Bacteriocins and Their Food Applications

Bacteriocins
and their Food

Applications
H. CHEN AND D.G. HOOVER
ABSTRACT: Over the last 2 decades, a variety of bacteriocins, produced by bacteria that kill or inhibit the growth of other
bacteria, have been identified and characterized biochemically and genetically. This review article focuses on the ecology
of bacteriocins, determination of bacteriocin activity, biosynthesis of bacteriocins, and mode of action. Bacteriocin production and modeling are discussed in the article. Nisin is discussed in some detail in this article since it is currently the
only purified bacteriocin approved for food use in the U.S. and has been successfully used for several decades as a food
preservative in more than 50 countries. For activity spectra and food applications, the review article focuses primarily on
class I and class IIa bacteriocins produced by lactic acid bacteria (LAB) given their development as food preservatives.
Introduction
Ever since the era of Louis Pasteur and Robert Koch, there has
been scientific recognition of an essential need to control detrimental microorganisms in our environment. The discovery of
penicillin by Alexander Fleming in 1929 opened the door for use
of therapeutic antibiotics by the medical and veterinary communities to combat specific disease-causing organisms. Although
therapeutic antibiotics are prohibited for use in foods, the utilization of antagonistic additives with preservative or antimicrobial
properties has since become a trademark approach in food safety
and preservation. In foods and beverages, addition of antimicrobial compounds to processed products has become a traditional
weapon in the food preservation arsenal.
Comprising a subgroup within the far larger body of commercial food preservatives are the bacteriocins. Bacteriocins are produced by bacteria and possess antibiotic properties, but bacteriocins are normally not termed antibiotics in order to avoid confusion and concern with therapeutic antibiotics that can potentially
illicit allergic reactions in humans (Cleveland and others 2001).
Bacteriocins differ from most therapeutic antibiotics in being proteinaceous and generally possessing a narrow specificity of action
against strains of the same or closely related species (Tagg and
others 1976). Bacteriocins are ribosomally synthesized polypeptides possessing bacteriocidal activity that are rapidly digested by
proteases in the human digestive tract (Joerger and others 2000).
Bacteriocins are a heterogenous group, characteristically selected
for evaluation and use as specific antagonists against problematic
bacteria; however, their effectiveness in foods can become limited for various reasons, and cost remains an issue impeding broader use of bacteriocins as food additives. Hence, not only do
searches continue for new and more effective bacteriocins, but
also development is ongoing for optimization of existing bacteriocins to address both biologic and economic concerns.
Even though the bacteriocin nisin has been used as a food preservative compound in other countries since the 1950s, the 1988
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FDA approval for use of nisin in pasteurized processed cheese

spreads established the legal precedent in the U.S. for application
of bacteriocins as food additives (Federal Register 1988). Nisin remains the most commercially important bacteriocin, although
other bacteriocins have been characterized and developed for
possible approval and use (Chikindas and Montville 2002). Because LAB and their metabolites have been consumed in high
quantities by countless generations of people in cultured foods
with no adverse effects, the LAB continue as the preferred source
for food-use bacteriocins, either in the form of purified compounds or growth extracts. A crude bacteriocin fermentate can be
obtained by growing the bacteriocin-producing LAB on a complex substrate. The crude fermentate contains other substances besides the bacteriocin. The term purified bacteriocin implies that
the bacteriocin is not a crude bacteriocin fermentate and is the
only antimicrobial substance contained in the purified preparation. Given the use of LAB as starter cultures in the manufacture of
fermented foods, they are obvious candidates for genetic modification should industrial strains lack genes regulating synthesis of
desirable bacteriocin(s). In addition to a sterling safety record and
a public perception of LAB as health aids, many bacteriocins from
these GRAS (generally regarded as safe) bacteria are effective
against key gram-positive pathogens of importance in foodborne
illness, including Listeria monocytogenes, a hardy, pathogenic
bacterium common in the environment and difficult to control in
foods. For these reasons, research interest in bacteriocins has continued unabated over the past 25 y and does not appear to be
waning significantly.
Ecology of Bacteriocins
On an evolutional basis, it appears that the ability to synthesize
one or more bacteriocins has been a highly advantageous characteristic. A clear opportunity for survival and proliferation of an organism can be envisioned if it can eliminate a competing organ-
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Bacteriocins and their food applications . . .

ism in its ecological niche, where competition can be quite intense given the diversity of species and rapid growth of bacteria
(Dykes 1995). Low-molecular-weight antibiotics (for example, tetracyclines), lytic agents, toxins, bacteriolytic enzymes, bacteriophage, and metabolic by-products, such as organic acids, hydrogen
peroxide, and diacetyl, also function in a somewhat similar capacity, but nonetheless the capability to produce bacteriocins and
producer-cell immunity occurs abundantly in prokaryotes, both
eubacteria and archaebacteria. Bacteriocins play a fundamental
role in bacterial population dynamics even though the degree of
bacteriocin interactions is so complex at the ecological and evolutionary levels in mixed populations (such as biofilms) that much
remains uncertain.
As noted by Riley (1998), examination of bacteriocins in natural
settings, such as Lactobacillus plantarum in green olive fermentations, Escherichia coli in guinea pig conjunctivae, and Streptococcus mutans in the human oral cavity, have indicated that the competitive advantage is substantially increased for bacteriocin-producing cells against bacteriocin-sensitive bacteria in the same environments. In the L. plantarum example, a bacteriocin-producing
strain was used to ferment Spanish-style green olives (Ruiz-Barba
and others 1994). The bacteriocin producer maintained itself at elevated levels over the course of the 12-wk fermentation. When a
bacteriocin-negative variant of the same strain was used in the fermentation, the background lactobacilli outcompeted the variant
to the extent that the variant was not detected after 7 wk.
In this light, mathematical models have been devised to evaluate the interaction between bacteriocin producers and sensitive
varieties. Most ecological modeling of bacteriocins has been with
colicins (bacteriocins produced by Escherichia coli and usually
showing activity against other strains of E. coli and very closely
related members of the Enterobacteriaceae). Induction usually occurs under stressful conditions such as nutrient depletion or overcrowding (Riley and Gordon 1999). Colicins differ from bacteriocins from gram-positive bacteria in the sense that they have 3
general mechanisms of action: channel formation in the cytoplasmic membrane, (the common mechanism found with gram-positive bacteriocins), degradation of cellular DNA, and inhibition of
protein synthesis. It is estimated that about 30% of the natural
populations of E. coli produce bacteriocins (Riley 1998). Over 25
colicin types have been identified (Pugsley 1984). It is also estimated that about 70% of the cells in a population of E. coli are
resistant to any one colicin, and 30% are resistant to all colicins
produced in a population with the remaining cells colicin-sensitive (Smarda 1992). The relative numbers of colicin-producing
cells have been found dependent on the energy costs associated
with colicin synthesis (Riley and Gordon 1999).
Determination of Bacteriocin Activity

Demonstration of antagonism from 1 strain of bacteria against
another is very common. Bacteriocins are only one category of
substances produced by bacteria that are inhibitory to other bacteria. As early as 1676, Antonie van Leeuwenhoek documented
antibiosis in which the product from one microorganism inhibited
growth of another (Joerger and others 2000). Louis Pasteur and J.F.
Joubert reported the inhibitive effect of common urine bacteria on
Bacillus anthracis in 1877. As mentioned earlier, in addition to
bacteriocins, inhibitors produced by bacteria include clinical or
therapeutic low-molecular-weight antibiotics, lytic agents, toxins,
bacteriolytic enzymes, bacteriophage, and other metabolic byproducts, such as hydrogen peroxide and diacetyl. Also, in any
ecological niche, one type of bacteria can be more competitive
than another regarding nutrient uptake or sensitivity to an environmental factor, such as available oxygen. With such a wide
range of possible inhibitory products or conditions, it is no wonAvailable at http://www.ift.org/publications/crfsfs
83
der that some bacteriocin-like activities may not be caused by

bacteriocins at all. When investigating bacteriocins synthesized by
LAB, one must always be aware of the presence of relatively high
amounts of organic acids, and take appropriate steps to determine
or eliminate acidic effects.
A common means for screening bacteriocin activity is the use
of agar media contained in petri plates. Mayr-Harting and others
(1972), Piddock (1990) and Parente and Riccardi (1999) have reviewed a number of these methods for detection and measurement of bacteriocin activity. There are many variations, most of
them derivatives of the spot-on-lawn approach, which can involve an agar overlay. The direct simultaneous tests are the simplest. In this approach, producing and indicator cultures are incubated concurrently before examination for zones of inhibition
around the growth of producing strains. An example is the addition of indicator cells to tempered, molten agar used to prepare
pour plates in which the producing strain is spot- or streak-inoculated onto the hardened agar (Sabine 1963). Deferred methods allow separation of the independent variables of time and conditions of incubation for the producing and indicator strains and are
often more sensitive than direct tests (Tagg and others 1976).
Kekessy and Piguet (1970) described a procedure in which the
producing and indicator strains were each grown on different optimal media. The producing culture was spot-inoculated onto a
spread plate, and after growth, the agar mass was aseptically dislodged with a spatula from the petri dish bottom and transferred
to the lid of the dish. A soft agar overlay seeded with the indicator
was then poured over the inverted agar. Following re-incubation,
bacteriocin-positive cultures displayed a halo of clearing in the
lawn around the original button of growth. This assay minimizes
the effects of acids and bacteriophage, since the producing and
indicator strains are physically separated by an agar layer.
Media composition is an important factor in plate assays, but
the issue goes beyond nutritional requirements of the producing
and target organisms. The gelling agents used in solid media can
interfere with diffusion of the bacteriocin, thus limiting effectiveness of these screening methods (Lindgren and Clevstrom 1978).
Other compounds can impede results. For example, Hoover and
others (1989) found that -glycerol phosphate (a buffering agent)
in M17 agar in the soft agar overlay of the Kekessy-Piguet methods, interfered with the zones of inhibition caused by Pediococcus acidilactici PO2 when compared to other agar media and
other buffering agents. However, Spelhaug and Harlander (1989)
experienced no such difficulties showing bacteriocin antagonism
with Pediococcus pentosaceus FBB61 and FBB63-DG2 in M17glucose agar. Possibly, different bacteriocins were produced by
these strains or the methodologies used were subtly different.
Mass spectrometry has been adapted for the rapid detection of
pediocin, nisin, brochocins A and B, and enterocins A and B from
culture supernatants by Rose and others (1999). The method is
called matrix-assisted laser desorption/ionization time-of-flight
mass spectroscopy (MALDI-TOF MS) and was originally devised
for the examination of large molecules, such as biopolymers. In
the protocol, a 30-s water wash of the supernatant is intended to
remove interfering compounds; however, at the time of publication, this step of the procedure requires improvement to eliminate
the contaminating and interfering substances found in foods that
prevent accurate identification of the bacteriocins.
PCR methods have been used to detect genes responsible for
bacteriocin production and regulation in bacterial cultures. Rodriguez and others (1995) demonstrated the amplification of a 75bp gene fragment of the lactocin S structural gene in 7 bacteriocinogenic strains of lactobacilli isolated from fermented sausages.
In another work, Garde and others (2001) detected the genes necessary for synthesis of lacticin 481 and nisin using PCR techniques with specific probes in an isolate of L. lactis subsp. lactis
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CRFSFS: Comprehensive Reviews in Food Science and Food Safety

