Bacteriocins: Biological tools for bio-preservation and shelf-life extension

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International Dairy Journal 16 (2006) 1058–1071

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Review

Bacteriocins: Biological tools for bio-preservationand shelf-life extension

Lucy H. Deegana, Paul D. Cottera, Colin Hilla,c,�, Paul Rossb,c

aMicrobiology Department, University College Cork, Cork, IrelandbMoorepark Biotechnology Centre, Teagasc, Ireland

cAlimentary Pharmabiotic Centre, Cork, Ireland

Received 28 April 2005; accepted 19 October 2005

Abstract

The lactococcal bacteriocin named nisin (or group N inhibitory substance) was first marketed in England in 1953 and since then has

been approved for use in over 48 countries. The successful development of nisin from an initial biological observation through regulatory

approval to commercial application is a model that has stimulated significant resurgence in bacteriocin research in recent years, but

similar success is yet to be repeated on the same scale. In spite of this sobering fact, we remain convinced that bacteriocins can be

exploited in foods in a variety of imaginative and commercially significant applications in bio-preservation and shelf-life extension.

However, in order to fully realise this potential, it is necessary to understand the biology of bacteriocins; in particular, to elucidate

structure–function relationships, production, immunity, regulation and mode of action. In this paper, we will discuss some of the

advances, made mainly with other lactococcal bacteriocins, in improving food safety, food quality and preventing food spoilage.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Spoilage; Pathogen; Inhibitor; Food grade

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1059

2. Classification of lactic acid bacteria bacteriocins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1060

2.1. Class I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1060

2.2. Class II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1060

2.3. Bacteriocin production and export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1060

2.4. Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1061

2.5. Mode of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1061

3. Using bacteriocins to improve food safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1061

3.1. Bacteriocin preparations as ingredients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1062

3.2. Bacteriocin-producing cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064

4. Bacteriocins in combination with additional hurdles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065

5. Bacteriocins for improving food quality and flavour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067

6. Conclusion/future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1068

e front matter r 2006 Elsevier Ltd. All rights reserved.

airyj.2005.10.026

ing author. Microbiology Department, University College Cork, Cork, Ireland. Tel.: +353 21 4901373; fax: +35321 4903101.

ess: [email protected] (C. Hill).

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ARTICLE IN PRESSL.H. Deegan et al. / International Dairy Journal 16 (2006) 1058–1071 1059

1. Introduction

Bacterial fermentation of perishable raw materials hasbeen used for centuries to preserve the nutritive value offood and beverages over an extended shelf-life. In anumber of food fermentations, the key event is theconversion of sugars to lactic acid by lactic acid bacteria(LAB, which include the genera Lactococcus, Streptococ-

cus, Lactobacillus and Pediococcus, among others). Lacticacid and other end products of LAB metabolism, includinghydrogen peroxide, diacetyl, acetoin and other organicacids, act as bio-preservatives by altering the intrinsicproperties of the food to such an extent as to actuallyinhibit spoilage microorganisms (Fig. 1). While the role ofthese metabolic end products has long been appreciated,the contribution of LAB-derived bacteriocins may fre-quently have been overlooked. The widespread ability ofLAB to produce bacterocins implies an important biolo-gical role maintained over many generations and theprecise nature of this role has been the subject of intensiveresearch in recent times. Bacteriocin production could beconsidered as advantageous to the producer as, in sufficientamounts, these peptides can kill or inhibit bacteriacompeting for the same ecological niche or the samenutrient pool (Fig. 1). This role is supported by the factthat many bacteriocins have a narrow host range, and arelikely to be most effective against related bacteria compet-ing for the same scarce resources. Although bacteriocins

Acid

Nutrient

Bacteriocin

H2O2, Diacetyl, other metabolites

Target bacterium

Producer bacterium

Fig. 1. Lactic acid bacteria can produce a number of antimicrobial

compounds, but bacteriocins are often the most potent inhibitors of

related bacteria. A bacteriocin producer (top) can be identified by the

zones of inhibition produced in a lawn of sensitive indicator cells.

are produced by many Gram-positive and Gram-negativespecies, those produced by the LAB are of particularinterest to the food industry (Nettles & Barefoot, 1993),since these bacteria have generally been regarded as safe(GRAS status). Furthermore, as the majority of bacter-iocin-producing LAB are natural food isolates, they areideally suited to food applications. Although variousmethods other than bacteriocin production are employedfor the preservation of food/beverages, an increasingly,health conscious public may seek to avoid foods that haveundergone extensive processing or which contain chemicalpreservatives. Therefore, the production of bacteriocins byLAB is not only advantageous to the bacteria themselvesbut could also be exploited by the food industry as a tool tocontrol undesirable bacteria in a food-grade and naturalmanner, which is likely to be more acceptable toconsumers.To date, the only commercially produced bacteriocins

are nisin (or group N inhibitory substance), produced byLactoccocus lactis, and pediocin PA-1, produced byPediococcus acidilactici, marketed as NisaplinTM (productdescription-PD45003-7EN; Danisco, Copenhagen, Den-mark) and ALTATM 2431 (Kerry Bioscience, Carrigaline,Co. Cork, Ireland), respectively. Nisin is used in over 48countries, has Food and Drug Administration approvaland NisaplinTM is sold as a natural food protectant. Nisinhas been shown to be effective in a number of foodsystems, inhibiting the growth of a wide range of Gram-positive bacteria, including many important foodbornepathogens such as Listeria monocytogenes (Tagg, Dajani, &Wannamaker, 1976). It is used predominantly in cannedfoods and dairy products and is especially effective whenutilised in the production of processed cheese and cheesespreads where it protects against heat-resistant spore-forming organisms such as those belonging to the generaBacillus and Clostridium. This has particular significance inthe case of preventing contamination with Clostridium

botulinum as there can be serious repercussions resultingfrom toxin formation by this species. There are also anumber of yet-to-be commercialised bacteriocins reportedin the scientific literature, such as lacticin 3147 and lacticin481, which have shown the potential for exploitation asnatural food bio-preservatives and flavour enhancers.Intensive studies to elucidate the fundamental structural

and functional properties of bacteriocins have beenvaluable. However, applied research carried out with aview to determining the impact of food components andprocessing methods on the structure, solubility and activityof bacteriocins is of extreme importance when consideringpotential food applications. In addition to the ongoingstudy of existing bacteriocins, the discovery of newbacteriocins, combined with imaginative developmentsregarding their application, can only be beneficial andwill increase the likelihood that the use of these peptidescan be optimised to fulfil their potential in food applica-tions. This review will focus mainly on evaluating bothnon-commercialised LAB bacteriocins and those that are

ARTICLE IN PRESSL.H. Deegan et al. / International Dairy Journal 16 (2006) 1058–10711060

already used within the food sector to improve food safetyand quality.

