Nisin As A Food Preservative - Part 1 - Physicochemical Properties, Antimicrobial Activity, and Main Uses
Nisin As A Food Preservative - Part 1 - Physicochemical Properties, Antimicrobial Activity, and Main Uses
Nisin As A Food Preservative - Part 1 - Physicochemical Properties, Antimicrobial Activity, and Main Uses
To cite this article: Adem Gharsallaoui, Nadia Oulahal, Catherine Joly & Pascal Degraeve (2015): Nisin as a Food Preservative:
Part 1: Physicochemical Properties, Antimicrobial Activity, and Main Uses, Critical Reviews in Food Science and Nutrition, DOI:
10.1080/10408398.2013.763765
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Dynamique Microbienne aux Interfaces Alimentaires), Equipe Mixte d’Accueil n°3733, IUT
Abstract
Nisin is a natural preservative for many food products. This bacteriocin is mainly used in dairy
and meat products. Nisin inhibits pathogenic food borne bacteria such as Listeria monocytogenes
and many other Gram-positive food spoilage microorganisms. Nisin can be used alone or in
combination with other preservatives or also with several physical treatments. This article
reviews physicochemical and biological properties of nisin, the main factors affecting its
antimicrobial effectiveness, and its food applications as an additive directly incorporated into
food matrices.
preservation.
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Corresponding author:
adem.gharsallaoui@univ-lyon1.fr
Phone (+33) 4 74 47 21 44
Fax. (+33) 4 74 45 52 53
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1. Introduction
reduced contents of ingredients and additives that usually serve to inhibit microbial growth
(Schillinger et al., 1996). Nisin, a small peptide produced by Lactococcus lactis ssp. lactis,
attacks the cell wall and causes lysis of the target microorganisms. Nisin is currently industrially
produced and is used for specific applications such as prevention of spore germination and
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growth of pathogenic bacteria contaminating the surface of food products. This bacteriocin has
been commercialized for the first time in the 50’s to inhibit the outgrowth of Clostridium
tyrobutyricum responsible for late cheese blowing (García et al., 2010). Lactic acid bacteria
(LAB) naturally produce nisin in raw milk and dairy products (Perin et al., 2012), however LAB
growth for nisin industrial production requires complex nutrition conditions which increases
production costs and complicates the purification steps. This explains the fact that commercial
nisin preparations (not exceeding generally 2.5 wt% pure nisin content) are standardized to 106
IU g-1 with denatured milk proteins and NaCl. If one gram of pure nisin contains 40 106 IU, a
preparation purity. It is noteworthy that the International Unit (IU) corresponds to the amount of
nisin able to inhibit one cell of Streptococcus agalactiae in 1 mL of broth (Tramer and Fowler,
1964).
Nisin is the only bacteriocin approved as a food preservative, which explains its
increasingly common use in food industry. This use is governed by the "FAO / WHO Codex
Committee on Milk and Milk Products" which has accepted the use of nisin as a food additive in
a concentration not exceeding 12.5 mg of pure nisin per kilogram (Ross et al., 2002). Nisin has
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been approved as GRAS (Generally Recognized As Safe) by the US Food and Drug
Administration (FDA) since 1988 because it has long been used in food preservation (E234)
without being involved in health problems. According to the FDA rules, this bacteriocin can be
used at a maximum concentration of 250 ppm in cheese products in order to prevent Clostridium
botulinum growth (Federal Register, 1988). Nisin is effective against several pathogenic Gram-
positive bacteria such as Listeria monocytogenes and C. botulinum, but also against some Gram-
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negative pathogens such as Escherichia coli and Salmonella spp. when combined with chelators
causing the alteration of the cell wall that becomes permeable which promotes contact between
Regarding this latter aspect, only the uses of “free nisin” will be discussed while nisin
2. Nisin structure
The classification of bacteriocins produced by LAB evolves with the research progress in
this field. It is based on several criteria: molecular weight, posttranslational modification, and
(Ennahar et al., 2000). The simplest classification includes bacteriocins into three classes: class I
(lantibiotics); class II (small, heat-stable and non-lantibiotics peptides); and class III (large and
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heat-sensitive proteins). The majority of bacteriocins produced by the bacteria associated with
food belong to classes I and II. The most important class of bacteriocins is lantibiotics which
contain an unusual amino acid: lanthionine (Lan). Because of this unusual amino acid and its
specific biological activity, these peptides are named lantibiotics. More than 25 lantibiotics have
been described in literature but the most important and most studied is nisin (Jack and Jung,
2000).