from raw milk cheese.
Rodriguez and others (1998) detected enterocin AS-48 from enterococci isolated from milk and dairy products using dot-blot
and colony hybridization. Genes encoding for synthesis of AS-48
have been sequenced (Martinez-Bueno and others 1994) and a
PCR technique developed for rapid detection of these genes in
isolated strains (Joosten and others 1997). Colony hybridization
allowed for a more rapid technique in which cultures are spotted
on MRS agar, incubated, and colony growth is transferred to
membranes for lysis with subsequent hybridization using a DNA
probe.
In a flow cell, Mugochi and others (2001) developed a rapid
and sensitive method for detection of bacteriocins in fermentation
broth. Low concentrations of potassium ions were measured, so
that released potassium ions from a bacteriocin-sensitive indicator strain directly correlated to concentrations of crude bacteriocin present in fermentation broth injected into the cell. This method compared very well to a conventional agar well diffusion assay.
Classification of Bacteriocins
First discovered by Gratia in 1925, principe V was produced
by 1 strain of E. coli against another culture of E. coli. The term
colicine was coined by Gratia and Fredericq (1946); bacteriocine was used by Jacob and others (1953) as a general term for
highly specific antibacterial proteins. The term colicin now implies a bacteriocidal protein produced by varieties of E. coli and
closely related Enterobacteriaceae (Konisky 1982).
Bacteriocins (as colicins) were originally defined as bacteriocidal proteins characterized by lethal biosynthesis, a very narrow
range of activity, and adsorption to specific cell envelope receptors (Jacob and others 1953). Later, the recognized association of
bacteriocin biosynthesis with plasmids was added to the description. The definition has since been modified to incorporate the
properties of bacteriocins produced by gram-positive bacteria
(Tagg and others 1976). Bacteriocins from gram-positive bacteria
commonly do not possess a specific receptor for adsorption although exceptions exist (Gravesen and others 2002b), are most
frequently of lower molecular weight than colicins, have a broader range of target bacteria with different modes of release and cell
transport, and possess leader sequences cleaved during maturation (Jack and others 1995; James and others 1991; Riley 1998).
Today, bacteriocidal peptides or proteins produced by bacteria
are typically referred to as bacteriocins. Usually, to demonstrate
the proteinaceous nature of a newly characterized bacteriocin,
sensitivity to proteolytic enzymes such as trypsin, -chymotrypsin,
and pepsin is an expected demonstration. Evaluation for use as a
food additive requires estimation of its heat resistance given the
widespread use of thermal processing in food production.
Over the years, several publications have reviewed colicins,
bacteriocins, bacteriocins from LAB, and applications of specific
bacteriocins. Examples include Reeves (1972), Franklin and Snow
(1975), Hardy (1975), Tagg and others (1976), Konisky (1982),
Klaenhammer (1988, 1993), Jack and others (1995), de Vos and
others (1995), Sahl and others (1995), Venema and others
(1995c), Abee and others (1995), Nes and others (1996), and
Cleveland and others (2001).
Most of the bacteriocins from LAB are cationic, hydrophobic,
or amphiphilic molecules composed of 20 to 60 amino acid residues (Nes and Holo 2000). These bacteriocins are commonly
classified into 3 groups that also include bacteriocins from other
gram-positive bacteria (Klaenhammer 1993, Nes and others
1996). Examples of bacteriocins from these 3 classes are summarized in Table 1.
Lantibiotics (from lanthionine-containing antibiotic) are small
84
Table 1Examples of bacteriocins

Bacteriocins
Producer
Hurst 1981
Mortvedt and others 1991
Allgaier and others 1986
Kellner and others 1988
Piard and others 1992
Altena and others 2000
Sahl and Bierbaum 1998
Henderson and others 1992;

Motlagh and others 1992
sakacin A
L. sake
Holck and others 1992
sakacin P
L. sake
Tichaczek and others 1992
leucocin A-UAL 187 Leuconostoc gelidum
Hastings and others 1991
mesentericin Y105 Leuconostoc mesenteroides Hechard and others 1992
enterocin A
Enterococcus faecium
Aymerich and others 1996
divercin V41
Carnobacterium divergens Metivier and others 1998
lactococcin MMFII L. lactis
Ferchichi and others 2001
Class IIb
lactococcin G
L. lactis
Nissen-Meyer and others
1992
lactococcin M
L. lactis
van Belkum and others 1991
lactacin F
Lactobacillus johnsonii
Allison and others 1994
plantaricin A
Lactobacillus plantarum
Nissen-Meyer and others
1993a
plantaricin S
L. plantarum
Jimenez-Diaz and others
1995
plantaricin EF
L. plantarum
Anderssen and others 1998
plantaricin JK
L. plantarum
Anderssen and others 1998
Class IIc
acidocin B
Lactobacillus acidophilus
Leer and others 1995
carnobacteriocin
Carnobacterium piscicola Worobo and others 1994
A
divergicin A
C. divergens
Worobo and others 1995
enterocin P
E. faecium
Cintas and others 1997
enterocin B
E. faecium
Nes and Holo 2000
Class III
helveticin J
Lactobacillus heleveticus
Joerger and Klaenhammer
1986
helveticin V-1829
L. helveticus
Vaughan and others 1992
(<5 kDa) peptides containing the unusual amino acids lanthionine (Lan), -methyllanthionine (MeLan), dehydroalanine, and dehydrobutyrine. These bacteriocins are grouped in class I. Class I is
further subdivided into type A and type B lantibiotics according to
chemical structures and antimicrobial activities (Moll and others
1999; van Kraaij and others 1999; Guder and others 2000). Type
A lantibiotics are elongated peptides with a net positive charge
that exert their activity through the formation of pores in bacterial
membranes. Type B lantibiotics are smaller globular peptides and
have a negative or no net charge; antimicrobial activity is related
to the inhibition of specific enzymes.
Small (<10 kDa), heat-stable, non-lanthionine-containing peptides are contained in class II. The largest group of bacteriocins in
this classification system, these peptides are divided into 3 subgroups. Class IIa includes pediocin-like peptides having an N-terminal consensus sequence -Tyr-Gly-Asn-Gly-Val-Xaa-Cys. This
subgroup has attracted much of the attention due to their antiListeria activity (Ennahar and others 2000b). Class IIb contains
bacteriocins requiring 2 different peptides for activity, and class
IIc contains the remaining peptides of the class, including sec-dependent secreted bacteriocins.
The class III bacteriocins are not as well characterized. This
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References
Class I-type A lantibiotics

nisin
Lactococcus lactis
lactocin S
Lactobacillus sake
epidermin
Staphylococcus epidermidis
gallidermin
Staphylococcus gallinarum
lacticin 481
L. lactis
Class I-type B lantibiotics
mersacidin
Bacillus subtilis
cinnamycin
Streptomyces cinnamoneus
ancovenin
Streptomyces ssp.
duramycin
S. cinnamoneus
actagardin
Actinoplanes ssp.
Class IIa
pediocin PA-1/AcH Pediococcus acidilactici
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group houses large (>30 kDa) heat-labile proteins that are of lesser
interest to food scientists. A 4th class consisting of complex bacteriocins that require carbohydrate or lipid moieties for activity has
also been suggested by Klaenhammer (1993); however, bacteriocins in this class have not been characterized adequately at the
biochemical level to the extent that the definition of this class requires additional descriptive information (Jimenez-Diaz and others 1995; McAuliffe and others 2001).
Genetics, Biosynthesis and Mode of Action

Organization of gene clusters: genetics and biosynthesis
Bacteriocins are ribosomally synthesized. The genes encoding

bacteriocin production and immunity are usually organized in
operon clusters (Nes and others 1996; Sahl and Bierbaum 1998;
McAuliffe and others 2001). For linear unmodified bacteriocins,
which include the plantaricins, carnobacteriocins, and sakacins,
it appears that specific inducing peptides or peptide pheromones
stimulate synthesis of bacteriocins that are usually located on the
same gene cluster (Quadri and others 1997; Brurberg and others
1997; Anderssen and others 1998). Bacteriocin gene clusters can
be located on the chromosome, as in the case of subtilin (Banerjee and Hansen 1988) and mersacidin (Altena and others 2000),
plasmids, as in the case of divergicin A (Worobo and others 1995)
and sakacin A (Axelsson and Holck 1995), or transposons, as in
the case of nisin (Rauch and de Vos 1992) and lacticin 481 (Dufour and others 2000).
The lantibiotic biosynthesis operons generally contain genes
coding for the prepeptide (LanA - the abbreviation lan refers to
homologous genes of different lantibiotic gene clusters), enzymes
responsible for modification reactions (LanB,C/LanM), processing
proteases responsible for removal of the leader peptide (LanP),
the ABC (ATP-binding cassette), superfamily transport proteins involved in peptide translocation (LanT), regulatory proteins (LanR,
K), and proteins involved in producer self-protection (immunity)
(LanI, FEG). This information was gathered from genetic analysis
of several lantibiotics, epidermin (Schnell and others 1992, Bierbaum and others 1996; Geissler and others 1996), nisin (Buchmann and others 1988; Mulders and others 1991; de Vos and others 1995), subtilin (Banerjee and Hansen 1988; Klein and others
1992; Klein and others 1993, Klein and Entian 1994), lacticin 481
(Piard and others 1993; Rince and others 1997; Uguen and others
2000), and mersacidin (Bierbaum and others 1995; Altena and
others 2000).
The genetic regulation of the class II bacteriocins, lactococcins
A, B, and M (Holo and others 1991; van Belkum and others 1991;
Stoddard and others 1992; van Belkum and others 1992; Venema
and others 1995b), pediocin PA-1/AcH (pediocin PA-1 and AcH
are the same molecule; the name of pediocin PA-1 is more commonly used) (Marugg and others 1992; Motlagh and others 1992;
Bukhtiyarova and others 1994; Venema and others 1995a), and
plantaricin A (Diep and others 1994; 1995; 1996) have been studied. Genes encoding the biosynthesis of class II bacteriocins share
many similarities in their genetic organizations, consisting of a
structural gene coding for precursor peptide, followed immediately by a dedicated immunity gene and genes for an ABC-transporter and an accessory protein. In some cases, regulatory genes have
been found. The accessory proteins are essential for the export of
class II bacteriocins. No counterparts of these accessory proteins
in lantibiotics have been found (Nes and others 1996; Sablon and
others 2000). For more elaborate reviews on the genetics of lantibiotics and nonlantibiotics see Klaenhammer (1993), Jack and
others (1995), Nes and others (1996), Sahl and Bierbaum (1998),
van Kraaij and others (1999), Ennahar and others (2000b), Sablon
and others (2000), and McAuliffe and others (2001).
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Biosynthetic pathway
Most bacteriocins are synthesized as a biologically inactive

prepeptide carrying an N-terminal leader peptide that is attached
to the C-terminal propeptide. For lantibiotics, the serine, threonine, and cysteine residues in their propeptide parts undergo extensive post-translational modification to form Lan/MeLan. The
biosynthetic pathway of lantibiotics follows a general scheme (as
shown in Figure 1): formation of prepeptide, modification reactions, proteolytic cleavage of the leader peptide, and the translocation of the modified prepeptide or mature propeptide across the
cytoplasmic membrane. Cleavage of the leader peptide may take
place prior to, during, or after export from the cell. Based on the
biosynthetic pathway, 2 categories of genetic organization of lantibiotics, groups I and II, can be identified (Sahl and Bierbaum
1998; Guder and others 2000; McAuliffe and others 2001). This
classification scheme has nothing to do with the above classification scheme that divides lantibiotics into type A and type B lantibiotics, since group I and II lantibiotics can be either type A or
type B lantibiotics. For example, lacticin 481, which belongs to
group II according to this genetic organizational scheme, is a type
A lantibiotic. In the production of the group I lantibiotics, as in
the case of nisin, epidermin, subtilin, and Pep5, the dehydration
reaction is presumably catalyzed by the LanB enzyme, while
LanC is involved in the thioether formation. The modified prepeptide is processed by a serine protease LanP and translocated
through the ABC-transporter LanT. In contrast, lantibiotics of
group II, as in the case of cytolysin, lacticin 481, and mersacidin,
are very likely modified by a single LanM enzyme (van Kraaij and
others 1999; McAuliffe and others 2001), and processing takes
place concomitantly with transport by LanT(P). Lactocin S is the
exception to this classification. It is modified by a single LanM
enzyme and processing takes place prior to export and may therefore represent a new group (Skaugen and others 1997).
Class II bacteriocins (Table 1) are synthesized as a prepeptide
containing a conserved N-terminal leader and a characteristic
double-glycine proteolytic processing site, with the exception of
Figure 1A schematic diagram of the biosynthesis of lantibiotics: (1) Formation of prebacteriocin; (2) The prebacteriocin is
modified by LanB and LanC, translocated through a dedicated
ABC-transporter LanT and processed by LanP, resulting in the
release of mature bacteriocin; (3) Histidine protein kinase (HPK)
senses the presence of bacteriocin and autophosphorylates;
(4) The phosphoryl group (P) is subsequently transferred to the
response regulator (RR); (5) RR activates transcription of the
regulated genes; and (6) Producer immunity mediated by immunity proteins, LanI, and dedicated ABC-transport proteins,
LanFEG.
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85