2. Classification of lactic acid bacteria bacteriocins

Bacteriocins can be distinguished from peptide antibioticsin that they are ribosomally synthesised rather thansecondary metabolites (Hurst, 1981) and the genes respon-sible for production and immunity are generally foundclustered in operons (Jack, Bierbaum, Hiedrich, & Sahl,1995). While as many as five classes of bacteriocins havebeen proposed (Cotter, Hill, & Ross, 2005a; Klaenhammer,1993; Nes et al., 1996), the majority fall into Classes I and II.As these bacteriocins feature predominantly in this review,the following section will describe their biosynthesis, export,immunity and mode of action.

2.1. Class I

Class I LAB bacteriocins (reviewed by Chatterjee, Paul,Xie, & van der Donk, 2005) are small ðo5 kDaÞ, heat-stable peptides that are extensively modified after transla-tion, resulting in the formation of characteristic thioetheramino acids lanthionine (Lan) and methyllanthionine(MeLan) (Jack & Sahl, 1995). These arise via a two-stepprocess: firstly, gene-encoded serine and threonine can besubject to enzymatic dehydration to give rise to dehydroa-lanine (Dha) and dehydrobutyrine (Dhb), respectively.Subsequently, thiol groups from neighbouring cysteinesattack the double bond of Dha or Dhb yielding either Lanor MeLan, respectively. This condensation between twoneighbouring residues results in the formation of covalentlyclosed rings within the formerly linear peptide, conferringboth structure and functionality (Ingram, 1969).

The LAB lantibiotics were initially divided into twosubclasses based on structural similarities. Subclass Iaincluded relatively elongated, flexible and positivelycharged peptides and they generally act by forming poresin the cytoplasmic membranes of sensitive target species.The prototypic lantibiotic nisin is a member of this group.Subclass Ib peptides are characteristically globular, morerigid in structure and are either negatively charged or haveno net charge (Klaenhammer, 1993). They exert theiraction by interfering with essential enzymatic reactions ofsensitive bacteria. For example, the non-LAB IIb-typelantibiotic mersacidin interferes with cell wall biosynthesisby binding to the peptidoglycan precursor lipid II (Brotz,Bierbaum, Leopold, Reynolds, & Sahl, 1998). Morerecently, the subclassification of lantibiotics on the basisof mode of action has been somewhat confused by thediscovery that nisin is active through dual mechanisms. Inaddition to pore formation, it can also inhibit cell wallbiosynthesis through the binding of lipid II—a step whichin fact probably precedes pore formation (Breukink et al.,1999; Weidemann et al., 2001). Furthermore, the existenceof a number of two-component lantibiotics that functionthrough the combined activity of two peptides has further

confused the classification of these peptides. To reflect thisconsiderable diversity within the group, the lantibioticshave been subclassified into 11 subgroups based on thealignment of the unmodified structural peptides (Cotter,Hill, & Ross, 2005b). These are named to reflect theprototypic lantibiotic in each case, i.e. nisin, epidermin,streptin, pep5, lacticin 481, mersacidin, LtnA2, cytolysin,lactocin S, cinnamycin and sublancin.

2.2. Class II

Class II bacteriocins are also small ðo10 kDaÞ and heatstable but do not contain Lan residues. A number ofdifferent subclasses of Class II bacteriocins have beensuggested (Diep & Nes, 2002; Nes et al., 1996; van Belkum& Stiles, 2000), but their heterogeneous nature makessubclassification difficult. Two subclasses are common toall classification systems: the subclass IIa pediocin-like (orListeria-active) and the subclass IIb two-componentbacteriocins. Among the former group, pediocin AcH/PA-1 was the first to be extensively studied. Members ofthe pediocin-like peptides show a high degree of homology(40–60%) when the corresponding amino acid sequencesare aligned. In particular, the cationic N-terminal domaincontains the homologous region YGNGVXCXXXXCXVor ‘‘pediocin box’’ with the two cysteine residues forming adisulphide bridge (Eijsink, Skeie, Middelhoven, Brurberg,& Nes, 1998). Class IIb refers to two-component bacter-iocins that require two peptides to work synergistically(Nissen-Meyer, Holo, Harvastein, Sletten, & Nes, 1992).Singly these peptides have little or no activity and thereappears to be no sequence similarities between comple-mentary peptides. Both lactacin F and lactococcin G aremembers of this subgroup.

2.3. Bacteriocin production and export

The genes responsible for bacteriocin production arefrequently associated with mobilisable elements, e.g. on thechromosome in association with transposons or onplasmids. As is the case for most bacteriocins, thelantibiotics are initially synthesised with an N-terminalleader peptide. In general, the pre-peptide (genericallydesignated LanA) is modified by the action of otherproteins encoded by the bacteriocin gene cluster beforeexport. The formation of Lan bridges is also enzymemediated. In some cases (e.g. nisin), a LanB proteinperforms the initial dehydrations of serines and threonines,while LanC is required for Lan formation (reviewed byMcAuliffe, Ross, & Hill, 2001). Alternatively, somelantibiotics (e.g lacticin 481) have a single enzyme, LanM,which performs both of these tasks. In the special case oftwo-component lantibiotics (e.g. lacticin 3147), two LanMenzymes may be required, one for each peptide (McAuliffe,Hill, & Ross, 2000, McAuliffe et al., 2001). Export oflantibiotics is through a dedicated ATP-binding cassette(ABC) transporter, LanT, consisting of two domains, one


which acts to recognise the pre-peptide and one thathydrolyses ATP to fuel export (Higgins, 1992; Walker,Saraaste, Runswick, & Gay, 1982). For subclass Ia lantibio-tics, a serine protease (LanP) removes the leader peptidebefore or after export from the cell (GeiXler, Gotz, & Kupke,1996; Meyer et al., 1995), whereas the leaders of subclass Iblantibiotics, which contain a double glycine cleavage site, arecleaved concomitantly with export by LanT.