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lanthionine, and β-methyllanthionine (Figure 1) that form thioether bridges in five positions.
Unlike other proteins, nisin has no absorbance at 280 nm because it does not contain aromatic
amino acids. Nisin is a cationic polypeptide, hydrophobic and heat stable. The nisin molecular
weight is 3 510 Da but this peptide is capable of forming dimers (7 000 Da, more stable dimer)
and tetramers (14 000 Da). Several types of nisin have been identified. The main variants are
called A, Z, and Q (Figure 2) and possess different biological activities. However, nisin A and Z
are the most active forms that are often marketed. These two variants (A and Z) differ by one
amino acid at position 27: histidine for nisin A and asparagine for nisin Z. This substitution
proteolytic enzymes, and antimicrobial action spectrum (Sonomoto et al., 2000). However, this
last study has shown that nisin Z is more soluble than nisin A in pHs near neutrality because
asparagine has a more polar side chain than that of histidine. This structural difference does not
affect antimicrobial activity but it changes some properties: the nisin Z is more soluble and its
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structural stability which in turn depend on pH. Indeed, nisin is more soluble and more stable
under acidic conditions and has a solubility of 12 wt% at pH 2.5 and 4 wt% at pH 5.0. This
solubility is close to zero when the pH reaches and exceeds neutrality (Hurst, 1981). Similarly,
the antimicrobial activity is stronger at acidic pH (hydrochloric acid solution pH 2.5 for
example) and gradually decreases with increasing pH which can be explained by an irreversible
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modification of the molecular structure of nisin (Hurst, 1981). This structural modification may
remarkably take place at pHs > pI (isoelectric point) (~ 8-9) and occurs primarily through the
Nisin activity is highly stable at low temperatures (during freezing for example) but this
activity can be lost when the peptide is heated for a long time. In addition, nisin thermostability
is largely related to pH. For example, the antibacterial activity of nisin is completely retained at
al., 1991). As a general rule, thermal stability of nisin increased with decreasing pH (Rollema et
al., 1995). This can be explained by the presence of the five thioether bridges.
and ficin that are able to break its peptidic chain. However, other enzymes such as trypsin,
pepsin and carboxypeptidase have no significant effect on its antimicrobial effect (Chollet et al.,
2008).
used in food systems that have different values of pH, ionic strength, viscosity, and fat content…
Therefore, the structure, composition, pH, and shelf life of the considered food matrix should be
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example, food matrices for which the risk of growth of pathogenic bacteria such as Listeria
monocytogenes is the highest have a pH close to neutrality: one must keep in mind that both
solubility and antimicrobial activity of nisin are lower at these pH values than at acidic pH.
Many of the nisin sensitive bacteria, such as Listeria monocytogenes and Clostridium
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botulinum, are known to be pathogenic. In fact, nisin has antimicrobial activity directed primarily
against Gram-positive bacteria and in particular the spore forming ones. For spore-forming
bacteria, nisin is able to inhibit both the vegetative forms and the outgrowth of their spores.
Other studies also suggest the ability of nisin to inhibit the germination of Bacillus and
Clostridium spores (Hurst, 1981; Venema et al., 1995). Furthermore, nisin can also inhibit some
Listeria, Pediococcus, and Micrococcus. It should also be noted that nisin has no inhibitory
activity against yeast cells, filamentous fungi, viruses and Gram-negative bacteria. In normal
circumstances, Gram-negative bacteria are usually resistant to nisin mainly due to their
The nisin action mechanism is widely studied (McAuliffe et al., 2001) and consists in the
adsorption on the target cell surface and destabilization of the cytoplasmic membrane structure.
This adsorption involves electrostatic interactions between nisin having a net positive charge and
the negatively charged membrane phospholipids (Martin et al., 1996). These electrostatic
interactions are also due to the hydrophilic character of the C-terminal extremity of the
polypeptide. The hydrophobicity of the N-terminal extremity of nisin acts subsequently to allow
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the insertion of nisin in the lipid cell membrane leading to its permeabilization. The effectiveness
of this integration depends on the nature and content of the cell membrane phospholipids which
may explain differences in sensitivity between target bacterial strains. The release of the essential
cytoplasm components, and/or cell lysis, results in the bacterium death (McAuliffe et al., 2001).
