvantages 239
immunomagnetic separation (IMS) biochemical reactions used in 248T
labelled 1892 comparisons with Enterotube and BBL
monoclonal see monoclonal antibodies Crystal systems 248-249, 2487
to moulds 281 method and evaluation 239
polyclonal 626 principle 245
comparison with monoclonal Abs protocol 245-246
627T MI-50 CHL 251
production 626-627 API system 223
radioimmunoassay (RIA) 628 fungi 23 1
synthetic, as biosensors 269 microflora in fermented foods 251-252
use in direct epifluorescent filter technique API YEAST-IDENT 231
529 API-ZYIM system, rapid 252
antibody-antigen reaction appertization, preservation methods 1571-
analytical use 627-628 1572
biospecific interactions analysis 275 apple juice
types 627 centrifugation, microbes removed 1684,
antibody-microcolony epifluorescence 1684T
microscopy (MEM) 2179 for cider 421
antifungal agents, Botrytrs control 282 preparation 421
antigenis) 625, 625 composition 426
antibody binding 627 contaminants 422T

apple juice (continued)
Escherichia coli 0 1 5 7 : H 7 650, 651
microbiology 422-423
nitrogenous compounds 426
sulphur dioxide role 422-423
sulphur dioxide treatment 426
apple juice-gelatin medium 2369T
apple juice-yeast extract medium 2369T
appressoria 853
Approved Quality Arrangement (AQA)
1842
aquatic animals see fish; seafood; shellfish
aqueous acetone, aflatoxin extraction 1528,
l527T
ARBOR database 178
Arc AIB system 560
Archaea (archaebacreriai 178
cell membranes, water activity effect 546
lipids 1335-1336
methanogens 1335
Arcobacter 336, 341, 50-54
aerotolerance 50
biochemical tests 52
Campylobacter differences 50, 50
Campylobacter jejuni similarities 5 3
characteristics of genus 50-51
classification 50
detection methods 50
DNA-based fingerprinting 5 2
polymerase chain reaction 52, 52, 51F
distributionisources 50, 51T
foods 52,52F
gastroenteritis 341
human infections 52-53
importance in foods, chicken and poultry
53
importance in livestockifoods 52-53
cattle and beef 52-53
swine and pork 5 3
inactivation methods 5 3
isolation methods 51
serotyping 52
species 5 1
identification 5 2
taxonomy 50
rhermotolerance 5 3
Arcobacter bzttzleri 50
identification 52
inactivation methods 5 3
isolation 345-346
in poultry 53
rRNA 5 2
Arcobacter cryaerphilus 50
Arcobacter nitrofigilis 50
Arcobacter skrrrowii 50, 52
arginine
deamination, Vagococcz~sidentification
2219
metabolism 1293, 1294F
arginine deaminase pathway 1291
aromatics 732-733
aromatic substrates, energy release (aerobic)
1278-1279
Arrhenius plot 552, 552F
arterial pumping, meat curing 1263
arthritis, reactive 336
Arthrobacter 54-61, 308
bacteriophage 58
biotechnological potentialities 60-61
in clinical specimens 6 1
culture media 56-57, 56T
ecology 56
electrotransformation 5 8
general characteristics 54-56, 55T
genes and genetics 58
glycogen storage 5 7
heterotrophic nitrification 5 7
isolation 56-57
from oil spills 6 1
by selective media 56T
metabolic properties 56T
metabolism and enzymes 57-58
Arthrobacter (continued)
Micrococcus separation 56
nutritional versatility 56
pcd plasmid 58
polysaccharides 5 7
proteolytic actions 5 7
psychrotrophic strains, in milk 60
remediation of ground water 5 9
role in foods 58-61
biodegradation of pesticides 5 9
meat, eggs and fish 59-60
milk and cheese 60
vegetables 5 9
species 54, 54T
groups 54
new 61
sensu stricto 541, 55T
strain Q36, genes 58
Arthrobacter agilis
Characteristics 1345, 1346T
differentiation 1347T
Arthrobacter citreus 54
Arthrobacter cumininsit sp. noti 61
Arthrobacter globiformis 54
metabolic properties 56T
Arthrobacter nicotianae 54
as biosensor 270
inhibition of Listeria 60
metabolic properties 56T
Arthrobacter sulfztreus 54
Arthrobacter ureafacieiis, proteinase 5 7
Arthrobacter woluwensis sp. nov 6 1
arrhrofactin 61
arplsulphatase, mycobacteria 1507
asci 890F
Ascodesinis sphaerospora 1403F
Ascomycetes 861, 855
classification 899
basis 889-891
commercial importance 891-893, 8927,
892T
defining teatures 887-889
Deuteromycete relationship 887, 888F
eukaryotic 887-893
general features 889
reproduction 887-889, 888F
sexual 888F
see also yeast(sj
Ascomycota 862-868
Ascomycotina 887-893
ascorbates, as curing agents 1263
L-ascorbic acid
as antioxidant 960
nitrous acid reaction 1767
production 960
Gluconobacter 960
ascospores 855, 887, 888F,890F
Aspergillus 62
characteristics of fungi producing 889T
species 890T
ascus 862
aseptic packaging 2195
filling procedures 1029-1030
sterility detection by ultrasound see
sterility testing
aseptic processing, UHT 1024
Asia
fermented fish products 756-757
fermented milk oroducts 798-805.799T
oriental foods, moulds application'2114-
2116,2114T
origin of fermented foods 736
aspartate amino acids, synthesis 1296,
1296F
aspartate aminotransferase, Rhodotorula
1902
aspartate protease, in cheese maturation 391
aspartic acid 1292, 1292
deamination 1293
synthesis 1295
Xspergillaceae, antibiotics produced 1320
aspergillic acid 78, 76F
INDEX I vii
aspergillic acid (continued)
Aspergilloides 856, 1647
importance in food 1651
aspergillosis 65, 66
Aspergillus 62-66, 863, 864, 869
aflatoxin production 70-71, 1512, 1540
amylase production 907
antigens, ELISA 281
appearance on cultural media 856
in cereals during storage 2046
characteristics 889T, 894-895
classification 894-895
conidial structures 891F, 891F
detection methods 62
diseases associated 63, 65
enzymes and organic acids from 65, 65T
in foods and feeds 63, 64T
genetic engineering 907
identification
fatty acids for 229
media 62-63
of species 63
identification keys 6 3
isolation methods 62-63
morphological characteristics 62, 62F
mycotoxin production 63, 65, 64T, 1520
see also aflatoxin production (above)
parasexual hybridization 909-910
species 62, 64T
teleomorphic genera 62, 63, 63, 64T
see also individual Aspergillus species
Aspergillus awamori ? ? ? ? ? , mammalian
protein synthesis 915
Aspergillus differential medium (ADM) 75
Aspergillus fischeri, cultivation medium
724T
AsberPihs flavus 69. 72-79
'afl&xin'production 71,72,77-78,1325,
1514, I 5 4 0
detection 75
types 74F
as allergen 78-79
a s animal pathogen 78-79
biology 73-74
changes to resemble A . oryzae 69-70
chromosomes 6 7
colonization of seeds after inlur, 74
conidia 76
conidiophores 76, 73F
detection in foodsifeeds 75-77
DNA methods 76-77
ELISA 75
growth media 75
differentiation from A. oryzae 70
differentiation from other species 75-77
diversity 74-75
ecological benefits 79
economic significance 77-79
in fermented meat products 747-748
freezing effect 843
growth, appearance 72F
growth media 75
habitat 73-74
insects relationship 79
interactions with hosts 74
morphological characteristics 75-76, 73F,
77T
mycotoxin production 1325
see also aflatoxin production (above)
phialides 76
as plant pathogen 73-74
post-harvest contamination 74
pre-harvest contamination 74
as saprophyte 73-74
sclerotia 74, 76
strains (5. and L.j and isolates 75
summary 79
toxins produced 77, 76F
vegetative compatibility groups (VCGs)
74-75
Aspergillus flavus and parasrticus agar
(AFPA) 75
I viii INDEX
Aspergtllirs f l m u s (continued)
Aspergillus f l a w s group 66
Aspergillus fumigatits 79
Aspergillus nidulans
A,$fAl gene 914
chromosome 6 7
cultivation medium 724T
homologous recombination 913
nuclei 922
pyrC gene 913
sterigmatocystin production 71
transformation by recombinant D S A 914
Aspergillus niger 710F
chromosome 67
citric acid synthesis 706-707, 707-714,
708F
enzy-mes, applications 915
glucoamylase 68
gluconic acid synthesis 6, 715
importance in food industr) 2057
Asheivillus nomius 72
'afl&Jxins73, 1514
morphological characteristics 77T
Asmrnilirrs o c h r ~ c e u sochratoxin
, 15 14
Aspergillus oryzae 66-72, 1327
aflatoxin not produced 71, 1326, 1327
applicationsiuses 66
Characteristics 66-67
chromosome 67
conidia 67, 69