class IIc bacteriocins, which are produced with a typical N-terminal signal sequence of the sec-type and processed and secreted
through the general secretory pathway (Leer and others 1995;
Worobo and others 1995). Unlike the lantibiotics, class II bacteriocins do not undergo extensive post-translational modification.
Following the formation of prepeptide, the prepeptide is processed to remove the leader peptide concomitant with export
from the cell through a dedicated ABC-transporter and its accessory protein (Nes and others 1996; Ennahar and others 2000b).
The biosynthetic pathway of class II bacteriocins is shown in Figure 2.
Several functions of the leader peptides have been proposed.
They may serve as a recognition site which directs the prepeptide
towards maturation and transport proteins, protect the producer
strain by keeping the lantibiotic in an inactive state while it is inside the producer, and interact with the propeptide domain to ensure a suitable conformation essential for enzyme-substrate interaction (van der Meer and others 1994; van Belkum and others
1997; Sablon and others 2000; McAuliffe and others 2001).
Post-translational modification, activation and transport
Ingram (1969; 1970) first proposed a 2-step post-translational

modification reaction of a pre-lantibiotic leading to formation of
Lan/MeLan. Initially, the hydroxyl amino acids, serine and threonine, are dehydrated to yield 2,3-didehydroalanine and 2,3-didehydrobutyrine, respectively. Some dehydrated amino acids do not
contain cysteine residues and remain as such in the mature peptide; others undergo an intramolecular Michael addition reaction
that involves the thiol groups of neighboring cysteine residues
and the double bonds of the didehydroamino acids, resulting in
the formation of thioether bridges. Following the modification reactions, the modified pre-lantibiotics undergo proteolytic processing to release the leader peptide that leads to activation of the
lantibiotic. For group I lantibiotics, the leader peptide is removed
by a serine protease, LanP, and, depending on the location of
LanP, this can take place before or after the peptide is exported
from the producing cell via a dedicated ABC-transporter, LanT. For
example, the proteases LanP of epicidin 280 and Pep5 (Sahl and
Figure 2A schematic diagram of the biosynthesis of class II

bacteriocins: (1) Formation of prebacteriocin and prepeptide
of induction factor (IF); (2) The prebacteriocin and pre-IF are
processed and translocated by the ABC-transporter, resulting
in the release of mature bacteriocin and IF; (3) Histidine protein kinase (HPK) senses the presence of IF and autophosphorylates; (4) The phosphoryl group (P) is subsequently transferred to the response regulator (RR); (5) RR activates transcription of the regulated genes; and (6) Producer immunity.
86
Bierbaum 1998) are located intracellularly so that proteolytic processing takes place within the cell. In contrast, the proteases of nisin (van der Meer and others 1993) and epidermin (Geissler and
others 1996), which are located extracellularly, activate the lantibiotics only after export by the ABC-transporter. The ABC-transporter contains 500 to 600 amino acids and is characterized by 2
membrane-associated domains. The N-terminal domain consists
of 6 membrane-spanning helices that can recognize the substrate
and form its pathway across the membrane, while the cytoplasmic
C-terminal domain contains 2 ATP-binding domains with the conserved ATP-binding or Walker motif. ATP hydrolysis, which likely
occurs at the ATP-binding domains, provides energy for the export
process (Fath and Kolter 1993; McAuliffe and others 2001). The
LanB and LanC enzymes, together with LanT transporter, probably
form a multimeric membrane-associated complex (Siegers and
others 1996; Kiesau and others 1997). For group II lantibiotics,
which possess a conserved double-glycine cleavage site, proteolytic processing takes place concomitantly with export through
a hybrid ABC-transporter. This unique ABC-transporter possesses
an N-terminal protease domain of approximately 150 amino acid
residues that cleaves the double-glycine leader (Nes and others
1996; Sablon and others 2000). This is exemplified in Figure 3.
Substantial similarities exist between the leader peptides of class
IIa and b and those of group II lantibiotics. Both contain the characteristic double-glycine cleavage site (Nes and others 1996; Ennahar and others 2000b). The conservation of the cleavage site
strongly suggests that the mechanism of processing and translocation of class IIa and b bacteriocins is very similar to that of the
group II lantibiotics. Class IIc bacteriocins are processed by a signal peptidase during translocation across the cytoplasmic membrane.
Regulation of biosynthesis
The biosynthesis of lantibiotics and nonlantibiotics is usually

regulated through well-known 2-component regulatory systems.
These regulatory systems consist of 2 signal-producing proteins, a
membrane-bound histidine protein kinase (HPK), and a cytoplasmic response regulator (RR) (Stock and others 1989; Parkinson
1993; Nes and others 1996). In this signal transduction pathway,
HPK autophosphorylates the conserved histidine residue in its intracellular domain when it senses a certain concentration of bacteriocin in the environment. The phosphoryl group is subsequently transferred to the conserved aspartic acid residue on the RR receiver domain and the resulting intramolecular change triggers
the response regulator to activate the transcription of the regulated
genes. These regulated genes include the structural gene, the export genes, the immunity genes, and in some cases, the regulatory
genes themselves (Kuipers and others 1998).
For nisin and subtilin, the bacteriocin molecule itself apparently
acts as an external signal to autoregulate its own biosynthesis via
Figure 3ABC-transporter with an N-terminal protease domain.
86
7/3/2003, 11:39 AM

signal transduction (Kuipers and others 1995; Guder and others
2000). In contrast, most class II bacteriocins produce a bacteriocin-like peptide with no antimicrobial activity and use it as an induction factor (IF) to activate the transcription of the regulated
genes. The IF is a small, heat-stable, cationic and hydrophobic
peptide that is first synthesized as prepeptide with a double-glycine leader sequence. A dedicated ABC-transporter specifically
cleaves the leader peptide of IF concomitant with export of the
mature peptide from the cell. The secreted IF acts as an external
signal that triggers transcription of the genes involved in bacteriocin production (Nes and others 1996; Ennahar and others 2000b).
Producer immunity
Two systems of lantibiotic immunity in the producing cell have

been identified. Protection can be mediated by immunity proteins, LanI, and dedicated ABC-transport proteins, LanFEG, which
can be encoded on multiple open-reading frames (Reis and others
1994; Siegers and Entian 1995; Peschel and Gotz 1996; Saris and
others 1996; McAuliffe and others 2001). These 2 immunity systems work synergistically to protect producing cells from their
own bacteriocin (Klein and Entian 1994). LanI, which is most
likely attached to the outside of the cytoplasmic membrane, probably confers immunity to the producer cells by preventing pore
formation by the bacteriocin. LanFEG apparently acts by transporting bacteriocin molecules that have inserted into the membrane back to the surrounding medium and thus keeping the concentration of the bacteriocin in the membrane under a critical
level.
For class II bacteriocins the immunity gene usually codes for a
dedicated protein that is loosely associated with the cytoplasmic
membrane. Quadri and others (1995), using Western blot (immunoblot) analysis, indicated that the major part of the immunity
protein CbiB2 of carnobacteriocin B2 is found in the cytoplasm
and that a smaller proportion is associated with the membrane.
Abdel-Dayem and others (1996) have demonstrated that the majority of the immunity protein MesI of mesentericin Y105 is in the
cytoplasm, with only a small proportion detected in the membrane. The immunity protein, which is cationic and ranges in size
from 51 to 254 amino acids, provides total immunity against the
bacteriocin. Interaction of the immunity protein with the membrane appears to protect the producer against its own bacteriocin
(Nissen-Meyer and others 1993b; Venema and others 1994; Nes
and Holo 2000).
Mode of action
The anionic lipids of the cytoplasmic membrane are the primary receptors for bacteriocins of LAB for initiation of pore formation (Abee 1995; Moll and others 1999). Conductivity and stability of pores induced by lantibiotics may be heightened by docking
molecules (lipid II, the peptidoglycan precursor), while in the
case of class II bacteriocins, receptors in the target membrane apparently act to determine specificity (Venema and others 1995b;
1995c). Class I bacteriocins may induce pore formation according
to a wedge-like model, and class II bacteriocins may function by
creating barrelstave-like pores or a carpet mechanism whereby
peptides orient parallel to the membrane surface and interfere
with membrane structure (Moll and others 1999).
Gravesen and others (2002b) examined the resistance of L.
monocytogenes to class IIa bacteriocins and found a relationship
to a specific recognition site. Eight mutants of L. monocytogenes
resistant to class IIa bacteriocins, such as pediocin PA-1 and leucocin A, were isolated and studied. The common mutation found
in all the resistant mutant strains was to a subunit of an enzyme in
a mannose-specific phosphoenolpyruvate-dependent phosphotransferase system regulated by the 54 transcription factor. It was
interpreted that resistance to these bacteriocins in L. monocytogeAvailable at http://www.ift.org/publications/crfsfs
87
nes and other gram-positive bacteria was fundamentally related to

this one common site regardless of the strain, the type of class IIa
bacteriocin, or the environmental conditions used in examination.
It was suggested that mutation to this specific subunit located in
the membrane changed the target recognition site of the class IIa
bacteriocins.
Lacticin 3147 is a lantibiotic composed of 2 components, 2
peptides termed lac 1 and lac 2 (McAuliffe and others 1998); both
components are required for full antagonistic activity that is a result of the formation of ion-specific pores in the membrane of
gram-positive target cells. McAuliffe and others (2000) demonstrated that lacticin 3147 apparently requires a separate modification enzyme for each of the prepeptides of lac 1 and lac 2. Other
known 2-component bacteriocins are lacticin F (Abee and others
1994), lactococcin G (Nissen-Meyer and others 1992), and thermophilin 13 (Marciset and others 1997).
Nisin is often compared to a surface-active cationic detergent
in that adsorption to the bacterial cell envelope is the necessary
first step of membrane disruption. This is then followed by the inactivation of sulfhydryl groups. Bruno and others (1992) showed
nisin to completely dissipate the basal proton-motive force of L.
monocytogenes Scott A, and other strains of L. monocytogenes
were found to be equally sensitive to nisin, exhibiting a similar
deteriorative response of the pH gradient and membrane potential. Other class I bacteriocins of LAB behave in a similar manner.
Carnocin UI49 acts upon the cytoplasmic membrane in a fashion
very similar to nisin (Stoffels and others 1994). Ion permeability
or channel formation in cytoplasmic membranes caused by bacteriocins of L. acidophilus was demonstrated using artificial lipid
membranes (Palmeri and others 1999). Increases in membrane
conductance were detected and channels with elementary conductance of 68 to 70 pS were measured by applying external voltages of different polarity.
Lactocin 27, produced by L. helveticus LP27, was described in
earlier research by Upreti and Hinsdill (1975) as a bacteriocin
whose effect was bacteriostatic and adsorbed equally well to sensitive and resistant cells, resulting in the termination of protein
synthesis. However, there was appreciable effect on DNA and
RNA syntheses and ATP levels. Zajdel and others (1985) found
the lactostrepcin, Las 5, to immediately block syntheses of DNA,
RNA, and protein, but these responses were probably a secondary
reaction to severe membrane disruption and loss of intracellular
constituents. For Las 5, both sensitive and insensitive cells adsorbed the bacteriocin equally well. Protoplasts of sensitive
strains were not affected by Las 5, indicating that cell membrane
receptors were required for the action of the bacteriocin.
Andersson (1986) demonstrated that resistant gram-negative
cells of E. coli, Erwinia carotovora, P. aeruginosa, and S. marcescens could be made sensitive to the bacteriocin of a strain of L.
plantarum by transforming the gram-negative cells to spheroplasts. Subsequent work has shown inactivation of gram-negative
pathogens with bacteriocins from gram-positive bacteria with the
use of chelating agents (that is, EDTA) that function to diminish
the barrier properties provided by the outer LPS membrane of
gram-negative bacteria (Stevens and others 1991; Shefet and others 1995; Scanell and others 1997; Helander and others 1997).
Additional stress to gram-negative bacteria as in a hurdle approach (simultaneous application of several processes or barriers)
also can enhance the effectiveness of a bacteriocin. Cutter and Siragusa (1995) showed that when used with EDTA, citrate, or lactate, nisin (2000 IU/ml) was effective against gram-negative bacteria (E. coli O157:H7 and Salmonella enterica serovar Typhimurium). Ganzle and others (1999) demonstrated that curvacin A
and nisin in combination with low pH, greater than 5% NaCl, or
propylparabene also leads to an increased sensitivity of Salmonella enterica and E. coli towards these bacteriocins. Such work sug-
7/3/2003, 11:39 AM
87

gests that combinations of bacteriocins, processes, and other additives applied in an appropriate manner can significantly broaden
the target spectrum of some LAB bacteriocins against gram-negative bacteria.
Production and Modeling