Association between nisin hydrophobic patches and hydrophobic bacterial membranes was
modeled using computer simulation to predict the most favorable interactions for an optimum
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antimicrobial activity. Probably, it is the hydrophobic part inserted into cell membrane that forms
pores (Lins et al., 1999). One of the several modes of nisin action proposed in literature is given
in Figure 3. Nisin initially forms a complex with Lipid II, a precursor molecule in the synthesis
of peptidoglycan forming the bacterial cell walls. This complex then inserts itself into the
cytoplasmic membrane forming pores and allows the efflux of essential cellular components
5.1. Legislation
Several bacteriocins produced by LAB have been purified, characterized and studied for
their antibacterial properties. For example, pediocin (Class IIa) possesses a confirmed
antilisterial activity (Naghmouchi et al., 2007) and could have several potential applications in
food industries. However, nisin is the only bacteriocin which use is currently permitted in food.
This peptide was added to the list of food additives under the European number E234 (EEC,
1983). The authorization of the use of this bacteriocin has many reasons: (i) the peptide is easily
degradable with intestinal proteases, (ii) it presents no risk to human health, and (iii) does not
alter the organoleptic and sensory properties of foods. The legislation concerning the maximum
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nisin amount used differs from one country to another: for example, if nisin can be added to the
cheese without any limit in the United Kingdom, Australia and France, the amount added should
not exceed 100 IU g-1 in Belgium, 500 IU g-1 in Argentina, and 10 000 IU g-1 in the USA
(Cleveland et al., 2001). More data concerning the maximum authorized nisin levels are given in
Table 1. Finally, it is important to highlight that nisin can not be regarded as a "natural"
preservative when used in concentrations higher than those naturally found in foods fermented
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Several strategies can be considered for application of nisin for food preservation: (i)
inoculation of food with a nisin-producing strain, (ii) use of a fermented product with a nisin-
preservative, and (iv) addition of encapsulated nisin and/or immobilization of nisin in solid
matrices (beads, gels, or films) to control its release and protect it from degradation by
proteolytic enzymes. Food composition and intrinsic and extrinsic parameters during
manufacture, storage, and distribution will determine the appropriate method to be used. In this
section we will focus on the direct addition of free nisin to food products.
Nisin can be used in a wide range of liquid or solid foods chilled or stored at room
temperature. According to the target microorganisms, areas of nisin use can be classified into
spore forming Gram-positive bacteria such as Clostridium botulinum. Nisin is not active against
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other microorganisms and should therefore not be used as a unique preservation agent unless the
microflora that is likely to contaminate the food consists mainly of Gram-positive bacteria.
Nisin should preferably be added as an aqueous solution and mixed with food during
manufacture. When nisin is added in powder form, one must ensure proper distribution in the
food. Alternatively, nisin can be used to decontaminate food surfaces by spraying. During
multistage production, nisin must be added during the final step so that it retains its full activity.
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When making cheese, for example, nisin is added during heating at the same time as melting
salts. In practice, nisin is often incorporated into or sprayed on the surface of these products in
the form of dry commercial powder containing a small amount (2.5 wt%) of pure nisin.
The used nisin amount depends on many factors: food composition, heat treatment
intensity, used packaging, pH, storage time before consumption, and storage conditions. Nisin is
often added to acidic foods, but it remains relatively active in a pH range up to 8 (Liu and
Hansen, 1990). Nisin is often used for the preservation of pasteurized milk, aged cheeses, and
canned soups and vegetables. Nisin can also be used to complement other preservation
treatments. In foods stored in cans, nisin is used in addition to heat pasteurization treatments to
successfully counter heat resistant spores of flat-sour thermophilic bacteria. When nisin is used,
one must be careful to the presence of certain chemical compounds that can alter its biological
activity. Indeed, it was shown that titanium dioxide and sodium metabisulphite degrade nisin and
inhibit its activity by oxidation of disulfide bridges. Besides, the antibacterial activity of nisin is
bacteria proliferation have been widely published and reviewed (Deegan et al., 2006). However,
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in the next sections we will summarize some recent common uses of nisin and the related
For meat preservation, nitrates are traditionally used: their partial conversion into nitrites
by microbial nitrate reductases in fermented meat products allows to prevent Clostridium spp.
growth. Direct addition of nitrites is an alternative. The reaction of nitrites with secondary
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amines in meat products can produce significant levels of nitrosamines under certain conditions.