cyclopiazonic acid 1514
detection methods 69-71
differenriation from A. flavzts 70
distribution 66
enzymes produced 68
applications 915
genes 68, 69T
genetics 67
GRAS status 66
growth 67
hl-phae (mycelia) 67
identification methods 69-71
molecular biologJ- 70
importance in food industry 67-69
fermented foods 66, 67-69
morphological characteristics 67, 69, 67F
mycotoxins 68-69
non-aflatoxigenicit!; molecular
characterization 71
seed cultures 68
so>-sauce production 65
taxonomy 66, 69-70, 70T
Aspergillits parmiticus 69, 72
aflatoxins 72-73, 74F,1325, 1514
biology and habitat 73
groarh media 75
identitication 70
interactions with hosts 74
morphological characteristics 77T
mycotoxin production 1325
as plant pathogen 73-74
as saprophyte 73-74
Aspergillus pentcilliotdes
in cereals during storage 2046
cereal spoilage 2046
Aspergillus restrictus, cereal spoilage 2046,
2046
Aspergillus section Flaui 66
Aspergdhs s o p 65, 69, 1327
identification 70
inability to produce aflatoxins 1326, 1327
aspertoxin 78
asphyxiation 1002-1003
assays, nucleic acid-based 1599-1609
Association of Official Analytical Chemists
(-\O.\C) 1102
Escherichia colt 0 157 immunoassays
2227,22281,2228T
evaluations of Bacillus detection methods
I55
mycotoxin detection 1527, 1528
PCR commercial t e i t validation 1639
Association of Official Analytical Chemists
(AOAC) (continued)
Salinone/la detection method 1967
Assurance Pol\ clonal Enzi me Immunoassa\
for E. coh 0 1 5 7 2223-2225,2231,
2228T
astaxanthin 730, 730F
astro\ iruses 2265, 2274
morphology 2275
ATB system 240
atomic force microscope 1419, 1419F
atomic force microscopy (AFhI) 1418-1425
colloid probe technique 1421
contact mode 1419-1420
in food microbiology 1421-1424
bacterial, yeast and animal cells 1422-
1423
forces of interaction measurement
1424-1425, 1424F, 1424F
macromolecular components of cells
1423-1424
surfaces 1421-1422. 1422F
viruses 1423
force-distance curves 1420-1421, 1421F
future prospects 1425
image analysis 1420
imaging in liquids (double layer mode)
1420
intermittent contact mode 1420
non-contact mode 1420
principles 1419-1421. 1419F
tip geometry 1420
uses 1418-1419
ATP see adenosine triphosphate (ATPj
ATP bioluminescence l6-17,209, 220,
1475,2170
advantages 80, 86-87, 97, 101
disadvantages 108
summary 108
applications 16-17, 2170
in food processing plants 98-101
ATP from non-microbial sources 220
in beverages 104
differentiation (from microbial ATP)
98,220
minimization 81, 82
bacteriophage I! sin-release 208-209,
210,209F, 209F
in beverage microbiology 101-109
accelerated forcing tests 107-108
adenylate kinase methods 107
background ATP measurement I05
benefits 101, 108
brewing yeast vitalit)- analysis 102-104
contamination analysis 105
conramination prevention 104-108
detection limits 105-106
disadvantages 108
fail and pass I05
filtered samples 104, 105
need for rapid testing 101
quality assurance I 0 1
reagents and instruments 101-102
reagent storage 102
sample preparation 104-105
test kits 102, 102T
use of Microstar system 107
comparison with XK-based assay 18F,
19F, 20F
concentration-dependent transition
phenomenon 98
correlation with plate counts 82, 83F, 83F,
84F,84F, 83T, 2170
advantages of ATP bioluminescence
101
beef and poultry 87T
hygiene monitoring 97-98
hygiene monitoring of milk
transporters 99F
cutoff limits 87, 87F
in dairy industry 88-94
assay for psychrotroph proteases 91
.ATP bioluminescence (continued)
in dairy industry (continued)
detection of inhibitory substances 93
interpretation of results 90, 91
monitoring of starter culture activity
92-93
other applications 93-94
r a a milk quality assessment 89-91,
907
screening of hygiene of farms and milk
tankers 89
shelf-life prediction of dairy products
91, 92T
somatic cell count and mastitis control
93
sterility testing of UHT dairy products
91-92, 92T
tests 90-91, 90T, 90T, 90T
definition 80
disadvantages 86-87, 97-98
environmental effects 98
sampling problems in hJ-giene
monitoring 97
effects of chemical sanitizers on 98, 98T
in hygiene monitoring 94-101, 220
adequacy of monitoring kits 96-97
advantages 9 7
cleaning procedures in institutions 95,
95F
cutoff value criteria 96
data expressioniinterpretation 96
dry cleaning methods effect 100
evaluations of commercial kits 96
in food processing plants 98-101
limitations 97-98
manufacturers of kits 95T, 220
methods 95-98
reagents and instruments 96
sensitivity 96-97, 9 7 1
theoretical limits of detection (TDL)
96,97T
inadequate for current regulations 8 7
limitations 17, 97-98, 2170
luminometers see luminomerer
manufacturers of kits 931, 95T, 102T
in meat industry 80-88
assays 81-85
BactoFoss (automation) 83
€scherichia colt O l 5 7 : H 7 detection
84-85
finished meat products 82-83
hygiene monitoring 99-100
meat homogenization problems 82
poultrl- hygiene monitoring 99
raw meat materials 8 1-82
'rinse-bag' method 82
role in meat processing 85-86, 85
sterile sponge method 82
total viable counts on meat 82
principle 80-81, 88-89, 9 5
procedure 82F
aseptic technique 86
rapid detection of microbes in food 1893
rapidishort turnover time 80, 86. 95, 101
reaction 80-81, 81F. 88-89: 94
reagent checking 102, 103T
reagent storage 102
real-time testing 85-86, 9 7
sensitivity 82, 96-97, 97T. 209
methods to increase 97, 107
technique 2170
total viable microbial count relationship
86-87
use in hygiene monitoring 1 7
ATP:citrate lyase 1300, 719
Rhodotorula 1901
ATP synthetase system 1287
audit
laboratory 1129, 1132
maintenance of accreditation schemes
1133
Aureobusidtum 109-112, 869, 895

Aureobasidium (continued)
characteristics of genus 109-110, 1 1 0 1
colony appearance 109
conidiogenesis 109
detection methods 110
immunological 110
plating 1IO, 112F
enzymes 111 T
in foods 109-110
fruits and vegetables 109
survival in reduced water activity 110
unacceptable levels 110
Aureobasidium pullulans
characteristics 109-110, 11OT
conidiation 109
control 112
enzymes 1IO
food additives produced 111 T
fruit spoilage l i 2
importance to consumer 112
importance to food industry 111-112
merabolismhutrition 110
opportunistic mycosis 112
pullulan production 110
Aureobasidium pullulans var. melanogenum
109
Australia
food hygiene regulations 1841-1842
regulatory systems for process hygiene
1833T
autoclaves 1126-1127
gravity displacement 1126, 1127F
pressure cooker 1126, 1126F
autolysin 448, 933-934
autolysis
bacteria 1474
fish 813, 814
yeast 2032
autolytic genes 1474
autonomously replicating sequences (ARSj
912
autoradiography, microautoradiography
2180
hutotrack system 107
auxotrophic markers 91 1
auxotrophic microorganisms 173, 1280
avenacins
antimicrobial compounds 1577
spoilage reduction 1577-1578
avidin 629
antimicrobial chelating agent 1574-1575
food application 1586
mode of action 1584
occurrence 1582-1583
properties I584
structure 1583
avocado, antimicrobial compounds 1576-
1577
a,> see water activity
Azobacter vrvelandii 1289
azo compounds 202
azoreductases 202
B gamma phage sensitivity 132
BAhX Pathogen Detection svstem 1636-
1637
Bach process, microwaves 1039
bacilli 159-160, 159F
Bacillus 113-119
aerobic endospore-forming 113
carbon catabolite repression 117, 117F
cell wall composition 114-115
central regulitory component (CcpA)
117. 117F
characteristics of genus 113-114
colonial morphology 149
detection by cultural techniques 149-158
aciduric flat sour spore-formers 152,
155,ljlT
advantages and limitations 155
Bacillus (continued)
detection by cultural techniques jcont.)
collaborative evaluations/validations
155
diluenrsisolutions used 156
flat sour spore-formers 155
formulations of media 151
heatine of samules 151-152
u
incubation of samples 152
media 149-150,156, l j O T
media for confirmation 157-158
media for enumeration 156-157
mesouhilic aerobic soore-formers 152.
