In spite of modern advances in technology, the preservationof foods is still a debated issue, not only for developing countries(where implementation of food preservation technologies areclearly needed) but also for the industrialized world. Amelio-ration of economic losses due to food spoilage, lowering thefood processing costs and avoiding transmission of microbialpathogens through the food chain while satisfying the growing
⁎ Corresponding author. Área deMicrobiología, Departamento de Ciencias de laSalud, Facultad de Ciencias Experimentales, Universidad de Jaén, Campus LasLagunillas s/n. 23071-Jaén, Spain. Tel.: +34 953 212160; fax: +34 953 212943.
consumers demands for foods that are ready to eat, fresh-tasting,nutrient and vitamin rich, and minimally-processed and pre-served are major challenges for the current food industry. Theextent of microbiological problems in food safety was clearlyreflected in the WHO food strategic planning meeting (WHO,2002): (i) the emergence of new pathogens and pathogens notpreviously associated with food consumption is a major concern;(ii) microorganisms have the ability to adapt and change, andchanging modes of food production, preservation and packaginghave therefore resulted in altered food safety hazards.
The empirical use of microorganisms and/or their naturalproducts for the preservation of foods (biopreservation) has beena common practice in the history of mankind (Ross et al., 2002).The lactic acid bacteria (LAB) produce an array of antimicrobial
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substances (such as organic acids, diacetyl, acetoin, hydrogenperoxide, reuterin, reutericyclin, antifungal peptides, andbacteriocins (Holzapfel et al., 1995; El-Ziney et al., 2000;Holtzel et al., 2000; Magnusson and Schnürer, 2001). Bacter-iocins are ribosomally synthesized antimicrobial peptides orproteins (Jack et al., 1995). LAB produce a variety ofbacteriocins, most of which can be grouped in one of the classesproposed by Klaenhammer (1993). The structure, biosynthesis,genetics and food application of LAB bacteriocins have beenreviewed recently (Cleveland et al., 2001; O'Sullivan et al.,2002; Chen and Hoover, 2003; Cotter et al., 2005; Fimland et al.,2005; Deegan et al., 2006; Drider et al., 2006).
The bacteriocins produced by LAB offer several desirableproperties that make them suitable for food preservation: (i) aregenerally recognised as safe substances, (ii) are not active and non-toxic on eukaryotic cells, (iii) become inactivated by digestiveproteases, having little influence on the gut microbiota, (iv) areusually pH and heat-tolerant, (v) they have a relatively broadantimicrobial spectrum, against many food-borne pathogenic andspoilage bacteria, (vi) they show a bactericidal mode of action,usually acting on the bacterial cytoplasmic membrane: no crossresistance with antibiotics, and (vii) their genetic determinants areusually plasmid-encoded, facilitating genetic manipulation.
The accumulation of studies carried out in recent years clearlyindicate that the application of bacteriocins in food preservationcan offer several benefits (Thomas et al., 2000): (i), an extendedshelf life of foods, (ii) provide extra protection during tem-perature abuse conditions, (iii) decrease the risk for transmissionof foodborne pathogens through the food chain, (iv) amelioratethe economic losses due to food spoilage, (v) reduce theapplication of chemical preservatives, (vi) permit the applicationof less severe heat treatments without compromising food safety:better preservation of food nutrients and vitamins, as well asorganoleptic properties of foods, (vii), permit the marketing of“novel” foods (less acidic, with a lower salt content, and with ahigher water content), and (viii) they may serve to satisfyindustrial and consumers demands. In this respect some of thetrends of the food industry in Europe, such as the need toeliminate the use of artificial ingredients and additives, thedemands for minimally-processed and fresher foods, as well asfor ready-to-eat food or the request for functional foods andnutraceuticals (Robertson et al., 2004) could be satisfied, at leastin part, by application of bacteriocins. The present review willaddress different aspects related to food preservation by bac-teriocins including factors influencing bacteriocin activity infood systems, hurdle technology, and the impact of recentadvances in molecular biology and the analysis of bacterialgenomes on bacteriocin studies and application.
2. Bacteriocins and food systems
2.1. Supplementing foods with bacteriocins
Foods can be supplemented with ex situ produced bacteriocinpreparations, or by inoculation with the bacteriocin-producerstrain under conditions that favour production of the bacteriocinin situ (Schillinger et al., 1996; Stiles, 1996). In the first case,
bacteriocin preparations obtained by cultivation of the producerstrain in a fermentor at industrial scale followed by adequaterecovery and processing can be added as partially purified orpurified concentrates, which would require specific approval aspreservatives from the legislative point of view. So far, nisin isthe only bacteriocin licensed as a food preservative (E234).Many preliminary studies on the activity of bacteriocins in vitroor in food systems are carried out with partially-purified prep-arations obtained from cultured broths. In most cases, a lowconcentration of bacteriocin is often recovered, which limits theefficacy of such preliminary tests.
Ex situ produced bacteriocins can also be added in the formof raw concentrates obtained by cultivation of the producerstrain in a food-grade substrate (such as milk or whey). Theresulting preparations may be regarded as food additives oringredients from the legal point of view, since some of theircomponents may play a recognised function in the food (such asincrease in protein content, or thickening). They also contain thecell-derived antimicrobial metabolites (such as lactic acid) andbacteriocins, affording an additional bioprotectant function. Inaddition to already-marketed concentrates such as ALTA™2341 or Microgard™, other milk-based preparations have beendescribed recently such as lacticin 3147 (Morgan et al., 1999;Guinane et al., 2005) and variacin (O'Mahony et al., 2001).
Ex situ produced bacteriocins can also be applied in the formofimmobilized preparations, in which the partially-purified bacte-riocin or the concentrated cultured broth is bound to a carrier. Thecarrier acts as a reservoir and diffuser of the concentrated bac-teriocin molecules to the food ensuring a gradient-dependentcontinuous supply of bacteriocin. The carrier may also protect thebacteriocin from inactivation by interaction with food compo-nents and enzymatic inactivation. Moreover, the precise localizedapplication of bacteriocin molecules on the food surface requiresmuch lower amounts of bacteriocin (compared to applicationin the whole food volume), decreasing the processing costs. Avariety of methods have been proposed for bacteriocin immobi-lization, including adsorption to the producer cells (Yang et al.,1992; Mattila et al., 2003; Ghalfi et al., 2006), silica particles orcorn starch powder (Coventry et al., 1996; Dawson et al., 2005),liposome encapsulation (Degnan and Luchansky, 1992), andincorporation on gel coatings and films of different materials suchas calcium alginate, gelatin, cellulose, soy protein, corn zein,collagen casings, polysaccharide based films, cellophane, siliconcoatings, polyethylene, nylon or other polymer plastic films(Daeschel et al., 1992; Cutter and Siragusa, 1995b; Ming et al.,1997; Siragusa et al., 1999, Natrajan and Sheldon, 2000; Gill andHolley, 2003; Scannell et al., 2000a; Ko et al., 2001; Dawsonet al., 2002; Franklin et al., 2004; Luchansky and Call, 2004;Guerra et al., 2005; Lungu and Johnson, 2005). In most cases,immobilized bacteriocin preparations are applied on the surface ofthe processed food to avoid post-process contamination andsurface proliferation of unwanted bacteria. A recent advance inthis field is the use of immobilized bacteriocins in the devel-opment of antimicrobial packaging. A polyethylene film contain-ing immobilized bacteriocin 32Y from L. curvatus reduced viablecounts of L. monocytogenes during storage in the packaged porksteak and ground beef as well as in frankfurters (Mauriello et al.,
Table 1Bacteriocin efficacy in foods: limiting factors
Food-related factors–Food processing conditions–Food storage temperature–Food pH, and bacteriocin unstability to pH changes–Inactivation by food enzymes–Interaction with food additives/ingredients–Bacteriocin adsorption to food components–Low solubility and uneven distribution in the food matrix–Limited stability of bacteriocin during food shelf life
The food microbiota–Microbial load–Microbial diversity–Bacteriocin sensitivity–Microbial interactions in the food system
The target bacteria–Microbial load–Bacteriocin sensitivity (Gram-type, genus, species, strains)–Physiological stage (growing, resting, starving or viable but non-culturablecells, stressed or sub-lethally injured cells, endospores...)
–Protection by physico-chemical barriers (microcolonies, biofilms, slime)–Development of resistance/adaptation
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2004; Ercolini et al., 2006). Similarly, a nisin-containing cel-lophane coating reduced viable counts of total aerobic bacteria infresh veal meat stored at 8 °C (Guerra et al., 2005), and an activepackage obtained from nisin-treated film reduced viable counts ofM. luteus ATCC 10,240 cells in broth as well as in raw milk andpasteurized milk during storage (Mauriello et al., 2005).Therefore, the use of antimicrobial films containing bacteriocinscan improve the quality and safety and prolong the shelf-life offood products.
In situ bacteriocin production offers several advantagescompared to ex situ production regarding both legal aspects andcosts. Lowering the costs of biopreservation processes may behighly attractive, especially for small economies and developingcountries, where food safety may be seriously compromised(Holzapfel, 2002). Many studies have focused on the selectionand development of bacteriocinogenic cultures for food ap-plication (Ross et al., 2000; Työppönen et al., 2003; Peláez andRequena, 2005; Foulquié Moreno et al., 2006; Leroy et al.,2006). The use of bacteriocinogenic cultures requires carefulselection of strains that are well-adapted to the particular foodenvironment in which they will be used and able to grow underthe food processing and/or storage conditions and to produceenough bacteriocin amounts as to inhibit the target pathogenic orspoilage bacteria. Therefore, it is necessary to implement theright experimental approaches to select bacteriocin-producingstrains that are suitable for use in food production. The strainproperties and the amount of bacteriocin produced could beimproved by heterologous expression of bacteriocin genes(Rodríguez et al., 2003; Zhou et al., 2006), and the precisemoment of bacteriocin production could also be tailored byusing inducible production systems (Zhou et al., 2006).
Bacteriocinogenic strains can be used either directly as startercultures, as adjunct or co-cultures in combination with a starterculture, or as protective cultures (especially in the case of non-fermented foods). When used as a starter culture, the bac-teriocinogenic strain must be able to carry out the desired fer-mentation process optimally besides being able to produceenough bacteriocin amounts to afford protection. In some cases,bacteriocin production may also serve to increase the implan-tation capacity, competitiveness and stability of the starter(Todorov et al., 1999). Adjunct cultures do not need to contributeto the fermentation, but they must not interfere with the primaryfunction of the starter culture. For this reason, bacteriocin re-sistance of the starter culture may be a key factor. This may beachieved by selection of natural resistant mutants, by adaptationthrough repeated subcultivation with increasing bacteriocinconcentrations, or by genetic modification. Nevertheless, some-times this may not be necessary as the bacteriocinmay just not beactive on the starter culture (as may be the case of many of thebacteriocins that predominantly show antilisterial activity) or thismay be much more tolerant to the bacteriocin than the targetbacteria in the food system. Differences in inoculum density, afaster growth rate of the starter or a delayed bacteriocin productionmay also permit the starter to grow without interference from thebacteriocinogenic adjunct culture. As an example, inoculation ofmilk with an enterocin AS-48 producer enterococcal strain asadjunct culture in combination with a commercial starter culture
for cheese manufacture had no effect on growth of the starter orthe physicochemical properties of the produced cheese. At thesame time, enough bacteriocin was produced in the cheese toensure inhibition of Bacillus cereus (Muñoz et al., 2004).
Bacteriocinogenic protective cultures can be used to inhibitspoilage and pathogenic bacteria during the shelf life period ofnon-fermented foods. A protective culture may grow and pro-duce bacteriocin during refrigeration storage of the food, and/orduring temperature abuse conditions. In the first case, growthof the protective cultures must have a neutral impact on thephysicochemical and organoleptic properties of the food, whileunder temperature abuse conditions the protective culture mayeven act as the predominant spoiler, ensuring that pathogenicbacteria do not grow and that the spoiled food is not consumed(Holzapfel et al., 1995).
2.2. Factors influencing the efficacy of bacteriocins in foodsystems
Comparisons of data obtained in culture media with thoseobtained in food systems reveal that the efficacy of bacteriocins isoften much lower in the later (Schillinger et al., 1996). Sometime,at least ten-fold higher bacteriocin concentrations must be addedto foods in order to achieve an equivalent inhibitory effect. Theefficacy of bacteriocins in foods will greatly depend on a numberof food-related factors (Table 1) that in most cases involveinteraction with food components, precipitation, inactivation, oruneven distribution of bacteriocin molecules in the food matrix.As an illustrative example, application of nisin in meat productsfaces several limitation derived from its interaction withphospholipids emulsifiers and other food constituents (Henninget al., 1986; Jung et al., 1992; Aasen et al., 2003), poor solubilityat pH above 6.0, and inactivation by formation of a nisin-glutathione adduct (Rose et al., 2003). However, inactivation is
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lower in cooked meats due to the loss of free sulphydryl groupsduring cooking as a result of the reaction of glutathione withproteins (Stergiou et al., 2006).
