BACILLUS CEREUS 123
B
BACILLUS CEREUS
A Christiansson, Swedish Dairy Association, Lund,
Sweden
Copyright 2002, Elsevier Science Ltd. All Rights Reserved
Introduction
Bacillus cereus is an aerobic sporeforming bacterium,
whose spores are commonly present at low levels in
raw milk. Until the 1960s, the presence of Bac. cereus
in pasteurized milk and cream clearly affected the
product quality, owing to coagulation (sweet curd-
ling) of milk and formation of ¯akes in cream when
added to coffee (`bitty cream'). These defects were
caused by contamination of milk by Bac. cereus, as
the result of poorly cleansed equipment (e.g. milk
cans) at the farm and in dairy factories and a lack of
adequate refrigeration. Nowadays, these problems
are rarely seen in countries where milk is kept at
temperatures below 6  C. However, where pasteur-
ized milk is stored at higher temperatures, Bac. cereus
may still be a limiting factor for keeping quality.
Bacillus cereus can produce several enterotoxins
causing both diarrhoea and vomiting. There are few
dairy-related cases, but milk and cream have been
incriminated in both types of illnesses.
Characteristics
Morphology and Cultivation
Bacillus cereus is a Gram-positive, rod-shaped bac-
terium, which is motile with peritrichous ¯agella.
The cells tend to grow in chains but may occur singly
as well. The length of the bacterium varies between
3 and 5 mm and the diameter is greater than 1 mm.
Spores are oval or cylindrical, their location is central
or paracentral/subterminal, and they do not distend
the cell. A typical trait of the Bac. cereus group is the
presence of storage granules, poly-b-hydroxybutyrate
(PHB), in the cytoplasm, which are easily seen by
phase contrast microscopy. Bacillus thuringiensis,
Bac. mycoides and Bac. anthracis have similar char-
acteristics. Bacillus cereus forms colonies with typical
appearance on agar media, generally with a dull or
frosted, greyish/whitish surface. Bacillus mycoides
forms rhizoid colonies. Widely used selective agar
media for cultivation from food are MYP and
PEMBA. The detection of Bac. cereus is based on the
absence of mannitol fermentation and a positive egg-
yolk reaction (lecithinase). Bacillus cereus can also be
counted on blood agar with polymyxin added,
where the production of colonies with clear zones of
haemolysis and a very sharp margin provides a useful
diagnostic feature.
Physiology
Bacillus cereus is a versatile microorganism with
respect to growth substrates. Most strains produce
proteases that can degrade casein and gelatin as
well as enzymes for starch hydrolysis. Enzymes such
as lecithinase and sphingomyelinase which degrade
phospholipids and lipase which degrades triglycerides
can also be produced. Sweet curdling of milk is due
to a protease and `bitty cream' to phospholipase
activity. Several carbohydrates are utilized by Bac.
cereus, e.g. glucose, fructose, trehalose, N-acetyl-
glucosamine and maltose; other carbohydrates are
utilized only by certain strains, e.g. salicin, cellobiose,
inositol and mannose. The majority of strains do not
grow on lactose. Mannitol is generally not utilized.
Bacillus cereus is in general VP positive (Voges±
Proskauer test) and utilizes citrate, but not urea. Most
strains can reduce nitrate.
The minimum growth temperature differs among
strains and is generally not lower than 5±6  C, al-
though a few strains have been shown to grow at
4  C. An increase in temperature from 6 to 9  C mark-
edly affects the growth rate among psychrotrophic
(psychrotolerant) isolates (Table 1). Some strains
have temperature minima as high as 10±15  C. The
optimum growth temperature is 30±37  C and upper
limit of growth maximum between 37  C and 48  C.
124 BACILLUS CEREUS

Table 1 Growth of Bacillus cereus (log cfu mlÿ1) in pasteurized milk at

various storage temperatures

Days of storage Storage temperature


6 C 7 C 8 C 9 C

1 Ð Ð Ð Ð
2 Ð Ð Ð Ð
3 Ð Ð Ð Ð
4 Ð Ð 0.0 1.0
5 Ð Ð 1.0 2.4
6 Ð 0.5 2.0 3.7
7 0.2 1.3 3.0 5.0
8 0.6 2.0 4.0 nd
9 1.0 2.8 5.0 nd
10 1.4 3.5 nd nd

Average data for pasteurized milk from 10 Swedish dairy plants in August. One milk
package was collected from each plant and the milk from each package was divided
aseptically into four aliquots that were incubated in glass bottles in water baths with
accurate temperature regulation (  0.1  C).
A dash indicates a value of less than log 0.
nd, not determined.
Original data from A. Christiansson.