li5, l5lT
procedures 151-154
procedures in food samples 151T
roue suores 150, 152-153,155,15lT
sample size 151
sample type 150
see also specific Bacillus species
detection of environmental changes 117
enzymes 113, 116T
reactions 116-117
food-borne illness 141-143
characteristics 144T
see also Bacillus cereus
food spoilage I50
acid foods 1009
flat sour 128, 150
see also Bacillus stearothertnophilus;
canned foods
gene regulation 117
genetic diversit). 113, 114
gene transfer 115-116
genome 114, 115T
identification of species of public health
interest 154T
mesophilic aerobic spore-formers 149
detection 152, 155, 1 5 l T
in milk 1444
non-pathogenic strains 113
pathogenicity 117-1 18
phylogenetic tree 114, 114F
products 113, 116-117, 116T
species
characteristics 116T
importance in food industry 149T
spores, in food samples 150
sporulating, isolation 115
sporulation 113, 115
thermophilic flat sour spore-formers 149
toxins 146
detection 141-149, 148
see also Bacillus cereus
Bacillus aerogenes 598
see also Enterobacter aeropenes
Bacillus anthracis 129-135
capsule 130, 133, 132F
characteristics of species 129-131
classification 129
control 129
detection 131-134, 134, 1 3 3 1
antigen-based methods 132
preliminary tests 132
presumptive tests 132-133
gene, capsule s)-nthesis 130
haemolysis absence 132
herbivore infections 129
human infections
meningitis 129-130
see also anthrax
importance to consumers 134-135
importance to food industry 134
isolation, WHO protocol 132, 133FT
motility absence 132
as obligate pathogen 129
penicillin sensitivity 132
regulations relating to 134
5-layer proteins (Ea1 and Sap) 131
spores 129
germination requirements 129
INDEX I ix
Bacillus anthracis (continued)
toxin 130
edema factor (EF) 130-131
genes 131
lethal factor (LFj 130, 131
protective antigen (PA) 130, 131, 132
toxins 118
virulence, detection 133
virulence factors 130
coordinate regulation 131F
Bacillus brevis
food-borne illness 142, 143. 144T
gramicidin synthetase synthesis 1332
Bacillus cereus 119-124
Bacillus thtiringiensis toxin gene transfer
118
cereolysin 120, 131
certified reference materials (CRMsi
18981. 1 8 9 8 1
characteristics of species 119-121, 121-
122, 1340T
colony morphology 165F
detection methods 121-123, 153
confirmatory tests 121-122
cultural techniques 155, 155
direct plating 121, 153
enrichment 153
in foods 121
most probable number 121, 155
presumptive tests 153
specific tests 122-123
differentiation from other species 121
enzymes 120
food-borne illness 119, 119-120, 123.
123, 136, 141-142, 143, 837
characteristics 144T
diarrhoeal syndrome 120, 141, 143
emetic syndrome 119-120, 141, 146
foods associated 119, 123
incidence 142
risk factors 124, 142
symptoms and signs 123, 141, 146
food-borne pathogen 150
genes 119
haemolysin 120, 122, 146
structure 146
haemolysis 154
importance to consumers 124-125
importance to food industry 124
infant formulae contamination 123
infectious dose 142
in liquid egg products 571
milk contamination 123, 124
counts in pasteurized milk 1886
most probable number (MPN)technique
121, 155
motility 122, 154
pathogenicity
to humans 118
to insects 118
phylogenetic relationships 119
protein toxin crystal formation 121, 122
regulations relating to 123
rhizoid growth 122, 154
shellfish contamination 2004-2005
in sous-vide products 1341, 1342
spores 142, 143
germination 119
survival at low remueratures 123
structure, haemolysin 145
toxin crystal production 154
toxin detection 122, 145, 146-148, 147T
diagnostic kits 148
various methods 148
in wtYo 147-148
in vivo 146, 147T
toxins 119-120, 120, 120T, 145T
amount produced 143
bce7 gene 145
diarrhoeal enterotoxin 143
emetic 120-121, 146
enterotoxin T 1 4 5
I x INDEX
Bacillus cereus (continued)
toxins (continued)
factors affecting 146
genes 119
mechanism of action 143
non-haemolytic enterotoxin complex
145
production 122-123
structure 145-146
various 146
virulence, assessment 122
virulence factors 119, 119-120
Bacillus cloacae see Enterobacter cloacae
Bacillus cocovenenans see Burkholderia
cocovenennns
Bacillus Genetic Stock Center 135
Bacillus licheniformis
amylase gene in Zymomonas mobilis
2372
detection 154
food-borne illness 142, 142-143, 144T
growth in bakery products 150
Bacillus megaterium, spores, germination
172
Bacillus mesentericus, propionic acid action
1781,1782
Bacillus mycoides 121
Bacillus popilliae, pathogenicity 118
Bacillus pumilus, food-borne illness 142,
143, 144T
Bacillus sporothermodurans
heat resistance data 1027T
UHT processes and milk contamination
1027
Bacillus stearothermophilus 124-129
aerobic thermophilic spore-former 126
characteristics of species 124-126, 127,
124T
detection methods 126-128
specific tests 127-128
distribution and sources 125
enzymes 126, 127T
in foods 125
food spoilage
fish 811
flat-sour spoilage 1010
growth requirements 124
heat resistance 126
importance to consumer 128
importance to food industrl- 128, 150
regulations relating to 128
spores
in canned foods in tropical areas 128
in canneries 128
germination 125-126
heat-resistance 125, 124T
heat-shocked 125-126
immobilized, uses 126
inactivation 125
as indicator of sterilization 126
‘process-resistant’ 126
prolonged heating effect 128
regulations 128
risk factors associated 128
sodium chloride effect 1726
strains 126
transmission electron microscopy 1417F
Bacillus subtilis 113, 135-141
ABC-transporters 138
bread spoilage 2050
capsule 135
characteristics of species 135-136
chromosome 135
detection 136-137, 154
DNA uptake, natural competence 138,
139
exoproteins 138
food-borne illness 136, 142, 142, 144T
foreign proteins in 139
genes, categories 114
genome 113, 1157, 135, 135
growth 136-137
Bacillus subtilis (continued)
growth conditions 136
supplements 136
growth in bakery products 150
as host for genes 116
importance to food industry 138-139
logarithmic growth, end (To)138, 138F
Marburg strain (168) 135, 136
as model organism 114
non-pathogenicity 135, 136
plasmids 140, 140
copy number 140
instability 140
proteins secreted 138-139
secretion process 139
proteome 114, 136
recombinant strains 136
regulatory aspects 117
safety aspects 136
selective markers 137, 137T
spores
culture recovery from 138
heat-resistance 137
long-term storage 137-138, 137F
sporulation 135, 137, 137F
stages 169F
strain construction 139-140
homologous recombination 140, 140F
transformation 139-140
subtilisin 135
toxin, heat-stable 136
tryptophan auxotrophy 136
Bacillus subtilis var. niger, detection,
adenylate kinase assay 22, 22F
Bacillus thuringiensis 118
Bacillus cereus relationship 119, 143
Bt toxin 118
characteristics 121
concerns over virulence 119
food-borne illness 142, 143
characteristics 144T
pathogenicity and insect control 118
toxin genes from Bacillus cereus 119
bacon
laser radiation 1181-1182, 1182F
storage, Arthrobacter role 59
vacuum packaged 1264
bacteraemia, Proteus 1859-1860
bacteria 158-183, 580
165 rDNX 178-179
acid resistance 1735
adaptation
to acids 1736
to redox potential and p H changes
559-561.559F
adenvlate kinase molecules 1 7
adheiion see bacterial adhesion
age of culture, effect of freezing 848
antigens 1 7 7
in apple juice 422T
atomic force microscopy 1422-1423
autolytic 1474
bioluminescence, rapid detection in food
1893
biomass estimation, instruments 220-221
as biosensors 270-271, 271T
capsule 164
characteristics 792-793
formation and functions 164
Nordic fermented milks 792, 792F
staining 1388
see also individual bacterial species
carotenes production 730
cell concentration measurement 686
cell division, structural changes 166
cell membrane 161-162, 161F
acid diffusion 1733-1734
functions 161-162
permeability increase by essential
oilsiphenolics 1718-1719
protein binding 1719
zones 162, 161F
bacteria (continued)
cell membrane (continued)
see also cell membrane
cell organization 160-166
cell sorting using flow cytometry 828-829
cellular contents and inclusions 164-166
cellular differentiation 1 6 7
cell wall 162
acid-fast 163, 176
Bacillus 114-115
composition and taxonomy 176-177
disruption by sorbate 1773
freezing effect 843
Gram-negative bacteria 162-163,
163F, 176, 176F
Gram-positive bacteria 162, 163F, 176,
177, 176F
ion-exchange system 162
phenolics and essential oils effects
1718-1719
Staphylococcus 2063-2064
strength 162
see also specific bacterial geneva
chemotaxonomy 176-177
chromosome 166
see also specific genera
classification, phylogenetic 178-183
1 6 s rDNA 178-179
advantageiobjective 1 8 0
application of results 179-183
laboratory procedures 179
limitations 182-183
link with traditional classification 180-
182
classification, traditional 173-178
groups based on energy sources 173
groups based on oxygen need 173,
174T