Foods are complex ecosystems with a range of microbialcompositions. These vary from (commercially) sterile foods toraw or fermented foods. In commercially sterile foods, micro-organisms from post-process contamination may easily prolif-erate because of the lack of competitors. Under these conditions,the efficacy of the bacteriocin will depend more directly on themicrobial load of the contaminant, and a higher bacteriocinconcentration will always be required in order to inactivate ahigher number of target cells. In raw foods, the autochthonousmicrobiota may counteract development of potential pathogens.However, a complex food microbiota may frequently cause alower efficacy of bacteriocins due to the presence of resistantbacteria (such as Gram-negatives) and/or the higher chances forproduction of inactivating enzymes (such as proteases). Inaddition, the bacteriocin may not always target the desiredbacterial group (for example, bacteriocin inactivation of theLAB responsible for food fermentation would be clearly un-desirable). In processed foods, microorganisms can also befound in a variety of physiological stages, which may greatlyinfluence bacteriocin sensitivity. As an example, cells that arenot actively growing may be more resistant to bacteriocins, andchanges of the target organisms in response to environmentalstress factors may also result in decreased bacteriocin sensitivity.Inactive bacterial forms (endospores) may also be resistant tobacteriocins, although processing treatments may trigger sporegermination and outgrowth, increasing bacteriocin sensitivity.Therefore, the efficacy of bacteriocins in food will greatly de-pend on the food microbial composition and microbial phys-iological stage (Table 1). It should also be considered thatmicroorganisms are seldom distributed homogeneously asplanktonic cells in the food matrix. Most probably, they willtend to formmicrocolonies in a solid or particulate food, or growin the form of slime-covered biofilms on food surfaces. Theprotective effect of biofilms against antimicrobial substances inthe food industry is well documented (Kumar and Anand, 1998),and may clearly limit the efficacy of bacteriocins as well.
Fig. 1. Influence of different factors on the efficacy of
Variations in strain sensitivities and development of strainresistance/adaptation are also of major concern for application ofbacteriocins. For example, not all strains of L. monocytogenesshow the same degree of sensitivity to antilisterial bacteriocins(Martínez et al., 2005). Bacteriocin-resistant L. monocytogeneshave been reported to appear at frequencies of 10−3 to 10−9 fornisin, depending on the strain (Ming andDaeschel, 1993; Daviesand Adams, 1994; Mazzotta and Montville, 1997; Martínez etal., 2005), of 10−4 to 10−6 for the class IIa bacteriocins leucocinsA, B, E and sakacin A (Dykes and Hastings, 1998), and of 10−3
to 10−4 for mesenterocin 52, curvaticin 13 and plantaricin C19(Rekhif et al., 1994). The fitness costs of bacteriocin resistancemay vary greatly depending on the strain, the bacteriocin and theenvironmental conditions (Gravesen et al., 2002a). Most wor-rying, the developed resistance to one bacteriocin may alsoafford protection against other bacteriocins, and the observedcross-resistance between pediocin-like bacteriocins has beenattributed to a general mechanism of resistance (Gravesen et al.,2002b). The needs to find novel bacteriocins showing no crossresistance with existing ones are clearly patent.
In addition to the factors influencing the effectiveness ofbacteriocins as antimicrobials in food systems, factors influenc-ing bacteriocin production are of most importance when usingbacteriocinogenic cultures (Fig. 1). The use of bacteriocinogeniccultures faces several limitations directly related to the producerstrain such as (i) the spontaneous loss of the bacteriocinogenictrait, (ii) the susceptibility of the producer strain to infection bybacteriophages, (iii) antagonism of other bacteria towards theproducer strain, (iv) inadequacy of producer strain as a starter,(v) difficulties for genetic manipulation and transfer of bac-teriocinogenic trait to suitable starters, and, the most important,(vi) a low capacity for bacteriocin production in the food system(which will depend greatly on the food storage conditions, thecapacity of the producer strain for implantation and proliferation,or the induction of bacteriocin production). In situ bacteriocinproduction may also depend greatly on physicochemical factors(such as pH, temperature, aw, CO2, O2, redox potential, timeof incubation…) as well as food-related factors (such as thefood structure — fluidity, particulate matter, emulsions…, —
in situ bacteriocin production for biopreservation.
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buffering capacity, composition — available nutrients, addi-tives, antimicrobials…— and processing conditions— freezingand thawing, pressure homogeneization or other procedures thatmay indirectly damage bacterial cells, as well as thermal treat-ments and other treatments intended to reduce the microbialload). Since the bacteriocinogenic strain will thrive within a(more or less complex) microbial population in the food eco-system, bacteriocin production may also depend greatly onmicrobial-related factors such as the microbial load anddiversity, the microbial interactions occurring in the food duringstorage (such as the competition for nutrients or the productionof other antagonistic substances), and the physiological state ofthe bacteria (Holzapfel et al., 1995; Rodríguez et al., 2003;Devlieghere et al., 2004). All these factors will be sensed by thebacteriocinogenic strain which, in turn, will also have a directinfluence on the food environment through the consumption ofand competition for nutrients, production of metabolites andmodification of the foodmicrobial balance through the producedbacteriocin. Therefore, bacteriocin production in food must beunderstood as a dynamic process where the different interactionschange over time, dictating the ultimate result as far as foodpreservation (Fig. 1).
Several illustrative examples may explain how bacteriocinproduction can change dramatically by altering environmentalconditions, and how optimum production may require a certaincombination of influencing factors (Leal-Sánchez et al., 2002).A suboptimal temperature (22 °C) and a moderate NaCl stress(0.65 M) stimulated bacteriocin production by Lactobacilluspentosus B96 (Delgado et al., 2005). Amylovorin production byLactobacillus amylovorus DCE 471 was stimulated by NaCl(Neysens et al., 2003) as well as carbon dioxide (Neysens and DeVuyst, 2005). Several other stress factors (such as ethanol,oxygen, competing microbiota, etc.) may also stimulate bacte-riocin production.
Bacteriocin production is often an inducible trait that dependson cell density and concentration of the inducer (which may bethe bacteriocin itself). Therefore, a minimum concentration ofinoculum is often required. The interactions of the inductionfactor with the food matrix (such as adsorption or inactivation)may have a great influence on bacteriocin production. On theother hand, the food matrix may also facilitate the concentrationof the induction factor around cells or microcolonies formed bythe bacteriocinogenic culture in the food. The presence of com-peting microorganisms can be an environmental factor stimu-lating production of LAB bacteriocins such as lactacin B(Barefoot et al., 1994) or divercin (Sip et al., 1998). Similarly,specific Gram-positive bacteria activate both plantaricin NC8(PLNC8) production by Lactobacillus plantarum NC8 and thePLNC8-autoinducing activity (Maldonado et al., 2004).
Since bacteriocin production is linked to cell growth, it mayalso depend on factors affecting this parameter (such as in-hibitory substances like salt or nitrite) or the lack of availablenutrients (such as manganese in the case of many LAB). As anexample, the production of enterocins A and B by Enterococcusfaecium CTC492 was significantly inhibited by sausage in-gredients and additives, with the exception of nitrate (Aymerichet al., 2000). The addition of sodium chloride and pepper de-
creased production by 16-fold. The temperature and pHinfluenced enterocin production, with optima between 25 and35 °C, and from 6.0 to 7.5 of initial pH (Aymerich et al., 2000).Bavaricin A production was negatively affected at NaClconcentrations of 3% or higher, while cells were still growing(Larsen et al., 1993). Growth and sakacin production in meatsausages by Lactobacillus sakei CTC 494 was affected nega-tively by salting and curing with nitrite as well as manganeselimitation (Leroy and De Vuyst, 1999, 2005). Similarly, growthand curvacin A production by Lactobacillus curvatus LTH 1174were negatively affected by nitrite (even by a concentration aslow as 10 ppm), and also by sodium chloride (Verluyten et al.,2003, 2004a). The effect on bacteriocin production was evendetected at low NaCl concentrations where there was no growthinhibition, and NaCl was shown to interfere with bacteriocininduction (Verluyten et al., 2004a). Growth and curvacin Aproduction were also negatively affected by some species (es-pecially by nutmeg) in ameat system (Verluyten et al., 2004b). Amodel was proposed to illustrate the influence of environmentalfactors on sakacin production in a meat system (Leroy and DeVuyst, 2003, 2005).
3. Bacteriocins and hurdle technology
The concept of hurdle technology began to apply in the foodindustry in a rational way after the observation that survival ofmicroorganisms greatly decreased when they were confrontedwith multiple antimicrobial factors (Leistner, 1978; Leistner andGorris, 1995; Leistner, 2000). After exposure of a bacterialpopulation to a single antimicrobial factor there is often a het-erogeneous response, depending on the intensity of treatment aswell as many other factors. A fraction of the population mayreceive a lethal dose of the antimicrobial factor, leading to celldeath. The remaining fraction may survive due to several rea-sons: (i) receiving a sub-lethal dose; (ii) showing an increasedresistance because of its physiological state (e.g. stationaryphase cells, or cells already stressed in response to other un-favourable environmental conditions), and (iii) cells naturallyresistant to the antimicrobial agent. Sub-lethally injured cells aswell as cells with increased resistance may repair the damagecaused by the antimicrobial agent and survive, having a goodopportunity to develop mechanisms of resistance or adaptationthat would make them immune to further challenges with theparticular antimicrobial factor. By contrast, when cells are ex-posed to a combination of antimicrobial factors, the intensity ofdamage may be higher since some of the antimicrobial factorsmay act on the same cellular target. The repair of multipledamages may also require much higher energy costs, leading toenergy exhaustion and cell death. Therefore, the probabilities forsurvival and proliferation for cells confronted with multiplehurdles are very low. In addition, the synergy between differentantimicrobial factors may allow the use of lower doses comparedto their individual application.
Over 60 potential hurdles have been described to improvefood stability and/or quality (Leistner, 1999). The application ofbacteriocins as part of hurdle technology has received greatattention in recent years (Chen and Hoover, 2003; Ross et al.,
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2003; Deegan et al., 2006), since bacteriocins can be usedpurposely in combination with selected hurdles in order toincrease microbial inactivation (Fig. 2). The combination ofhurdles to be applied will depend greatly on the type of food andits microbial composition. This must be carefully considered,since different hurdles usually have different effects onthe members of a microbial community. As an example, foodacidification may select for aciduric bacteria while heat treat-ment may select for endospore formers. Also, elimination ofsome members of the population may provide a more fa-vourable environment for others, due to the lack of competi-tion. The interaction of different antimicrobial factors may alsomodify their individual antimicrobial spectra. For example,Gram-negative bacteria may become sensitized to bacteriocinsand other molecules upon exposure to hurdles that destabilisethe bacterial outer membrane, as will be discussed further on.
3.1. Combination of bacteriocins with chemical substances andnatural antimicrobials
Previous studies have shown that the presence of NaClenhanced the antimicrobial action of bacteriocins such as nisin,leucocin F10, enterocin AS-48 and others (Harris et al., 1991;Thomas and Wimpenny, 1996; Mazzotta et al., 1997; Parenteet al., 1998; Ananou et al., 2004). However, nisin activity wasalso antagonized by low concentration of NaCl (Bouttefroyet al., 2000). Sodium chloride also decreased the antilisterialactivity of acidocin CH5 (at 1–2%; Chumchalová et al., 1998),lactocin 705 (at 5–7%; Vignolo et al., 1998), leucocins 4010 (at2.5% NaCl; Hornbæk et al., 2004), pediocin (at 6.5% NaCl;Jydegaard et al., 2000), curvacin (Verluyten et al., 2002) andCarnobacterium piscicola A9b bacteriocin (at 2–4% NaCl;Himmelbloom et al., 2001). The protective effect of sodiumchloride may be due to interference with ionic interactionsbetween bacteriocin molecules and charged groups involved inbacteriocin binding to target cells (Bhunia et al., 1991). Sodiumchloride may also induce conformational changes of bacter-
Fig. 2. Application of bacteriocins
iocins (Lee et al., 1993) or changes in the cell envelope of thetarget organisms (Jydegaard et al., 2000).