The genes required for the processing and export ofClass II bacteriocins differ somewhat from those forlantibiotics due to the absence of post-translationalmodifications. For the purpose of this review, the genesassociated with pediocin PA-1/AcH production, i.e. pedA,B, C and D, are used as an example. Like the majority ofClass II bacteriocins, pediocin PA-1/AcH is synthesised asa pre-peptide, with a leader peptide containing a conserveddouble glycine cleavage site, encoded by pedA. PedB isresponsible for producer self-protection and its role isdiscussed further below. The ABC transporter (encoded bypedD) along with an essential accessory protein (PedC) areinvolved in cleavage of the leader in combination withexport of the bacteriocin (Havarstein, Diep, & Nes, 1995).

2.4. Immunity

Self-evidently, bacteriocin producers must protect them-selves from the action of their bacteriocins. This isaccomplished by the production of dedicated immunityproteins. Two types of immunity have been described forlantibiotics, one reliant on a specific immunity protein,LanI, while the other depends on a separate dedicatedmulti-component ABC transporter (LanEFG). Somelantibiotic clusters have only the single immunity protein,as is the case for Pep5 (Reis & Sahl, 1991), whereas others,such as nisin, possess both mechanisms (Kuipers,Beerthuyzen, Siezen, & de Vos, 1993; Seigers & Entian,1995). It is thought that the LanI protein antagonises theaction of lantibiotics, somehow preventing the insertion ofthe lantibiotic in the cell membrane, while it has beensuggested that the ABC transporter works by expelling theantimicrobial peptide from the cell or the membrane(Peschel & Gotz, 1996; Stein, Heinzmann, Dusterhaus,Borchert, & Entian, 2005). Class II bacteriocins generallyhave a single-cell membrane-associated immunity proteinthat provides complete immunity (Quadri et al., 1995).Based on the study of carnobacteriocin B2 (Class IIa), itappears that the majority of the immunity protein islocated inside the cytoplasm and that it may interact withanother protein, perhaps a receptor, at the cytoplasmic sideof the cell membrane (Quadri et al., 1995; Sprules,Kawulka, & Vederas, 2004).

2.5. Mode of action

While LAB bacteriocins can work via different mechan-isms to exert an antimicrobial effect, the cell envelope isgenerally the target. Nisin has a broad spectrum of activity

against most Gram positives and also inhibits the out-growth of Bacillus spp. and Clostridium spp. spores (Gross& Morell, 1971), while in contrast lactococcin A is narrowspectrum, being active only against other lactococcalstrains (Stoddard, Petzel, van Belkum, Kok, & McKay,1992). The initial electrostatic attraction between the targetcell membrane and the bacteriocin peptide is thought to bethe driving force for subsequent events. While manybacteriocins have been shown to induce pore formationin sensitive microorganisms, the mechanism of action ofnisin has been studied in greatest detail. Nisin forms poresthat disrupt the proton motive force and the pHequilibrium causing leakage of ions and hydrolysis ofATP resulting in cell death (Benz, Jung, & Sahl, 1991; Sahl,Kordel, & Benz, 1987). Other lantibiotics that also formpores include lacticin 3147, Pep5, subtilin and epidermin(Brotz et al., 1998, Kordel, Benz, & Sahl, 1988, McAuliffeet al., 1998; Schuller, Benz, & Sahl, 1989). However, it haslong been recognised that nisin also interferes with cell wallbiosynthesis (Linnett & Strominger, 1973; Reisinger,Seidel, Tschesche, & Hammes, 1980). It has now beenestablished that this phenomenon is mediated by the abilityof nisin to bind lipid II, a peptidoglycan precursor, thusinhibiting cell wall biosynthesis. Such binding is alsointrinsic to the ability of nisin to form pores. Thepossession of dual mechanisms of action renders nisinactive at nM concentrations (Breukink et al., 1999). Aspointed out previously, mersacidin also binds to lipid II,although at a different position to that of nisin, and doesnot exhibit pore-forming activity (Brotz et al., 1998).Class II bacteriocins predominantly act by forming

pores, causing dissipation of the cell membrane, depletionof intracellular ATP and leakage of amino acids and ions.The mannose permease EIIman

t of the phosphotransferasesystem, a sugar uptake/phosphorylation system, has beensuggested as a putative receptor for Class IIa bacteriocinsbased on a number of lines of evidence. This theory wasinitially fuelled by the existence of spontaneous bacter-iocin-resistant mutants displaying reduced expression ofcertain subunits, which constitute EIIman

t , i.e. subunit IIABwas found not to be produced in a leucocin A-resistantL. monocytogenes (Ramnath, Beukes, Tamura, & Hastings,2000), while mutation of mptD, encoding another mem-brane subunit of EIIman

t , in a strain of L. monocytogenes

also led to resistance to mesentericin Y105 (Dalet,Cenatiempo, Cossart, Hechard, & the European ListeriaGenome Consortium , 2001). However, the role of EIIman

t

as a target for Class IIa bacteriocins has not been fullyelucidated.

3. Using bacteriocins to improve food safety

Bacteriocins have often been mooted as potentiallyvaluable biological tools to improve the food safety andreduce the prevalence of foodborne illnesses. It is usuallysuggested that bacteriocins should not be used as theprimary processing step or barrier to prevent the growth or

ARTICLE IN PRESS

Live culture Ingredient

Fermented food Non-fermented food

Fig. 2. Bacteriocins can be incorporated directly into fermented foods by

using a bacteriocin producer as a starter or adjunct culture. Alternatively,

the producer can be used to make a food-grade fermentate, which can be

dried to make a powdered ingredient. This powder can be then

incorporated into either fermented or non-fermented foods.