Since some nitrosamines are carcinogenic, there is an increasing interest for food preservatives
such as nisin, which could replace, even partially, nitrites. However, as mentioned previously in
this review, nisin has particular physico-chemical properties influencing especially its stability
and biological activity. Thus, when nisin is in contact with meat matrix, it will have an
antimicrobial activity that heavily depends on the meat characteristics. The use of nisin in meat is
glutathione S-transferase catalyzed reaction (Rose et al., 1999). This glutathione inactivation is
lower in cooked meat due to the loss of free sulphydryl groups, which catalyze the reaction
between glutathione and proteins, during heating process (Stergiou et al., 2006). Nisin can also
be inactivated by proteolytic enzymes generally found in fresh meat (Rose et al., 1999). In
addition to glutathione and the presence of proteolytic enzymes, nisin can easily interact with the
meat fats what can reduce its antimicrobial efficacy. In this context, Deegan et al. (2006)
explained the fact that nisin is more effective in dairy than in meat products in terms of
interactions between nisin and phospholipids. Davies et al. (1999) studied the effect of fat and
phospholipids on nisin effectiveness and showed that low fat content was correlated with higher
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antimicrobial activity of nisin. Some examples of nisin uses in meat based foods, concentrations,
and target microorganisms are summarized in Table 3. As a general rule, nisin stability in meat
systems during storage depends on four main factors: temperature, pH, presence or absence of
Nisin addition to milk during cheese making without lactic fermentation allows
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controlling microbial contamination without nisin alteration during pasteurization. Nisin inhibits
the outgrowth of C. botulinum spores in cheese spreads (Cleveland et al., 2001). In cheese
inoculated with 104 CFU (colony forming unit) g-1 Listeria monocytogenes, a 3 log reduction in
germination for 3 months during cheese storage at 5 °C. In addition, several species of Bacillus
have been inhibited by the use of 5 mg kg-1 of nisin (Plockova et al., 1996). Study of the shelf
life of Ricotta cheese type showed that the use of 2.5 mg L-1 of nisin can inhibit the growth of L.
monocytogenes for more than 8 weeks. In addition, measuring the concentration of residual nisin
in cheese showed that the loss does not exceed 32% after incubation at 6-8 °C for 10 weeks
(Davies et al., 1997). More examples concerning nisin uses for dairy products preservation are
gathered in Table 4.
salmon (Oncorhynchus keta) was studied after treatment with nisin and radio frequency heating
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or after treatment with antimicrobial chemicals or moderate heating (Al-Holy et al., 2005).
Authors found that nisin combined with either radio frequency or moderate heat, inhibited
without changing the visual quality of treated products. Elotmani and Assobhei (2004) evaluated
the inhibition of microbial flora of sardine with nisin and lactoperoxidase, and observed the
effectiveness of combining nisin lactoperoxidase in inhibiting the fish spoilage microbiota. More
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recently, nisin was used to reduce the surface numbers of L. monocytogenes, Salmonella and
native microflora on vacuum-packed raw shrimps stored at 4 °C (Wan Norhana et al., 2012).
Cells of E. coli and L. innocua, used as models for foodborne pathogens, were inoculated into
apple or carrot juice (7 log CFU/mL) containing 0 or 10 IU/mL nisin (Pathanibul et al., 2009). In
this study, a small amount of nisin (0.25 mg L-1) was added to apple and carrot juices before
HHP treatment. No additional inactivation effect was observed in combination with high
pressure against E. coli K12 cells. However, synergy effects were observed in the case of L.
innocua under the same conditions. In another study, Xu et al. (2007) suggested that a mixture of
nisin with citric acid and grapefruit seed extract could significantly inhibit tested bacteria (three
strains of Salmonella spp. and three strains of L. monocytogenes) and prolong shelf life of fresh-
cut ready-to-eat vegetables like cucumber and lettuce. Sun et al. (2012) suggested that nisin can
be successfully used for the preservation of fermented vegetable products like beer and wine
because it inhibits contaminating bacteria but not influence yeast responsible for the ethanol
fermentation. Finally, it is important to note in this section that the major part of the available
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literature concerning nisin applications for the preservation of vegetable products uses nisin in
It has been shown that the effectiveness of nisin increased when combined with other
molecules such as lysozyme (Chung and Hancock, 2000), some lactates (Nykänen et al., 2000),
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essential oils (Razavi Rohani et al., 2011), and listeriophages (Dykes and Moorhead, 2002). In
particular, some authors have evaluated the nisin antimicrobial activity in combination with other
bacteriocins (Gálvez et al., 2008). A 31 days increase of brined shrimp shelf life was observed
after nisin Z in both crude and purified forms addition. Bouttefroy and Millière (2000) tested
combinations of nisin and curvaticin 13 produced by L. curvatus SB13 to prevent the regrowth of
resistant cells of L. monocytogenes, believing that this combination induced an inhibitory effect
higher than that of a single bacteriocin. Aasen et al. (2003) studied the interactions of sakacin P
and nisin with the constituents of cold-smoked salmon, cold-cut chicken, and raw chicken. These
authors concluded that due to the amphiphilic nature of these peptides, they can be adsorbed on
food macromolecules and undergo proteolytic degradation, which may limit their use as
preservatives. Over 80% of added sakacin P and nisin were quickly adsorbed by the food matrix
proteins. In non heat treated foods, proteolytic activity caused a rapid degradation of
bacteriocins. Less than 1% of the total activity remained after 1 week storage of cold-smoked
salmon, and even less in raw chicken. In heat processed foods, bacteriocin activity was stable for
over 4 weeks. No significant differences were observed between sakacin P and nisin, but less
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Lysozyme is an enzyme commonly added to milk during cheese making with the aim of
inhibiting Bacillus genus bacteria but has no effect on nisin producers like Lactococcus strains.