Reduction of nitrite content by addition of bacteriocins maybe beneficial in the food industry. The combinations of nisin andnitrite delayed botulinal toxin formation in meat systems andshowed increased activity on clostridial endospores outgrowth(Rayman et al., 1981, 1983; Taylor et al., 1985) and also onLeuconostoc mesenteroides and L. monocytogenes (Gill andHolley, 2003). Addition of nitrite also increased the anti-listeriaactivity of bacteriocinogenic lactobacilli in meat (Hugas et al.,1996) and the activities of enterocin EJ97 against L. mono-cytogenes, Bacillus coagulans and Bacillus macroides (Garcíaet al., 2003, 2004a,b) and enterocin AS-48 against B. cereus(Abriouel et al., 2002).
Organic acids and their salts can potentiate the activity ofbacteriocins greatly, while acidification enhances the antibacte-rial activity of both organic acids and bacteriocins (Jack et al.,1995; Stiles, 1996). The increase in net charge of bacteriocins atlow pH might facilitate translocation of bacteriocin moleculesthrough the cell wall. The solubility of bacteriocins may alsoincrease at lower pH, facilitating diffusion of bacteriocin mol-ecules. Buncic et al. (1995) found that the sensitivity ofL. monocytogenes to nisin (400 IU/ml) increased in combinationwith lactate. Further reports have confirmed the increasedantibacterial activity of nisin in combination with sodium lactatein several food systems (Scannell et al., 1997; Nykänen et al.,2000; Long and Phillips, 2003; Ukuku and Fett, 2004). A nisin-sorbate combination showed increased activity against Listeria(Avery and Buncic, 1997), and B. licheniformis (Mansour et al.,1998). In the production of Ricotta-type cheeses, the combina-tion of nisin with acetic acid and sorbate controlled L. mono-cytogenes contamination over a long period storage (70 days) at6–8 °C (Davies et al., 1997). Lacticin 3147 activity also in-creased in combination with sodium lactate or sodium citrate(Scannell et al., 2000b), as well as did pediocin AcH activity incombination with sodium diacetate and sodium lactate (Uhartet al., 2004). Activity of enterocin AS-48 against B. cereus in
as part of hurdle technology.
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rice gruel was also potentiated by sodium lactate (Grande et al.,2006). Lactic acid, sodium lactate and peracetic acid (as well asseveral other chemical compounds) increased AS-48 activity fordecontamination of L. monocytogenes in sprouts (CoboMolinoset al., 2005). When nisin and pediocin were used in combinationwith chemical compounds to combat L. monocytogenes in freshproduce, best results were obtained for nisin with phytic acid(Bari et al., 2005).
Chelating agents permeate the outer membrane (OM) ofGram-negative bacteria by extracting Ca2+ and Mg2+ cationsthat stabilize lipopolysaccharide of this structure, allowing bac-teriocins to reach the cytoplasmic membrane (Stevens et al.,1991; Vaara, 1992; Schved et al., 1994; Helander et al., 1997).The enhanced effect of chelators such as EDTA, disodiumpyrophosphate, trisodium phosphate, hexametaphosphate orcitrate and bacteriocins against Gram-negative bacteria has beendemonstrated for nisin both under laboratory conditions and infoods (Stevens et al., 1991; Cutter and Siragusa, 1995a,b;Carneiro De Melo et al., 1998; Boziaris and Adams, 1999; Fangand Tsai, 2003). Brochrocin C and enterocin AS-48 also showedincreased antimicrobial activity on EDTA-treated Gram-nega-tive bacteria (Abriouel et al., 1998; Gao et al., 1999; Ananou etal., 2005). Sensitization of Gram-negative bacteria to bacter-iocins by other chelators such as maltol or ethyl maltol has beenreported (Schved et al., 1996). Sodium lactate or sodium citratein combination with nisin showed increased antimicrobialactivity against Arcobacter butzleri in chicken due to theirchelating effect (Long and Phillips, 2003). Chelating agents canalso enhance the activity of bacteriocins on Gram-positivebacteria. A combination of nisin and sodium polyphosphateshowed an increased activity against L. monocytogenes (Buncicet al., 1995). More recently, an increased activity of chrisin (acommercial nisin preparation) in combination with EDTA hasbeen reported against several Gram-positive bacteria (Gill andHolley, 2003). The combination of sodium tripolyphosphate andenterocin EJ97 showed increased activity against B. coagulansand B. macroides (García et al., 2003, 2004a).
Other antimicrobial compounds such as ethanol can act syn-ergistically with nisin to reduce the survival of L. monocytogenes(Brewer et al., 2002). Sub-lethal concentrations of nisin(30 IU/ml) and monolaurin (100 μg/ml) in combination actedsynergistically on B. licheniformis vegetative cells and sporeoutgrowth in milk (Mansour et al., 1999). Synergism was alsoobserved for the sucrose fatty acid esters sucrose palmitateand sucrose stearate and nisin against several strains ofL. monocytogenes, B. cereus (cells and spores), L. plantarumand Staphylococcus aureus, but not against Gram-negativebacteria (Thomas et al., 1998). Reuterin also showed a sig-nificant synergistic effect on L. monocytogenes and a slightadditive effect on S. aureus after in combination with nisin(100 IU/ml), although the antimicrobial effect of reuterinagainst Gram-negative pathogens was not enhanced, though(Arqués et al., 2004).
Essential oils and their active components, the phenoliccompounds are also attractive natural preservatives (Burt, 2004).When used in combination with bacteriocins, the dose of addedphenolic compounds could be lowered thereby decreasing their
impact on the food flavour and taste. Nisin acted synergisticallywith carvacrol, eugenol or thymol against B. cereus and/orL. monocytogenes (Pol and Smid, 1999; Periago et al., 2001;Yamazaki et al., 2004). Combinations of nisin with carvacrol,eugenol, or thymol resulted in synergistic action against Bacillussubtilis and Listeria innocua, while nisin and cinnamic acid hadsynergistic activity against L. innocua, but only additive againstB. subtilis (Olasupo et al., 2004). Carvacrol (0.5 mM) was usedto enhance the synergy found between nisin and a pulsed electricfield treatment (PEF) against vegetative cells of B. cereus in milk(Pol et al., 2001a,b). The combination of nisin and cinnamonaccelerates death of Salmonella Typhimurium and EscherichiacoliO157:H7 in apple juice (Yuste and Fung, 2004). The naturalvariant nisin Z also acted synergistically with thymol againstL. monocytogenes and B. subtilis (Ettayebi et al., 2000). Theantimicrobial activity of enterocin AS-48 against S. aureus cellin vegetable sauces was potentiated significantly in combinationwith the phenolic compounds carvacrol, geraniol, eugenol,terpineol, caffeic acid, p-coumaric acid, citral and hydrocin-namic acid (Grande et al., 2007).
Combinations of bacteriocins have also been tested in orderto increase their antimicrobial activities. The simultaneous use ofnisin with pediocin AcH (Hanlin et al., 1993) or with leucocinF10 (Parente et al., 1998) as well as lactacin B or lactacin F withnisin or pediocin AcH, and lactacin 481/pediocin AcH (Mulet-Powell et al., 1998) provides a greater antibacterial activity thaneach bacteriocin separately. Simultaneous or sequential addi-tions of nisin (50 IU/ml) and curvaticin 13 (160 AU/ml) alsoinduced a greater inhibitory effect against L. monocytogenesthan each bacteriocin individually (Bouttefroy and Millière,2000). The simultaneous use of two or more bacteriocins couldbe useful not only to lower the added bacteriocin doses, but alsoto avoid regrowth of bacteriocin-resistant/adapted cells. How-ever, development of cross-resistance should be carefullyconsidered, especially for the combinations of bacteriocinsbelonging to the same class, as it has been shown that a generalmechanism may account for high-level resistance to class IIabacteriocins in L. monocytogenes (Gravesen et al., 2002a,b).
An increased antibacterial activity can also be achievedby combination of bacteriocins with other (non-bacteriocin)antimicrobial proteins or peptides. Nisin and lysozyme actedsynergistically against Gram-positive bacteria, including spoil-age lactobacilli and S. aureus (Chung and Hancock, 2000;Nattress and Baker, 2003). The spectrum of the nisin-lysozymecombination could be extended to Gram-negative bacteria byaddition of chelating agents (Gill and Holley, 2000) The com-bined addition of nisin and the lactoperoxidase system (LPS) hada strong antimicrobial effect against L. monocytogenesOhio and(to a lesser extent) L. monocytogenes Scott A in skim milk after24 h at 30 °C (Zapico et al., 1998), against L. monocytogenesATCC 15313 in skim milk at 25 °C for at least 15 days(Boussouel et al., 2000), and also against fish spoilage flora(Elotmani and Assobhei, 2004). Lactoferrin (and its partial-hydrolysis derivative lactoferricin) is another natural protein(which is found in milk and other secretions) with antimicrobialactivity due to its iron-binding capacity and polycationic nature(Ellison, 1994). The combination of nisin and lactoferrin showed
58 A. Gálvez et al. / International Journal of Food Microbiology 120 (2007) 51–70
increased antilisterial activity when tested in culture media(Branen and Davidson, 2004). Synergy between eukaryoticantimicrobial peptides and bacteriocins has also been investi-gated. The antimicrobial activity of pleurocidin (an antimicro-bial peptide from fish) against E. coli was greatly enhanced bypediocin PA-1, sakacin P, and curvacin A (Lüders et al., 2003).Although no potential application has been proposed for theobserved synergy, the available synthetic pleurocidin couldprobably be used in combination with bacteriocins for selectiveinhibition of Gram-negative bacteria in fish.
3.2. Bacteriocins and heat treatments
Bacteriocins can be used to reduce the intensity of heattreatments in foods without compromising microbial inactiva-tion. Nisin and heat act synergistically against L. plantarum andL. monocytogenes (Mahadeo and Tatini, 1994; Ueckert et al.,1998), reducing the heat resistance of L. monocytogenes in milk(Maisnier-Patin et al., 1995) and in cold-pack lobster meat(Budu-Amoako et al., 1999). Nisin-resistant L. monocytogenescells grown in the presence of nisin were more sensitive to heat at55 °C than wild-type cells (Modi et al., 2000). The efficacy ofenterocin AS-48 was higher on S. aureus cells sub-lethallyinjured by heat due to the lower concentration of remainingviable cells and to the cell damage induced by the heat treatment(Ananou et al., 2004). Bacteriocins can also provide anadditional protection during food storage against proliferationof endospores surviving heat treatments. Moreover, it has beendemonstrated that the intensity of heat treatments againstbacterial endospores can be lowered in combination with nisinas well as with enterocin AS-48 (Beard et al., 1999; Wandling etal., 1999; Grande et al., 2006), saving costs in the heat treatmentand decreasing the impact of heat on the food. Sub-lethal heathas been shown to sensitize Gram-negative bacteria to severalbacteriocins such as nisin or pediocin AcH (Kalchayanand et al.,1992; Boziaris et al., 1998), enterocin AS-48 (Abriouel et al.,1998; Ananou et al., 2005), or jenseniin G (Bakes et al., 2004),extending their spectrum of action. Highest sensitization wasreported for combined treatments of bacteriocins, heat and achelating agent (Abriouel et al., 1998; Ananou et al., 2005).
3.3. Bacteriocins and modified atmosphere packaging
Modified atmosphere packaging (MAP) is frequently used inthe food industry to prolong the shelf life of perishable foodproducts. MAP may be defined as “the enclosure of food prod-ucts in gas-barrier materials, in which the gaseous environmenthas been changed” (Young et al., 1988). Prolongation of the shelflife of food by MAP is based on retardation of intrinsic foodchanges and inhibition of spoilage microbiota. In a modifiedatmosphere, the dissolved CO2 will determine growth inhibitionof microorganisms (Devlieghere et al., 1998). Gram-negativebacteria are generally more sensitive to CO2, while lactic acidbacteria are much more resistant (Farber, 1991; Church, 1994).Since Gram-negative bacteria are usually not sensitive tobacteriocins, MAP and bacteriocins are therefore two comple-mentary hurdles of advantage to food spoilage.
Fang and Lin (1994a,b) found that growth of L. monocyto-genes was completely inhibited on pork immersed in 10 IU/mlnisin and packed in 80% CO2/20% air during 30 days of storageat 4 °C. Activity of nisin as well as ALTA™ 2341 againstL. monocytogenes also increased in cold smoked salmon pack-aged under vacuum as well as under a 100% CO2 atmosphere(Nilsson et al., 1997; Szabo and Cahill, 1999). A cocktail ofseven L. monocytogenes isolates was inhibited by nisin as wellas by ALTA™ 2341 under a 100% CO2 atmosphere, but notunder 100% N2, or 40% CO2/60% N2 (Szabo and Cahill, 1998).It has been reported that nisin and CO2 atmosphere actedsynergistically on the cytoplasmic membrane of L. monocyto-genes by enhancing membrane permeabilization (Nilsson et al.,2000).