In order to make commercial use of bacteriocins economically
feasible, optimization of yield during production is necessary. In a
study of nisin, pediocin PA-1 from P. acidilactici, leuconocin
Lcm1 by Leuconostoc carnosum, and sakacin A by L. sake Lb
706, Yang and Ray (1994) found that in growth media, the key
factors were maintenance of optimal pH and supplementation of
the medium with specific nutrients for each strain or species.
Conditions that favored high cell density resulted in a comparable
increase in bacteriocin yield, which is indicative of a primary metabolite. To further enhance yield and purity of LAB bacteriocins
from industrial fermentations and lower production costs, Coventry and others (1996) demonstrated that nisin, pediocin PO2,
brevicin 286, and piscicolin 126 could be produced significantly
free of contaminating proteins (92 to 99%) by using food-grade
diatomite calcium silicate and several desorbing agents as compared to original culture supernatants and ammonium sulfate
preparations. To lower production costs, improvements in speed
and yield of purification methods for bacteriocins from culture
broth continue (Guyonnet and others 2000). Carolissen-Mackay
and others (1997) have reviewed protocols for purification of bacteriocins from LAB.
Models have been devised to predict the commercial effectiveness of bacteriocins based on information gathered in test systems
in order to optimize the application of bacteriocins. Blom and
others (1997) designed a method for simultaneous examination of
the effect of intrinsic factors (pH, concentration of indicator cells,
agar, soy oil, and sodium chloride concentration) on the diffusion
and efficacy of bacteriocins using agar plates. Sakacins A and P,
piscicolin 61, pediocin PA-1, and nisin were each found to have
a unique intrinsic factor effect profile, although pH and the concentration of indicator cells affected all of the bacteriocins similarly. Except for a commercial preparation of nisin, the other bacteriocins were synthesized using genetically engineered strains of
Lactobacillus sake Lb790.
Parente and others (1998) used logistical regression analysis of
categorical data to generate predictive models for the probability
of survival for L. monocytogenes compared with leucocin F10
and nisin in tryptic soy broth with 0.6% yeast extract. Nisin had a
significant effect on the probability of survival, but supplementation with leucocin F10 was necessary to eliminate L. monocytogenes. Lower pH values in the broth decreased survival while
NaCl and EDTA demonstrated a limited effect. In a somewhat different approach, Pleasants and others (2001) modeled the growth
of L. monocytogenes L70 in response to L. sake 706, a producer
of sakacin A. In de Man, Rogosa, and Sharp broth it was found
that the L. sake strain grew much faster than L. monocytogenes
L70 at reduced pH. Excretion of sakacin A by its producer rapidly
reduced the presence of L. monocytogenes in mixed culture. It
was felt that the model could accurately predict growth and interaction of the 2 bacteria in the test medium.
Resistance
Realistically, the presence of an antibacterial substance in a given environment will eventually select for varieties of bacteria resistant to the antagonistic component. As found with therapeutic
antibiotics in the environment, bacteriocin-resistant mutants do
occur. Gravesen and others (2002a) examined the responses of a
number of strains of L. monocytogenes to pediocin PA-1 and ni88
sin, and found a wide range of resistances to the 2 bacteriocins

occurring naturally. The influence of environmental stress (reduced pH, low temperature, and the presence of sodium chloride) appeared bacteriocin-specific; that is, these stresses did not
influence the frequency of resistance to pediocin PA-1, but the
frequency of nisin resistance was significantly reduced. Also of interest, the stability of the phenotype of nisin resistance varied substantially, while resistance to the pediocin was stable with ongoing growth of L. monocytogenes. Fitness costs as measured by reduced growth rate in pediocin-resistant mutants was demonstrable, but nisin-resistant mutants showed limited growth rate reductions. The bacteriocin-resistant mutants of L. monocytogenes
were not more sensitive to the applied environmental stresses
than wild-type strains; in a model sausage system growth differences were minimal.
The genes for synthesis of pediocin PA-1 were transferred into
nisin-producing L. lactis FI5876 by Horn and others (1998, 1999).
As a result, both pediocin PA-1 and nisin A were synthesized concurrently in this strain of Lactococcus. Production levels of pediocin PA-1 were at the same levels as found in the original producer
of the pediocin, P. acidilactici 347. Since pediocin PA-1 and nisin
A are unrelated bacteriocins, use of L. lactis FI5876 as a starter
culture in fermented dairy products should prevent the emergence of any bacteriocin-resistant isolates of L. monocytogenes
because the frequency for emergence of a strain resistant to both
peptides should be low.
Modi and others (2000) evaluated resistance to nisin by L.
monocytogenes to ask the question if spontaneous nisin resistance leads to increased heat resistance relative to the wild-type
strains. In the absence of nisin, there was no significant difference
in heat resistance as shown by D values. When nisin-resistant L.
monocytogenes were grown in the presence of nisin at 55 C, the
cells became more sensitive to heat. When nisin-resistant cells
were subjected to a combined treatment of nisin and heat, there
was a synergistic effect of inactivation. After exposure to heat, nisin-resistant cells once again became sensitive to the effects of nisin. In the case of L. moncytogenes, resistance to nisin does not
appear to be particularly stable, nor impart an undesirable effect
of heightened heat resistance.
Nisin
At one time it was an accurate statement to say that colicins
were the most studied and most understood of the bacteriocins;
however, it is now safe to say that the bacteriocins produced by
gram-positive bacteria are the most investigated group of antibacterial peptides, given their potential for commercial applications
in foods and other products. Nisin has spearheaded this popularity because of its relatively long history of safe use and its documented effectiveness against important gram-positive foodborne
pathogens and spoilage agents.
It was not unusual for early cheesemakers to observe failed or
slow fermentations. While infection with bacteriophage was a
major contributer to this problem, it was recognized that some
dairy streptococci could inhibit others to cause problems in
cheesemaking. In 1928, Rogers and Whittier first published on
the implied inhibitive effect of nisin in which Streptococcus lactis
(now Lactococcus lactis subsp. lactis) inhibited Lactobacillus bulgaricus (Rogers 1928; Rogers and Whittier 1928). Whitehead and
Riddet (1933) described inhibitory streptococci in milk. The
name nisin was coined by Mattick and Hirsch (1947) from N inhibitory substance since L. lactis was originally classified as
Lancefield serological group N Streptococcus. Early efforts to
identify a practical application for nisin included testing its effectiveness for treating mastitis in dairy herds (Taylor and others
1949). Hirsch and others (1951) first examined the potential of ni-
88
7/3/2003, 11:39 AM

Table 2Activity spectra of some Class I and Class IIa bacteriocins
Bacteriocins
Class I
acidocin J1132
Strain
Activity spectra
Lactobacillus
acidophilus JCM
1132
Active against different species of Lactobacillus (9/32)*.

Tahara and others 1996
Not active against Lactococcus (0/6), Pediococcus (0/5),
Streptococcus (0/7), Listeria monocytogenes (0/4),
Bacillus spp. (0/7), and Staphylococcus spp. (0/2).
Active against different species of Enterococcus (4/4),
Ryan and others 1996
Lactobacillus (14/14), Lactococcus (17/18), Leuconostoc
(1/1), Pediococcus (3/3), Streptococcus (2/2),
L. monocytogenes (1/1), Listeria innocua (1/1),
Staphylococcus aureus (1/1), Bacillus spp. (2/2) and
Clostridium spp. (2/2).
Cintas and others 1998
Lactobacillus (8/8), Lactococcus (1/3), Leuconostoc (1/1),
Pediococcus (3/3), L. monocytogenes (5/5), L. innocua (1/1),
Staphylococcus (6/6), Bacillus cereus (1/1), and Clostridium
spp. (5/5).
Cintas and others 1998; Meghrous and
others 1999
Pediococcus (4/4), L. monocytogenes (14/14),
L. innocua (2/2), Listeria grayi (1/1), Listeria ivanovii (1/1),
Listeria murrayi (1/1), Listeria seeligeri (1/1),
Listeria welchimeri (1/1), and Staphylococcus spp. (7/7).
Prevents outgrowth of Bacillus spp. and Clostridium spp.
and bactericidal to their vegetative cells.
Gonzalez and others 1994
Pediococcus (2/2), Streptococcus (2/2), Staphylococcus
carnosus (1/1), Bacillus spp. (2/3), and Clostridium spp. (2/2).
Not active against L. innocua (0/1).
Marciset and others 1997
Streptococcus (1/1), L. monocytogenes (1/1), L. innocua (1/1),
S. carnosus (1/1), Bacillus spp. (2/2), and Clostridium spp. (2/2).
Prevents outgrowth of spores of B. cereus and Clostridium botulinum.
lacticin 3147
Lactococcus lactis
DPC3147
lactocin S
Lactobacillus
sake L45
nisin
Lactococcus lactis
subsp. lactis
plantaricin C
Lactobacillus
plantarum LL441
thermophilin 13
Streptococcus
thermophilus SFi13
Class IIa
acidocin A
Lactobacillus
acidophilus TK9201
bavaricin A
Lactobacillus
sake MI401
curvacin A
Lactobacillus
curvatus LTH1174
divercin V41
Carnobacterium
divergens V41
enterocin A
Enterococcus
faecium CTC492
lactococcin
MMFII
mesentericin
Y105
Lactococcus lactis
MMFII
Leuconostoc
mesenteroides Y105
mundticin
Enterococcus
mundtii ATO6
pediocin PA-1
Pediococcus
acidilactici PAC 1.0
89
References

Lactobacillus (13/32), Pediococcus (2/7), Streptococcus
(8/13), and L. monocytogenes (5/5). Not active against
Bacillus subtilis (0/6) and S. aureus (0/2).
Lactobacillus (11/25), Lactococcus (5/15), Leuconostoc
(4/7), Pediococcus (2/5), and L. monocytogenes (9/10).
Not active against Carnobacterium (0/1), Streptococcus
(0/2), Brochothrix thermosphacta (0/1), Bacillus spp. (0/7),
and Staphylococcus spp. (0/5).
Active against different species of Carnobacterium (3/3),
Enterococcus (1/2), Lactobacillus (10/23), Lactococcus
(1/12), Pediococcus (5/8), L. monocytogenes (7/7),
L. innocua (1/1), and L. ivanovii (1/1). Not active against
Leuconostoc (0/3) and Clostridium spp. (0/12).
Lactobacillus (2/5), Pediococcus (2/2), L. monocytogenes
(1/1), L. innocua (1/1), and L. ivanovi (1/1). Not active
against Lactococcus (0/1) and Leuconostoc (0/3).
(4/4), and L. innocua (2/2).
Lactobacillus (2/2), Lactococcus (2/6), and L. ivanovi (1/1).
Lactobacillus (1/5), Leuconostoc (2/3), Pediococcus (2/2),
L. monocytogenes (1/1), L. innocua (1/1), and L. ivanovi
(1/1). Not active against Lactococcus (0/1)
Enterococcus (2/2), Lactobacillus (2/2), Leuconostoc (2/2),
Pediococcus (2/2), L. monocytogenes (1/1), and L. innocua
(1/1). Prevents the outgrowth of spores and vegetative
cells of C. botulinum.
Enterococcus (2/3), Lactobacillus (23/31), Lactococcus
(1/14), Leuconostoc (3/4), Pediococcus (8/11), L.
monocytogenes (12/12), L. innocua (2/2), L. ivanovii (1/1),
Staphylococcus spp. (2/6), B. cereus (1/1), and Clostridium spp.
(4/17).
Kanatani and others 1995
Larsen and others 1993
Eijsink and others 1998
Guyonnet and others 2000
Aymerich and others 1996