The minimum pH for growth is 4.3±4.9 and the
upper limit is 9.3. However, in the presence of
organic acids the minimum pH is higher, e.g. pH 5.6
in 0.1 mol lÿ1 lactate. Although Bac. cereus grows
best under aerobic conditions, anaerobic growth by
fermentation of, for example, glucose or by anaerobic
respiration with nitrate is possible. Bacillus cereus is
able to grow in media with up to 7% NaCl if other
conditions are optimum. Minimum water activity for
growth is 0.92±0.95.
Spores
Spores are formed on a variety of growth media under
aerobic conditions on starvation. The presence of
manganese and magnesium ions stimulates sporu-
lation. Sporulation is a fairly lengthy and complicated
process, occurring in the late logarithmic and early
stationary phase of growth. Even under favourable
conditions sporulation may take up to 16±24 h to
complete. Spores are never formed as a result of
chilling if nutrients are available, i.e. refrigeration of
milk does not induce sporulation. For example, high
levels of spores are not found in refrigerated pas-
teurized milk although the Bac. cereus counts may
reach 107 cfu mlÿ1. Milk diluted 1 : 50 with water is
still a good growth medium, but nutrients will be
depleted after growth and spores are formed abun-
dantly, particularly if the milk is present in thin lay-
ers. This is relevant to the cleaning process in a dairy
plant. The spores may germinate and grow out to
vegetative cells again under favourable conditions.
Germination is much faster than sporulation. The
germination rate is highly temperature dependent and
may occur within much less than an hour at favour-
able temperatures. In milk, it is stimulated by high-
temperature short-time (HTST) pasteurization, i.e.
heat treatment. The spores become activated and
substances that stimulate germination may be formed
as a result of the heat treatment. Increased pasteur-
ization temperature in the range of 72±85  C will
lead to activation and germination of more spores.
However, initiation of growth in refrigerated milk
will occur only after a lag phase of several days.
The heat resistance of Bac. cereus spores is com-
paratively low. However, there is considerable vari-
ation in heat resistance among strains. Although
Bac. cereus spores are not inactivated by HTST pas-
teurization, they are easily killed during UHT treat-
ment. Typical D-values at 100  C are in the range of
0.3 to 10 min. For comparison, D100  C for Bac. stearo-
thermophilus has been estimated at approximately
3000 min. Generally, psychrotrophic strains tend
to be less heat resistant than mesophilic, e.g. with
D-values of 2±9 min at 90  C. Vegetative cells are
easily killed by pasteurization.
Milk-Borne Illness
Bacillus cereus is a common contaminant in many
food types, including milk, and is a signi®cant cause
of foodborne illness worldwide. It can cause diar-
rhoea and/or vomiting when food containing large
numbers of the organism is consumed. The symptoms
are generally mild and transient, lasting no more
than 24 h, generally without sequelae. Two types of
outbreak are known.