The combination of lysozyme and nisin caused severe cell damage as the authors have observed
by scanning electron microscopy compared to samples treated with nisin alone (Chung and
Hancock, 2000). These damages reflect the action of lysozyme. In addition, nisin-lysozyme
Staphylococcus aureus, an effect that reflects the nisin action mechanism. Thus, nisin and
lysozyme appear to demonstrate synergy against Gram-positive bacteria because they reinforce
the action of each other to kill bacteria. The nisin activity and that of supernatant of a Bacillus
Staphylococcus aureus, Micrococcus flavus, and Bacillus cereus (He and Chen, 2006). However,
this study also showed that nisin activity and that of the used supernatant begin within the first
monocytogenes ATCC 15313 in skim milk and effectiveness of this combination allows to have
maximum inhibitory effect was observed when nisin was added at the beginning and then LPS
was added 4 h later which shows that the addition order is important in the mechanism of action
on the cell target membrane. Indeed, nisin forms pores in the membrane after interaction with
phospholipids, whereas LPS produces a molecule, hypothiocyanite, which reacts with thiol
groups of some proteins important for the viability of pathogenic bacteria, which inactivates the
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enzyme systems (Boots and Floris, 2006). The action mechanisms of these two factors can
Combination of nisin (2.5 mg L-1) and monolaurin (250 mg L-1) induces a bactericidal
synergistic effect on vegetative cells of 4 tested Bacillus species after 5 days at 37 °C (Mansour
and Millière, 2001). This bactericidal effect is due to both regrowth and sporulation inhibition.
Nisin combination with lactic acid has increased nisin effectiveness to inhibit some Gram-
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(Ariyapitipun et al., 2000). Nykänen et al., (2000) tested the combination of nisin and sodium
lactate in the control of L. monocytogenes in cold-smoked trout, believing that nisin and sodium
lactate injected into smoked fish decreased the number of L. monocytogenes cells by 3.3 to 1.8
The combination of nisin and other bacteriocins with other treatments and other chemicals has
also been already well reviewed (Gálvez et al., 2007; Gálvez et al., 2008).
Nisin addition during heat treatment could be an effective way to increase the shelf life of
food products and to use milder treatments (Al-Holy et al., 2012; Li et al., 2012), which help to
preserve the organoleptic and sensory properties of these foods. In fact, during sterilization
treatments, nisin is known to influence the microorganism’s thermal resistance by changing the
value of the D constant (decimal reduction time). In the presence of nisin (25 mg L-1), the
average D value of B. cereus in milk is reduced by more than 40% in a temperature range of 80-
100 °C (Penna and Moraes, 2002). Wandling et al., (1999) showed that the decimal reduction
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time of B. stearothermophilus ATCC 12980 spores was reduced by 13 and 21% in the presence
Wirjantoro et al. (2001) showed that the addition of nisin (1.875 or 3.75 mg L-1) to milk
before sterilization increases the shelf life with or without refrigeration. In this study, no
microbial growth was observed in the milks treated at 117 °C for 2 s after storage at 10 or 20 °C
for 1 year in addition to their best sensory properties compared to UHT-treated milk. Budu-
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Amoako et al. (1999) evaluated nisin activity in combination with heat treatment to inhibit
monocytogenes populations, while heat treatment or nisin alone resulted in reductions from 1 to
3 log.