groups based on temperature 173-174
nucleic acids 175
objectives 175
phylogenetic method links 180-182
clumping 934
colony formation and characteristics 166-
167
surface topology 167, 165F
composition 159T, 160T
conductance changes 575
conjugation 934-935, 934F
crystalline surface layers 163-164
cultivation conditions, optimization by
electrical techniques 582
cultures, turbidity measurement 685
cytosol 164-165
damage due to freezing see freezing
death
growth phase-dependence 1735,
1735F
kinetics 1735, 1735F
timescale of preservative action 1713
death curves, non-thermal 1704, 1704F
death rate
heat killed bacteria 1012
modelling 1704
definition 173
destruction
kinetics 1464
manothermosonication 1463-1464,
1465
see also microbial inactivation
detection time ( D T ) 576-577, 585
diversity 158
doubling time 2035T
D values 1340, 1341
ecology in food see ecology
effect of freezing on 842-843
effect of rehydration 535-536
encapsulated, Nordic fermented milks
792
endospores see endospores
envelope 160
methanogens 1336

bacteria (continued)
envelope (continued)
S-la) er 1336
structure 161-164
F’ and F strains 934
fat and lipid composition 718,720T
in fermented foods 249T
fish 807
flagella see flagella
flavours produced by 7331
food poisoning due to 835
see also food poisoning
food spoilage by see spoilage of food
G+C values see DNA, G+C content
generation time, calibration of
impedimetric technique 587
genetic engineering see genetic
engineering
genetics 929-940
gene transfer 934-938
genus 175
glycocalyx, fermented milk products 792
growth 205
after rehydration 536
environment-dependence 1709-1710
factors influencing 542-543
freezing effect 846-847
as function of environment and models
1708-1710
lag phase 543-544,550,665
limits 550-551
low p H foods 561-562
at low temperatures 845,845T
minimumimaximum p H 5581,1729F
minimum temperatures 846T
minimum water activity 841T
modified atmosphere packaging effect
414-415
normal profiles 665F
optimization 548
phases and effect of freezing 842
reaction rates and 551-552
redox dependence 557F
requirements 542-545
sous-vide products 1341-1342
submerged fermentations 665-666,
665F
suppression by salt 1724-1725
temperature control 548
temperature effect 575,845
temperature interaction with other
factors 550
temperatures for 840
tolerance of low water activity 542,
543T
water activity levels 542,542T
growth curve 548,548F,665F,1709F
maximum carrying capacity 548,548F
growth limit models 1706,1706F
growth rate 1723
absolute and specific 1709F
Arrhenius plot 552,552F
carbon dioxide effect 559
comparison of food-borne bacteria
55OF
effect of freezing 848
effect of water activity 543,543F
environmental factors effect 543,544F
fastest-growing organisms 549-550
in food 548
interactions of factors 551,551F
modelling 1704,1709F
models 1707T
optimum temperature 549
solute tolerances 174F
temperature effect 549-550,552-553,
549F
see also temperature
harvesting, centrifugation application
1685-1686
heat resistance 1340,1340,1341
higher taxa 175
bacteria (continued)
high-frequency recombination (hfr)
strains 934
identification 174,175
electrical techniques 582
inactivation see bacteria, destruction;
microbial inactivation
inhibition of undesirable microbes
by fermentation 1726
by salt 1725,1726-1727
in intestine see gastrointestinal flora
intracellular structures, water activity
effect 545-546
lipids 1299
lysis
by bacteriophage 203-204
PCR sample preparation 1479
by phage lysins 1473
marine, tetrodotoxin production 1674
metabolism, temperature effect 553-555
mineral uptake 1313-1314
morphology 159-160,l59F
environmental influences 160
variations and flow cytometry 828
nomenclature 174,174
nucleoid 166
organelles 165
origin of term 173
outer membrane in Gram-negative cells
163
pathogen detection by phage-based
techniques see bacteriophage-based
techniques
periplasm 163
phage adherence 1471
phage as viability indicator 205
phage interactions see bacteriophage
phage-resistant mutants 1472
phage typing see phage typing
oili 164.934
polysomes 165
preservatives active against 1712T
protective cultures, meat preservation
1271
replication 205,933-934
replication rate, phage rate comparison
208,208F
reproduction, binary fission 933-934
riboflavin production 730
secondary metabolites 1328-1334
see also secondary metabolites
selective adsorption 1696-1699
single-cell protein see single-cell protein
S-layer 163-164
atomic force microscopy 1423
slime 792
composition 793T
determinants affecting production 793
production process 793-794
production rate 794
sourdough bread 300
spore-forming 168
see also endospores; spore-formers
spores see endospores, bacterial
sporulation 543-544
starter cultures see starter cultures
storage granules 166
structure 158,1591
sublethal injury due to freezing 844
recovery 844
surface-ripening, as starter culture 20851
survival at low temperatures 847-848
see also psychrophiles
taxonomy 174
chemical 176-177
classical and numerical 175
genetic methods 175
major taxa 177-178
serology 176
toxin production 543-544
INDEX I xi
bacteria (continued)
transduction see transduction
transformation 935-936,935F
competence 935
‘type strains’ 174
viability
freezing effect 847,847F
freezing rates and 841
staining for 830-832
viable cell counts
alternative methods 219-220
method 219
virulence factors 1472
viruses see bacteriophage
water activity
inhibitory levels 1724T
requirements 542,542T,1723
tolerance of low levels 542,543T
yield, water activity effect 544
bacterial adhesion
conditioning layer of organic materials
1693
control by polymer technologies 1692-
1699
free energy change 1693
importance 1692
inhibition 1692-1696
hydrophilic surface polymers 1693-
1695
low surface energy polymers 1695
mobile surface polymers 1695-1696
polymers for retardation 1692-1693
polymer structures 1694T
thermodynamic treatments 1693
selective adsorption of bacteria 1696-
1699
see also adherence; biofilms; polymers
bacteria-specific adsorbents 1696-1698
bactericidal barriers, bacteriocins as 190
bactericides, ultrasound 1463-1464
bacteriocins 183-191,1711
advantages/disadvantages 188-189
applications 185
as bactericidal barriers 190
Bacteroides 202
Brevibacterium /mens 309
cost-effectiveness issues 190
definition and description 184-186
detection 184,184F
effect in fermented meat products 746
effect on Bifidobacterium 1356
enterococci producing 623-624
Enterococcus faecalis 1367
Enterococcus faecrum 1367
fast- and slow-acting 190
future prospects 190
genetics 189
GRAS status 184,190
harvesting 189-190
hurdle technology 1074
hydrophobicity 189
lactic acid bacteria 2103-2104
lactobacilli 1136,1136T
Lactobacillus acidophilus 1153-1154,
1153T
Lactobacillus brevis 1149,1 l5OT
Lactococcus lactis 1169
Leuconostoc 1193-1194
as markers for food-grade cloning vectors
919
meat preservation 1271
Moraxella 1491
mutants resistant to 189
natural antimicrobials 1573-1574
as natural food preservatives 185
as natural products 188
origin of term 184
pediocin-like 187-188
potential uses 188
production 189-190
Proteus 1863
safety aspects 189
I xii INDEX

bacteriocins (continued) bacteriophage (continued) Bacteroides thetaiotaomicron

in starter cultures 2103-2104 in starter cultures (cotztiuued) characteristics 199T
strains producing 184 test kits 2093 polysaccharide fermentation 201, 201T
‘super producers’ 1573-1574 yoghurt culture contamination 788 Bacteroides uniformis 199T
thermostability 188-189 Streptococctts thermophilus 21 32 Bacteroides vtilgatus 199T
toxicity trials 189 Streptomyces 2138 BactimediaO 2094
types and classes 185, 1 8 5 1 structure 203, 1470 BactoFoss system 83, 83F
see also nisin: other specific bacteriocms survival wirhout hosts 2092 procedure 83F
Bacteriological Analytical Manual (BAM) taxonomy 1470, 1471F raw milk assessment 90
Salmonella detection 1967 temperate 1471 bactofugation 1685
Salmonella enteritidis detection 1939 toxin synthesis 1333 Bacillus cereus spore removal 123
bacterioohaee 203., 936.2264. 1469-1471
I
transduction by 936-937 Clostridium tyrobutyricum spores 457
in adenylate kinase assay for bacteria 22- typing see phage typing Bacrofuge 1682, 1683, 1684F
23,22F, 23F use to control pathogens in foods 1472- Bactometer 220, 574T, 584T
adsorption 2091.2105. 1471. 1473 1473 bactoprenol 1303
ampli’fication technique see phage psychrotrophic pathogens 1473 Bacto Rogosa SL Broth 1156, 1156T
amplification yoghurt starter culture contamination 788 BactoScan 529
appearance 1470F bacteriophage A511 206 Bactotherm 1683
applicability for bacterial pathogen bacteriophage-based techniques 203-210 BACTRXC 5741, 584T
detection 204-205 advantagesidisadvantages 204T bagoong756-757