The Diarrhoeal-Type Outbreak
The illness is characterized by a fairly long incubation
period of between 8 and 22 h. Watery diarrhoea is
extremely common, together with abdominal cramps,
rectal spasms, and moderate nausea. Vomiting is
rare. The duration of illness is generally 12±24 h. The
delayed onset of symptoms indicates that the illness is
most likely due to growth of Bac. cereus in the small
intestine, since the toxin(s) are very susceptible to
inactivation by low pH and degradation by proteases.
Preformed toxin in food will thus be inactivated to a
large extent in the stomach and ileum. Foods asso-
ciated with diarrhoeal outbreaks generally contain
high numbers of Bac. cereus i.e. 105±108 gÿ1 food.
Foods thought to be responsible for diarrhoeal out-
breaks include meat products, soups, vegetables,
puddings and milk products.
The Emetic-Type Outbreak
This type of illness is characterized by a short incuba-
tion period of between 0.5 and 5 h. The rapid onset of
nausea and vomiting is due to a preformed toxin in
the food. Abdominal cramps and diarrhoea occur
occasionally. Recovery is rapid, i.e. within 6±24 h.
The levels of Bac. cereus in suspect food can vary from
between a few thousand to more than 5  1010 gÿ1,
although levels are generally high. Fried or cooked
rice is typically involved in outbreaks, but milk-borne
cases are also known.
Toxins
The nature of the enterotoxins produced by Bac.
cereus has remained elusive for decades. However,
during the last 10 years the knowledge about these
toxins has increased considerably.
At least three types of enterotoxins capable of
causing diarrhoea have been identi®ed. Two of these,
haemolysin BL (HBL) and nonhaemolytic entero-
toxin (NHE), are protein toxins consisting of three
subunits each. All three subunits are needed for
full activity. Both toxins have been isolated from
Bac. cereus strains involved in food poisoning. The
third toxin, enterotoxin T, is a single protein toxin.
Additional toxins have been described but their
involvement in foodborne illness is uncertain. Bacil-
lus thuringiensis and Bac. mycoides are also able to
produce similar enterotoxins. With regard to food
safety, there is therefore no need to differentiate
between these species in the Bac. cereus group as far
as milk products are concerned. The toxins are heat
labile and considered to be inactivated by heating
above 60  C for 5 min.
The emetic toxin is a cyclic peptide, cereulide,
which contains 12 modi®ed amino acids and
BACILLUS CEREUS 125
resembles the ionophore valinomycin. The molec-
ular weight of the toxin is 1.2 kDa; it is quite heat
resistant and cannot be destroyed even by heating
at 121  C for 1 h. The emetic toxin is a more serious
health hazard than the diarrhoeal toxins and has
been the cause of death in rare cases.
In addition to enterotoxins several proteases,
phospholipases and haemolysins may have a role in
the pathogenesis of Bac. cereus.
Outbreaks Related to Dairy Products
Outbreaks related to dairy products are few; some of
the cases are presented in Table 2. Both diarrhoeal
and emetic symptoms have been recorded. The data
given in Table 2 indicate that young people and the
elderly may be at a higher risk than the general popu-
lation. Several factors may explain why milk-borne
cases are uncommon. Milk is generally kept at
refrigeration temperature and growth of Bac. cereus is
slow, thus the risk of exposure to high levels of
bacteria is limited, although signi®cant. Toxin pro-
duction is limited at low temperature, however,
temperature abuse will increase the risk of toxin
production. Sweet curdling often occurs when the
product contains 106±107 Bac. cereus mlÿ1 of milk,
and there is a decreased probability of consumption.
Toxin production ability varies strongly between
strains. Some strains are able to produce several dif-
ferent toxins. It is possible that only a few strains with
a greater ability to produce toxin and with additional,
as yet unknown, pathogenicity factors, are able to
cause illness. If so, illness would go unnoticed in most
cases, since the number of strains with different
properties in milk is very large, and illness could be
due to growth of a strain in only a limited number of
milk packages.
Incidence in Dairy Products
Vegetative Bac. cereus are found in raw milk at levels
of less than 10 mlÿ1 up to a few hundred. These cells
are killed by pasteurization. Spores are found at levels
of less than 10 lÿ1 to a few thousand, i.e. at much
lower levels. There is a marked seasonal variation in
psychrotrophic spores, with highest levels in the
summer and early autumn.
Milk and Cream
The number of Bac. cereus in pasteurized milk and
cream depends on the quality of the raw milk, the
hygiene at the dairy plant, the storage temperature
of the product, and the age of the product at the
sampling time. Bacillus cereus grows slowly at tem-
peratures below 6  C and if stored below this
126 BACILLUS CEREUS