The current trend is the use of nisin in combination with new non-thermal preservation
techniques such as high hydrostatic pressure (HHP) (Pathanibul et al., 2009; Zhao et al., 2012) or
pulsed electric fields (PEF) treatments (Nguyen and Mittal, 2007). This explains the abundance
of literature in this field. HHP treatments can inactivate Gram-negative bacteria such as
Escherichia coli and Pseudomonas fluorescens but Gram-positive ones such as Listeria innocua
seem to be more resistant (Black et al., 2005). In this latter study, viability of several bacteria
was evaluated in skim milk treated at pressures of 250-500 MPa for 5 min at 20 °C in the
presence of 0, 6.25, or 12.5 mg L-1of nisin. The combination of the two treatments gave higher
inactivation than that obtained for each treatment separately for both Gram-negative and Gram-
positive bacteria. Nisin addition to milk causes damage to bacteria cell membranes and increases
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After treatment of milk containing 12.5 mg L-1 at 500 MPa for 5 min, a greater reduction
(8 log) was observed for Listeria innocua and Lactobacillus viridescens. Taken separately, the
same physical treatment led to a 3.8 log reduction while the same amount of nisin caused a 1.5
log reduction. Since nisin is effective against Gram-positive bacteria, a sensitization by nisin of
In fact, the microbial reduction of pathogenic bacteria such as Staphylococcus aureus or Listeria
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monocytogenes achieved by the combination of HHP treatments and nisin, indicates that, the
microbiological safety of food could be improved, while little changing organoleptic properties
Combination of 500 MPa and nisin was the most effective treatment to inactivate
indigenous microorganisms of goat cheese (Capellas et al., 2000). The number of aerobic
mesophilic microorganisms in the cheese subjected to combined treatment was lower than that
obtained in the case of only nisin treated cheese due to the effect of high pressure on cells
sensitization. However, this effect was not additional because there are some populations that
may be inactivated by both HHP and nisin. Above 150 MPa, Escherichia coli became sensitive
to nisin when the bacteriocin was added before HHP treatment, but HHP-treated cells remained
insensitive to nisin when it was added after treatment. The authors considered that HHP
treatment can sensitize Escherichia coli to nisin by inducing a transient permeabilization of the
outer membrane that does not involve physical disruption and is immediately restored after the
products. The behavior of several foodborne bacteria in a meat model system containing several
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bacteriocins including nisin after pressurization (400 MPa, 10 min, 17 °C) and during cold
storage has been studied (Garriga et al., 2002). Among the bacteria studied, Staphylococcus was
the least sensitive to pressure but in the presence of nisin this bacterium showed a lower cell
number during storage at 4 °C than in the presence of other bacteriocins. Greater inactivation of
E. coli (> 6 log) in the presence of nisin has been registered and the number of surviving cells
remained unchanged for 61 days during storage at 4 °C. Masschalck et al. (2000) have succeeded
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to inactivate the mutant E. coli MG1655 strain, known for its high resistance to high hydrostatic
pressure, by using a relatively low pressure (400 MPa) in the presence of nisin (2.5 mg L-1). At
room temperature, this treatment was able to achieve 6 log reductions. In this work, a
hypothetical mechanism of "pressure-promoted uptake" has been proposed to explain the outer
membrane permeabilization under pressure by lipophilic cationic peptides like nisin or enzymes
like lysozyme.
Gao and Ju (2008) studied the combined effects of pressure (300-700 MPa maintained
from 7.5 to 17.5 min at temperatures ranging from 30 to 70 °C) and nisin (0-8 mg L-1) on the
inactivation of C. botulinum 33A spores. They have pinpointed the optimum process parameters
for a 6 log reduction of spores: pressure of 545 MPa; temperature of 51 °C; pressure holding
The effect of combined HHP and nisin treatments on microbial inactivation in liquid
whole egg was studied by Lee et al. (2003). The addition of nisin (0.5-20 mg L-1) before
pressurization treatments significantly increased the lethal effects of high pressure against
Listeria seeligeri (up to 5 log-reduction of their population). On the other hand, individual effects
of HHP and nisin on Listeria were almost negligible, and therefore the observed reductions were
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considered to be due to the synergistic action of nisin and HHP. However, study of the effect of
the combination of nisin with HHP on E. coli showed exactly the same inactivation level by
HHP alone, which was interpreted by the authors in terms of protection of Gram-negative
Lee and Kaletunç (2010) studied effects of nisin (5 mg L-1), HHP (100-150 MPa), and
their combination on the cellular components of two pathogenic strains of Salmonella Enteritidis
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increases the sensitivity of the two strains to nisin. Indeed, high pressure caused alterations in the
outer cell membrane thereby facilitating nisin penetration in the cell. The results obtained by
DSC were used to compare the different final states of the cells obtained under different
treatment conditions starting from the same initial state. The apparent enthalpy of each strain did