3.4. Bacteriocins and pulsed electric fields
Pulsed electric field (PEF) technology is a non-thermal pro-cess where microbial inactivation is achieved by application ofhigh-voltage pulses between a set of electrodes (Vega-Mercadoet al., 1997). The effects of PEF resemble bacterial electro-poration, but the higher intensity of this treatment causes severedamage to the bacterial cell membrane. Although thistechnology can only be applied to pumpable food products, ithas gained attraction in recent years as an individual treatmentor in combination with other hurdles such as bacteriocins. Sincemost bacteriocins act on the bacterial cytoplasmic membrane,the combined application of bacteriocins and PEF is expected toelicit increased bactericidal effects. Moreover, bacteriocinscould also provide an additional hurdle against survivors fromPEF treatments, such as sub-lethally injured cells or bacterialendospores (Fig. 3). PEF could also be applied to extend theantimicrobial spectrum of bacteriocins, since PEF disrupts thebacterial outer membrane allowing bacteriocin molecules toreach the bacterial cytoplasmic membrane target (Fig. 3). Theefficacy of the combined treatments of PEF and bactericiocinsin food preservation depends on several factors related to thePEF treatment (such as field strength, number of pulses, waveform or pulse duration), the food microbial load, compositionand physiological stage, the added bacteriocin, and otherenvironmental factors (Wouters et al., 2001; Bendicho et al.,2002; Heinz et al., 2002). All of these may have an influence onthe numbers and types of bacteria surviving the combinedtreatment and, most important, their proliferation during theshelf life period of the processed food. For this reason, par-ticular applications of bacteriocins and PEF must be studied indetail for each type of food and target bacteria.
Several investigations have thrown light on the effectivenessof combined treatments of bacteriocins and PEF treatments infood systems (Table 2). Exposure of L. innocua to nisin inliquid whole egg following PEF treatment exhibited an additiveeffect on inactivation of the microorganism (Calderón-Mirandaet al., 1999a). A synergistic effect was observed as the electricfield intensity, number of pulses and nisin concentrationincreased both in liquid whole egg and in skim milk(Calderón-Miranda et al., 1999a,b). L. innocua treated byPEF-nisin in skimmed milk exhibited an increase in the cell wall
Fig. 3. Effects of pulsed electric fields in combination with bacteriocins on microbial populations.
Table 2Reported effects on the application of nisin and pulsed-electic fields for bacterialinactivation
Observed effects References
–Increased inactivation of M. luteus in phosphatebuffer
Dutreux et al. (2000)
–Increased inactivation of P. aeruginosa Santi et al. (2003)–Increased inactivation of S. aureus in skim milk Sobrino-Lopez and
Martin Belloso (2006)–Increased inactivation of L. innocua in liquidwhole egg, skim milk, and liquid wheyprotein concentrate
–Increased inactivation of E. coli in simulatedmilk ultrafiltrate media. Observed synergismwith reduced water activity
Terebiznik et al., 2000,2002
–Increased inactivation of B. cereus vegetativecells (more efficient in buffer than in skim milk).Observed synergism with carvacrol
Pol et al., 2000, 2001a,b
–Inactivation of Salmonella in orange juice.Observed synergism with lysozyme
Liang et al. (2002)
–Inactivation of E. coli O157:H7 in fresh applecider. Observed synergism with cinnamon
Iu et al. (2001)
–Inactivation of L. plantarum in model beer Ulmer et al. (2002)
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roughness, cytoplasmic clumping, leakage of cellular material,and rupture of the cell walls and cell membranes (Calderón-Miranda et al., 1999c). It has been reported recently that theefficiency of the combined treatment of nisin and PEF in liquidwhey protein concentrate was strongly dependent on thesequence of application, since exposure to nisin after PEFproduced a lower effect on L. innocua inactivation. Thisbehaviour was mainly attributed to changes in the cell envelopeand to modifications of the medium caused by PEF application(Gallo et al., 2007).
The application of nisin clearly enhanced the lethal effect ofPEF treatment on other Gram-positive bacteria such as Micro-coccus luteus cells in phosphate buffer (Dutreux et al., 2000),L. plantarum in model beer (Ulmer et al., 2002), S. aureus inskim milk (Sobrino-Lopez and Martin Belloso, 2006), andvegetative cells of B. cereus (Pol et al., 2000). The synergybetween nisin and PEF treatment against resting vegetative cellsof B. cereus was enhanced by carvacrol (0.5 mM). Nisinshowed less activity against B. cereus in milk compared tobuffer (Pol et al., 2001a).
Bacterial endospores are refractile to PEF treatments, andincorporation of bacteriocins into the food may provide anadditional hurdle against surviving endospores. Treatment ofB. cereus spores with nisin and/or PEF treatment did not lead todirect inactivation of the spores or increased heat sensitivity as aresult of sub-lethal damage. In contrast, germinating sporeswere found to be sensitive to PEF treatment. Nisin treatmentwas more efficient than PEF treatment for inactivatinggerminating spores (Pol et al., 2001b). Accordingly, bacter-iocins could be applied as an additional hurdle againstendospores surviving PEF treatment, provided that bacteriocinmolecules remain active in the treated food.
Several studies have shown that the efficacy of PEF againstGram-negative bacteria can be enhanced by nisin. When PEFtreatment was applied to Salmonella cells in orange juice in thepresence of nisin (100 U/ml), lysozyme (2,400 U/ml), or amixture of nisin (27.5 U/ml) and lysozyme (690 U/ml), cellviability loss was increased by an additional 0.04 to 2.75 logcycles. The combination of nisin and lysozyme had a morepronounced bactericidal effect (by at least 1.37 log cycles) thaneither nisin or lysozyme alone (Liang et al., 2002). Similarly,
PEF treatment combined with cinnamon or nisin triggered celldeath of E. coli O157:H7 in fresh apple cider, resulting in areduction in viable counts of 6 to 8 log cycles (Iu et al., 2001).Although nisin was totally inactivated by PEF treatment insimulated milk ultrafiltrate media, a 4-log cycle reduction ofinoculated E. coli cells was accomplished with nisin (ca. 1,000IU/ml) and three pulses of 11.25 kV/cm or 500 IU/ml for fivepulses of the same intensity (Terebiznik et al., 2000). Nisin-PEFinactivation of E. coli in simulated milk ultrafiltrate media wasenhanced by water activity reduction. Decreasing water activityto 0.95 with NaCl and applying PEF at 5 kV/cm (a non-lethalintensity when no other hurdle is used) with the further additionof nisin (1200 IU/ml) resulted in a 5-log cycle reduction of thebacterial population (Terebiznik et al., 2002). Nisin also in-creased PEF effectiveness against Pseudomonas aeruginosa.At high PEF intensities (i.e., 11 kV/cm), the inhibitory effect ofnisin increased with the number of pulses applied (Santi et al.,2003). In conclusion, addition of nisin can improve the effi-cacy of PEF treatments against vegetative cells of foodborne
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pathogenic and spoilage Gram-positive and Gram-negativebacteria. Presumably, similar effects are to be expected for otherbacteriocins, although experimental data are needed to supportthis conclusion. The stability of bacteriocins to PEF treatmentsis another issue to be addressed, since remaining bacteriocinactivity may provide an important hurdle against survivors(such as bacterial endospores) after PEF treatment.
3.5. Bacteriocins and high hydrostatic pressure (HHP)
High hydrostatic pressure (HHP) is an innovative foodprocessing and preservation method that causes injury andkilling of microbial cells (Kalchayanand et al., 1994; Farkas andHoover, 2000; Patterson, 2000; Ray, 2002). A variety ofpressure-treated products, such as ready-to-to eat chicken meat,sliced ham, fresh whole oysters, jams, fruit juices, andguacamole, are now commercially available. During pressuri-zation, the disruption of H-bonds, ionic bonds and hydrophobicinteractions of the macromolecules adversely affects theirstructures and functions (Hoover, 1993). The sub-lethal damageis initiated by membrane phase transitions (Kato and Hayashi,1999), affecting mainly ATP-generating and transport proteins.Cell death caused by HHP increases with pressure and so doesthe synergism with bacteriocins. Since most bacteriocins act onthe bacterial cytoplasmic membrane it can be hypothesized thatthe observed synergy between bacteriocins and HHP resultsfrom cumulative damage to this structure. However, bacterio-cin–membrane interaction during phase transition at high pres-sure has never been studied. A tailing effect is often observedafter application of HHP treatments (Kalchayanand et al.,1998a), indicating that cell death occurs as a consequence ofmultiple events or cumulative cell damage. Sub-lethally injuredvegetative cells surviving HHP treatment may develop pressureresistance, as has been previously reported for E. coli (García-Graells et al., 1998) and L. monocytogenes (Karatzas andBennik, 2002). The increased cell damage caused by combinedtreatments of HHP and bacteriocins could prevent the tailingeffect, providing an additional hurdle against selection of pres-sure-resistant vegetative cells.
The bactericidal effect of HHP (as well as its negative effectson food constituents) increases along with the temperature,which determines the different modalities of treatment, eg. coldHHP pasteurisation (ca. 5 °C), HPP-assisted pasteurisation (ca.40 °C), or HHP-assisted sterilisation (ca. 90 °C). The food pH isalso an influencing factor, and bacteria are usually more resistantto HHP in low acid foods. Addition of bacteriocins could im-prove the efficacy of HHP treatments in foods, compensating forthe required increase in pressure or temperature.
The bacterial type and physiological stage (e.g. vegetive cellsor endospores) may have great influence on HHP efficacy (Chenet al., 2006). Although bacteriocins are generally inactive onGram-negative bacteria, HHP transiently sensitizes Gram-nega-tive bacteria through outer membrane damage, increasing thepossibilities for application of bacteriocins in food preservation(Kalchayanand et al., 1994; Hauben et al., 1996; Masschalcket al., 2001; Black et al., 2005). HHP also induces a morepersistent sensitisation of Gram-negative bacteria to small dif-
fusible antimicrobial molecules (García-Graells et al., 1998,2000), which may act synergistically with other hurdles as well.Bacterial endospores are resistant to HHP treatments currentlyapplied to foods (Smelt, 1998), although HHP treatments caninduce endospore germination. Addition of bacteriocins as asecond hurdle against surviving endospores could also improvethe safety and shelf life of HHP-processed foods (Shearer et al.,2000).
Several studies have described the combined effects of bac-teriocins and HHP on bacteria (Table 3). Nisin in combina-tion with HHP showed strong synergistic effects againstL. plantarum, E. coli and L. monocytogenes (ter Steeg et al.,1999; Farkas et al., 2003). HHP transiently sensitized Gram-negative bacteria to nisin, and a mechanism of pressure-pro-moted uptake of antimicrobial proteins and peptides wasproposed to explain this sensitisation (Masschalck et al., 2001).
Pediocin AcH also increased decimal reductions caused byHHP on food spoilage and pathogenic bacteria in peptonesolution (Kalchayanand et al., 1998b), and increased cell lysis inL. mesenteroides through cell wall degradation (Kalchayanandet al., 2002). The synergistic activity of pediocin AcH in com-bination with nisin was greatly enhanced during pressurization(Kalchayanand et al., 1998a,b, 2004a). A combination of nisin/pediocin AcH added to the plating mediumwas also effective onkilling Clostridium spores induced to germinate by HHPtreatment (Kalchayanand et al., 2004b).
A key issue in the efficacy of HHP treatments is the protectiveeffect afforded by the food. For example, milk and meat (havinga complex composition as well as a pH closer to neutrality) exerta grater protection against HHP. Inactivation of E. coliMG1655was reduced from 7 logs at 400 MPa in phosphate buffer to only3 logs at 700MPa in milk, but addition of lysozyme (400 μg/ml)and nisin (400 IU/ml) to the milk increased the lethality oftreatment (Garcia-Graells et al., 1999). Combining HHP andnisin (500 IU/ml) also increased inactivation of bacteria asso-ciated with milk (Black et al., 2005). Similarly, the combinationof lacticin 3147 and HHP increased the viability loss of S. aureusand L. monocytogenes in milk and in whey (Morgan et al.,2000), and residual lacticin still showed inhibitory effect in thefood, inactivating and preventing growth of sub-lethally injuredcells. Since HHP may have unwanted effects on milkcomponents (Trujillo et al., 2002), bacteriocin-HHP treatmentscould serve to decrease the intensity of HHP treatment withoutcompromising microbial inactivation.