Table 2 Outbreaks of milk-borne illness caused by Bac. cereus

Product Year Country Patients Symptoms Analytical data

Unpasteurized milk 1972 Romania 221 school children Diarrhoea and abdominal 20 million Bac. cereus mlÿ1 in
(heated and then kept cramps after 8±11 h milk; Bac. cereus found in
at room temperature children's faeces
overnight)
Cream, pasteurized 1975 England Two 15-year-old Vomiting after 8±10 h. 5 million Bac. cereus gÿ1
girls One girl had in cream
diarrhoea
Milk, pasteurized 1981 Denmark 1-year-old boy Vomiting after 1.5 h, 2.6 million Bac. cereus mlÿ1 in
no diarrhoea milk. Remaining milk was
sweet curdled 1 h after
consumption
Milk powder, infant 1981 Chile 35 neonate Diarrhoea Bac. cereus found in stool
formula children cultures
Human breast milk 1981 India Child, 6 months Diarrhoea, occasional Bac. cereus found in
vomiting breast milk
Milk, pasteurized 1988 The Netherlands 42 elderly people Nausea and vomiting 0.4 million Bac. cereus mlÿ1
after 2±14 h in milk
UHT milk 1991 Japan 201 people Vomiting in 95%, Milk distributed at
(process failure) average after 5 h. room temperature
Diarrhoea in 55%

Compiled from Shinagawa (1993), Cohen et al. (1984), Van Netten et al. (1990) and Christiansson (1992).

temperature there will not be a quality problem,
unless the `sell by' date is set at several weeks. After
storage for 7 days at 7  C, the incidence of Bac. cereus
can typically vary between 5% and 90% (winter and
summer) at less than 10±105 cfu mlÿ1 (re¯ecting dif-
ferences in dairy hygiene). When stored below 5  C,
Bac. cereus is rarely detected unless there is a hygiene
problem in the dairy plant.
Fermented Milks and Cheese
Bacillus cereus is rapidly inactivated in traditional
yoghurt manufacture as well as in the manufacture of
fermented milk with lactococci. Some growth is
possible within the ®rst few hours of fermentation.
Multiplication in semi-hard cheese is likewise
restricted to the ®rst hours in the cheesemaking
process. Inhibition occurs as a result of the presence
of lactic acid at pH 5.6, but other inhibitors are also
active. As the pH is lowered, vegetative Bac. cereus will
die, whereas spores that have not germinated may still
be present. When present in fermented milk and
cheese products, Bac. cereus seldom exceeds 100 gÿ1.
Milk Powder
Bacillus cereus is frequently found in milk powder
and infant formulae. The frequency of isolation varies
between 30% and 100% of samples taken from all
over the world. Under certain conditions, there
may be some opportunity for growth of Bac. cereus
in the evaporation process. Most samples contain
less than 10 cfu gÿ1, but samples with more than
103 cfu gÿ1 have been found. These increased levels
are due to hygiene problems in the factory or raw
milk with a high degree of contamination. High levels
of Bac. cereus in infant formulae may constitute a
health risk. In general, 10±100 Bac. cereus gÿ1 is set
as an acceptable level for these foods.
Source
At the Farm
Bacillus cereus is a ubiquitous microorganism. The
spores are present in soil from 102 cfu gÿ1 to more than
105 cfu gÿ1. Consequently, food products of plant
origin frequently contain Bac. cereus spores. Soil is an
important source of contamination of milk. There is a
marked seasonal variation in the spore content of raw
milk, with higher levels during the pasture period,
when the teats of the cow may be contaminated with
soil. Dirty teats that are not cleansed before milking
are an important contamination source, particularly
during wet weather. Bacillus cereus is able to grow
and sporulate on insuf®ciently cleaned milking
equipment, so equipment may be a secondary source
of contamination. Used bedding material and feed
may also contain spores of Bac. cereus.
In the Dairy Plant
There has been considerable disagreement as to
whether the occurrence of Bac. cereus in dairy prod-
ucts is caused by recontamination of milk at the dairy
plant or by contamination at the farm. To some