Arthrobacter 58 bacterial pathogen detection 205 Baird-Parker (BP) agar 2067, 2073-2074
bacterial interactions in food 1469-1470, ltrx-bacteriophage see lux-bacteriophage bakery products
1472 lysin-release ATP bioluminescence 208- Bacillus growth 150
concentrations for contact 1472 209,209F, 209F modified-atmosphere packaging 4 1 1
bacrerial resistance see bacteriophage, Listeria detection 210 effect on spoilage 414
resistance phage amplification 207-208 nisin addition 195
bacterial taxonomy 175 see also phage amplification propionic acid addition 1781
as bacterial viability indicator 205 bacteriophage fFSW 1160 shelf-life 195-196
Bacterordes 202 bacteriophage J1 1160 sorbate addition 1772
Bacteroides fragilis 2278 bacteriophage 1 9 3 7 s p oi I a g e
burst size 2091-2092, 2105 bacteriophage P22 1853F bacterial 2055
characteristics 203-204, 1470, 1471F transduction 936-937 fungal 2060
concentrations in foods 1472 bacteriophage PL1 1160 staphylococcal food poisoning 2078
defective 936 bacteriophage T4 1472 see-alio bread
discovery 1469 transmission electron microscopy 1417F baking
as disinfectants 1473 ‘bacteriophage test,’ Salmonella 526 bread 289
distributionisources 1469 bacteriostatic compounds, meat confectionery products 474
DNA packaging 936 preservation 1271 Saccharomyces cerevisiae role 1920-1 922
faecal contamination indicator 2284 Bacterium monocytogenes see Listeria yeast see Saccharomyces cerevisiae
fermented milks from Northern Europe see also bread-making
nzonocytogenes
794 Bacteroides 198-203 baking industry, nisin application 187
gene product detection 205 anaerobe 1 9 8 , 2 0 0 , 2 0 0 balao-balao 757
generalized transduction by 936-937, antibiotic resistance 198 Balkan endemic nephropathy 1519, 1541-
936F 1542, 1654
bacteriocins and bacteriophage 202
harmful effects 1470 ballistoconidia 855, 899
bile salt metabolism 202
helper 937 Bantu beer 1921
capsules 200 barrier technology see hurdle technology
host specificity 1471 classification and characteristics 198, 200 bases, nucleic acid 930, 930F
induction of temperate phage 1471 cultivation 200 Basidiobolus ranarum 883
infecting food pathogens 1470 media 200,200T Basidiomycetes 868
infection effect on foods in gastroinrestinal tract edible species 868T
butanol-acetone fermentation 447 200-202 Basidiomycota 868
detection by electrical techniques 582 glycosidases 201 basil, effects 1721
infection cycle 203-204 polysaccharide breakdown 200-201 Basipetospora 863, 895
infection process 1471 protein metabolism 201 characteristics 8 8 9 7
lactic acid bacteria 2105-2107 xenobiotic and carcinogen metabolism conidial structures 891F
Lactobacillus bulgaricus 1141 201-202 batch processes
Lactobacillus casei 1159-1160, 1161T Flavobacterium relationship 821 fermentation see fermentation
Lactococcus lactis 1169-1170, 2106F glycosidases 201 microbial ecology of foods and 548, 548
lux see lux-bacteriophage importance in agriculture and food pasteurization 1033-1034, 1033F
lysins see lysins production 202 BAX screening system 226
lysogenic cycle 1471 lipopolysaccharide 198, 200 Bayes theorem 2167
lysogenic phage 1170, 2106-2107 nitroreductases 202 BB factors 1356-1358
lytic cycle 2092, 2105-2107, 1471 pathogenicity 202 BBL agar 1156, 1156T
lytic phage 1169-1170 proteinases 201 BBL Campyslide 348, 348T, 350T
Propionibacterium 1853 species 198 detection limits and sensitivity 351T,
receptors 1471 characteristics 199T 351T
recombinant 205, 205F Bacteroides distasonis 199T protocols 348-349
replication 204F, 2091-2092, 1470-1471 Bacteroides eggertii 199T BBL Crystal system 240
rate, bacterial rate us 208, 208F Bacteroides fragilis biochemical reactions used in 248T
rates 205 catalase and superoxide dismutase comparison with other systems 248-249,
requirements 205 production 200 248T
in water 1472 characteristics 199T principle 245
resistance 1472 enterotoxin 202 protocol 247
genetic engineering 2099 faecal contamination indicator 2284 BBL Enterotube system
lactic acid bacteria 920 pathogenicity 202 biochemical reactions used in 248T
Lactococcus lacris 1169-1170 phage 2278 comparison with other systems 248-249,
starter cultures 939, 2106-2107 protein metabolism 201 248T
in starter cultures 2090, 2091-2092 Bacteroides uodosus 1418F principle 245
contraliprevention 2094, 2107 Bacteroides ovatus protocol 246-247
management 2092T characteristics 199T BBMB-lactate medium 455T
testing 2094-2095 polysaccharide fermentation 201 B cells, mitogen, from fermented milks 797

BC motilitj medium. composition I 5 8
BDE VIA kit 148
beach peas, Pantoen infection 1629
bead mill, high-speed 698, 699F
bean curd 2098
beef
Arcobacter importance 52-53
microbiological analysis using BactoFoss
83
see also meat
beer
accelerated forcing tests 107-108
acidification, Acetobacter role 7
ATP bioluminescence. sample preparation
104
Bantu 1921
brewing see brewing
fermentation 1926-1927
Saccharomyces rereuisiae role 1921
yeasts see Saccharomyces cerevisiae;
yeastis), brewer's
see aiso brewing
filtration 1679
lager see lager
Iambic, Brettanomyces role in 305
from millet 766
nisin application 187, 197
nutrient composition 766T
production 1921
brewing yeast energy levels 103
Lvort, constituents 677T
see also brewing; wort
sorghum see sorghum beer
spoilage
Lactobncillits brevis 1149, 1149T
Fedtococcus 1646
Torulopsis 2 1 48
Zymomonas 2370
starter cultures 2089
sterility analysis 106F, 106F
beer-glucose medium 2369T
bees, Gluconobacter in 957
beet molasses, ethanol fermentation 2371
benches, for laboratories 1124
bench tops, in laboratories 1124-1125
benomyl-containing medium 1 4 9 7 1
benzaldehyde 732-733
oroduction 732-733
be&o(a)pyrene 1742
benzoic acid 961, 1754-1757
antimicrobial action 1756-1757
mechanisms 1756-1757
p H effect 1756, 1756
speciesistrain tolerance 1756-1757
appiicationsiuses 562
assimilation and transformation by
Rhodotoritla 1904
behariour in foods 1755-1756
chemical properties 1755-1756, 1756
enzymes inhibited by 1756
GRAS status 1755
ionized and non-ionized 1756
maximum levels used 1756
as preservative 1754-1757
foods preserved 1755-1756, 1755T
important criteria 1755T
interaction with other preservatives
1757
limitations 1754
mechanism of action 1713
spectrum of activity 1712T
temperature effect 1757
regulations affecting 1755
spectrum of activity 171 1
toxicology and regulatory aspects 1716
uptake by microbes 1756
benzylpenicillin 1321, 1321T
Bergeyella 821
Berlin process 298
Best Manufacturing Practices (BMP) 972
beta-lactam antibiotics 1320F
betalains 732
betuloside 735
beverages
alcoholic see alcoholic be\-erages
Bifidobacteriitm in 216
Brettariomyces contaminationieffects 305
clarification 1679
contamination prevention. ATP
bioluminescence role 104-108
Debaryomyces significance 5 1 8 1
fermented 2098
Brettanomyces contaminationieffects
305
Lactobacillus casei role 116 I
nutritional aspects 765-766
Saccharomyces cerevisiae role 1920-
1922
from sorghum and millet 759-767
see also alcoholic beverages; beer;
sorghum beer; wine
microbial stability, assessment 101
microbiology. ATP bioluminescence role
101-109
see also ATP bioluminescence
natamvcin application 1779-1780
smoke-processed 1738
from sorghum and millet 759-767
spoilage
Saccharomyces ceret'iszae 1922-1 923
sorghum beer 764, 765T
see also beer; wine
sterility testing 106, 106F, 106F
thermoduric spoilage organisms 101
see also beer; soft drinks; wine
BIXcore 275
instrument 277
bias, models 1705
Bzfidobacteritim 2 10-2 17, 1 35 5-1 360
acid production 1356
actions 215
anaerobic growth conditions 216
antibiotics for selection 216-217
bacteriocins effect on 1356
breast-feeding and intestinal levels 214
cell morphology 1357F
characteristics of species 780T
for fermented milks 1377T
colonization of intestine 214, 1356
factors affecting 1356
culture stock/isolates characterization
1359-1360
development after birth 1358
discovery 211, 1355
enumeration 216-217
enzymatic characteristics 1375T
fermented milks using 7 7 7 2 780T, 1374T
in foodsibeverages 216
in gastrointestinal tract 1367
colon 212
counrs and supplementation 1358-
1359
depletion and effects of 1356
ecology 212, 214
role 1355-1356
genus description 2 l l T
growth media for 216
growth promoting factors 1356, 1358,
1358T
health benefits (implied) 215-216, 215T,
1374
health-promoting activities 1358-1360
historical perspective 211-212
inhibitory effects on pathogenic bacteria
216
isolation methods 216-217
lactobacilli similarity 212
metabolism 1356
as probiotic 1138, 1374,2085
effects 212
products containing 1359
rapid identification 217
INDEX I xiii
Bifidobactertum (coiztintted)
species 211, 212, 1355-1356, 1357F
descriptions 213T
survival after consumption 1359
taxonomy 211-212