Combined bacteriocin-HHP treatments have successfullyapplied for inhibition of foodborne pathogens in cheese. Sur-vival of L. monocytogenes Scott A in cheeses made from rawmilk that were previously inoculated with nisin and otherbacteriocin-producer strains decreased as the intensity of HHPtreatment increased (Arqués et al., 2005). Nisin-HHP has alsoshown to increase inactivation of Bacillus and Clostridium en-dospores (Roberts and Hoover, 1996; Stewart et al., 2000). InMato' cheese, combining nisin with high pressure improvedthe biocidal effect on spores and aerobic mesophilic bacteria(Capellas et al., 2000). In model cheeses submitted to agermination cycle of 60 MPa at 30 °C for 210 min, followedby a vegetative cells destruction cycle of 300 or 400 MPa at
Table 3Reported effects on the application of bacteriocins and high tydrostatic pressure (HHP) for bacterial inactivation
Bacteriocin Observed effects References
Nisin Increased inactivation of B. coagulans, B. subtilis and Clostridium spores Roberts and Hoover, 1996;Stewart et al., 2000
Increased bactericidal activity and spectrum (E. coli, S. enteritidis, S. typhimurium, S. sonnei, S. flexneri,P. fluorescens and S. aureus)
Masschalck et al. (2001)
Increased inactivation of bacteria associated with milk (E. coli, P. fluorescens, L. innocua, andL. viridescens)
Garcia-Graells et al., 1999;Black et al., 2005
Increased sensitivity of pressure-resistant E. coli Garcia-Graells et al. (1999)Strong synergistic effects against L. plantarum and E. coli at reduced temperature ter Steeg et al. (1999)Improved bactericidal effect on spores and aerobic mesophilic bacteria in cheese Capellas et al. (2000)Increased inactivation of B. cereus spores and inhibition of the surviving fraction in cheese López-Pedemonte et al. (2003)Increased inactivation of L. monocytogenes Scott A in cheese inoculated with a nisin-producing strain Arqués et al. (2005)Increased inactivation of E. coli and L. innocua in liquid whole egg Ponce et al. (1998)In a meat model system, nisin reduced viable counts of E. coli, reduced growth of S. aureus, andsuppressed slime-producing bacteria
Garriga et al. (2002)
Pediocin AcH Increased inactivation of food spoilage and pathogenic bacteria (S. aureus, L. monocytogenes,S. typhimurium, E. coli O157:H7, L. sakei, L. mesenteroides, S. liquefaciens, and P. fluorescens)suspended in peptone water
Kalchayanand et al. (1998b)
Increased cell lysis through cell wall degradation in L. mesenteroides Kalchayanand et al. (2002)Reduction of L. monocytogenes viable counts and inhibition of proliferation during storage in meat modelsystem
Garriga et al. (2002)
Pediocin AcH+nisin Increased inactivation of S. aureus, L. monocytogenes Scott A, Salmonella Thyphimurium and E. coliO157:H7
Kalchayanand et al., 1998a,b,2004a
Killing of Clostridium spores induced to germinate Kalchayanand et al. (2004b)Sakacin K, enterocins A
and BReduction of L. monocytogenes viable counts and inhibition of proliferation during storage in meat modelsystem
Garriga et al. (2002)
Lacticin 3147 Increased inactivation of L. monocytogenes and S. aureus in milk and in whey Morgan et al. (2000)
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30 °C for 15 min the presence of nisin significantly increasedinactivation of B. cereus spores and reduced proliferation of thesurviving fraction measured 24 h and 15 d after HHP treatment(López-Pedemonte et al., 2003).
In liquid whole egg, HHP inactivation of E. coli andL. innocua improved significantly with nisin addition. A reduc-tion of almost 5 log units in E. coli counts and more than 6 logunits for L. innocua was reported at 450 MPa and 5 mg/l ofnisin, and the two microorganisms were not detectable afterone month of storage at 4 °C (Ponce et al., 1998).
The application of HHP to fresh meat products may result in acooked-like aspect, and sometimes the products may develop arubbery consistency (Murano et al., 2002). Bacteriocin additioncould improve the efficacy of HHP treatments at lower pressurewithout adverse effects on meat (Hugas et al., 2002). In a meatmodel system, while added bacteriocins (enterocins A and B,sakacin K, pediocin AcH or nisin) had no influence on killingand survival of Salmonella enterica, a greater inactivation ofE. coli (N6 logs) was reported in the presence of nisin (Garrigaet al., 2002). Nisin also kept staphylococci at significant lowlevels during storage and reduced slime-producing LAB belowthe detection limit. Although the bacteriocin-HHP treatmentfailed to inhibit proliferation of L. monocytogenes during stor-age in the case of nisin, counts of listeria remained belowdetection levels in samples treated with sakacin K, enterocins Aand B as well as pediocin AcH (Garriga et al., 2002). Theseresults clearly indicate that each particular bacteriocin may showdifferent effects in HHP-combined treatments depending on thetarget bacteria. Presumably, the combination of more than onebacteriocin and HHP could provide better results when the target
consists of a mixture of different bacteria with varying sen-sitivities to bacteriocins.
High pressure homogeneisation (HPH) is another processingtreatment applied to certain types of food. It was observedthat E. coli became sensitive to lysozyme and nisin duringhomogeneisation at pressures above 150 MPa when thesecompounds were added before the HPH treatment, but cellstreated by HPH remained insensitive for lysozyme and nisinadded after that treatment (Diels et al., 2005). Similarly to HHP,HPH may sensitise E. coli to lysozyme and nisin by inducing atransient permeabilisation of the outer membrane that does notinvolve a physical disruption and that is immediately repairedafter the process. Accordingly, the risks of foodborne pathogensin HPH-treated foods could be lowered by an optimised in-corporation of bacteriocins in foods before HPH treatment.
3.6. Bacteriocins and other non-thermal treatments
In spite of the many other non-thermal treatments currentlyunder study for food processing application, only a few reportshave been published on their combination with bacteriocins.Irradiation offers a great potential for application in food pre-servation (Farkas, 1998, 2006). The spectrum of applications offood irradiation could be expanded in combination with bac-teriocins, especially if the radiation dose can be lowered, sincelow-dose gamma irradiation has less unwanted effects on foodand may also have better acceptance among consumers. It wasreported that the combined application of pediocin (as ALTA™2341) and low-dose irradiation (2.3 kGy) had an increased anti-microbial effect on L. monocytogenes on frankfurters (Chen et al.,
62 A. Gálvez et al. / International Journal of Food Microbiology 120 (2007) 51–70
2004). One of the main limitations of gamma irradiation isthe increased resistance of bacterial endospores (Farkas, 1998),requiring the application of combined treatments for a highereffectiveness. In sous vide meals inoculated with spores ofpsychrotrophic B. cereus, a combined treatment of nisin, heat(90 °C, 10min) and gamma irradiation (5 kGy)markedly reducedthe number of survivors for at least 42 days at an abuse tem-perature of 10 °C (Farkas et al., 2002).
Other non-thermal treatments such as pulsed magnetic fields(PMF) have also been tested in combination with bacteriocins.However, a combined treatment of PMF with EDTA and nisinhad no effect on E. coli (San Martín et al., 2001).
4. Bacteriocins and recent advances in molecular biologyand genome studies
Advances in molecular biology and molecular microbialecology have provided new valuable tools to study microorgan-isms in food ecosystems, such as the determination of theirbacteriocinogenic potential, the capacity for proliferation andinhibition of unwanted bacteria, or the response to stress factors.The distribution of bacteriocin-encoding genes among foodisolates is a common issue that can be easily be resolved bymolecular techniques. As an example, Maldonado et al. (2002)established that the plantaricin S operon is widely distributedamong wild-type L. plantarum strains from olive fermentationsby PCR amplification and hybridization with specific probes.Similarly, Ben Omar et al. (2004) analyzed the incidence ofstructural genes for several known bacteriocins among foodisolates of Enterococcus faecalis and E. faecium by PCR am-plification, and Faye et al. (2004) studied the distribution ofpropionicin T1 genes among propionibacteria in a similar way.PCR amplification with specific primers for bacteriocin genesmay be used to follow the predominance of an inoculated strainin food fermentation, as shown for sakacin-P producing L. sakeiduring production of fermented sausages (Urso et al., 2006a) andL. gasseri K7 in semi-hard cheese (Matijašiæ et al., 2007).Similarly, multiplex PCR targeting both bacteriocin genes andspecies-specific genes can serve for precise identification ofbacteriocinogenic strains in a single analysis. Nisin-producinglactococci and divercin 41 producing C. divergens V41 wereidentified rapidly by this procedure (Moschetti et al., 2001;Connil et al., 2002). Multiplex PCR was also used successfullyto follow implantation of V41 strain in cold smoked salmon(Connil et al., 2002). Other procedures such as analysis ofRAPD-PCR profiles (Ryan et al., 2001; Matijašiæ et al., 2007),REP-PCR (Foulquié-Moreno M.R., Verluyten J., VancanneytM., Adriany T., Leroy F., Swings J., and De Vuyst L., unpub-lished) or PFGE analysis (Moschetti et al., 2001) have also beenused to evaluate strain implantation in food systems and theimpact of bacteriocinogenic starters on the food quality from themicrobiological point of view. A recent study addressed the useof DGGE to evaluate simultaneously the impact of a bacte-riocinogenic LAB on foodborne pathogens inoculated in afermented food as well as on the overall microbiological profileof the fermentation (Díaz G., Ben Omar N., Abriouel H., LucasR., Martínez Cañamero M., and Galvez A, unpublished).
Real-time PCR could be used to follow survival of targetbacteria in the presence of added or in situ produced bacteriocinwhile simultaneously determining the growth of bacteriocino-genic strains. This approach could provide additional data in-dependent of the biases introduced by sample preparationprocedures and the pressure of selective media currently used forenumeration of microorganisms. In other cases, it could solvethe problems of differentiation of closely related bacteria inmixed populations. Grattepanche et al. (2005) used this methodto evaluate the impact of the nisin Z-producing L. lactis subsp.lactis biovar. diacetylactis strain UL719 on Lactococcuscremoris in milk fermented with mixed cultures, avoiding theproblem to distinguish the two bacterial populations in dif-ferential media when they were in the same order of magnitude.Real-time PCR could also provide more precise information oncells sub-lethally injured by bacteriocin molecules. Similarly,DNA-based technology could be used to follow the expressionof bacteriocin genes in food systems, as well as the influence ofenvironmental conditions on gene expression and the stressresponse of target bacteria to the produced or added bacteriocinin food. In a recent work, expression of sakacin P structural genesspA by the L. sakei strain I151 in sausages was studied in orderto determine the influence of the production procedure for fer-mented sausages on bacteriocin production (Urso et al., 2006b).This alternative method could overcome the biases introducedby the currently used extraction procedures on the determinationof the amounts of bacteriocin produced in the food.
The use of fluorescence-based technology (such as fluores-cence in-situ hybridisation—FISH—, confocal laser microsco-py, or fluorescence ratio imaging microscopy—FRIM—) couldalso provide valuable information, for example, on the dis-tribution of bacteriocinogenic strains within the food matrix(Fernández de Palencia et al., 2004) or the heterogeneous re-sponse of bacterial populations to bacteriocins (Hornbæk et al.,2006). Other suggested applications could be to study thedistribution of target bacteria in the food both in the absence andin the presence of bacteriocin pressure and the generation ofgradients and protected niches as a function of bacteriocin con-centration. These techniques may facilitate the study of theeffects of bacteriocins in food systems at the level of singlecells, providing a completely new scenario picture of bacteriocineffects.
Another line of research on bacteriocin application may risefrom genomic studies. Classical methods for detection of pro-duced bacteriocins may underestimate the bacteriocinogenicpotential of LAB due to several factors such as the influence ofenvironmental conditions on bacteriocin production, the induc-ible character of many bacteriocins, and the loss of theproduction capacity (which may be caused by gene mutation,gene loss or genetic rearrangements). However, the analysis ofcomplete genomes may reveal the presence of potential bac-teriocin genes and new bacteriocins independently of the pro-ducer capacity of strains (Nes and Johnsborg, 2004). As anexample, in silico analyses of the probiotic strain Lactobacillusacidophilus NCFM predicted a chromosomal locus for lactacinB, a class II bacteriocin (Altermann et al., 2005). Similarly,while a limited number of bacteriocins have been described in
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Streptococcus thermophilus (Gilbreth and Somkuti, 2005 andreferences cited therein), analysis of the genome sequencesavailable revealed two loci predicted to be related to bacterio-cin production (Hols et al., 2005). The first locus (lab, forlantibiotic) contains genes that are similar to genes generallyfound in lantibiotic production loci. However, it is not clearwhether this locus is really involved in lantibiotic production.The second locus is similar to the bacteriocin-like peptide (blp)locus described in S. pneumoniae and displays the typicalcharacteristics of a Class II bacteriocin locus. The blp loci of theS. thermophilus strains also contain genes encoding bacteriocin-like peptides (i.e., lpD, blpU, blpE, blpF in strain LMD9, blpU,blpK in strain LMG18311, and blpK in strain CNRZ1066 (Holset al., 2005). However, nonsense mutations detected in some ofthe regulatory genes indicate that this locus is not functional atleast in some strains. The data available suggest that bacteriocinloci in S. thermophilus could be exploited by genetic engineeringin order to develop new bacteriocinogenic strains of technolog-ical interest. Similarly, genome sequencing of L. sakei 23 K hasrevealed that this strain carries a 6.5 kb region containing partof the spp gene cluster responsible for sakacin P production(Møretrø et al., 2005) described previously in L. sakei Lb674(Hühne et al., 1996). Since spp homologue genes (mainly sppKand sppR homologues) seem to be widely distributed amongL. sakei, it has been suggested that these genes could be used as afingerprinting region in typing methods and/or as a marker forL. sakei strains (Møretrø et al., 2005).