extent this is due to the impossibility of detecting the
low levels of spores in raw milk, whereas Bac. cereus
is easily detected in pasteurized milk after storage. It is
now generally agreed that the original contamination
occurs at the farm from soil. The seasonal variation in
the occurrence of Bac. cereus in dairy products kept at
temperatures above 6  C can to a large extent be ex-
plained by the increased contamination rate of milk
during the grazing period. However, additional con-
tamination may occur from the dairy plant equip-
ment. Since spores survive pasteurization, they will be
present in the milk throughout the dairy process.
Spores of Bac. cereus are very hydrophobic and will
attach to the surfaces of equipment where they may
germinate and form bio®lms at sites that are dif®cult
to clean. Several strain-typing methods have recently
become available and could be helpful in ®nding
contamination sites in dairy plants. Contamination of
milk by Bac. cereus has been demonstrated in silo
tanks, pasteurizers, milk pipelines with bad welding,
and packaging machines.
Control
The Farm
At the farm, measures to control Bac. cereus include
careful teat cleansing before milking and proper
cleaning and disinfection of the milking equipment.
Since the teats become dirty with soil when the cows
are outdoors during the grazing period, it is essential
that they are clean before attaching the teat-cups.
During the indoor season, high levels of Bac. cereus
spores may be found in used bedding material, if not
replaced daily, and may contaminate the teats. It is
likely that the best cleansing routine includes the use
of one moistened cloth per cow, which has been
found to be the most ef®cient method for removing
clostridial spores. In addition, the milking equipment
must be cleaned carefully after milking. Teat liners
and other rubber material must be replaced regularly
since aged rubber with cracks can harbour milk res-
idues where Bac. cereus can propagate and sporulate.
The Dairy Plant
At the dairy factory, cleaning and maintenance is
essential. Attention must be given to the correct
concentrations of cleaning agents, suf®ciently high
cleaning temperature and ¯ow rates during cleaning,
since spores are dif®cult to remove and kill. The
spores are not killed by disinfection with hot water,
but sodium hypochlorite at pH 6±7 is effective.
Regular replacement of gaskets and other rubber
parts is important.
BACILLUS CEREUS 127
In Dairy Products
The best way to control Bac. cereus levels in pas-
teurized milk and cream is to keep a low storage
temperature throughout the chain from the dairy
plant to the customer. Below 5±6  C, growth of most
strains of Bac. cereus is insigni®cant, but if the
storage temperature is higher, the `sell by' date must
be shortened accordingly. Suitable time/temperature
combinations may be found by storage tests. Seasonal
variation, occurrence of recontamination at the dairy
plant, as well as possible moderate temperature abuse
by the customer, must be taken into consideration
when choosing the recommended last consumption
date of the products.
Milk powder is microbiologically stable and growth
of Bac. cereus cannot occur, however, contamination
of milk powder with Bac. cereus is frequent. Milk
powder is frequently used in infant formulae and in
infant foods and when they are reconstituted it is
important that the product is consumed shortly after
preparation unless it is cooled to below 8  C. Spores
of Bac. cereus are able to germinate rapidly at the
reconstitution temperature and will grow rapidly if
the product is kept at room temperature. Young
children may be more susceptible to the toxins pro-
duced than adults.
See also: Biofilm Formation. Hygiene in Dairy
Production and Processing. Milking and Handling of
Raw Milk: Milking Hygiene; Effects of Storage and
Transport on Milk Quality. Milk Powders: Types and
Manufacture. Pasteurization of Liquid Milk Products:
Principles, Public Health Aspects. Sterilization of Milk
and Other Products.
Further Reading
Becker H, Schaller G, von Wiese W and Terplan G (1994)
Bacillus cereus in infant foods and dried milk products.
International Journal of Food Microbiology 23: 1±15.
Christiansson A (1992) The toxicology of Bacillus cer-
eus. International Dairy Federation Bulletin 275: 30±35.