L.H. Deegan et al. / International Dairy Journal 16 (2006) 1058–10711062

survival of pathogens, but rather that they could providean additional hurdle to reduce the likelihood of foodbornedisease. There already exist many control measures withinthe food industry to prevent or minimise bacterialcontamination, including good manufacturing practices,effective sanitation and hygiene measures with respect toraw materials, the food plant, the food products, the foodprocessing personnel (Moberg, 1989) and other basicfundamentals of an effective Hazard Analysis CriticalControl Point (HACCP) programme. These measuresfacilitate the identification, evaluation and control of foodsafety hazards (National Advisory Committee on Micro-biological Criteria for Foods (NACMCF), 1998). How-ever, despite these precautions, foodborne outbreaks dooccur alarmingly frequently. L. monocytogenes is ofparticular concern to the food industry and susceptibleconsumers that include pregnant women, infants, immuno-compromised individuals and the elderly. L. monocytogenes

is ubiquitous in the environment and is extremely resilient,surviving refrigeration temperatures and high salt concen-trations. Statistics for Wales and England show 194hospital cases of listeriosis in 2000, of which 68 resultedin death (Adak, Long, & O’Brian, 2002). Within the UnitedStates, the microorganism is estimated to be responsible for2500 cases, resulting in 500 deaths annually (www.cdc.gov/ncidod/dbmd/diseaseinfo/listeriosis_t.htm). A policy ofzero tolerance in ready to eat foods has been implementedin the United States for L. monocytogenes, but outbreakscontinue to occur. A recent outbreak associated withcontamination of hot dogs resulted in 101 cases and 21deaths (www.cdc.gov/ncidod/disease/foodborn/lister.htm)and the recall of 500; 000 lbs of contaminated hot dogsand meats (Donnelly, 2001). This highlights the consider-able burden this food pathogen can place on consumersand the food industry. It would seem particularlyimportant to provide an additional hurdle in food toprevent such outbreaks, and bacteriocins would be aneconomically feasible option. L. monocytogenes is not theonly concern, there exists a substantial list of foodpathogens that result in foodborne illness every year,including many Gram-negative pathogens such as Escher-

ichia coli VTEC 0157, Campylobacter and Salmonella

among others (Adak et al., 2002). Although the nature ofthe Gram-negative cell wall restricts the activity of LABbacteriocins, bacteriocins may be used in combination withother treatments, such as high hydrostatic pressure (HHP),to increase their effectiveness. Thus, bacteriocins may bebest applied when providing an extra obstacle to preventthe growth of pathogenic and spoilage bacteria, especiallyin situations where contaminationcould occur post-pro-duction.

There are at least three ways in which bacteriocins can beincorporated into a food to improve its safety (Fig. 2), i.e.,using a purified/semi-purified bacteriocin preparation as aningredient in food, by incorporating an ingredient pre-viously fermented with a bacteriocin-producing strain, orby using a bacteriocin-producing culture to replace all or

part of a starter culture in fermented foods to produce thebacteriocin in situ. The use of purified bacteriocins is notalways attractive to the food industry, as in this form theymay have to be labelled as additives and require regulatoryapproval. Nisin is utilised as an additive and was assignedthe number E234 (EEC, 1983 EEC commission directive83/463/EEC). The two other alternatives (fermentedingredient/starter culture) do not require regulatoryapproval or preservative label declarations. These optionsare frequently regarded as more attractive routes throughwhich bacteriocins can be incorporated into a food. Thesetwo options are described in greater detail below and anumber of representative examples, primarily relating tothe use of nisin, pediocin PA-1/AcH and lacticin 3147, aredescribed.

3.1. Bacteriocin preparations as ingredients

Bacteriocins can be incorporated into foods as aconcentrated, though not purified, preparation made withfood-grade techniques. For example, NisaplinTM is pre-pared by initially fermenting non-fat milk with a nisin-producing Lactococcus lactis. The resulting fermentate issubsequently concentrated and separated, spray dried andmilled to yield small particles. The end product (consistingof nisin ð1� 106 IUg�1Þ, 74.4% NaCl, 23.8% denaturedmilk solids and 1.7% moisture) has found many applica-tions in food processing and a large body of data on the useof nisin in food systems has been generated over the past 50years. While nisin has been found to be extremely effectiveas an additive to prevent spoilage and increase shelf-life ina number of foods (Chen & Hoover, 2003; Choi & Park,2000; Cutter & Siragusa, 1995; Davies et al., 1999) itseffectiveness has been more variable with respect to otherapplications (Bell & De Lacy, 1985; Jones, 1974; Jung,Bodyfelt, & Daeschel, 1992; Scott & Taylor, 1981a,b). Thefact that situations have been described where nisin is

http://www.cdc.gov/ncidod/dbmd/diseaseinfo/listeriosis_t.htm

http://www.cdc.gov/ncidod/dbmd/diseaseinfo/listeriosis_t.htm

http://www.cdc.gov/ncidod/disease/foodborn/lister.htm


ineffective highlights why it would be attractive to explorethe use of other bacteriocins. When investigating novelcandidates, there are many considerations that willdetermine their usefulness in food systems. One of themost significant criteria is an ability to withstand thermalprocessing. Thermal processing is used extensively withinthe food manufacturing process and can have adverseeffects on the bio-active capability of a bacteriocin,potentially rendering it less effective. The chemical andphysical properties of a food, e.g. pH, and fat content, canalso have a significant role in the suitability of a particularbacteriocin. This is exemplified by the fact that nisin is veryactive at acid pH, but loses activity above pH 7 (Delves-Broughton, Blackburn, Evans, & Hugenholtz, 1996),whereas another lactococcal lantibiotic, lacticin 3147,retains activity at neutral pH and significantly is particu-larly heat stable at low pH (McAuliffe et al., 1998). Itshould also be noted that nisin is generally not as effectivein the preservation of meat as it is in dairy products. This isthought to be due to interference by meat components suchas phospholipids that limit its activity, especially wherethere may be a high-fat content. The corollary is also true

2

0

0 2 4 6 0

100 200 0

4

6

8

10

12

14

Sur

viva

l (%

)

Infant formula Cotta

Time (min)

Log

cfu.

mL-1

8

7

6

5

4

3

Time (days

Cottage cheese

(a)

(b)

Fig. 3. Control of Listeria monocytogenes using (a) lacticin 3147 powder as an

producing culture (black) and non-producing culture (white).