Many bacteriocins are encoded by small genes that are oftenomitted in the annotation process of bacterial genomes (De Jonget al., 2006). In addition, bacteriocins and their accessory pro-teins are often encoded by poorly conserved ORFs. The iden-tification of genes that are functionally similar but have limitedor no sequence homology is often a problem in genome datamining. A web server (BAGEL) that identifies putative bac-teriocin ORFs in a DNA sequence using novel, knowledge-based bacteriocin databases and motif databases has been re-cently created (De Jong et al., 2006). BAGEL is freely accessibleat: http://bioinformatics.biol.rug.nl/websoftware/bagel. Hope-fully, this software will help researchers to identify novel bac-teriocin-related genes in the upcoming LAB genome sequences.
A recent data mining study demonstrated that the genome ofPediococcus pentosaceus ATCC 25745 contains a gene clusterthat resembles a regulated bacteriocin system (Diep et al.,2006). The gene cluster has an operon-like structure consistingof a putative pediocin-like bacteriocin gene (termed penA),a potential immunity gene (termed peiA) as well as geneticdeterminants involved in bacteriocin transport and regulation.Nevertheless, the accessory gene involved in transport and theinducer gene involved in regulation are missing, which makesthis bacterium a poor bacteriocin-producer. Cloning of penAand peiA in a L. sakei host that contains the complete apparatusfor gene activation, maturation and externalization of bacter-iocins confirmed the production of this new and potent bac-teriocin, termed penocin A (Diep et al., 2006).
Based on the genome sequences and gene identification, itwill be possible to develop adequate methodologies (such asmicroarray technology) to study the global response of bacteria
to bacteriocins. In one example, by using microarrays for globalanalysis of gene expression, it has been shown that lactococcin972 induces a cell-envelope stress response in L. lactismediatedby the two-component system KinD/LlrD (Martínez et al.,2006). Transcriptome analysis can also reveal new and relevantdata on the biology of bacteriocins such as the mechanismsof resistance/adaptation. Strains of L. lactis that are naturallyadapted or transiently resistant to nisin can be readily obtainedby subcultivation with sub-lethal nisin amounts. The adaptationis lost upon subcultivation without nisin. Transcriptome analysisof adapted strains revealed a significant up/down regulation of95 genes belonging to several main functional categories: (i) cellwall synthesis, (ii) central and energy metabolism, (iii)phospholipid- and fatty acid metabolism, (iv) gene regulation,(v) transport, (vi) stress, and (vii) miscellaneous or unknownfunctions (Kok et al., 2005). The comparative transcriptomeanalysis of nisin-sensitive and nisin-resistant L. lactis concludedthat nisin resistance is a complex phenotype, involving variousdifferent mechanisms, mainly (i) preventing nisin from reachingthe cytoplasmic membrane, (ii) reducing the acidity of theextracellular medium, thereby stimulating the binding of nisin tothe cell wall, (iii) preventing the insertion of nisin into themembrane, and (iv) possibly transporting nisin across themembrane or extruding nisin out of the membrane (Krameret al., 2006). Studies similar to these are clearly needed in orderto elucidate the mechanisms that may be involved in adaptationof foodborne pathogens upon exposure to bacteriocin pressure infood systems.
Overall, the set of methodologies that have emerged in recentyears provide an arsenal of barely unexplored tools that couldexpand the potential of bacteriocinogenic strains for food ap-plication and improve our understanding on the global effects ofbacteriocins in food ecosystems, allowing a more rational ap-plication of these natural antimicrobial hurdles in foods.
5. Conclusions
A large number of bacteriocins from LAB have been char-acterized to date, and many different studies have indicated thepotential usefulness of bacteriocins in food preservation. Bac-teriocins are a diverse group of antimicrobial proteins/peptides,and therefore are expected to behave differently on differenttarget bacteria and under different environmental conditions.Since the efficacy of bacteriocins in foods is dictated by envi-ronmental factors, there is a need to determinemore precisely themost effective conditions for application of each particularbacteriocin. The use of novel preservation technologies offersnew opportunities for application of bacteriocins as part ofhurdle technology, as has been demonstrated for PEF and HHP.However, the combined application of many other technolo-gies (such as ultrasonication, irradiation, microwave and ohmicheating, or pulsed light) still remains unexplored. Bacteriocino-genic cells may also act as living factories in foods. The anti-microbial effects of bacteriocins and bacteriocinogenic culturesin food ecosystems must be understood in terms of microbialinteractions. The application of a microbial ecology approachmay provide a more realistic portrait of the complex interactions
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occurring in food systems that ultimately lead to microbialinactivation, survival or adaptation to environmental stress. Theuse of molecular microbial ecology methodologies may help tounderstand better the biology of bacteriocins and food microbialecosystems at cellular and molecular levels. The study ofbacterial genomes and other related aspects such as global geneexpression analysis can provide valuable information on thebacteriocinogenic potential of LAB. Genomic studies can alsothrow light on other issues of great interest in food applicationsuch as the global response of ecosystems to bacteriocins or thedevelopment of adaptation or resistance.
References
Aasen, I.M., Markussen, S., Møretrø, T., Katla, T., Axelsson, L., Naterstad, K.,2003. Interactions of the bacteriocins sakacin P and nisin with foodconstituents. International Journal of Food Microbiology 87, 35–43.
Abriouel, H., Valdivia, E., Gálvez, A., Maqueda, M., 1998. Response of Salmo-nella choleraesuis LT2 spheroplasts and permeabilized cells to the bacteriocinAS-48. Applied and Environmental Microbiology 64, 4623–4626.
Abriouel, H., Maqueda, M., Gálvez, A., Martínez-Bueno, M., Valdivia, E.,2002. Inhibition of bacterial growth, enterotoxin production, and sporeoutgrowth in strains of Bacillus cereus by bacteriocin AS-48. Applied andEnvironmental Microbiology 68, 1473–1477.
Altermann, E., Russell, W.M., Azcarate-Peril, M.A., Barrangou, R., Buck, B.L.,McAuliffe, O., Souther, N., Dobson, A., Duong, T., Callanan, M., Lick, S.,Hamrick, A., Cano, R., Klaenhammer, T.R., 2005. Complete genomesequence of the probiotic lactic acid bacterium Lactobacillus acidophilusNCFM. Proceedings of the National Academy of Sciences of the USA 102,3906–3912.
Ananou, S., Valdivia, E., Martínez Bueno, M., Gálvez, A., Maqueda, M., 2004.Effect of combined physico-chemical preservatives on enterocin AS-48activity against the enterotoxigenic Staphylococcus aureus CECT 976strain. Journal of Applied Microbiology 97, 48–56.
Ananou, S., Gálvez, A., Martínez-Bueno, M., Maqueda, M., Valdivia, E., 2005.Synergistic effect of enterocin AS-48 in combination with outer membranepermeabilizing treatments against Escherichia coli O157:H7. Journal ofApplied Microbiology 99, 1364–1372.
Arqués, J.L., Fernández, J., Gaya, P., Nuñez,M., Rodríguez, E.,Medina,M., 2004.Antimicrobial activity of reuterin in combination with nisin against food-bornepathogens. International Journal of Food Microbiology 95, 225–229.
Arqués, J.L., Rodríguez, E., Gaya, P., Medina, M., Nuñez, M., 2005. Effect ofcombinations of high-pressure treatment and bacteriocin-producing lacticacid bacteria on the survival of Listeria monocytogenes in raw milk cheese.International Dairy Journal 15, 893–900.
Avery, S.M., Buncic, S., 1997. Antilisterial effects of a sorbate-nisin combinationin vitro and on packaged beef at refrigeration temperature. Journal of FoodProtection 60, 1075–1080.
Aymerich, M.T., Artigas, M.G., Garriga, M., Monfort, J.M., Hugas, M., 2000.Effect of sausage ingredients and additives on the production of enterocins Aand B byEnterococcus faeciumCTC492. Optimization of in vitro productionand anti-listerial effect in dry fermented sausages. Journal of AppliedMicrobiology 88, 686–694.
Bakes, S.H., Kitis, F.Y.E., Quattlebaum, R.G., Barefoot, S.F., 2004. Sensitizationof Gram-negative and Gram-positive bacteria to jenseniin G by sublethalinjury. Journal of Food Protection 67, 1009–1013.
Barefoot, S.F., Chen, Y.R., Hughes, T.A., Bodine, A.B., Shearer, M.Y., Hughes,M.D., 1994. Identification and purification of a protein that inducesproduction of the Lactobacillus acidophilus bacteriocin lactacin B. Appliedand Environmental Microbiology 60, 3522–3528.
Bari, M.L., Ukuku, D.O., Kawasaki, T., Inatsu, Y., Isshiki, K., Kawamoto, S.,2005. Combined efficacy of nisin and pediocin with sodium lactate, citricacid, phytic acid, and potassium sorbate and EDTA in reducing the Listeriamonocytogenes population of inoculated fresh-cut produce. Journal of FoodProtection 68, 1381–1387.
Ben Omar, N., Castro, A., Lucas, R., Abriouel, H., Yousif, N.M., Franz, C.M.,Holzapfel, W.H., Perez-Pulido, R., Martinez-Cañamero, M., Galvez, A.,2004. Functional and safety aspects of Enterococci isolated from differentSpanish foods. Systematic and Applied Microbiology 27, 118–130.
Bhunia, A.K., Johnson, M.C., Ray, B., Kalchayanand, N., 1991. Mode of actionof pediocin AcH from Pediococcus acidilactici H on sensitive bacterialstrains. Journal of Applied Bacteriology 70, 25–33.
Black, E.P., Kelly, A.L., Fitzgerald, G.F., 2005. The combined effect of highpressure and nisin on inactivation of microorganisms in milk. InnovativeFood Science and Emerging Technologies 6, 286–292.
Boussouel, N., Mathieu, F., Revol-Junelles, A.-M., Millière, J.-B., 2000. Effectsof combinations of lactoperoxidase system and nisin on the behaviour ofListeria monocytogenes ATCC 15313 in skim milk. International Journalof Food Microbiology 61, 169–175.
Bouttefroy, A., Millière, J.-B., 2000. Nisin-curvaticin 13 combinations foravoiding the regrowth of bacteriocin resistant cells of Listeria mono-cytogenes ATCC 15313. International Journal of Food Microbiology 62,65–75.
Bouttefroy, A., Mansour, M., Linder, M., Milliere, J.-B., 2000. Inhibitory com-binations of nisin, sodium chloride, and pH on Listeria monocytogenesATCC 15313 in broth by an experimental design approach. InternationalJournal of Food Microbiology 54, 109–115.
Boziaris, I.S., Adams, M.R., 1999. Effect of chelators and nisin produced in situon inhibition and inactivation of Gram negatives. International Journal ofFood Microbiology 53, 105–113.
Boziaris, I.S., Humpheson, I., Adams, M.R., 1998. Effect of nisin on heat injuryand inactivation of Salmonella enteritidis PT4. International Journal of FoodMicrobiology 43, 7–13.
Branen, J.K., Davidson, P.M., 2004. Enhancement of nisin, lysozyme, andmonolaurin antimicrobial activities by ethylenediaminetetraacetic acid andlactoferrin. International Journal of Food Microbiology 90, 63–74.
Brewer, R., Adams, M.R., Park, S.F., 2002. Enhanced inactivation of Listeriamonocytogenes by nisin in the presence of ethanol. Letters in AppliedMicrobiology 34, 18–21.
Budu-Amoako, E., Ablett, R.F., Harris, J., Delves-Broughton, J., 1999. Combinedeffect of nisin and moderate heat on destruction of Listeria monocytogenes incold-pack lobster meat. Journal of Food Protection 62, 46–50.
Buncic, S., Fitzgerald, S., Bell, C.M., Hudson, R.G., 1995. Individual andcombined listericidal effects of sodium lactate, potassium sorbate, nisin andcuring salts at refrigeration temperatures. Journal of Food Safety 15,247–264.