not change after nisin addition under atmospheric pressure while under high pressure a reduction
López-Pedemonte et al., (2003) studied the effect of HHP on the inactivation of Bacillus
cereus ATCC 9139 spores inoculated in model cheeses made from raw milk, and the effects of
nisin addition (0.05 and 1.56 mg L-1). At a pressure of 400 MPa, highest inactivation (~ 2.4 log
CFU g-1) was obtained in the presence of nisin (1.56 mg L-1), while lysozyme (22.4 mg L-1) was
not able to increase spores sensitivity to high pressure. When studying the sensitivity of spores of
Bacillus subtilis and Clostridium sporogenes PA 3679, nisin showed a synergistic effect with
pressurization at high temperatures and acidic pH for both types of studied spores. This effect
was most clear at a pressure of 404 MPa, 45 °C, pH 4.0 for 15 min. Under these conditions, the
number of B. subtilis spores decreased by 3 log and a further reduction of 3.1 log was observed
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in the presence of nisin (Stewart et al., 2000). Furthermore, HHP treatment may also improve the
efficiency of nisin on the inactivation of some spores by increasing their permeability after the
germination process. The number of Bacillus cereus spores in a traditional curd cheese was
significantly reduced when nisin addition was followed by two HHP cycles, a cycle to induce
spore germination and a second to destroy vegetative cells (López-Pedemonte et al., 2003).
Several other studies have been conducted in order to explain the synergy resulting from
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the combination of HHP and nisin. Among the proposed hypotheses, the pore formation by nisin,
followed by its parallel orientation and subsequent binding to the membrane, may increase the
et al., 1999). Synergistic effects can also be attributed to sub-lethal damage due to cell wall
leakage and/or outer membrane, for Gram-negative bacteria, by high pressures which could
facilitate the nisin access to the cytoplasmic membrane (Hauben, et al., 1996).
Other physical treatments such as pulsed electric fields were also used in combination
with nisin. PEF damage cell walls and membranes that lose their barrier function. Observations
between cells treated with nisin, with PEF, and those treated by the combination of both
(Calderón-Miranda et al., 1999). L. innocua cells subjected to PEF in skimmed milk containing
nisin (0.925 mg L-1) showed an increase in the cell wall width. When applying the highest
electric field intensity used in this study (50 kV cm-1), a cell elongation was observed. The
combination of PEF and nisin has an additional effect on the morphological damages of L.
inactivation is a consequence of the cell membrane breakdown and loss of its functionality. This
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synergistic effect is not consistent with other studies that even show that nisin has no additional
lethal effect during treatment with PEF. Indeed, Terebiznik et al. (2000) explain that the outer
cell wall shrinkage and the cytoplasmic membrane tear facilitate nisin entry in the cytoplasm and
loss of its ability to form pores because of the internal pH (alkaline) and chemical potential
(negative).
Nilsson et al. (1997) tested the combination of nisin with a CO2 atmosphere for the control of L.
monocytogenes in smoked salmon. Results showed that the combination of nisin and CO2 in the
followed by a lag phase of 8 to 20 days when 500 and 1000 IU nisin/g are used, respectively.
However, despite the importance of this non-thermal technique, few studies are published on this
subject.
When nisin is directly added to foods, its effectiveness can be altered by the physicochemical
properties of the system such as high pH, high fat content, and the presence of large particles.
These parameters can generate interactions with nisin, precipitation, inactivation, or non-uniform
distribution within the food. Nisin effectiveness is thus generally lower in food systems than in
growth media. To reach the same efficiency, it is often necessary to add a nisin amount about ten
times higher than that in a culture medium (Gálvez et al., 2007). Nisin effectiveness depends also
on the food microbial properties like the type of microflora contaminating the food and the
properties of the targeted bacteria. The growth phase of microorganisms contaminating the food
can influence their sensitivity and therefore the nisin effectiveness. Indeed, cells that are not in a
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growth phase may be more resistant. In addition, cells that have already adapted to changes in
their environment are often insensitive to bacteriocins. The effectiveness of nisin can also be
EDTA is a chelator of divalent cations (especially Ca2+ and Mg2+) that contribute to the
antimicrobial agents such as nisin. Nisin, in the presence of EDTA, has a high activity against
Gram-negative bacteria such as Salmonella (Tu and Mustapha, 2002). The use of some surface
active molecules such as Tween 80 helped considerably to keep nisin activity in half-whole milk
(Jung et al., 1992). These results were subsequently interpreted by the fact that the amphiphilic
Tween 80 is capable of moving nisin from the water/fat interface thereby enhancing its
adsorption to cell membranes of bacteria (Bhatti et al., 2004). In addition, it is important to note
that nisin can chemically degrade during the production and storage of some food products like
cheese (Schneider et al., 2011). In fact, it has been shown that nisin is susceptible to the acid
catalyzed addition of a water molecule at the double bond of the unsaturated aminoacids.