in that nisin has been shown to be effective in thepreservation of Bologna-type sausages with a lower fatcontent (Davies et al., 1999).The Class IIa bacteriocin pediocin PA-1/AcH has

received attention due to its ability to protect fresh andfermented meat from contamination by L. monocytogenes.Its use seems especially appropriate when combined withmodified atmosphere packaging (MAP). In fact, the use ofMAP to package meat under higher levels of carbondioxide to reduce the incidence of Gram-negative spoilagebacteria can be especially conducive towards bacteriocinproduction as it favours the proliferation of non-spoilageLAB that can contribute to increasing the shelf-life ofrefrigerated meats (Hitchener, Egan, & Rogers, 1982; Shaw& Harding, 1984; Stiles & Hasting, 1991). The LABmicroflora found in chilled meat predominantly consists oflactobacilli and pedicocci, and significantly many pediocinPA-1/AcH-producing strains have been isolated from meat(Bhunia, Johnson, & Ray, 1988; Gonzalez & Kunka, 1987;Rodriguez et al., 1997). Pediocin PA-1/AcH has also beentested as a bio-active ingredient in various dairy systems,thus demonstrating its effectiveness over a range of

1 2 3

100 200 0 75 150

ge cheese Yoghurt

)

Surface of mould cheese

ingredient at 0 (black), 5 (grey) and 10% (white), and (b) a lacticin 3147-


different pH and in varying levels of fat (for a review seeRodriguez, Martinez, & Kok, 2002). Lacticin 3147 has alsobeen produced and tested as a bio-preservative. Aproducing strain, Lc. lactis DPC3147, was used to fermentreconstituted demineralised whey (10% solids), which waspasteurised, concentrated and spray dried to produce a bio-active lacticin 3147 powder (Morgan, Galvin, Kelly, Ross,& Hill, 1999). This powder was subsequently tested toinvestigate its effectiveness against L. monocytogenes ScottA and Bacillus cereus in natural yoghurt, cottage cheeseand soup showing the potential of lacticin 3147 as an aid toeliminate pathogenic organisms (Fig. 3). Furthermore,lacticin-containing powder has also been tested in infantmilk formula, which is of particular significance as infantsare more vulnerable to the effects of listeriosis. Ominously,counts of L. monocytogenes increased 700% in controlsamples over a 3 h period, whereas the addition of either 15or 10% lacticin powder resulted in 99% kill after the sameperiod (Fig. 3). Although there is no doubt that lacticin3147 powder has the ability to kill pathogenic organisms inthe foods tested, the optimisation of lacticin 3147 powderto increase specific activity is needed, as it may not befeasible to replace 10% of the product with lacticin powder(Morgan et al., 1999, Morgan, Galvin, Ross, & Hill, 2001).Large-scale production of this lantibiotic could be possiblethrough immobilisation of DPC3147 to calcium alginatebeads to improve the stability and productivity ofbacteriocin over conventional cell-free fermentations in alab setting. Some initial investigations in this regard haveyielded promising results (Scannell et al., 2000a,b).Variacin, a bacteriocin produced by Kocuria varians, hasalso been used to produce a milk-based fermentedingredient and was used to evaluate its effectiveness againstB. cereus in chilled dairy products, vanilla and chocolatedesserts. The fermented ingredient was tested using either1, 3 or 10% and over a range of abusive temperatures 8, 12and 30 �C. A correlation between temperature abuse andthe level of bio-active ingredient required to be effectivewas very evident. Although the use of the fermentate atlower percentages showed potential at lower temperatures,the use of 10% fermentate was more effective overall(O’Mahony, Rekhif, Cavadini, & Fitzgerald, 2001).Further, showing the much needed investment intooptimising the production of these and other bio-activefermentates.

3.2. Bacteriocin-producing cultures

The possibility of including live bacteriocin-producingLAB in foods has been touched on previously in thisreview. This system of incorporating a bacteriocin-produ-cing culture into a food gives it its own built in bio-preservation, thereby returning to a more natural methodof shelf-life extension and improving the safety of food (fora review see O’Sullivan, Ross, & Hill, 2002).

There can, however, also be problems associated withthe utilisation of bacteriocin-producing cultures in fermen-

ted foods, the most obvious being a lack of compatibilitybetween the bacteriocin-producing strain and other cul-tures required in the fermentation. This was found to be thecase for nisin-producing cultures, which inhibit the startercultures required for cheese making. In an attempt toovercome this problem, the viability of using nisin-producing starters was assessed, but unfortunately thesestarters suffered from poor acid development, possessedinadequate proteolytic activity and often displayed anenhanced susceptibility to bacteriophage attack. As aconsequence, without intervention or troubleshooting,nisin-producing cultures appear to be unsuitable as a bio-preservative in cheese production. However, the manipula-tion of multi-strain starters can be adopted by usingbacteriocin-producing strains in concert with other culturespossessing desirable traits such as efficient acid production(Lipinska, 1973). The use of cultures to produce bacter-iocins in situ as a means of bio-preservation has received agreat deal of interest in recent times. The inhibition ofL. monocytogenes in dry-fermented sausage using lyophi-lised bacteriocin-producing cultures as a protective adjuncthas been investigated. To reflect realistic pathogencontamination levels, sausage meat was artificially spikedwith a four strain cocktail at 10�2–10�3 cfu g�1. Lb.

curvatus was more successful than Lc. lactis, as following4 h fermentation, L. monoctogenes was reduced too10 cfu g�1. Also recovery proceeding enrichment wasnot possible after day 8 of drying. Lc. lactis also decreasedlisteria to below the detectable limit after day 15 of drying.Furthermore, pH was not regarded as a factor inelimination of L. monocytogenes in this study as therewas no significant difference between samples containingbacteriocin-producing cultures and those that did notcontain these cultures (Benkerroum et al., 2005).The utilisation of Carnobacterium strains to combat

listerial contamination in cold-smoked salmon (CSS) hasbeen studied and has shown potential, but due to theemergence of resistance to Class II bacteriocins there hasbeen an increased interest in the use of bacteriocin-negativeCarnobacterium strains as protective cultures (Nilsson et al.,2004). However, a recent study highlights the potential ofC. divergens V41 for effective bacteriocin production in situfor bio-preservation of CSS. C. divergens when comparedwith two other bacteriocin-producing Carnobacterium strainswas more efficient as during a 4-week period of vacuumstorage levels of L. monocytogenes were substantially reducedto o50 cfu g�1 regardless of the pathogens sensitivity. Inaddition, this strain colonised the CSS to a higher level thanthe other strains, which could be a contributing factor to itseffectiveness against all L. monocytogenes strains tested. Thisstudy also shows the different sensitivities to bacteriocinsthat can be encountered with pathogens (Brillet, Pilet,Prevost, Bouttefroy, & Leroi, 2004). The strains used in thisstudy were isolated directly from salmon in the environmentand salmon products, which is undoubtedly an importantaspect when considering the use of a bacteriocin to beapplied to a certain food system.