Burt, S., 2004. Essential oils: their antibacterial properties and potentialapplications in foods— a review. International Journal of Food Microbiology94, 223–253.
Calderón-Miranda, M.L., Barbosa-Canovas, G.V., Swanson, B.G., 1999a.Inactivation of Listeria innocua in liquid whole egg by pulsed electric fieldsand nisin. International Journal of Food Microbiology 51, 7–17.
Calderón-Miranda, M.L., Barbosa-Canovas, G.V., Swanson, B.G., 1999b.Inactivation of Listeria innocua in skim milk by pulsed electric fields andnisin. International Journal of Food Microbiology 51, 19–30.
Calderón-Miranda, M.L., Barbosa-Canovas, G.V., Swanson, B.G., 1999c.Transmission electron microscopy of Listeria innocua treated by pulsedelectric fields and nisin in skimmed milk. International Journal of FoodMicrobiology 51, 31–38.
Capellas, M., Mor-Mur, M., Gervilla, R., Yuste, J., Guamis, B., 2000. Effect ofhigh pressure combined with mild heat or nisin on inoculated bacteria andmesophiles of goats' milk fresh cheese. Food Microbiology 17, 633–641.
Carneiro De Melo, A.M.S., Cassar, C.L., Miles, R.J., 1998. Trisodiumphosphate increases sensitivity of Gram-negative bacteria to lysozyme andnisin. Journal of Food Protection 61, 839–844.
Chen, H., Hoover, D.G., 2003. Bacteriocins and their food applications.Comprehensive Reviews in Food Science and Food Safety 2, 82–100.
65A. Gálvez et al. / International Journal of Food Microbiology 120 (2007) 51–70
Chen, C.-M., Sebranek, J.G., Dickson, J.S., Mendonca, A.F., 2004. Combiningpediocin with post-packaging irradiation for control of Listeria mono-cytogenes on frankfurters. Journal of Food Protection 67, 1866–1875.
Chen, H., Guan, D., Hoover, D.G., 2006. Sensitivities of foodborne pathogens topressure changes. Journal of Food Protection 69, 130–136.
Chumchalová, J., Josephsen, J., Plocková, J.M., 1998. The antimicrobialactivity of acidocin CH5 in MRS broth and milk with added NaCl, NaNO3
and lysozyme. International Journal of Food Microbiology 43, 33–38.Chung, W., Hancock, R.E.W., 2000. Action of lysozyme and nisin mixtures
against lactic acid bacteria. International Journal of Food Microbiology 60,25–32.
Church, N., 1994. Developments in modified-atmosphere packaging and relatedtechnologies. Trends in Food Science & Technology 5, 345–352.
CoboMolinos, A., Abriouel, H., Ben Omar, N., Valdivia, E., Lucas, R., Maqueda,M., Martínez Cañamero, M., Gálvez, A., 2005. Effect of immersion solutionscontaining enterocin AS-48 on Listeria monocytogenes in vegetable foods.Applied and Environmental Microbiology 71, 7781–7787.
Connil, N., Prévost, H., Dousset, X., 2002. Production of biogenic amines anddivercin V41 in cold smoked salmon inoculated with Carnobacteriumdivergens V41, and specific detection of this strain by multiplex-PCR.Journal of Applied Microbiology 92, 611–617.
Cotter, P.D., Hill, C., Ross, R.P., 2005. Bacteriocins: developing innateimmunity for food. Nature Reviews Microbiology 3, 777–788.
Coventry, M.J., Gordon, J.B., Alexander, M., Hickey, M.W., Wan, J., 1996. Afood-grade process for isolation and partial purification of bacteriocins oflactic acid bacteria that uses diatomite calcium silicate. Applied andEnvironmental Microbiology 62, 1764–1769.
Cutter, C.N., Siragusa, G.R., 1995a. Population reductions of Gram-negativepathogens following treatments with nisin and chelators under variousconditions. Journal of Food Protection 58, 977–983.
Cutter, C.N., Siragusa, G.R., 1995b. Treatments with nisin and chelators toreduce Salmonella and Escherichia coli on beef. Journal of Food Protection58, 1028–1030.
Davies, E.A., Adams, M.R., 1994. Resistance of Listeria monocytogenes to thebacteriocin nisin. International Journal of Food Microbiology 21, 341–347.
Davies, E.A., Bevis, H.E., Delves-Broughton, J., 1997. The use of thebacteriocin, nisin, as a preservative in ricotta-type cheeses to control thefood-borne pathogen Listeria monocytogenes. Letters in Applied Microbi-ology 24, 343–346.
Dawson, P.L., Carl, G.D., Acton, J.C., Han, I.Y., 2002. Effect of lauric acid andnisin-impregnated soy-based films on the growth of Listeria monocytogeneson turkey bologna. Poultry Science 81, 721–726.
Dawson, P.L., Harmon, L., Sotthibandhu, A., Han, I.Y., 2005. Antimicrobialactivity of nisin-adsorbed silica and corn starch powders. Food Microbiol-ogy 22, 93–99.
Deegan, L.H., Cotter, P.D., Hill, C., Ross, P., 2006. Bacteriocins: biologicaltools for bio-preservation and shelf-life extension. International DairyJournal 16, 1058–1071.
Degnan, A.J., Luchansky, J.B., 1992. Influence of beef tallow and muscle on theantilisterial activity of pediocin AcH and liposome-encapsulated pediocinAcH. Journal of Food Protection 55, 552–554.
De Jong, A., van Hijum, S.A., Bijlsma, J.J., Kok, J., Kuipers, O.P., 2006.BAGEL, a web-based bacteriocin genome mining tool. Nucleic Acids 34(Web Server issue), W273–W279.
Delgado, A., Brito, D., Peres, C., Arroyo-López, F.N., Garrido-Fernández, A.,2005. Bacteriocin production by Lactobacillus pentosus B96 can beexpressed as a function of temperature and NaCl concentration. FoodMicrobiology 22, 721–726.
Devlieghere, F., Debevere, J., van Impe, J., 1998. Concentration of carbondioxide in the water-phase as a parameter to model the effect of a modifiedatmosphere on microorganisms. International Journal of Food Microbiology43, 105–113.
Devlieghere, F., Vermeiren, L., Debevere, J., 2004. New preservationtechnologies: possibilities and limitations. International Dairy Journal 14,273–285.
Diels, A.M.J., De Taeye, J., Michiels, C.W., 2005. Sensitisation of Escherichiacoli to antibacterial peptides and enzymes by high-pressure homogenisation.International Journal of Food Microbiology 105, 165–175.
Diep, D.B., Godager, L., Brede, D., Nes, I.F., 2006. Data mining andcharacterization of a novel pediocin-like bacteriocin system from thegenome of Pediococcus pentosaceus ATCC 25745. Microbiology 152,1649–1659.
Drider, D., Fimland, G., Héchard, Y., McMullen, L.M., Prévost, H., 2006. Thecontinuing story of class IIa bacteriocins. Microbiology and MolecularBiology Reviews 70, 564–582.
Dutreux, N., Notermans, S., Góngora-Nieto, M.M., Barbosa-Cánovas, G.V.,Swanson, B.G., 2000. Effects of combined exposure ofMicrococcus luteus tonisin and pulsed electric fields. International Journal of FoodMicrobiology 60,147–152.
Dykes, G.A., Hastings, J.W., 1998. Fitness costs associated with class IIabacteriocin resistance in Listeria monocytogenes B73. Letters in AppliedMicrobiology 26, 5–8.
Ellison, R.T., 1994. The effects of lactoferrin on gram-negative bacteria. In:Hutchens, T.W., Lönnerdal, B., Rumball, S. (Eds.), Lactoferrin-Structureand Function. Plenum Press, New York, pp. 71–87.
Elotmani, F., Assobhei, O., 2004. In vitro inhibition of microbial flora of fish bynisin and lactoperoxidase system. Letters in Applied Microbiology 38,60–65.
El-Ziney, M.G., Debevere, J., Jakobsen, M., 2000. Reuterin. In: Naidu, A.S. (Ed.),Natural Food Antimicrobial Systems. CRC Press, London, pp. 567–587.
Ercolini, D., Storia, A., Villani, F., Mauriello, G., 2006. Effect of a bacteriocin-activated polythene film on Listeria monocytogenes as evaluated by viablestaining and epifluorescence microscopy. Journal of Applied Microbiology100, 765–772.
Ettayebi, K., El Yamani, J., Rossi-Hassani, B., 2000. Synergistic effects of nisinand thymol on antimicrobial activities in Listeria monocytogenes and Ba-cillus subtilis. FEMS Microbiology Letters 183, 191–195.
Fang, T.J., Lin, L.-W., 1994a. Growth of Listeria monocytogenes and Pseudo-monas fragi on cooked pork in a modified atmosphere packaging/nisincombination system. Journal of Food Protection 57, 479–485.
Fang, T.J., Lin, L.-W., 1994b. Inactivation of Listeria monocytogenes on rawpork treated with modified atmosphere packaging and nisin. Journal of Foodand Drug Analysis 2, 189–200.
Fang, T.J., Tsai, H.-C., 2003. Growth patterns of Escherichia coli O157:H7in ground beef treated with nisin, chelators, organic acids and theircombinations immobilized in calcium alginate gels. Food Microbiology 20,243–253.
Farber, J.M., 1991. Microbiological aspects of modified-atmosphere packagingtechnology—a review. Journal of Food Protection 54, 58–70.
Farkas, J., 1998. Irradiation as a method for decontaminating food: a review.International Journal of Food Microbiology 44, 189–204.
Farkas, J., 2006. Irradiation for better foods. Trends in Food Science &Technology 17, 148–152.
Farkas, D.F., Hoover, D.G., 2000. High pressure processing. Journal of FoodScience 65, 47–64 (Supplement).
Farkas, J., Polyák-Fehér, K., Andrássy, É., Mészáros, L., 2002. Improvement ofmicrobiological safety of sous-vide meals by gamma radiation. RadiationPhysics and Chemistry 63, 345–348.
Farkas, J., Andrassy, E., Meszaros, L., Simon, A., 2003. Increased salt- andnisin-sensitivity of pressure-injured bioluminescent Listeria monocytogenes.Acta Microbiologica et Immunologica Hungarica 50, 331–337.
Faye, T., Brede, D.A., Langsrud, T., Nes, I.F., Holo, H., 2004. Prevalence of thegenes encoding propionicin T1 and protease-activated antimicrobial peptideand their expression in classical propionibacteria. Applied and Environ-mental Microbiology 70, 2240–2244.
Fernández de Palencia, P., de la Plaza,M.,Mohedano,M.L.,Martinez-Cuesta,M.C.,Requena, T., López, P., Peláez, C., 2004. Enhancement of 2-methylbutanalformation in cheese by using a fluorescently tagged Lacticin 3147 producingLactococcus lactis strain. International Journal of Food Microbiology 93,335–347.
66 A. Gálvez et al. / International Journal of Food Microbiology 120 (2007) 51–70
Fimland, G., Johnsen, L., Dalhus, B., Nissen-Meyer, J., 2005. Pediocin-likeantimicrobial peptides (class IIa bacteriocins) and their immunity proteins:biosynthesis, structure, and mode of action. Journal of Peptide Science 11,688–696.
Foulquié Moreno, M.R., Sarantinopoulos, P., Tsakalidou, E., De Vuyst, L.,2006. The role and application of enterococci in food and health.International Journal of Food Microbiology 106, 1–24.
Franklin, N.B., Cooksey, K.D., Getty, K.J., 2004. Inhibition of Listeriamonocytogenes on the surface of individually packaged hot dogs with apackaging film coating containing nisin. Journal of Food Protection 67,480–485.
Gallo, L.I., Pilosof, A.M.R., Jagus, R.J., 2007. Effect of the sequence of nisinand pulsed electric fields treatments and mechanisms involved in theinactivation of Listeria innocua in whey. Journal of Food Engineering 79,188–193.
Gao, Y., van Belkum, M.J., Stiles, M.E., 1999. The outer membrane of gram-negative bacteria inhibits antibacterial activity of brochocin-C. Applied andEnvironmental Microbiology 65, 4329–4333.
García,M.T., BenOmar, N., Lucas, R., Pérez-Pulido, R., Castro, A., Grande,M.J.,Martínez-Cañamero,M., Gálvez, A., 2003. Antimicrobial activity of enterocinEJ97 on Bacillus coagulans CECT 12. Food Microbiology 20, 533–536.