actinomycetes differences 21 1
vitamin production 1358
Btfidobactertzrm acidophilus, ice-cream
making 1359
Bifidobactetkm adolescentis 213T, 780T,
1356, 1358
Bifidobacterium urzgulatum 213T
Bifidobactertum antmalzs 213T. 1357F
Bi$dobacterium asteroides 213T
Bifidobactertum hifidum 211. 213T. 1356
'characteristics hOT, 1377T
in fermented milks 1359, 1359, 1360F
characteristics 1 3 7 7 1
preparation 779
ice-cream making 1359
Bifidobacterium bourn 21 3T
Bifidobacterizim breve 213T, 780T
Bzfidobacterium cateiiulatirm 213T
Bifidobacterium choerimtm 213T
Bifidobacterium coryrzeforme 213T
Bzfidobacteriutn cuniculi 2 1 3 1
Bifidobacterium dentiuni 213T
pathogenicity 212
Bifidobacteriirm globosum 213T
Bifidobacterium iiidicum 213T
Bifidobacterium infantis 213T, 1357F
characteristics 780T, 1377T
in fermented milks 1359
Bifidobacterium lactis 1356
Bzfidobactertum lactzs BB 12 1786
Bifidobacterium longunt 213T, 1358. 1357F
characteristics 780T, 1377T
in fermented milks 1359
characteristics 1 377T
probiotic product 1786
supplementation with 1359-1360
Bifidobacterittm magnum 213T
Bifidobacterium minimum 213T
Bifidobacterium pseudocatenulatum 213T
Bifidobacterizim pseudolongum 213T,
1357F
Bifidobacterium puliortrm 214T
Bifidobacterium subtzle 214T
Bifidobacterium suis 214T
Bifidobacterium thermophilum 214T
bifidogenic factors 1356-1358, 1374
bifidus factor 1374
Bifidus milk 781
bifidus yoghurr 781
Bifighurt 780
bile, conjugated xenobiotic secretion via 202
bile acid conjugates, metabolism by
Lactobaczllus acidophilus 1362
bile-aesculin test, Vagoroccus identification
221 8
bile salts, metabolism, Bacterozdes action
202
bile salts-brilliant green agar (BBG) 31-32
bile salts-brilliant green-starch agar 32
binary fission 933-934
bacteria 166
binomial names 174
biocatalysts
Ptchin pastoris 1691
Schizosaccharomyces pombe 1988
Zymomonas mobzlis 2372
biochemical identification techniques 218-
228,228
alternative methods for viable cell counts
219-220
areas of recent developments 218
Enterobacteriaceae, coliforms and E . coli
244-249
comparisons 248-249
principles and types of tests 245
protocols 245-247
wet and dry systems 244
I xiv INDEX
biochemical identification techniques
(contrnued)
food-poisoning organisms 237-244
agar-based kits 238-239
application range 241-244
dehydrated media kits 239-241
diagnostics kits 238-241
miniaturization 238
food spoilage yeastsimoulds 228-237
advantagesilimitarions 235-236
molecular methods vs 234-235
see also fungi, identification
instruments for microbial biomass 220-
22 1
microflora of fermented foods 249-252
miniaturized techniques 221-224
see also miniaturized microbiological
techniques
prediction of developments 227T
refinements of novel methods 224-227
genetic methods 226-227
immunology 224-226
relative interest in 218F
sample preparation improvements 218-
219
usual biochemical tests 221
see also indrvidual
microorgarzisms/techtzrques
biochips, D S A arrays 1603
biocides 1794-1801, 1826
chemical classification 1794-1795
effects on microorganisms 1800-1801
effluent and waste stream issues 1800
food rinses 1828
hand-sensitizers 1826
ideal characteristics 1 7 9 5 7
microbial resistance 1800
physicalichemical properties 1795-1 800
testing systems 1823-1824, 1824F
types used 1 7 9 5 1
see also disinfectants
biocontrol see biological control
biodegradation, Flavobacterium
applications 825
biodispersans 16
bioenergy 2187
biofilm bacteria 255T
adaptation to environmental stress 255-
256
adhesion 253-254,255
characteristics 253
resistance to antimicrobials, deep-lying
cells 257
surface properties 254
viable but non-culturable (VBNC) 256,
256F
biofilms 252-259,253F, 1692, 1828
architecrure 253, 254
coliform 608
composition on floors 255,255T
definition 252-253
dense confluenr 253
disinfectant resting and 1828
elimination by cleaningidisinfecrion 258-
259
formation 253,253-255, 1791-1792
adhesion 253-254,255
chlorine effectiveness 1798
colonization 254-255
conditioning 254
forces involved 254
Pseudomonas aeruginosa 1870
Shewanella putrefaciens 2013-2014
surfaces 1870
time required 254-255
Klebsiella production 1114
locationsisurfaces with 253
preventionireducrion 258, 1828
design and surface modificarion 258
dryness 258
surface texture 258
see also bacterial adhesion, inhibirion
biofilms (contrnnedi bioparticles (contrnued)
properties 255-257 motion in travelling wave electric fields
active but non-culturable cells 256, 266-267
256F orienration (induced motion) 260
adaptation to environmental stresses oscillations 261
255-256 polarizability variations 263, 263F
adhesion strength 256 polarization in AC electric field 261, 261F
linked to exrracellular oolvmeric
I .
rotation 265
subsrances 255 separation by dielectrophoresis 262-263
resistance to anrimicrobials 256-257 surface charge 260
resistance to cleaning agents 256 torque generation 265, 265F
Pseudomonas aerziginosa 1870 biopesticides
removal 1692, 1870 Bacillus thuringiensts 119
testing by electrical techniques 582 see also biological control
substratum effect on biocide efficacy 257 biopharmaceutical industry, clean-in-place
BIOgardeB 780,2085 in 1815
biogenic amines 748 biophysical techniques 259-267
enterococci forming 1369-1370 basic concepts 260-261
Lactobacillus brevis forming 1150 see also dielectrophoresis (DEPj;
Leucouostoc forming 1193 electrorotation ( R O T )
in malolactic fermentation 2312 biopolymers 1693
production fractionation 264
meat spoilage 1266-1267 biopreservative, Pedrococcns role 1646
organiims 1366-1267 BioProbe 9 7
stored mear 1259 beer sterility analysis 106F
wine 2312 bioprocess 683
see also amines bioprotectire species, in meats 1271
Bioghurt 780 bioreactors
biohazard safety cabinets, in laboratories Alcaligenes in 4 0
1125 continuous high-cell-density, filtration use
Biokys 78 1 1680
biological conrrol membrane 1680, 1680F
Bacillus role 117-1 18 methanogenic 1336-1337, 1337F
Bacillus thuriizgiensrs 119 bioremediaGon
Trichoderma for fungal plant pathogens Acinetobacter 1 6
2 188-2 189 Alcaligenes 39-40
‘biological ennoblement’ 737 biosensors 268-278, 1894
biological enrichment of nutritional value acceptance by regulatory agencies and
250 users 278
advantages 278
biological remediation see bioremediation
biological value (BVj, single-cell protein Alcaligenes, for heavy metals 40
alcohol measurement 686
2040
Alteromonas ptitrefacreus 270
Biolog system 223, 232, 240-241
advantages 240 amperometric on-line 277
applications 269T
fungi 231-232
Arthrobacter nicotianae 270
microflora in fermented foods 252 bacreria 270-271, 2 7 1 7
bioluminescence
citations 268
adenylate kinase assay see adenylate Clostridrum acidrririci 270
kinase (XKj
Clostrrdrunz botnlinnm 275
ATP see ATP bioluminescence cyanide 270
bacterial 1893 definitions 268-276
comparison with PCR 1631T development and sales projections 268
definition 8 0 , 9 4 enzymes as 269-270,272, 686
Lactobacilltrs brevis detection 1146-1 1 4 7 fish freshness 270, 270
Leuconostoc 1191, 1 1 9 1 1 flow injection analysis and on-line systems
shellfish 2007 276-278,273F
biomass flow-through and on-line
algae 2024, 2026 future prospects 278
ATP as cell marker for 1 6 microprocessor-conrrolled 277
control in industrial fermenrations 685- future prospects 278
686 Gluconobacter role 957
estimarion, instruments 220-221 glucose oxidase 270
fungi 2036T see also glucose
measurement 80, 685-686 Hansenula unomala 270
calorimetry 685 hydrogen peroxide detection 277
instruments 220-221 industrial fermentation 686
production, Yurrowia lrpolytica 364 integrated multi-biosensors 686
single-cell prorein 2030 limitationsiproblems 278
veact
, ~~~
291 method of operation 686F
bioparticles 259 optical detection of D S X 274
application of AC field 261, 261F optical flow cells in 277
application of D C field 260-261, 260F peptides as 269
distribution of charge 260F Pseudomonas 270
innare electrical properries 260, 260F regeneration of activity 278
investigated by non-uniform XC electric Rhodococcus 270
fields 262T sensors 269T
levitation by dielectrophoresis 264 affinity (IXsys) 274
morion in inhomogeneous AC electric enzyme 272
field see dielecrrophoresis (DEP) with potentiometric transducers 272
motion in rorating electric fields see shelf life 278
electrorotarion (ROT) stability problem 278
 INDEX I xv

biosensors (continued) bone sour 2052-2053 botulism (continued)

Synechococcus 270
thermistor-based 276
tissue 270
transducers 268, 271-276, 272T
acoustic wave 274
amperometric 272-273
automated optical 274
conductance and capacitative 273-274
electrochemical 271-274, 272T
evanescent wave 275-276
with fibre optics 274-275
hybrid 274-275
optical 274-275, 272T
piezoelectric 274