Guyonnet and others 2000
Bennik and others 1998
Cintas and others 1998; Eijsink and

others 1998
Table 2 continued on next page
7/3/2003, 11:39 AM
89

Table 2continued
piscicocin V1a
Carnobacterium
piscicola V1
piscicocin V1b
Carnobacterium
piscicola V1
piscicolin 126
Carnobacterium
piscicola JG126
sakacin A
Lactobacillus
sake LB706
sakacin P
Lactobacillus
sake LB674

Pediococcus (1/1), L. monocytogenes (1/1), and L. innocua
(1/1). Not active against Lactococcus (0/1), B. cereus (0/1),
Clostridium spp. (0/3), and S. aureus (0/1).
Pediococcus (1/1), L. monocytogenes (1/1), and L.
innocua (1/1). Not active against Lactococcus (0/1),
B. cereus (0/1), Clostridium spp. (0/3), and S. aureus (0/1).
Pediococcus (1/2), Streptococcus (2/2), L. monocytogenes
(2/2), L. grayi (1/1), L. ivanovii (1/1), L. seeligeri (1/1),
and B. thermosphacta (1/1). Not active against Bacillus spp.
(0/5), Clostridium spp. (0/2), Lactococcus (0/3), Listeria
denitrificans (0/1), and Staphylococcus spp. (0/3).
Bhugaloo-Vial and others 1996
Bhugaloo-Vial and others 1996
Jack and others 1996
Aymerich and others 1996; Guyonnet

and others 2000
Aymerich and others 1996; Guyonnet
and others 2000
*the numbers inside the bracket represent Number of strains inhibited/number of strains tested.
sin as a food preservative. In 1957, nisin was reported to be commonly occurring in farmhouse cheese (Chevalier and others
1957). In that same year, Aplin and Barrett developed commercial
preparations for use in foods (Delves-Broughton and others
1996). Nisin-like substances were found to be commonplace
among cheese cultures (Hurst 1967), and now it is understood
that lactococci can produce other bacteriocins and inhibitory
substances in addition to nisin.
First elucidated by Gross and Morrell in 1971, nisin is a 34amino acid peptide. At least 6 different forms have been discovered and characterized (designated as A through E and Z), with
nisin A the most active type. Nisin Z is a natural variant nisin differing from nisin A with substitution of a histidine residue for an
aspartic acid. The most established commercially available form
of nisin for use as a food preservative is NisaplinTM, with the active ingredient 2.5% nisin A and the predominate ingredients
NaCl (77.5%) and nonfat dry milk (12% protein and 6% carbohydrate). Several companies market antimicrobial products containing nisin.
Nisin is a lantibiotic. The term lantibiotic comes from lanthionine-containing antibiotic as it contains unusual distinctive posttranslationally modified amino acids, thioether-bridged lanthionine and 3-methyllanthionine, and unsaturated 2,3-didehydroalanine and 2,3-didehydrobutyrine (van Kraaij and others 1999).
These unsaturated or dehydrated residues feature electrophilic
centers that can react with nearby nucleophilic groups (McAuliffe
and others 2001). Consequently, lantibiotics feature polycyclic
structures that are very important in the membrane insertion properties of the bacteriocin. These ring structures are believed to retain the rigidity of the peptide (Kuipers and others 1996) as well
as protect the bacteriocin from proteolytic enzymes and thermal
denaturation (Hurst 1981). Nisin is categorized both as a class I
bacteriocin and a type-A lantibiotic (that is, elongated peptides
with a net positive charge). Closely related lantibiotics not produced by LAB include subtilin from Bacillus subtilis and epidermin from Staphylococcus epidermidis. Like nisin, these peptides
function by disrupting membrane integrity.
Nisin usually has no effect on gram-negative bacteria, yeasts,
90
and molds, although gram-negative bacteria can be sensitized to

nisin by permeabilization of the outer membrane layer as caused
by sublethal heating, freezing, and chelating agents (DelvesBroughton and others 1996). Normally only gram-positive bacteria are affected, and these types include LAB, vegetative pathogens such as Listeria, Staphylococcus, and Mycobacterium, and
the sporeforming bacteria, Bacillus and Clostridium. The spores
of bacilli and clostridia are actually more sensitive to nisin than
their vegetative cells, although the antagonism is sporostatic, not
sporicidal, thus requiring the continued presence of nisin to inhibit outgrowth of the spores. Heat damage of spores substantially
increases their sensitivity to nisin, so that nisin is effective against
spores in low-acid, heat-processed foods, resulting in its use as a
processing aid in canned vegetables. The mechanism whereby nisin inhibits spore outgrowth is unclear, although it has been determined that the sporostatic action of nisin is caused by its binding
to sulfhydryl groups of protein residues (Morris and others 1984).
The mechanism of its sporostatic action is distinct from its bacteriocidal effect on the cytoplasmic membrane of vegetative cells.
Purified nisin has been evaluated for toxicological effect and
found harmless or at least with very low toxicity using rat and
guinea pig models (Frazer and others 1962; Shtenberg and Ignatev 1970). Its use is approved as a food additive in over 50 countries. It is probably safe to say that in most of these countries, nisin is the only bacteriocin authorized for use as a food preservative. International acceptance of nisin was given in 1969 by the
Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives (WHO
1969). The only other antibiotic-like compound with similar approval as a preservative is the surface-active antimycotic compound, pimaricin (Henning and others 1986). The FAO/WHO
Committee recommended a maximum daily intake of nisin for a
70-kg person to be 60 mg of pure nisin or 33000 Units (Hurst and
Hoover 1993); however, nisin is permitted in processed cheeses
in Australia, France, and Great Britain with no maximum limit. In
the U.S., the maximum limit is 10000 IU/g; in Russia, the maximum limit is 8000 IU/g, while in Argentina, Italy, and Mexico, the
limit is 500 IU/g for processed cheeses and other products (Chi-
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kindas and Montville 2002). Examples of international food products that legally can be amended with nisin are canned soups
(Australia), ice for storing fresh fish (Bulgaria), baby foods, baked
goods and mayonnaise (Czech Republic), and milk shakes (Spain)
(Hurst and Hoover 1993); however, the majority of approved
product types are dairy products (especially cheeses) and canned
goods. In the U.S., use of nisin-producing starter cultures has never been regulated, as lactococci are considered GRAS. As inferred
from the FDA Code of Federal Regulations (1988) regarding use of
bacteriocins as food additives, when natural antimicrobials (that
is, bacteriocins such as nisin) are used as food preservatives, the
producing culture must be considered a GRAS microorganism
(hence the search for food preservatives from LAB). The Food
Safety and Inspection Service (FSIS) of the USDA evaluates the
safety and effectiveness of bacteriocins in commodity segments
such as meat and poultry products. Thus depending on the type
of product, the FDA or FSIS approve use of novel bacteriocins before any applications are permitted (Chikindas and Montville
2002).
Activity Spectra and Properties of Class I and Class IIa

Bacteriocins
Activity spectra
Most of the class I bacteriocins have a fairly broad inhibitory

spectrum. They not only inhibit closely related bacteria, such as
species from the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Streptococcus, but also inhibit many less closely related gram-positive bacteria, such as L.
monocytogenes, Staphylococcus aureus, Bacillus cereus, and
Clostridium botulinum. Several bacteriocins in this class, such as
nisin and thermophilin 13, prevent outgrowth of spores of B.
cereus and C. botulinum. Interestingly, acidocin J1132 has a very
narrow inhibitory spectrum and sensitive strains are limited to
members of the genus Lactobacillus (Table 2), while at the other
extreme, plantaricin LP84 (produced by Lactobacillus plantarum
NCIM 2084) has demonstrated antagonism against E. coli (Suma
and others 1998).
Compared to class I bacteriocins, most class IIa bacteriocins
have comparatively narrow activity spectra and only inhibit
closely related gram-positive bacteria. In general, members of the
genera Enterococcus, Lactobacillus, Pediococcus are sensitive to
class IIa bacteriocins, and members of the genus Lactococcus are
resistant (Table 2). For example, Eijsink and others (1998) found
that pediocin PA-1 was active against different species of Enterococcus, Lactobacillus, and Pediococcus; however, only 1 out of
11 Lactococcus strains tested (Lactococcus lactis LMG 2070) was
sensitive to the bacteriocin. Some class IIa bacteriocins, such as
pediocin PA-1, have fairly broad inhibitory spectra and can inhibit some less closely related gram-positive bacteria, such as S. aureus and vegetative cells of Clostridium spp. and Bacillus spp.
Some class IIa bacteriocins, such as mundticin from Enterococcus
mundtii, even prevent the outgrowth of spores of C. botulinum
(Table 2).
As evident in Table 2, class IIa bacteriocins are generally active
against Listeria. Eijsink and others (1998) found that 9 strains of
Listeria tested, including L. monocytogenes, Listeria innocua, and
Listeria ivanovii, were very sensitive to 4 class IIa bacteriocins
(pediocin PA-1, enterocin A, sakacin P, and curvacin A). Moreover, the extent of sensitivity varied from strain to strain. The minimal inhibitory concentrations against L. monocytogenes for the
above 4 bacteriocins varied from 0.1 to 8 ng/ml; however, some
Listeria strains, such as L. monocytogenes V7 and L. innocua LB1,
have been found to be resistant to class IIa bacteriocins (enterocin
A, mesentericin Y105, divercin V41, and pediocin AcH) (Ennahar
91
and others 2000a).

In direct comparison, nisin has been shown to have wider inhibitory spectrum against strains of L. monocytogenes than pediocin PA-1 (Ennahar and others 2000a). Rasch and Knchel (1998)
found that only 2 out of 381 L. monocytogenes strains tested were
able to grow weakly when exposed to 500 IU of nisin/ml. The
majority of strains were able to grow at levels of 100 IU/ml but
not at 500 IU/ml. Although a large portion of the 381 strains tested (67.5%) were unable to grow at the lowest pediocin concentration tested (100 AU/ml), 20 strains grew normally at 1600 AU/
ml. Further studies indicated that a large portion of these 20 pediocin-resistant strains were able to grow normally at the highest
pediocin concentration tested (12800 AU/ml).
It might seem that bacteriocins with broader activity spectra
would always be preferable for use in food preservation, but under certain circumstances bacteriocins with narrower inhibitory
spectra may prove more desirable. For example, sakacin P, which
has limited activity against LAB but is nearly as effective as pediocin PA-1 against Listeria, might find application in LAB fermentation products that are prone to contamination by L. monocytogenes (Eijsink and others 1998).
Properties
Class I and class IIa bacteriocins are usually very stable at acidic pH (Table 3). For example, Rodriguez and others (2002) found
that pediocin PA-1 was perfectly stable after 21 d of storage at
15 C at pH 4 to 6; however, half of the activity was lost at pH 7.
Larsen and others (1993) found that bavaricin A was very stable at
pH 2.0 to 9.7, but storage of bavaricin A at pH 12.5 for 4 h resulted in the complete loss of activity. In addition, bacteriocins from
these 2 classes are heat stable at acidic pH. As pH increases, their
heat stability decreases. Jack and others (1996) found that heating
of piscicolin 126 for 120 min at pH 2 and 3 did not affect its bactericidal activity, while heating for 15 min at pH 4 or 5 reduced
its activity by 50%. In general, bacteriocins are usually sensitive
to proteolytic enzymes, such as trypsin, due to their proteinaceous nature.
Food Applications
Consumers have been consistently concerned about possible
adverse health effects from the presence of chemical additives in
their foods. As a result, consumers are drawn to natural and
fresher foods with no chemical preservatives added. This perception, coupled with the increasing demand for minimally processed foods with long shelflife and convenience, has stimulated
research interest in finding natural but effective preservatives.
Bacteriocins, produced by LAB, may be considered natural preservatives or biopreservatives that fulfill these requirements. Biopreservation refers to the use of antagonistic microorganisms or
their metabolic products to inhibit or destroy undesired microorganisms in foods to enhance food safety and extend shelflife
(Schillinger and others 1996).
Three approaches are commonly used in the application of
bacteriocins for biopreservation of foods (Schillinger and others
1996):
(1) Inoculation of food with LAB that produce bacteriocin in the
products. The ability of the LAB to grow and produce bacteriocin
in the products is crucial for its successful use.
(2) Addition of purified or semi-purified bacteriocins as food
preservatives.
(3) Use of a product previously fermented with a bacteriocinproducing strain as an ingredient in food processing.
Biopreservation of meat products
L. monocytogenes is a gram-positive, non-sporeforming faculta-
7/3/2003, 11:39 AM
91

Table 3Properties of some Class I and Class IIa bacteriocins
Bacteriocins
MW* (Da)
Properties
References
Class I
lacticin 3147A
lacticin 3147B
2847
3322
Ryan and others 1996; Guder and others

2000
nisin
3488
plantaricin C
3500
Heat stable at 100 C for 10 min at pH 5 or 90 C for 10 min at pH 7.