Lacticin 3147-producing strains have also been added tofoods. As the genetic determinants for lacticin 3147 areencoded on a large conjugative plasmid pMRC01, they canalso be transferred in a food-grade manner to manycommercially useful starter cultures (Coakley, Fitzgerald,& Ross, 1997). pMRC01 also contains genes that encoderesistance to bacteriophage attack, an industrially impor-tant trait in the manufacture of cheese. A lacticin 3147transconjugant was used successfully in a study where itseffectiveness against L. monocytogenes Scott A in cottagecheese was investigated (McAuliffe, Hill, & Ross, 1999).The ability of the producing strain to reduce numbers ofL. monocytogenes ð104 cfumL�1Þ artificially introducedinto cottage cheese (pH 5.2) at 4, 18 and 30 �C was testedover a duration of 1 week. Reductions of 99.9% were seenin cottage cheese held at 4 �C after only 5 days (Fig. 3),whereas at the elevated temperatures the reductionsoccurred more promptly. Mould-ripened cheese (pH6.5–8.0) is a favourable setting to support the growth ofL. monocytogenes. A lacticin 3147 transconjugant showed apositive protective effect, reducing L. monocytogenes 1000-fold when applied to the surface of the cheese (Ross,Stanton, Hill, Fitzgerald, & Coffey, 2000). In contrast, thisprotective effect was not apparent when a nisin-producingculture was used, most probably due to pH instability(Ross et al., 1999). These studies are representative ofsimilar observations associated with a variety of differentbacteriocin producers.

Recently, a food-grade strain has been developed toproduce both lacticin 3147 and lacticin 481. This strainaddresses both the food safety and food improvementaspects of bacteriocin production. Significantly, the killingeffect of this double producer was more pronounced, whentested against Lb. fermentum and L. monocytogenes, thaneither bacteriocin producer alone (O’Sullivan, Ross, & Hill,2003). The use of strains that produce multiple bacteriocinscould be advantageous to limit the potential emergence ofbacteriocin-resistant populations. Resistance can occurnaturally and it has been reported especially with regardsto Class IIa bacteriocins such as pediocin PA-1 andmesentericin Y105 among others (Ennahar, Sashihara,Sonomoto, & Ishizaki, 2000). Pediocin-acquired resistancehas been reported at levels of 10�4–10�6 (Dykes &Hastings, 1998), while nisin-resistant mutants occur at afrequency of 10�2–10�8 (Gravesen, Jydegaard Axelson,Mendes de Silva, Hansen, & Knochel, 2002; Mazzotta,Modi, & Montville, 2000, Ming & Daeschel, 1993). Incontrast, lacticin 3147-resistant strains have not been foundin the presence of high levels of the bacteriocin (Coakleyet al., 1997; Dodd, 1996; Klijn, 2001; Ryan, Meaney, Ross,& Hill, 1998). Given the manner in which bacteriocinsare likely to be used in food, i.e. as one of a numberhurdles, it is unlikely that the food industry will see theemergence of bacteriocin-resistant bacteria in the extent towhich antibiotic-resistant pathogens have emerged inhospitals. However, the utilisation of double bacteriocin-producing strains would provide an additional barrier to

ensure that the emergence of resistant populations is evenless likely.As stated previously, food producers and consumers

may frequently serendipitously benefit from the presence ofbacteriocin-producing LAB in foods. One particularlyinteresting study has demonstrated that of 48 differentvacuum-packed meat products, bacteriocin-producing co-lonies were isolated from 46% of the packages examined(Budde, Hornbaek, Jacobsen, Barkholt, & Koch, 2003).This study confirms that bacteriocin-producing cultures arefrequently found in some commercially available foods.In summary, the use of bacteriocin-producing cultures in

foods can be very beneficial. While the choice ofbacteriocin-producing culture may have to vary to meetwith the particular microenvironment associated withcertain foods, the net result of this technology can be theproduction of foods that have a built-in preservationsystem.

4. Bacteriocins in combination with additional hurdles

Hurdle technology refers to the manipulation of multiplefactors (intrinsic and extrinsic) designed to preventbacterial contamination or control growth and survival infood. A combination of preservation methods may worksynergistically or at least provide greater protection than asingle method alone, thus improving the safety and qualityof a food. While in certain foods intrinsic properties suchas high salt (see a review by Leistner, 2000) may provideadequate protection, the conscious addition of an extrahurdle(s) can ensure safety. The role of bacteriocins ashurdles in food systems has been documented in this, andother, reviews. However, it is also apparent that abacteriocin alone in a food is not likely to ensurecomprehensive safety. This is of particular significancewith regards to Gram-negative pathogenic bacteria that areprotected by the presence of an outer membrane. When theouter membrane is impaired by agents such as the food-grade chelating agent ethylene diamine tetraacetate(EDTA), which acts by binding to Mg2þ ions inlipopolysaccharide, the outer membrane is disruptedrendering Gram-negatives sensitive to bacteriocins (Ste-vens, Sheldon, Klapes, & Klaenhammer, 1991).Non-thermal treatments such as HHP and pulsed electric

field (PEF) represent other methods of food preservation.As these technologies are non-thermal, they are advanta-geous as they have little or no effect on food functionalityand nutritional qualities compared to thermal methodswhich affect vitamin content. As a consequence of usingthese non-thermal methods, the bacterial cell membranebecomes destabilised, thus interfering with essential cellfunctions (Jeyamkondan, Jayas, & Holley, 1999). While theutilisation of HHP or PEF alone is not always economic-ally viable, lower levels could be used if combined withother treatments such as bacteriocins. The benefits ofcombining nisin with HHP (Capellas, Mor-Mur, Gervilla,Yuste, & Guamis, 2000; Lopez-Pedemonte, Roig-Sagues,