potentiometric 271-272
sensitivity 268
surface plasmon resonance 275-276
thermal 276, 277
without fibre optics 274
typical sensors 268-271
antigens and other compounds 269
enzymes 269-270
immunoglobulin 268-269
microbial cells 270-271, 271T
whole cells 270-271, 271T
urea measurement 686
biospecific interactions analysis 275
biosynthesis, definition 1279
biotechnology
Acinetobacter applications 15-16
Avthrobacter applications 60-61
European Union safety standards 1837-
1840
fungi use 2036
Thevmus aquaticus value 2140-2141
wine-making 2310, 2309T
wine yeasts 2308T
see also genetic engineering; recombinant
DKA techniques
BioteCon Diaenostis FoodProof Kit svstems
1633-1g35, 1636T, 1638T ‘ metabolism 23 17-231 8
biotin 629
biosvnthesis and uptake 1315-1316
enzymes 1315
function 1315
operon regulation 1315-1316, 1316F
in PCR amplification product 1480,
1480F
requirements 1308
biotin protein ligase 1316
Biotrace Dairy Kit assay 92, 92T
biotransformation, by Acinetobacter 1 6
biotyping
Campylobactev 339
Clostridium perfririgens 4 3 9 1
Pseudomonas aeruginosa 1869
Staphylococcus aureus 2067
Vibrio cholerae 2245
bio-yoghurt 785
birds, control in manufacturing facilities 968
2’,7’-bis-(2-carboxyethyl)5[6)-
carboxyfluorescein acetoxymethyl-
ester (BCECF-AM) 831
bismuth sulphite agar (BSX) 1947
bisulphites 422F, 1750, 1753
‘bitty (broken) cream’ 123, 1444, 150
Biverticillium 856, 1649
importance in food 1652-1653
blanching of foods 846
blastospores 855, 900
bleach, effect on ATP bioluminescence 98
bleomycin, media containing, Aspergillus
parasiticus detection 75
blood agar base medium 6 4 2 7
blood films, Bacillus anthvacis detection 131
blood pressure
control, role of fermented milks 784
reduction, Lactobacillus casei role 1163
Boerhaave, Hermann, fermentation process
classification 1068
bone charcoal 134
bone taint 2052-2053 infant 460-461
bongkrek acid 1871, 1872, 1872F outbreaks 4 6 0 , 4 6 1 T
actions 1873 pathogenesis 460
production 1873, 1873T prevention 461
‘bootstrap’ method, phvlogenetic trees 179 see also Clostridium botulinum
Bordetella bovine spongiform encephalopathy (BSE)
Alcaligenes relationshipidifferentiation 283-288
41,411 in Britain 283-284
rapid diagnostic tests 4 1 in cattle, case numbers 285T
Bordet-Gengou (BG) agar 4 1 clinical signs 286
bornanes 734 diagnosis 2 8 6-2 8 7
’bot cook‘ 459 in Europe 285-286,285T
Botrvotmia 865. 895 exotic animal species infected 284T
Botryotrichum, characteristics 8 8 9 1 infectivity of agent 284. 287
Botrytis 279-283, 865, 869, 895 notifiable disease 285
characteristics of genus 279 pathogenesis 285
control 282 pathology 284, 286,283F
cultural characteristics 279 protection of humanianimal health 285
detectionienumeration methods 280 regulations after 285,285
acidified media 280 related diseases 284-285
preferred antibiotic method 280, 280 animals 284-285
staining method 280-281 humans 285
distinguishing characteristics 279-280, see also Creutzfeldt-Jakob disease
2807 (CJD)
fluorescence microscopy 281 brain-heart infusion agar (BHIA) 32
growth characteristics 279 brain-heart infusion broth and agar 6 4 2 1
immunological assays 28 1 branched DNA (bDNA) signal amplification
importance to food industry 281-282 1478, 1477F
species 279 Branhamella 1487
zones of growth 279 pathogenic species 1492
Botrytis aclada 282 taxonomy 1876
Botrytis cinerea 2317-2318 bread 288-301
advantages in food industry 282 ‘black’ 297
citric acid formation 282 frozen 843
detectioniindicators 23 17-231 8 ingredients 288, 289T
effect on winesisherry 282 leavened 288
enumeration methods 281, 282T quality
grape infection 2316-2317, 2317-2318 effect of ingredients 288, 2 8 9 1
characteristics of juice 2 3 1 8 1 role of wheat flour constituents 290T
grey mould (disease)due to 282 rising and yeast causing 2097-2098
rope in 150
by-products 2318 ropiness 293,294, 294T
noble rot due to 282 rye 289
sweet white wine production 2316-2317 sourdough see sourdough
Botrytis fabae 282 spoilage 293-294,294T, 2050
botrytized wines 2318-2319, 2 3 1 9 1 mould 293-294,294T
’botulinum cook’ prevention 293-294
canning process 1017 susceptibility 293-294
UHT 1027 staling 1749
botulinum toxin IBoNT) 429, 458 prevention 1749
antiserum production 465 types 289-290
detection 430-431,461-462, 463-465 from wheat flour 288-301
bioassays 463-464 bread-making 288-290, 290T
biosensors 275 baking 289
ELISA 462,464-465,463T biochemical actions of yeast 293
immunoassays 464-465 comparison of procedures 2 8 9 7
immunoassay sensitivity 465 continuous process 288, 289F, 2 8 9 1
mouse lethality assay 430, 461-462, effect of temperature changes 291F
463-464,462F fermentation 289
nonspecific reactions 465 historical aspects 295
in dried foods 534 Lactobacillus importance 1135-1136
in fermented meat products 746-748 mixing and dough development 289
haemagglutinin activity 430 overmixing 289
mechanism of action 430,430, 461, 463 role of additives 291T
progenitor toxin 430 starch fermentation 293, 294F
properties 461-462 steps 288-289,289F
in sous-vide foods 1343 yeast forms see Saccharomyces cerevisiae;
synthesis inhibition yeast, bakers’
by nitrite 1765 see also baking
by parabens 1760 breast-feedine. intestinal bifidobacterial
use as pharmaceutical 462 levels 274
see also Clostridium botulinum, toxins brem 770
botulism 429, 432, 458,463, 1333 brem cake 770
clinical features 460-461, 460F, 1020- brem wine 770
1021 brem wonogiri 770
diagnosis 461 Brettanomyces 302-308, 867, 869, 895
foods associated 429, 4 6 1 1 adherence to insects 307
fishifish products 809, 812 appearance 302F
sous-vide products 1339 aroma production 303
underprocessed food 1020-1021 assimilation 3 0 3 1
geographic distribution 460 characteristics of genus 302, 303T
I xvi INDEX
Brettanomyces (coiztinued)
cider contamination 425
Custer effect 303
detection methods 306-307, 307F
nested PCR 306-307
selective media 307
fermentation 305, 303T
fluorescent microscopy 303F
genomic analysis 304
genomic properties 304F
importance in fermented beveragesifoods
305
isolation 306
media 302
methods of control 307
mitochondrial genomes 304
nutritional requirements 303
petite mutants 303
physiologicalinutritional properties 302-
304
relationship to Dekkera 302
RFLP analysis of miDNA 304-305
sensitivity to sulphur dioxide 307
species 302
characteristics 303T
Brevibacteriuni 308-3 14
alcohol usage 311, 311T
applications 313
biochemical characteristics 310-312
metabolic end products 311-312
substrate utillization 310-31 1
carbonienergy sources 310T
carotenoid pigments 309, 310, 309F
catabolism of amino acids 308, 311
cellular morpho1og)- 309F
cheese surface growth characteristics
309-3 10
classification 308
control 309
enzymes
for cheese ripening 308
for proteolysis and lipolysis 312-313
genetic engineering 313
growth characteristics 308-309
pure culture characteristics 308-309
species 308
Brevibacterium lactofermentum 3 13
Brevibacteriuin h e n s 308, 310
amino acid utilization 311
antibiotic sensirivity 309
characteristics 309, 395
cheese
Brie preparation 1658-1659
growth on and effects 309-310, 311
isolation from 310
maturation 392
ripening 60, 379,2102
role in 395-396, 396F
colour formation 309
control 309
esters produced 733
th conditions 2102
linecin production 309
metabolism 396
in microcapsules 312
pH range for growth 309
pigment production 309, 310
proteinases 396
proteolytic enzymes 313
vitamin requirements 310
breweries
cylindroconical fermentation vessels
(CCFVs) 1175, 1176, 1175F
fermentation 1175
lager 1174F
brewing
hops and wort boiling 1175
lautering 1174-1175
milling and mashing 1173-1 174
process 1172-1175, 1 1 7 3 1
brewing (continued) Brucella (corztinuedi
sorghum beer detection methods (continued)
industrial methods 761-763, 761F, cultivation 322
761F serological identification 322-323
traditional methods 760-761, 761F discovery 324
see also sorghum beer importance to food industry 320-321
yeast see yeast, breLvers’ disease epidemiology 320
see also beer entry into food and transmission 320-
Brie 387,2103T 321
defects 392 fate during processingistorage 321
history 388 lipopolysaccharide 322
manufacture 379 morphology and physiology 320
bright green yellow fluorescence test (BGYF), 0 chain in LPS 322
for mycotoxins 153 1 pathogenicity and symptomatology 321-
brilliant green bile broth (BGBB), 322
Escherichia coli detection 637 p H range 325
brine 1741 rough strains 322
Debaryomyces etschellsii in 518 smooth strains 322-323
effect on microorganisms 402 species 319-320, 324
hams in 1728 survival and growth in milkimilk products
microflora and spoilage of foods 1264 325
strength 1741 temperature range 325
vegetables 1726-1727 temperature sensitivity 321