Cohen JV, Marmabio E, Lynch B and Moreno A (1984)
Bacillus cereus in food poisoning amid newborns.
Revista Chilena de Pediatrica 55: 20±25.
Donovan KO (1959) The occurrence of Bacillus cereus in
milk and on dairy equipment. Journal of Applied
Bacteriology 22(1): 131±137.
Granum PE and Lund T (1997) Bacillus cereus and its food
poisoning toxins. FEMS Microbiology Letters 157:
223±228.
IDF (1992) Bacillus cereus in Milk and Milk Products.
International Dairy Federation Bulletin no. 275.
Brussels: IDF.
IDF (2000) New Developments in Detection and Identi-
®cation of Spore-Forming Bacteria in Milk and Milk
128 BACTERIOCINS
Products. International Dairy Federation Bulletin no.
357. Brussels: IDF.
Kramer K and Gilbert J (1989) Bacillus cereus and
other Bacillus species. In: Doyle MP (ed.) Foodborne
Bacterial Pathogens, pp. 21±70. New York: Marcel
Dekker.
Langeveld LPM and Cuperus F (1980) The relation
between temperature and growth rate in pasteurized
milk of different types of bacteria which are important
to the deterioration of that milk. Netherlands Milk and
Dairy Journal 34: 106±125.
Meer RR, Baker J, Bodyfelt FW and Grif®ths MW (1991)
Psychrotrophic Bacillus spp. in ¯uid milk: a review.
Journal of Food Protection 54(123): 969±979.
Notermans S, Dufrenne J, Teunis P et al. (1997)
A risk assessment study of Bacillus cereus
BACTERIOCINS
C Hill and T O'Keeffe, University College, Cork,
Ireland
P Ross, Dairy Products Research Centre, Teagasc,
Cork, Ireland
Copyright 2002, Elsevier Science Ltd. All Rights Reserved
Antimicrobial Factors Produced by
Lactic Acid Bacteria
The lactic acid bacteria (LAB) ± including the genera
Lactobacillus, Lactococcus, Leuconostoc and Pedio-
coccus ± have long been used in fermentations to
preserve the nutritive qualities of various foods. The
primary function of a starter culture is the production
of lactic acid at a suitable rate to ensure a consistent
and successful fermentation. Other functions include
the production of ¯avour compounds such as diacetyl
and CO2 from citrate by mesophilic cultures, and
acetaldehyde from lactose by thermophilic cultures;
acting as a source of proteolytic enzymes during
growth in milk and ripening of many cheeses; and
®nally, contributing to the preservation of the fer-
mented product as a consequence of a number of in-
hibitory metabolites produced by the lactic cultures.
present in pasteurized milk. Food Microbiology 14:
143±151.
Shinagawa K (1993) Serology and characterization of
toxigenic Bacillus cereus. Netherlands Milk and Dairy
Journal 47: 89±103.
Stewart DB (1975) Factors in¯uencing the incidence of
Bacillus cereus spores in milk. Journal of the Society for
Dairy Technology 28(2): 80±85.
Van Netten P, van de Moosdijk A, van Hoensel P, Mossel
DA and Perales I (1990) Psychrotrophic strains of
Bacillus cereus producing enterotoxin. Journal of
Applied Bacteriology 69: 73±79.
Van Netten P and Kramer JM (1992) Media for the
detection and enumeration of Bacillus cereus in foods:
a review. International Journal of Food Microbiology
17(2): 85±99.
The major metabolite of lactic acid bacteria is lactic
acid. The production of this organic acid is respons-
ible for the associated drop in pH, which may be
suf®cient to inhibit the growth of many undesirable
microorganisms. In addition to a direct effect on the
pH, the undissociated form of the molecule can cause
the collapse of the electrochemical proton gradient of
susceptible bacteria, leading to bacteriostasis and
eventual death. Outside of its use in food fermenta-
tions, the main application of lactic acid in the food
industry is in the decontamination of meat and
poultry carcasses. Acetic and propionic acids are also
produced in small amounts by lactic acid bacteria.
They act in a similar manner to lactic acid and are
widely used as food additives; however, they are not
usually derived from LAB fermentations for this
purpose. They do play an important antimicrobial
role in some fermented foods, and it is known that
acetic acid has a synergistic antimicrobial effect when
present with lactic acid.
Diacetyl and acetaldehyde, as well as imparting
aroma and ¯avour to cultured dairy products,
also have an antimicrobial effect. Acetaldehyde
can inhibit cell division in Escherichia coli, and
diacetyl inhibits yeasts, Gram-negative and Gram-
positive bacteria. However, the use of the latter as