Trujillo, Capellas, & Guamis, 2003; Stewart, Dunne, Sikes,& Hoover, 2000) and to a lesser extent PEF (Pol, Mastwijk,Bartels, & Smid, 2000; Terebiznik, Jagus, Cerrutti, deHuergo, & Pilosof, 2000) has been demonstrated exten-sively. Lacticin 3147 was also used in concert with HHPat pressures of 150–275MPa to investigate the effectson Staphylococcus aureus and L. innocua. Using10; 000AUmL�1 and the lower pressure of 150MParesulted in a 2:1 log kill of S. aureus relative to a controllacking lacticin where a o0:5 log kill was observed. Amore pronounced 46 log kill was observed when lacticin3147 was combined with a higher pressure of 275MPa.Similar, although less marked, trends were seen forL. innocua (Morgan, Ross, Beresford, & Hill, 2000). Morerecently, the ability of seven bacteriocin-producing LABhave been investigated in combination with HHP at 300and 500MPa in raw cheese that had been contaminatedwith E. coli 0157:H7. In all cases, HHP- and bacteriocin-producing adjuncts where synergistic resulting in theincreased inactivation of E. coli 0157:H7 compared toeach hurdle individually. In particular, HHP at 300MPaon day 50 (of 60-day-old cheeses) in combination withadjuncts producing nisin A, bacteriocin TAB 57 andenterocin I or AS-48 resulted in the complete eliminationof E. coli 0157:H7 (Rodriguez, Arques, Nunez, Gaya, &Medina, 2005). Similarly, S. aureus was inactivated usingbacteriocin-producing cheese adjuncts with HHP, whichwas also synergistic (Arques et al., 2005).

Bacteriocins used in combination with organic acids orother antimicrobials can also result in enhanced inactiva-tion of bacteria. This phenomenon has been demonstratedfor pediocin following artificial contamination of beeffranks with 107 cfu g�1 L. monocytogenes; pediocin in theform of ALTA 2341 was used in combination with sodiumdiacetate (SD) and sodium lactate (SL) as dippingsolutions. It was found to be very effective in controllingL. monocytogenes in vacuum packed beef franks stored at4 �C. Pediocin ð6000AUÞ reduced levels of L. monocyto-

genes by o1 log cfu g�1 unit, following storage for 14 and21 days. This activity was enhanced by the addition ofantimicrobials SD and SL, after the same period of storageL. monocytogenes was reduced by 1.5 to 2:5 log cfu g�1,respectively (Uhart, Ravishankar, & Maks, 2004). In amore recent comprehensive study, nisin and pediocin areused in combination with a number of antimicrobialsincluding citric acid and potassium sorbate to combatL. monocytogenes on fresh-cut produce. The synergisticnature of hurdle technology was evident from this study asin nearly all cases combinations were better than nisin andpediocin alone. Nisin with phytic acid was most effectiveoverall at reducing L. monocytogenes (Bari et al., 2005). Ina study by Scannell et al. it was shown that lacticin 3147activity was improved in fresh pork sausage with either SLor sodium citrate (SC). In particular Clostridium perfrin-

gens was reduced by 3:1 log with only lacticin 3147 but wasnot recovered for up to 4 days with the addition of eitherSL or SC. The use of lacticin 3147 or nisin with SC reduced

Salmonella enterica var. Kentucky by �1 and 1:6 log,respectively, again highlighting that the use of combina-tions of preservation techniques can limit the risk posed byGram-negative pathogens (Scannell, Ross, Hill, & Arendt,2000). Bio-active packaging is a further potential applica-tion in which bacteriocins can be incorporated intopackaging destined to be in contact with food. This systemcombines the preservation function of bacteriocins withconventional packaging materials, which protects the foodfrom external contaminants. Spoilage of refrigerated foodsusually begins with microbial growth on the surface, whichreinforces the attractive use of bacteriocins being used inconjunction with packaging to improve food safety andimprove shelf-life (Collins-Thompson & Hwang, 2000).Bio-active packaging can be prepared by directly immobi-lising bacteriocin to the food packaging, or by addition of asachet containing the bacteriocin into the packaged food,which will be released during storage of the food product.Studies investigating the effectiveness of bio-active cellu-lose-based packaging inserts and a vacuum packagingpouch made with polyethylene/polyamide to improve shelf-life and safety aspects have proved promising. Whenconsidering bio-active packaging, the stability and theability to retain activity while immobilised to the packagingfilm is of vital importance. Studies with lacticin 3147 andnisin have shown that lacticin 3147 did not absorbeffectively to plastic, whereas nisin in the form ofNisaplinTM absorbed to the surface tightly, resulting ineffective bio-active films that reduced levels of non-starterLAB (NSLAB), L. innocua and S. aureus in vacuum andMAP packaged cheddar cheese, and also showed protec-tion towards ham that had been packaged under MAP.During this study, it was also demonstrated that the insertswere stable at ambient and refrigeration temperatures andthat the bio-active packaging for optimal protection mustbe in direct contact with the food. (Scannell et al., 2000b).While bacteriocins may also be incorporated into an ediblefilm or coating that can be applied to the food either bydipping or spraying, this approach has legislative implica-tions as it cannot be described as active packaging only. Ina study by Coma et al. whereby nisin was added to a film-forming solution, although, L. innocua and S. aureus wereinhibited, it was apparent that other constituents, such asstearic acid added to the film to improve water vapourbarriers, can have adverse effects on the activity ofincorporated bacteriocins (Coma, Sebti, Pardon,Deschamps, & Pichavant, 2001). Methods involving theincorporation of chelating agents such as EDTA (alreadydescribed in this review) could also be an option for activepackaging designed to provide protection against Gram-negative spoilage/pathogenic contamination. This has beeninvestigated by Siragusa, Cutter, and Willett (1999) andwas effective, when used with nisin, in inhibiting E. coli inbeef carcasses. It would seem apparent that this might bean effective way of delivering bacteriocins to the surface offood to improve preservation and shelf-life. Further, thecontinued research in this area taking into consideration


both the positive and negative aspects with new develop-ments in polymeric packaging could lead to an advancedsystem of bio-active packaging.

Thus, while many LAB bacteriocins possess significantantimicrobial qualities that could greatly enhance thesafety of a food, it may yet emerge that industrially theywill be most frequently applied as a ‘final-hurdle’ in a foodsystem where another hurdle(s) already exists to eliminatepathogens and spoilers that survive only in adventitiouscircumstances.