Sensitive to trypsin, -chymotrypsin, proteinase K, and pronase E,
resistant to pepsin.
Heat stable at 121 C for prolonged heating at pH 2. Become less
heat stable at pH 5-7. Sensitive to -chymotrypsin, resistant to
trypsin, elastase, carboxypeptidase A, pepsin, and erepsin.
Stable at room and low temperatures, heat stable at 100 C for
60 min or 121 C for 10 min. Most stable at acid and neutral pHs.
Sensitive to pronase, trypsin, and -chymotrypsin, resistant to
pepsin, proteinase K, -amylase, and lipase.
Class IIa
bavaricin A
3500-4000
lactococcin MMFII 4143

pediocin PA-1
4624
piscicolin 126
4416
Heat stable at 100 C for 60 min. Stable at pH 2.0 to 9.7. Sensitive

to pepsin, trypsin, pronase E, proteinase K and chymotrypsin A4,
resistant to catalase.
Heat stable at 70 C for 30 min. Stable at pH 5 to 8. Sensitive to
proteinase K, trypsin and papain, resistant to glucoamylase,
lipase, -amylase and lysozyme.
Stable at pH 4 to 6, becomes less stable as pH increases. Heat
stable at 80 C for 60 min or 100 C for 10 min. Sensitive to trypsin,
papain, ficin, -chymotrypsin, protease IV, XIV, and XXIV, and
proteinase K, resistant to phospholipase C, catalase, lysozyme,
DNAses, RNAses, and lipase.
Stable at pH 2 after 2-mo storage at 4 C. Heat stable at 100 C for
120 min at pH 2 to 3. Becomes less heat stable as pH increases.
Sensitive to -chymotrypsin, -chymotrypsin, protease type I, XIV,
XXIII, and trypsin, resistant to catalase, lipase, and lysozyme.
Hurst and Hoover 1993; Delves-Broughton

and others 1996
Gonzalez and others 1994
Larsen and others 1993

Rodrguez and others 2002
Jack and others 1996
*MW = molecular weight
tively anaerobic rod widely distributed in the natural environment. It can grow over a pH range of 4.1 to 9.6 and a temperature
range of 0 to 45 C. Moreover, it is relative resistant to desiccation
and can grow at aw values as low as 0.90. The ubiquitous nature
of L. monocytogenes, its hardiness and ability to grow at refrigeration temperatures and anaerobic conditions make it a threat to the
safety of foods. It is regarded as a major food safety problem because it can cause serious illnesses and death. The United States
government has the most rigid policy regarding L. monocytogenes
and set a zero tolerance level for L. monocytogenes in ready-toeat foods (Jay 1996; Ryser and Marth 1999). It has been detected
in a variety of foods and implicated in several foodborne outbreaks, such as turkey franks (Jay 1996). Many studies have been
carried out to control L. monocytogenes in meat products since it
is common within slaughterhouse and meat-packing environments and has been isolated from raw meat, and cooked and
ready-to-eat meat products (Ryser and Marth 1999).
The activity of nisin alone at concentrations of 400 and 800 IU/
g and in combination with 2% sodium chloride against L. monocytogenes in minced raw buffalo meat was examined by Pawar
and others (2000). Samples of the raw meat mince were inoculated with 103 CFU/g of L. monocytogenes and stored at 4 C. The
counts of L. monocytogenes in the control samples increased
from 3.0 log10 to 6.4 log CFU/g after 16-d storage; however, nisin
significantly inhibited the growth of L. monocytogenes. Addition
of nisin at a level of 400 IU/g increased the lag phase of L. monocytogenes, and at a level of 800 IU/g resulted in counts of L.
monocytogenes 2.4-log10 cycles lower than the control samples
after 16-d storage. When the storage temperature was increased to
37 C, the inhibition effects of nisin were less pronounced. Addition of 2% sodium chloride in combination with nisin was found
to increase the efficacy of nisin at both storage temperatures.
Sections of beef carcass were inoculated with approximately 4
log10 CFU/cm2 of Brochothrix thermosphacta, Carnobacterium di92
vergens, or L. innocua by Cutter and Siragusa (1994) to evaluate

the effectiveness of nisin to sanitize the surface of red meat carcasses. Spray treatments with water did not significantly alter the
bacterial populations; however, nisin spray treatments (5000 AU/
ml) reduced populations by 1.8 to 3.5 log10 CFU/cm2 at d 0 and
by 2.0 to 3.6 log10 CFU/cm2 after storage at 4 C for 1 d.
Fang and Lin (1994) found that addition of 10000 IU/ml of nisin to inoculated cooked tenderloin pork inhibited the growth of L
monocytogenes, but not Pseudomonas fragi. Nisin was found to
be more effective when used in combination with modified atmosphere packaging (MAP, 100% CO2, 80% CO2 + 20% air). MAP
and nisin (1000 or 10000 IU/ml) inhibited growth of both organisms, and this inhibitory effect for MAP/nisin combination system
was more pronounced at 4 C than at 20 C.
Murray and Richard (1997) evaluated the antilisterial activity of
nisin A and pediocin AcH in decontamination of artificially contaminated pieces of raw pork. Nisin A was considerably more efficient than pediocin AcH, but after 2 d of storage, surviving bacteria in meat treated with each bacteriocin resumed growth at a rate
similar to that of the control. Moreover, nisin was more stable
than pediocin AcH. The loss of effectiveness, especially of pediocin AcH, was attributed to rapid degradation by meat proteases.
In addition to nisin and pediocin, other LAB bacteriocins have
been examined to control the growth of Listeria. Examples include Laukov and others (1999), who examined the effectiveness
of enterocin CCM 4231 in controlling L. monocytogenes contamination in dry fermented Hornd salami. Addition of enterocin reduced the counts of L. monocytogenes by 1.7-log10 cycles immediately after addition of the bacteriocin (initial counts: 108 CFU/
ml). After 1 wk of ripening of the salami, the L. monocytogenes
count in the control (without enterocin added) reached 107 CFU/
g, while in the treated sample (with enterocin added) the count
was 104 CFU/g, a difference that was maintained after 2 and 3 wk
of ripening. Also, Hugas and others (1998) found that sakacin K, a
92
7/3/2003, 11:39 AM

Table 4Use of bacteriocinogenic protective cultures to control Listeria monocytogenes in meat products
Meat products
Meats
minced meat and comminuted cured raw
pork filled into casings
Vacuum packaged
minimally heat-treated beef cubes
wieners
frankfurters
Fermented
dry fermented sausage
dry fermented sausages
chicken summer sausages
salami
dry fermented sausage
turkey summer sausage
Modified atmosphere packaged
Brazilian sausage
Protective culture
References
Lactobacillus sake Lb 706
Schillinger and others 1991
Lactobacillus bavaricus MN
Pediococcus acidilactici JBL 1095
Pediococcus acidilactici JD1-23
Winkowski and others 1993

Degnan and others 1992
Berry and others 1991
Staphylococcus xylosus DD-34,

Pediococcus acidilactici PA-2, and
Lactobacillus bavaricus MI-401
Lactobacillus sake CTC494
Pediococcus acidilactici
Lactobacillus plantarum MCS
Pediococcus acidilactici PAC 1.0
Pediococcus acidilactici JBL 1095
Lahti and others 2001

Hugas and others 1995
Baccustaylor and others 1993
Campanini and others 1993
Foegeding and others 1992
Luchansky and others 1992
Lactobacillus sake 2a
Liserre and others 2002
bacteriocin produced by Lactobacillus sake CTC494, inhibited

the growth of L. innocua in vacuum-packaged samples of poultry
breasts and cooked pork, and in modified atmosphere-packaged
samples of raw minced pork. Schbitz and others (1999) determined the inhibitory capacity of a bacteriocin-like substance produced by Carnobacterium piscicola L103 against L. monocytogenes, and found that the bacteriocin completely inhibited L. monocytogenes in vacuum-packaged meat after 14 d of storage at 4 C.
Vignolo and others (1996) showed that lactocin 705 produced by
Lactobacillus casei CRL 705 inhibited the growth of L. monocytogenes in ground beef; however, when the producer strain was
added to the slurry, no significant inhibition was detected.
Degnan and others (1992) demonstrated the possibility of using
bacteriocinogenic cultures of Pediococcus acidilactici (pediocin
AcH producer) to control L. monocytogenes growth in vacuumpackaged all-beef wieners (Table 4). When wieners were surfaceinoculated with L. monocytogenes and P. acidilactici and vacuum-packaged, L. monocytogenes and the pediococci survived in
packages held at 4 C, but the pediococci did not produce acid or
pediocin during refrigerated storage. When packages were temperature-abused at 25 C for 8 d, total numbers of L. monocytogenes in the control (without pediococci added) increased by 3.2
log10 CFU/g. In contrast, L. monocytogenes was inhibited (average reduction of 2.7 log10 CFU/g) in packages inoculated with
pediococci. The onset of bacteriocin production coincided with
early logarithmic growth of pediococci and continued into the
late logarithmic phase.
The primary reasons that nitrite is commonly used in curing
meats is to stabilize red meat color and inhibit food spoilage and
poisoning organisms, such as C. botulinum; however, nitrite can
react with secondary amines in meats to form carcinogenic nitrosamines. This possible adverse health effect has prompted researchers to explore the potential of using bacteriocins as an alternative to nitrite. Rayman and others (1981) reported that a combination of 3000 IU/g of nisin and 40 ppm of nitrite almost completely inhibited outgrowth of Clostridium sporogenes spores in
meat slurries at 37 C for 56 d; however, in a later study (Rayman
and others 1983) they found that up to 22000 IU/g of nisin in
combination with 60 ppm of nitrite failed to prevent outgrowth of
C. botulinum spores in meat slurries at pH 5.8. Reducing the pH
was found to enhance nisin activity. For example, 8000 IU/g of
nisin in combination with 60 ppm of nitrite inhibited outgrowth
of the spores in pork slurries at pH 5.5. Taylor and others (1985)
showed that combinations of nisin and nitrite were able to delay
botulinal toxin formation in chicken frankfurter emulsions (pH
93
5.9 to 6.2). Control samples (no preservatives added) became toxic within 1 wk, while a combination of 4000 IU/g of nisin and
120 ppm of nitrite delayed toxin formation to 5 wk.
Biopreservation of dairy products
L. monocytogenes has been the documented cause of a number