brine curing 1264 see also brucellosis
cucumber 741 Brttcella abortus 319, 321, 1441
intermediate moisture foods 1100 behaviour during cheese
brined foods, spoilage bacteria 1741 manufactureistorage 3 2 5 1
British Calibration Service (BCS) 1130 epidemiology 3 2 7
British Standards 1128 in milkimilk producrs 1441
BS57501130 Brucella blood agar 200, 322
Brochothrrx 3 14-3 18, 3 17-3 18 Brucella canis 319, 320
bacteriophage specificity 316 Brucella maris 319-320
characteristics 3 14-3 15 Brucella melitensis 319, 324
distinguishing Characteristics 314T epidemiology 327
food spoilage, oxygen-modified Brucella neotomae 319
atmosphere 317, 317-318 B r z ~ e l l aovis 319, 320
growth, on meats 317 Brucella suis 319, 320, 321
importance to food industry 317-318 brucellosis 319, 326-328
isolation and enumeration 316 in animals 320, 320-321
from foodienvironment samples 316- control 21 1, 326
317 from butter and cream 1454
international guideline 317 clinical features 327-328
rapid detection 317 complications 322, 328
species 314 control and prevention 323-324
comparisons 31 6 epidemiology 320, 326-327
Brochothrix catnpestris 314 outbreaks due to milk products 328T
Brochothrix thermosphacta 314,314,1268- incidence 320-321
1269 incubation period 321-322, 327
characteristics 315 occupations associated 321
cooked cured meats 1268-1269 onset and clinical features 321-322
distribution 314 pathogenesis 321-322
enzymes 315 relapses, chronicity and recurrence 322
as facultative anaerobe 315 transmission 320, 320-321. 327
food spoilage 314-315, 317-318 aerosol inhalation 321
meat see meat spoilage (below) dietary practices associated 321
prevention 317 direct contact 321, 327
glycerol esterase 315 foods 327
in meat 316 treatment 323, 328
meat spoilage 1255, 1256, 1258 vaccines 323-324
fermented products 745 see also Brttcella
meat products 1266 brucellosis-free herds 326
modified atmosphere packaging 1270 brushes, cleaning 1848
substrates and end products 1258, BSE see bovine spongiform encephalopathy
1258T iBSEi
metabolism 315 bubbles
pH range for growth 315 manothermosonication 1464-1465
‘broken cream’ 123, 1444, 150 ultrasonic waves causing 1463
bromothymol blue (BTB) teepol agar 2251 buckets 1848
browning of foods budu 756
enzymatic 1752 buffered peptone water 1199-1200
non-enzymatic 1752 modified 2230
browning reaction, cheese 392 pre-enrichment for Salmonella 1948-
Brucella 319-324, 324-328 1950
characteristics 319, 320T buffers
in culture 322 bread-making 291T
chromosomes 319 for immunomagnetic separation of
classification 319 Salmonella 1 9707
controliprevention 325-326 for pour plate technique 2155, 2155T
on farms 326 buildings
pasteurization 325-326 construction and design 1803
detection methods 322-323 contamination risks 1793
commercial kits 322 design for hygienic operation 1791
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INDEX I xvii

buildings (continued) buttermilk 776, 1451 calcofluor white 831, 1388

exterior 1803 Bulgarian 779 calcofluor white Primulin 1387T
good manufacturing practice and 964 preparation 776 caldo-de-cana-picado, Zymomonas
hygienic processing and 1803-1 804 in Northern Europe 795 fermentation 2370
Bulgarian buttermilk 779 butyl parabens 1759T, 1761T calf rennin (calf chymosin), genetically
Burkholderia cepacia, phenotypicigenotypic butyrate engineered 939
characteristics 1872T formation in intestine 1786 caliciviruses 2271-2272
Bttrkholdevia cocouenenans 1871-8175 metabolism in gut 1354 calories, mycoprotein content 2042T
characteristics 1871 butyric acid calorimetry
control 1874-1875 fermentation 1285, 1287F biomass measurement 685
detectioniisolation 1875 formationiorodnction 1281 non-destructive sterility testing 2198-
phenotypicigenotypic characteristics Breuibacteri~m312 2199
1872T butanol-acetone fermentation 447- camalexin, mode of action 1581
significance in foods 1873-1874 448 Camembert cheese 387, 2103T
taxonomy 1871 Clostridium tyrobtityrictrm 453 defects 392
toxins 1871-1873 in intestine 1786 history 388
actions and symptoms 1873 butyric-butanol fermentation 1286, 1287F manufacture 379
biochemistry 1871-1 873 butyric esterase, Lactobacdltcs casei 1159 volatile compounds 2113T
control 1874-1875, 1874T butyric fermentation 1285, 1287F CAMP test 1196
control in fermented foods 1874 ’butyric late blowing,’ prevention by Listeria identification 1206, 1206F,
detecrion 1872-1873 lysozyme 1585-1586 1206T
effect on onion extract 1874, 1874T byssochlamic acid 329 Listeria inonocytogenes 1236
production 1873, 1873T Byssochlanzys 328-333, 863, 896 Campylobactev 335-352
Burkholderia cocot’enenans biovar ascocarp 328 adhesion method 338
farinofermentans 825, 1871 ascospores Avcobacter diiferences 50, 50
phenotypicigenotypic characteristics acceptable levels 332 bacteriophage 204
1872T heat inactivation 330, 330T characteristics 342, 50
toxins 1872, 1872F heat resistance 329, 332, 330T chemo-organotroph 336
2,3-butanediol, Klebsiella production 1114 inactivation methods 329, 330T detection by latex agglutination 347-352
2,3-butanedione see diacetyl soil as reservoir 333 advantages and limitations 347-348,
but an oI characteristics of genus 328-329, 330T, 351-352
genetically engineered C. acetobcrtylicunz 889T background 347-348
producing 450-451 commercial importance 892 comparison of protocols of tests 350,
mutant C. acetobcitylicuin producing 450, detection methods 329-332 35OT
45lT impedimetry and conductimetry 332, detection limits 351, 3 5 l T
toxicity 448 332F enrichment serology 591
butanol-acetone fermentation plating techniques 329-332, 331F points of application 350
bacteriophage infection of clostridia 447 distribution 328-329 principle and test types 348, 348F
butano1:acetone ratio 448 food spoilage 328-329, 892 protocols 348-350
cell recycling 449 fruits 333 regulations/guide~inesidirectjves 350
Clostridium spp. 446T products at risk 332 test comparisons 348T
continuous 449 importance to food industry 333 detection methods 341-347
development and product recovery 449- mycotcjxins 329, 332-333, 331T comparisons of media 345, 345T
450 patulin production 329 conductimerriciimpedimetric
factors affecting 447 pectolytic e n q m e s 329 technique 524
genetic strain improvement for 450-451 species 328 enrichment media 34, 344T
history 445-446 unacceptable levels 332 enrichment serology 591
industrial process 446-447, 447F Byssochlamys fulva 328 from environmental samples 345
industrial production 449 characteristics 330T FBP media 342
new substrates 449 fruit spoilage 333 inhibitors for plating media 342-343,
physiology 447-449 growth at low oxygen tensions 329 3431
recent progress in research 449-451 heat resistance of spores 329, 330T membrane filtration 343-344
spore formation and 448 Byssochlamys niuea 328 phage amplification 208, 208, 208F,
time course 447, 447F ascospores In milk 333 208F
see also acetone-butanol-ethanol (ABE) characteristics 330T rapid 346
fermentation fruit spoilage 333 selective media 342-343, 3 4 3 1
butter 1445,1450-1453 growth at low oxygen tensions 329 ecology 337
aflatoxins 1454 heat resistance of spores 330T enterotoxins 338-339
brucellosis from 1454 fimbriae 338
defects 1452T flagella 338
enterococci as indicators of poor hygiene
623
C general physiology 336-337
cabbage, acid see sauerkraut growth requirements 336-337
fat content 1450 cacao habitats 3 3 7
food poisoning outbreaks 1453-1454 fruit 467 historical aspects 33.5
herb 1451-1452 fermentation 467-469 identification 205
in ice cream 1083 fermentation procedures 467 infections 340, 341-342, 341-342
lactic 2103T harvesting 467 causative species 341
manufacture 1450-1452, 1450F, 1451T pod 467F enteritis, incubation period 335
NIZO method 1451 see a h cocoa gastroenteritis 34 1-342
microbiological standards 1453, 1453T cadaverine Guillain-Barr6 syndrome 335-336
microflora 1450-1452 Hafnia alvei production 975 reactive arthritides 336
public health concerns 1453-1455 in stored meat 1259 sources 341-342
rancidity 1453 ‘caged Fenton reaction’ 1465 iron requirements 338
salt 1451 cakes 474-479 isolation from faeces 345
soft spreadable 1452 see also bakery products isolation of less common species 345-346
sorbate additioniuse 1772 calcium, Pediococcns requirement 1643 isolation of thermophilic strains from
spoilage 1452-1453, 1452T calcium citrate 712 foods 344-345
starter cultures used 2084-2085 calcium dipicolnate (CaDPA) 168 methods of control 340
storage 1452-1453 calcium gluconate 714 morphology 336
problems 1452 isolation 715 oxygen requirements 336
whey cream 1451 calcium hypochlorite 1797 oxygen sensitivity 336
Butterfield’s phosphate 156 calcium propionate 1781 pathogenicity 338-339