5. Bacteriocins for improving food quality and flavour

Bacteriocins can also be used to improve the quality andsensory attributes of certain foods (Fig. 2). During themanufacturing of cheese, bacteriocin-producing LAB canbe utilised to control adventitious flora such as NSLABand also in the induction of cell lysis, thereby increasing therate of proteolysis in cheese (see a review by Guinane,Cotter, Hill, & Ross, 2005) The role of NSLAB in cheeseripening has not been entirely elucidated but it is apparentthat they are responsible for defects such as the formationof calcium lactate crystals (due to an ability to racemise L-lactate to D-lactate), slit formation and off-flavour devel-opment, but they are likely to also have a positive effect onflavour. Using bacteriocins as tools to influence thedevelopment of NSLAB in cheese could prove to be veryadvantageous by giving cheese manufacturers more controlover this normally uncontrolled flora. A lacticin 3147transconjugant, used as the starter culture in the produc-tion of low-fat cheddar cheese, proved to be efficient incontrolling NSLAB during ripening (Fenelon et al., 1999).The control of NSLAB in cheese at an elevated ripeningtemperature could prove to be very economically attractiveto the cheese industry.

Lactococci-producing lactococcin ABM (a combinationof three Class IIa plasmid-encoded bacteriocins) have anarrow spectrum with bacteriolytic and bacteriostaticeffects on sensitive lactococci (Morgan, Ross, & Hill,1995). Although these bacteriocins have a narrow spectrumand do not exhibit activity against pathogens, ABMproducers do possess substantial potential with respect toimproving cheese quality and flavour as the presence oflactococcin ABM accelerates the rate-limiting step ofreleasing intracellular proteinases and peptidases from thestarter culture, contributing to the flavour. A shortcomingassociated with the use of bacteriocins for lysing starterculture cells is reduced acidification, especially in caseswhere a bacteriocin- sensitive starter is employed. It canalso be extremely difficult to get the correct balancebetween lysis and appropriate levels of acidification. As aresult, a three starter system was developed using Lc. lactis

HP, Lc. lactis DPC3286 and a Streptococcus thermophilus

DPC1842 strain that is not susceptible to lactococcin ABMand which contributes to acid production (Morgan,O’Sullivan, Ross, & Hill, 2002). Although there arecommercial enzymes available to carry out the procedure

of starter cell lysis, they can be costly and inefficient,whereas bacteriocin-producing cultures can perform thisprocess without adverse cost implications and with animprovement in the overall quality and flavour of thecheese. Danisco have formulated a freeze-dried cultureof Pediococcus acidilactici marketed as CHOOZITTM

Lyo. Flav 43 (product description—PD203800-1EN).CHOOZITTM which can be used for ‘‘cheddar cheese andsemi-hard cheese’’ as an ‘‘adjunct, which accelerates andenhances strong and sweet flavour compounds, due to theproduction of bacteriocins.’’The ability to induce the release of intracellular enzymes

has also been associated with the lacticin 3147 producingstrain Lc. lactis IFPL359 resulting in the transamination ofbranched chain amino acids into the volatile compound 2-methylbutanal in cheese. The formation of this compoundis desirable in cheese production as it is responsible forflavour enhancement (Fernandez de Palencia et al., 2004).Similarly, a lacticin 481 (a medium spectrum lantibioticwith activity against other LAB and Cl. tyrobutryricum)producing adjunct has also shown great potential withrespect to the control of NSLAB and the acceleration ofstarter cell lysis without acid production by the starterbeing compromised (O’Sullivan, Ryan, Ross, & Hill, 2003).Avila et al. have also shown the potential of using lacticin481- or a plantaracin-producing adjunct with the highlypeptidolytic Lb. helveticus for the manufacture of cheesedue to the improvement of cell-free aminopeptidases(Avila, Garde, Medina, & Nunez, 2005).

6. Conclusion/future prospects

Civilisation has reaped the benefits of bacteriocinsunknowingly for 1000s of years, yet nisin is the onlybacteriocin bio-preservative that has received acceptance incountries worldwide. Knowing that other bacteriocins existand can work at least as effectively as nisin (if not more so)with respect to particular foods/target bacteria, thequestion is often posed why more have not been exploitedto the same extent. In addition to the difficulties that canarise with respect to legislation, there may be a reluctanceto commit financially to the development/formulation ofcommercial products akin to NisaplinTM. Despite this, itshould be apparent that a wide array of means existthrough which a bacteriocin can be incorporated into afood, all of which are at the disposal of the food sector.It would be naive to believe that bacteriocins represent

the ultimate solution to food safety problems. However,given the effectiveness of bacteriocins, the existence ofeconomically viable means through which they can beincorporated and a consumer desire for minimally pro-cessed food, they may represent an excellent alternative foruse in combination with other natural preservatives. Thereis a need to attract consumer attention to the existence ofnatural substances that can protect against food-relatedillness. The acceptance of probiotics by the consumer wasaided greatly when such bacteria were marketed as natural


cultures that aid in digestion/health. In the same way,bacteriocins and bacteriocin-producing cultures should beattractive, especially as a consequence of consumer distrustof chemical preservatives.

While the application of bioengineered/modified bacter-iocins may be counterproductive with respect to themarketing of bacteriocins as natural products, the creationof such derivatives has been beneficial with respect to ourunderstanding of these peptides. Such techniques could beutilised to improve the stability and production ofbacteriocins so they may be more applicable in foodproducts. There have already been examples of suchsuccesses, for example, a single amino acid change in nisinZ, whereby a methionine at position 21 was changed to alysine, resulted in a 5-fold increase in the solubility of thepeptide at pH 8 compared to wild-type nisin Z plus anextension of its antimicrobial spectrum to encompassGram-negative bacteria (Yuan, Zhang, Chen, Yang, &Huan, 2004). As some of the food matrices and processesemployed by the food industry can affect the activity/stability of bacteriocins, the generation of derivatives withenhanced features could be very rewarding from a foodsafety and economic perspective, but only if any associatedconsumer concerns could be addressed.

Continued research on bacteriocins will undoubtedlylead to our increased understanding, and with theemergence of new bacteriocins, new potential bio-preser-vatives. However, this potential will only be fulfilled ifbacteriocins are appropriately used and marketed success-fully.

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Bacteriocins: Biological tools for bio-preservation and shelf-life extension

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