Nisin use is also limited by the emergence of resistant strains and its ineffectiveness is not
genetically related to the nisin production itself. Nisin-resistance mechanisms are strain-specific.
Jarvis and Farr (1971) explained the resistance of Bacillus cereus by inactivation of nisin through
a reductase that acts on dehydroaminoacids. This resistance can also occur spontaneously in
some nisin sensitive mutant strains that are grown in the presence of this lantibiotic. The
mechanism of this resistance may reflect the synthesis of a lipoprotein able to "obstruct"
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membrane pores formed by nisin (Engelke et al., 1994). This resistance phenomenon, due to
genetic mutations, is a major problem that limits the use of nisin as a food preservative. For
example, Streptococcus thermophilus INIA 463 is a nisin sensitive strain but became resistant
after exposure to low nisin concentrations (1-3 IU mL-1) for less than 2 h. This resistance is due
to an increase in the thickness of the cell wall as revealed by transmission electron microscopy
(TEM). Results also showed that resistance disappears after 4 h of growth in skim milk (Garde et
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al., 2004). Some strains of L. monocytogenes showed resistance to nisin in acidic conditions and
a study has also demonstrated that this resistance does not depend only on pH but also the nature
Nisin use for food preservation may offer several advantages: (i) increasing the shelf life
of the product, (ii) reducing the transmission risk of pathogen food borne, (iii) reducing the use
of salts, acids, and other chemical preservatives, and (iv) permitting the use of soft treatments
which better preserve vitamins and organoleptic properties. However, the majority of articles
concerning nisin applications deals with its combination with other treatments. Because of the
rapid reduction of nisin inhibitory activity due to its degradation by proteolytic enzymes or its
interaction with fat compounds, current research works aim at improving nisin stability and
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L. monocytogenes growth
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Beef meat
Once vacuum packed, B.
cubes dipped Brochotrix Tu &
thermosphacta growth was
Round steak for 10 min in thermosphacta Mustapha
inhibited for more than 25
125 mg L-1 (2002)
days at 4 °C
nisin solutions
More than 68% of initially
No
Cooked added nisin still detected Reunanen &
11.25 mg kg-1 antimicrobial
sausage after 28 days storage at 6 Saris (2004)
activity assay
°C
Meat
emulsions Time to increase initial L.
(fresh lean monocytenes population
0.25 to 2.5 mg Listeria
beef, 20% from 104 UFC.g-1 to 107
kg-1 monocytogenes Pellicer et
w/w bovine UFC.g-1 at 20 °C increased al. (2011)
fat, 2.5% from 1 to 7 days
w/w NaCl)
Dipping for 8
Natural 90% reduction of C.
days in 50 mg Clostridium Wijnker et
sausage sporogenes spores
L-1 nisin sporogenes al. (2011)
casings compared with a control
solutions
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Nisin Target
Food concentratio microorganism Main observations References
n s
After an initial
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Exposure to subminimal
inhibitory concentrations
Streptococcus of nisin induced resistance Garde et al.
Skim milk £ 0.5 mg L-1
thermophilus to nisin (possibly caused (2004)
by changes in S.
thermophilus cell wall)
Processed 2.5-12.5 mg Nisin was effective in Delves-
cheese kg-1 Clostridium delaying or preventing Broughton
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- Up to 2 log cycles
£ 12.5 mg L-1 reduction in S. aureus
Minas Serro (nisin addition Staphylococcus count from the 7th day of
cheese to milk before aureus ripening Soares Pinto
enzymatic et al. (2011)
coagulation) - Decrease of the ripening
index
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Figure 2. Main natural nisin variants. The black-filled residues indicate the substituted residues
as compared with nisin A. The grey-filled residues indicate unusual amino acids.
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Figure 3. A schematic representation of the mechanism of action of nisin (from Breukink & de
Kruijff, 2006). First, nisin reaches the bacterial plasma membrane (a), where it binds to Lipid II
via two of its amino-terminal rings (b). This is then followed by pore formation (c), which
involves a stable transmembrane orientation of nisin. During or after assembly of four 1:1 (nisin:
Lipid II) complexes, four additional nisin molecules are recruited to form the pore complex (d).
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