of outbreaks associated with dairy products, such as pasteurized
milk (Fleming and others 1985) and cheese (James and others
1985), and nisin has been shown effective against L. monocytogenes in dairy products. Ferreira and Lund (1996) found that following inoculation of a nisin-resistant strain into long-life cottage
cheese at pH 4.6 to 4.7, the number of L. monocytogenes decreased approximately 1-log10 cycle during storage at 20 C for 7
d. Addition of nisin (2000 IU/g) to the cottage cheese increased
the rate of inactivation to approximately 3-log10 cycles in 3 d.
Davies and others (1997) determined the efficacy of nisin to control L. monocytogenes in ricotta-type cheeses over 70 d at 6 to
8 C. Addition of nisin (100 IU/ml) effectively inhibited the growth
of L. monocytogenes for a period of 8 wk or more (dependent on
cheese type). The control cheese contained unsafe levels of the
organism within 1 to 2 wk of storage. Zottola and others (1994)
used nisin-containing cheddar cheese that had been made with
nisin-producing lactococci as an ingredient in pasteurized process cheese or cold pack cheese spreads. The shelflife of the nisin-containing pasteurized process cheese (301 and 387 IU nisin/
g) was significantly greater than that of the control cheese
spreads. In cold pack cheese spreads, nisin (100 and 300 IU/g)
significantly reduced the numbers of L. monocytogenes, S. aureus, and heat-shocked spores of C. sporogenes. Another problem
in cheese production is the Clostridium-associated butyric acid
fermentation. Nisin is commonly added to pasteurized processed
cheese spreads to prevent the outgrowth of clostridia spores, such
as Clostridium tyrobutyricum (Schillinger and others 1996).
One application of lacticin 3147, a broad-spectrum, 2-component bacteriocin produced by L. lactis subsp. lactis DPC 3147, is
to control cheddar cheese quality by reducing non-starter LAB
populations during ripening (Ross and others 1999). Cheese manufactured with the lacticin 3147-producing transconjugant, L. lactis DPC4275, contained 2 log10 less non-starter LAB than control
cheese after 6 mo of ripening. Moreover, cheese manufactured
with 3 natural lacticin 3147-producing strains had no detectable
non-starter LAB over the same time period. In cottage cheese the
population of L. monocytogenes was reduced by 3-log10 cycles
over a 1-wk ripening period when it was manufactured with L.
lactis DPC4275; however, the number of Listeria in the control
7/3/2003, 11:39 AM
93

Table 5Hurdle technology to enhance food safety
Bacteriocins
In combination with heat

nisin
nisin
nisin, pediocin AcH
Inactivation effects
References
Nisin (1000 IU/g) enhances inactivation of Listeria monocytogenes in lobster

by mild heat (60 or 65 C).
Nisin (500 to 2500 IU/ml) enhances inactivation of Salmonella Enteritidis by
mild heat (55 C).
Both bacteriocins reduced the viability of gram-negative and gram-positive
bacterial cells surviving sublethal stresses.
Budu-Amoako and others 1999
In combination with chelating agents

nisin
When used with EDTA, citrate, or lactate, nisin (2000 IU/ml) is effective
against gram-negative bacteria (Salmonella Typhimurium and E. coli O157:H7).
in combination with modified atmosphere packaging (MAP)
nisin
When used with MAP and low temperature, nisin at a level of 400 IU/ml
increases the lag phase of L. monocytogenes, and at 1250 IU/ml prevents
its growth.
nisin
Combined use of MAP (100% CO2, 80% CO2 + 20% air) and nisin (1000 or
10000 IU/ml) inhibits growth of L. monocytogenes and Pseudomonas fragi.
In combination with antimicrobials
nisin
The combined use of potassium sorbate (0.3%) and nisin (400 IU/ml) inhibited
the growth of L. monocytogenes.
pediocin AcH
Synergistic effects between sodium diacetate (0.3 and 0.5%) and pediocin
(5000 AU/ml) against L. monocytogenes.
nisin
Synergist effect between sucrose fatty acid esters and nisin on inhibition of
gram-positive bacteria.
nisin
Carbon dioxide and nisin act synergistically against L. monocytogenes.
nisin
When combined with carvacrol (0.3 mmol /l), nisin (6 IU/ml) is more effective
in reducing the counts of Bacillus cereus than when it is applied alone.
nisin
Nisin (100 IU/ml) and monolaurin (0.25 mg/l) act synergistically against
Bacillus sp. vegetative cells in milk.
In combination with lactoperoxidase system
nisin
A synergistic and lasting bactericidal effect on L. monocytogenes between
nisin (100 or 200 IU/ml) and lactoperoxidase system.
nisin
Synergistic effect of nisin (10 or 100 IU/ml) and the lactoperoxidase system
on inactivation of L. monocytogenes in skim milk.
In combination with other bacteriocins
pediocin AcH
When used with nisin, lacticin 481, or lactacin F, pediocin AcH produced
synergistic effects.
leucocin F10
In combination with nisin, leucocin F10 provides greater activity against
L. monocytogenes.
curvaticin
Simultaneous or sequential additions of nisin (50 IU/ml) and curvaticin 13
(160 AU/ml) induces a greater inhibitory effect against L. monocytogenes
than the use of a single bacteriocin.
cheese, manufactured with a non-lacticin 3147-producing starter,

remained unchanged (104 CFU/g). The lacticin 3147-producing
transconjugant has also been used as a protective culture to inhibit Listeria on the surface of a mold-ripened cheese. Presence of the
lacticin 3147 producer on the cheese surface reduced the number
of L. monocytogenes by 3-log10 cycles (Ross and others 1999).
Biopreservation of seafood products
The effectiveness of bacteriocins and protective cultures to

control growth of L. monocytogenes in vacuum-packed coldsmoked salmon has been demonstrated by several researchers.
Katla and others (2001) examined the inhibitory effect of sakacin
P and/or L. sake cultures (sakacin P producer) against L. monocytogenes in cold-smoked salmon. The vacuum-packaged salmon
samples were incubated at 10 C for 4 wk. Sakacin P had an initial inhibiting effect on growth of L. monocytogenes while cultures
of L. sake had a bacteriostatic effect. When L. sake culture was
added to salmon together with sakacin P, a bacteriocidal effect
against L. monocytogenes was observed. Nilsson and others
(1999) showed that a nonbacteriocin-producing strain of C. piscicola was as effective as a bacteriocin-producing strain of C. piscicola in the inhibition of L. monocytogenes in vacuum-packed
cold-smoked salmon. They suggested that the growth inhibition of
94
Kalchayanand and others 1992
Cutter and Siragusa 1995

Szabo and Cahill 1998
Fang and Lin 1994
Buncic and others 1995

Schlyter and others 1993
Thomas and others 1998
Nilsson and others 2000
Periago and others 2001
Mansour and Millire 2001
Boussouel and others 2000

Zapico and others 1998
Mulet-Powell and others 1998

Parente and others 1998
Bouttefroy and Millire 2000
L. monocytogenes was probably due to the competitive growth of

C. piscicola that resulted in depletion of essential nutrients.
The inhibitory effect of nisin in combination with carbon dioxide and low temperature on the survival of L. monocytogenes in
cold-smoked salmon has also been investigated (Nilsson and others 1997). Addition of nisin (500 or 1000 IU/g) to salmon inoculated with L. monocytogenes and stored at 5 C delayed, but did
not prevent growth of L. monocytogenes in vacuum-packs. Numbers of L. monocytogenes increased to 108 CFU/g in vacuumpacked salmon in 8 d, whereas CO2 packing of cold-smoked
salmon resulted in an 8-d lag phase for L. monocytogenes with
numbers eventually reaching 106 CFU/g in 27 d. Addition of nisin
to CO2-packed cold-smoked salmon resulted in a 1- to 2-log10 reduction of L. monocytogenes followed by a lag phase of 8 and 20
d in salmon using 500 and 1000 IU nisin/g, respectively. The levels of L. monocytogenes remained below 103 CFU/g during 27 d
of storage at both concentrations of nisin.
In order to improve shelflife, brined shrimp are typically produced with the addition of sorbic and benzoic acids. Concerns
about the use of these organic acids have led researchers to explore the potential of using bacteriocins for their preservation. The
effectiveness of nisin Z, carnocin UI49, and a preparation of crude
bavaricin A on shelflife extension of brined shrimp was evaluated
94
Boziaris and others 1998
7/3/2003, 11:39 AM

Table 6Simultaneous application of bacteriocins and high hydrostatic pressure or pulsed electric field (PEF) to enhance food
safety
Bacteriocins
Inactivation effects
References
In combination with high hydrostatic pressure

nisin
Nisin (100 IU/ml) increases pressure (155 to 400 MPa) inactivation of
Escherichia coli, Salmonella Enteritidis, Salmonella Typhimurium, Shigella
sonnei, Pseudomonas fluorescens, and Staphylococcus aureus.
nisin
Nisin (62400 IU/ml) improves pressure (500 MPa) inactivation of cheese
indigenous microbiota.
nisin
Nisin (100 IU/ml) enhances pressure (200 to 600 MPa) inactivation of
pressure-resistant E. coli cells.
nisin
Nisin (1 or 10 IU/ml) in combination with pressure (404 MPa), heat and
reduced pH, enhances inactivation of spores of Bacillus subtilis and
Clostridium sporogenes.
nisin
Nisin (200 IU/ml) significantly improves pressure (450 MPa) inactivation
of E. coli.
nisin
Nisin (4000 IU/g) in combination with pressure (350 MPa ) and glucono-deltalactone (1%) extends the shelflife of poultry meat.
nisin
Nisin (>0.2 IU/ml) improves pressure (400 MPa) inactivation of Bacillus
coagulans spores.
pediocin AcH
Pediocin AcH (3000 AU/ml) enhances pressure (345 MPa) inactivation of
S. aureus, Listeria monocytogenes, S. typhimurium, E. coli O157:H7,
Lactobacillus sake, Leuconostoc mesenteroides, Serratia liquefaciens, and
P. fluorescens.
lacticin 3147
Lacticin 3147 (10000 or 15000 AU/ml) causes increased pressure
(150 to 275 MPa) inactivation of S. aureus and Listeria innocua in milk
and whey.
In combination with PEF
nisin
Nisin (2.4 IU/ml) enhances inactivation of vegetative cells of Bacillus cereus
by PEF treatment (16.7 kV/cm, 50 pulses each of 0.002-ms duration).
nisin
Nisin (10, 100 IU/ml) enhances inactivation of L. innocua in liquid whole
egg and skim milk by pulsed electric fields (30, 40, or 50 kV/cm, 10.6, 21.3,
and 32 pulses each of 0.002-ms duration).
by Einarsson and Lauzon (1995). Carnocin UI49 did not extend

the shelflife compared to control (10-d shelflife), while bavaricin A
resulted in a shelflife of 16 d. Nisin Z delivered a shelflife of 31 d.
The benzoate-sorbate solution was superior as it preserved the
brined shrimp for the entire storage period of 59 d.
In a study using vacuum-packed cold-smoked rainbow trout,
Nyknen and others (2000) examined the inhibition of L. monocytogenes and mesophilic aerobic bacteria by nisin, sodium lactate, or their combination. Trout samples were stored at 8 C for
17 d or at 3 C for 29 d. Both nisin and lactate inhibited the
growth of L. monocytogenes in smoked fish, but the combination
of the 2 compounds was even more effective. The combination of
nisin and sodium lactate injected into smoked fish decreased the
count of L. monocytogenes from 3.3 to 1.8 log10 CFU/g over 16 d
of storage at 8 C. The level of L. monocytogenes remained almost
constant (4.7 to 4.9 log10 CFU/g) for 29 d at 3 C in the samples
injected before smoking and which contained both nisin and sodium lactate.
Hurdle technology to enhance food safety
The major functional limitations for the application of bacteriocins in foods are their relatively narrow activity spectra and moderate antibacterial effects. Moreover, they are generally not active
against gram-negative bacteria. To overcome these limitations,
more and more researchers use the concept of hurdle technology
to improve shelflife and enhance food safety (Table 5 and 6). It is
well documented that gram-negative bacteria become sensitive to
bacteriocins if the permeability barrier properties of their outer
membrane are impaired. For example, chelating agents, such as
EDTA, can bind magnesium irons from the lipopolysaccharide
layer and disrupt the outer membrane of gram-negative bacteria,
thus allowing nisin to gain access to the cytoplasmic membrane
(Abee and others 1995).
95
Masschalck and others 2001

Capellas and others 2000
Masschalck and others 2000
Stewart and others 2000
Ponce and others 1998
Yuste and others 1998
Roberts and Hoover 1996
Kalchayanand and others 1998
Morgan and others 2000
Pol and others 2000

Caldern-Miranda and others 1999a, 1999b
It is well documented that nisin enhances thermal inactivation

of bacteria, thus reducing the treatment time and resulting in better food qualities (Table 5). For example, Boziaris and others
(1998) found that addition of nisin (500 to 2500 IU/ml) in media,
liquid whole egg, or egg white caused a reduction of required
pasteurization time of up to 35%. Budu-Amoako and others
(1999) found that nisin reduced the heat resistance of L. monocytogenes in lobster meat and significantly reduced the treatment
time compared with thermal treatment alone. The reduced heat
process resulted in significant reduction in drained weight loss
that would allow considerable cost savings.
The synergistic effect between bacteriocins and other processing technologies on the inactivation of microorganisms has also
been frequently reported in the literature (Table 5). Schlyter and
others (1993) reported synergistic effects between sodium diacetate and pediocin against L. monocytogenes in meat slurries. In
the control samples, counts of L. monocytogenes increased from
4.5 log10 to approximately 8 log10 CFU/ml within 1 d at 25 C and
within 14 d at 4 C. A listericidal effect (approximately 7-log10
CFU/ml difference compared to the control samples) was observed in treatments containing pediocin (5000 AU/ml) with 0.5%
diacetate at 25 C and pediocin with 0.3% diacetate at 4 C.
Zapico and others (1998) showed a synergistic effect of nisin (10
or 100 IU/ml) and the lactoperoxidase system on inactivation of L.
monocytogenes in skim milk. Addition of nisin and lactoperoxidase system resulted in counts of L. monocytogenes up to 5.6log10 cycles lower than the control milk after 24 h at 30 C, while
nisin alone had no effect on the counts. This synergistic inactivation was also observed in gram-negative bacteria, which are normally insensitive to these bacteriocins (Boziaris and others 1998).
The use of combinations of various bacteriocins has also been
shown to enhance antibacterial activity (Hanlin and others 1993;
Mulet-Powell and others 1998). When used in combination with
7/3/2003, 11:39 AM
95

nisin, leucocin F10 provides greater activity against L. monocytogenes (Parente and others 1998).
There has been continued interest in the food industry in using
nonthermal processing technologies, such as high hydrostatic
pressure (HP) and pulsed electric field (PEF) in food preservation.
It is frequently observed that bacteriocins, in combination with
these processing techniques, enhance bacterial inactivation (Table 6). In addition, gram-negative bacteria that are usually insensitive to LAB bacteriocins, such as E. coli O157:H7 and S. Typhimurium, become sensitive following HP/PEF treatments that induce sublethal injury to bacterial cells (Kalchayanand and others
1994). Studies in our laboratory also demonstrate that nisin enhances the pressure inactivation of spores of Bacillus coagulans,
Bacillus subtilis, and C. sporogenes (Roberts and Hoover 1996;
Stewart and others 2000).
Bacteriocins in packaging film
Incorporation of bacteriocins into packaging films to control

food spoilage and pathogenic organisms has been an area of active research for the last decade. Antimicrobial packaging film
prevents microbial growth on food surface by direct contact of
the package with the surface of foods, such as meats and cheese.
For this reason, for it to work, the antimicrobial packaging film
must contact the surface of the food so that bacteriocins can diffuse to the surface. The gradual release of bacteriocins from a
packaging film to the food surface may have an advantage over
dipping and spraying foods with bacteriocins. In the latter processes, antimicrobial activity may be lost or reduced due to inactivation of the bacteriocins by food components or dilution below
active concentration due to migration into the foods (Appendini
and Hotchkiss 2002).
Two methods have been commonly used to prepare packaging
films with bacteriocins (Appendini and Hotchkiss 2002). One is
to incorporate bacteriocins directly into polymers. Examples include incorporation of nisin into biodegradable protein films
(Padgett and others 1998). Two packaging film-forming methods,
heat-press and casting, were used to incorporate nisin into films
made from soy protein and corn zein in this study. Both cast and
heat-press films formed excellent films and inhibited the growth
of L. plantarum. Compared to the heat-press films, the cast films
exhibited larger inhibitory zones when the same levels of nisin
were incorporated. Incorporation of EDTA into the films increased
the inhibitory effect of nisin against E. coli. Siragusa and others
(1999) incorporated nisin into a polyethylene-based plastic film
that was used to vacuum-pack beef carcasses. Nisin retained activity against Lactobacillus helveticus and B. thermosphacta inoculated in carcass surface tissue sections. An initial reduction of 2log10 cycles of B. thermosphacta was observed with nisin-impregnated packaged beef within the first 2 d of storage at 4 C. After
20 d of refrigerated storage at 4 or 12 C (to simulate temperature
abuse), B. thermosphacta populations from nisin-impregnated
plastic-wrapped samples were significantly less than control
(without nisin). Coma and others (2001) incorporated nisin into
edible cellulosic films made with hydroxypropylmethylcellulose
by adding nisin to the film-forming solution. Inhibitory effect
could be demonstrated against L. innocua and S. aureus, but film
additives such as stearic acid, used to improve the water vapor
barrier properties of the film, significantly reduced inhibitory activity. It was noted that desorption from the film and diffusion into
the food required further optimization for nisin to function more
effectively as a preservative agent in the packaged food.
Another method to incorporate bacteriocins into packaging
films is to coat or adsorb bacteriocins to polymer surfaces. Examples include nisin/methylcellulose coatings for polyethylene films
and nisin coatings for poultry, adsorption of nisin on polyethylene,
ethylene vinyl acetate, polypropylene, polyamide, polyester,
96
acrylics, and polyvinyl chloride (Appendini and Hotchkiss 2002).

Bower and others (1995) demonstrated that nisin adsorbed onto
silanized silica surfaces inhibited the growth of L. monocytogenes. Nisin films were exposed to medium containing L. monocytogenes and the contacting surfaces were evaluated at 4-h intervals for 12 h. Cells on surfaces that had been in contact with a
high concentration of nisin (40000 IU/ml) exhibited no signs of
growth and many displayed evidence of cellular deterioration.
Surfaces contacted with a lower concentration of nisin (4000 IU/
ml) had a smaller degree of inhibition. In contrast, surfaces contacted with films of heat-inactivated nisin allowed L. monocytogenes to grow. L. innocua and S. aureus (along with L. lactis subsp. lactis) were also used in a study by Scannell and others (2000)
of cellulose-based bioactive inserts and antimicrobial polyethylene/polyamide pouches. Lacticin 3147 and nisin were the tested
bacteriocins. Although lacticin 3147 adhered poorly to plastic
film, nisin bound well and the bioactive film made with nisin was
stable for 3 mo with or without refrigeration. Bacterial reductions
of up to 2-log10 CFU/g cycles in vacuum-packed cheese were
seen in combination with modified atmosphere packaging (MAP)
with storage at refrigeration temperatures. Cellulose-based bioactive inserts were placed between sliced products of cheese and
ham under MAP. Inserts with immobilized nisin reduced L. innocua (starting inocula of 2 to 4 x 105 CFU/g) by >3 log10 CFU/g in
cheese after 5 d at 4 C, and by approximately 1.5 log10 CFU/g in
sliced ham after 12 d, while S. aureus (starting inocula of 2 to 4 x
105 CFU/g) was reduced by 1.5 and 2.8 log10 CFU/g in cheese
and ham, respectively. The efficacy of bacteriocins coatings on
the inhibition of pathogens has also been demonstrated in other
studies. For example, coating of pediocin onto cellulose casings
and plastic bags has been found to completely inhibit growth of
inoculated L. monocytogenes in meats and poultry through 12-wk
storage at 4 C. (Ming and others 1997). Coating of solutions containing nisin, citric acid, EDTA, and Tween 80 onto polyvinyl
chloride, linear low density polyethylene, and nylon films reduced the counts of Salmonella Typhimurium in fresh broiler
drumstick skin by 0.4- to 2.1-log10 cycles after incubation at 4 C
for 24 h (Natrajan and Sheldon 2000).
Although shelflife was extended in food products as populations of food spoilage organisms were reduced, the primary thrust
was towards control of specific anticipated pathogens in the
product. In this regard, Rhodia, Inc., is developing a casing to be
used in hot dog manufacture and other cooked meats (D. Willrett,
personal communication, 2002). The film harbors a proprietary
combination of bacteriocins, enzymes, and botanicals. The components have received regulatory clearance. The approach is to
cook the meat product while tightly contained within the bioactive casing or wrapper. The target is L. monocytogenes and results
are described as very promising. The added cost is considered
economically sound given the large product recalls experienced
by major meat brands as the result of product contaminated with
L. monocytogenes.
Closing Remarks
Although intensive studies over the last decade have greatly advanced our knowledge base about bacteriocins, further work is
needed before we are able to fully understand the molecular
mechanisms, structure-function relationships, and mechanisms of
action of bacteriocins. In the biosynthesis of lantibiotics, the function of the enzymes responsible for modification reactions is still
not clearly understood. The mechanism of producer immunity remains to be answered. Research in these areas is critical for the
effective applications of bacteriocins and would help develop
methods to genetically engineer bacteriocins with better activity,
solubility, and stability.
96
7/3/2003, 11:39 AM

Genetic engineering or chemical modifications of bacteriocins
to improve their activity and properties can be expected to persist
and possibly thrive. For example, Rollema and others (1995) improved the solubility and stability of nisin Z by replacing Asn-27
or His-31 with lysine; Johnsen and others (2000) improved the
stability of pediocin PA-1 at 4C and room temperature by replacing Met-31 with alanine, isoleucine or leucine; Miller and others
(1998) found that a pediocin PA-1 chimeric protein mutant displayed approximately 2.8-fold-higher activity against an indicator
strain, L. plantarum. Examples such as these indicate solutions to
some of the problems related to bacteriocin application; however,
getting the regulatory approval for the use of engineered bacteriocins would be very difficult if they are considered as new proteins
(Montville and others 1995).
Although many bacteriocins have been isolated and characterized, only a few have demonstrated commercial potential in food
application. At the time of this writing, nisin is the only purified
bacteriocin approved for food use in the U.S. It has been used as
a food preservative in more than 50 countries, mainly in cheese,
canned vegetables, various pasteurized dairy, liquid egg products,
and salad dressings (Guder and others 2000). The applications of
other bacteriocins in food preservation have been studied intensively. The use of pediocin PA-1 for food biopreservation has
been commercially exploited and is covered by several U.S. and
European patents (Ennahar and others 2000b; Rodriguez and others 2002). Fermentate containing pediocin PA-1, AltaTM, is commercially available and used as a food preservative to increase
shelflife and inhibit the growth of bacteria, especially L. monocytogenes in ready-to-eat meats (Rodriguez and others 2002). Lacticin 3147, which is active over a wider pH range than nisin, is expected to find applications in non-acid foods (Ross and others
1999).
Since bacteriocins for use as food preservatives have relatively
narrow activity spectra and are generally not active against gramnegative bacteria, it can be expected that nisin and other bacteriocins will continue to be incorporated and developed into hurdle
concept technologies for food preservation. The simultaneous application of bacteriocins and nonthermal processing technologies,
such as HP and PEF, to improve shelflife of foods is attractive since
foods produced using these nonthermal technologies usually have
better sensory and nutritional qualities compared with products
produced using conventional thermal processing methods.
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MS 20020567
The authors are with Dept. of Animal & Food Sciences, Univ. of Delaware,
Newark, DE 19716-2150. Direct inquiries to author Hoover (E-mail:
dgh@udel.edu).
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Bacteriocins and Their Food Applications

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Bacteriocins

and their Food

FDA approval for use of nisin in pasteurized processed cheese

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 2, 2003

2003 Institute of Food Technologists