García, M.T., Lucas, R., Abriouel, H., Ben Omar, N., Pérez, R., Grande, M.J.,Martínez-Cañamero, M., Gálvez, A., 2004a. Antimicrobial activity ofenterocin EJ97 against ‘Bacillus macroides/Bacillus maroccanus’ isolatedfrom zucchini purée. Journal of Applied Microbiology 97, 731–737.
García, M.T., Martínez Cañamero, M., Lucas, R., Ben Omar, N., Pérez Pulido,R., Gálvez, A., 2004b. Inhibition of Listeria monocytogenes by enterocinEJ97 produced by Enterococcus faecalis EJ97. International Journal ofFood Microbiology 90, 161–170.
García-Graells, C., Hauben, K.,Michiels, C.W., 1998. High pressure inactivationand sublethal injury of pressure-resistant Escherichia coli mutants in fruitjuices. Applied and Environmental Microbiology 64, 1566–1568.
Garcia-Graells, C., Masschalck, B., Michiels, C.W., 1999. Inactivationof Escherichia coli in milk by high-hydrostatic-pressure treatment incombination with antimicrobial peptides. Journal of Food Protection 62,1248–1254.
García-Graells, C., Valckx, C., Michiels, C.W., 2000. Inactivation of Escherichiacoli and Listeria innocua in milk by combined treatment with high hydrostaticpressure and the lactoperoxidase system. Applied and EnvironmentalMicrobiology 66, 4173–4179.
Garriga, M., Aymerich, M.T., Costa, S., Monfort, J.M., Hugas, M., 2002.Bactericidal synergism through bacteriocins and high pressure in a meatmodel system during storage. Food Microbiology 19, 509–518.
Ghalfi, H., Allaoui, A., Destain, J., Benkerroum, N., Thonart, P., 2006.Bacteriocin activity by Lactobacillus curvatus CWBI-B28 to inactivateListeria monocytogenes in cold-smoked salmon during 4 degrees C storage.Journal of Food Protection 69, 1066–1071.
Gilbreth, S.E., Somkuti, G.A., 2005. Thermophilin 110: a bacteriocin ofStreptococcus thermophilus ST110. Current Microbiology 51, 175–182.
Gill, A.O., Holley, R.A., 2000. Inhibition of bacterial growth on ham andbologna by lysozyme, nisin and EDTA. Food Research International 33,83–90.
Gill, A.O., Holley, R.A., 2003. Interactive inhibition of meat spoilage andpathogenic bacteria by lysozyme, nisin and EDTA in the presence of nitriteand sodium chloride at 24 °C. International Journal of Food Microbiology80, 251–259.
Grande, Ma.J., Lucas, R., Abriouel, H., Valdivia, E., Ben Omar, N., Maqueda,M., Martínez-Bueno, M., Martínez-Cañamero, M., Gálvez, A., 2006.Inhibition of toxicogenic Bacillus cereus in rice-based foods by enterocinAS-48. International Journal of Food Microbiology 106, 185–194.
Grande, Ma.J., Lucas, R., Abriouel, H., Valdivia, E., Ben Omar, N., Maqueda,M., Martínez-Cañamero, M., Gálvez, A., 2007. Treatment of vegetablesauces with enterocin AS-48 alone or in combination with phenoliccompounds to inhibit proliferation of Staphylococcus aureus. Journal ofFood Protection 70, 405–411.
Grattepanche, F., Lacroix, C., Audet, P., Lapointe, G., 2005. Quantification byreal-time PCR of Lactococcus lactis subsp. cremoris in milk fermented by amixed culture. Applied Microbiology and Biotechnology 66, 414–421.
Gravesen, A., Jydegaard Axelsen, A.-M., Mendes da Silva, J., Hansen, T.B.,Knøchel, S., 2002a. Frequency of bacteriocin resistance development andassociated fitness costs in Listeria monocytogenes. Applied and Environ-mental Microbiology 68, 756–764.
Gravesen, A., Ramnath, M., Rechinger, K.B., Andersen, N., Jansch, L.,Hechard, Y., Hastings, J.W., Knochel, S., 2002b. High-level resistance toclass IIa bacteriocins is associated with one general mechanism in Listeriamonocytogenes. Microbiology 148, 2361–2369.
Guerra, N.P., Macias, C.L., Agrasar, A.T., Castro, L.P., 2005. Development of abioactive packaging cellophane using Nisaplin as biopreservative agent.Letters in Applied Microbiology 40, 106–1610.
Guinane, C.M., Cotter, P.D., Hill, C., Ross, R.P., 2005. Microbial solutions tomicrobial problems; lactococcal bacteriocins for the control of undesirablebiota in food. Journal of Applied Microbiology 98, 1316–1325.
Hanlin, M.B., Kalchayanand, N., Ray, P., Ray, B., 1993. Bacteriocins of lacticacid bacteria in combination have greater antibacterial activity. Journal ofFood Protection 56, 252–255.
Harris, L.J., Fleming, H.P., Klaenhammer, T.R., 1991. Sensitivity and resistanceof Listeria monocytogenes ATCC 19115, Scott A, and UAL500 to nisin.Journal of Food Protection 54, 836–840.
Hauben, K.J.A., Wuytack, E.Y., Soontjens, C.C.F., Michiels, C.W., 1996. Highpressure transient sensitization of Escherichia coli to lysozyme and nisin bydisruption of outer membrane permeability. Journal of Food Protection 59,350–355.
Heinz, V., Alvarez, I., Angersbach, A., Knorr, D., 2002. Preservation of liquidfoods by high intensity pulsed electric fields-basic concepts for processdesign. Trends in Food Science & Technology 12, 103–111.
Helander, I.M., von Wright, A., Mattila-Sandholm, T.-M., 1997. Potential oflactic acid bacteria and novel antimicrobials against Gram-negative bacteria.Trends in Food Science & Technology 8, 146–150.
Henning, S., Metz, R., Hammes, W.P., 1986. New aspects for the application ofnisin to food products based on its mode of action. International Journal ofFood Microbiology 3, 135–141.
Himmelbloom, B., Nilsson, L., Gram, L., 2001. Factors affecting production ofan antilisterial bacteriocin by Carnobacterium piscicola strain A9b inlaboratory media and model fish systems. Journal of Applied Microbiology91, 506–513.
Hols, P., Hancy, F., Fontaine, L., Grossiord, B., Prozzi, D., Leblond-Bourget, N.,Decaris, B., Bolotin, A., Delorme, C., Ehrlich, S.D., Guédon, E., Monnet, V.,Renault, P., Kleerebezem, M., 2005. New insights in the molecular biologyand physiology of Streptococcus thermophilus revealed by comparativegenomics. FEMS Microbiology Reviews 29, 435–463.
Holtzel, A., Ganzle, M.G., Nicholson, G.J., Hammes, W.P., Jung, G., 2000. Thefirst low-molecular-weight antibiotic from lactic acid bacteria: reutericyclin,a new tetramic acid. Angewandte Chemie. International Edition 39,2766–2768.
Holzapfel, W.H., 2002. Appropriate starter culture technologies for small-scalefermentation in developing countries. International Journal of FoodMicrobiology 75, 197–212.
Holzapfel, W.H., Geisen, R., Schillinger, U., 1995. Biological preservation offoods with reference to protective cultures, bacteriocins and food-gradeenzymes. International Journal of Food Microbiology 24, 343–362.
Hornbæk, T., Brocklehurst, T.F., Budde, B.B., 2004. The antilisterial effect ofLeuconostoc carnosum 4010 and leucocins 4010 in the presence of sodiumchloride and sodium nitrite examined in a structured gelatin system.International Journal of Food Microbiology 92, 129–140.
Hornbæk, T., Brockhoff, P.B., Siegumfeldt, H., Budde, B.B., 2006. Twosubpopulations of Listeria monocytogenes occur at subinhibitory concen-trations of leucocin 4010 and nisin. Applied and Envirnomental Microbi-ology 72, 1631–1638.
Hugas, M., Neumeyer, B., PagCs, F., Garriga, F., Hammes, W.P., 1996. Dieantimikrobielle Wirkung von Bakteriozin-bildenden Kulturen in Fleischwa-ren: IT) Vergleich des Effektes unterschiedlicher bakteriozinbildenderLaktobazillen auf Listerien in Rohwurst. Fleischwirtschaft 76, 649–652.
Hugas, M., Garriga, M., Monfort, J.M., 2002. New mild technologies in meatprocessing: high pressure as a model technology. Meat Science 62, 359–371.
67A. Gálvez et al. / International Journal of Food Microbiology 120 (2007) 51–70
Hühne, K., Axelsson, L., Holck, A., Kröckel, L., 1996. Analysis of the sakacin Pgene cluster from Lactobacillus sakei Lb674 and its expression in sakacin-negative L. sakei strains. Microbiology 142, 1437–1448.
Iu, J., Mittal, G.S., Griffiths, M.W., 2001. Reduction in levels of Escherichiacoli O157:H7 in apple cider by pulsed electric fields. Journal of FoodProtection 64, 964–969.
Jung, D.-S., Bodyfelt, F.W., Daeshel, M.A., 1992. Influence of fat andemulsifiers on the efficacy of nisin in inhibiting Listeria monocytogenes influid milk. Journal of Dairy Science 75, 387–393.
Jydegaard, A.-M., Gravesen, A., Knøchel, S., 2000. Growth condition-relatedresponse of Listeria monocytogenes 412 to bacteriocin inactivation. Lettersin Applied Microbiology 31, 68–72.
Kalchayanand, N., Hanlin, M.B., Ray, B., 1992. Sublethal injury makes Gram-negative and resistant Gram-positive bacteria sensitive to the bacteriocins,pediocin AcH and nisin. Letters in Applied Microbiology 16, 239–243.
Kalchayanand, N., Sikes, A., Dunne, C.P., Ray, B., 1994. Hydrostatic pressureand electroporation have increased bactericidal efficiency in combinationwith bacteriocins. Applied and EnvironmentalMicrobiology 60, 4174–4177.
Kalchayanand, N., Sikes, A., Dunne, C.P., Ray, B., 1998a. Factors influencingdeath and injury of foodborne pathogens by hydrostatic pressure pasteuri-zation. Food Microbiology 15, 207–214.
Kalchayanand, N., Sikes, A., Dunne, C.P., Ray, B., 1998b. Interaction ofhydrostatic pressure, time and temperature of pressurization and pediocinAcH on inactivation of foodborne bacteria. Journal of Food Protection 61,425–431.
Kalchayanand, N., Frethem, C., Dunne, P., Sikes, A., Ray, B., 2002. Hydrostaticpressure and bacteriocin-triggered cell wall lysis of Leuconostoc mesenter-oides. Innovative Food Science and Emerging Technologies 3, 33–40.
Kalchayanand, N., Dunne, C.P., Sikes, A., Ray, B., 2004a. Viability loss andmorphology change of foodborne pathogens following exposure tohydrostatic pressures in the presence and absence of bacteriocins.International Journal of Food Microbiology 91, 91–98.
Kalchayanand, N., Dunne, C.P., Sikes, A., Ray, B., 2004b. Germination inductionand inactivation of Clostridium spores at medium-range hydrostatic pressuretreatment. Innovative Food Science and Emerging Technologies 5, 277–283.
Karatzas, K.A.H., Bennik, M.H., 2002. Characterization of a Listeriamonocytogenes Scott A isolate with intolerance towards high hydrostaticpressure. Applied and Environmental Microbiology 68, 3183–3189.
Kato, M., Hayashi, R., 1999. Effects of high pressure on lipids of biomembranesfor understanding high-pressure-induced biological phenomena. Bioscience,Biotechnology and Biochemistry 68, 1321–1328.
Klaenhammer, T.R., 1993. Genetics of bacteriocins produced by lactic acidbacteria. FEMS Microbiology Reviews 12, 39–86.
Ko, S., Janes, M.E., Hettiarachchy, N.S., Johnson, M.G., 2001. Physical andchemical properties of edible films containing nisin and their action againstListeria monocytogenes. Journal of Food Science 66, 1006–1011.
Kok, J., Buist, G., Zomer, A.L., van Hijum, S.A.F.T., Kuipers, O.P., 2005.Comparative and functional genomics of lactococci. FEMS MicrobiologyReviews 29, 411–433.
Kramer,N.E., vanHijum, S.A.,Knol, J.,Kok, J.,Kuipers,O.P., 2006. Transcriptomeanalysis reveals mechanisms by which Lactococcus lactis acquires nisinresistance. Antimicrobial Agents and Chemotherapy 50, 1753–1761.
Kumar, C.G., Anand, S.K., 1998. Significance of microbial biofilms in foodindustry: a review. International Journal of Food Microbiology 42, 9–27.
Larsen, A.G., Vogensen, F.K., Josephsen, J., 1993. Antimicrobial activity oflactic acid bacteria isolated from sourdoughs: purification and characteriza-tion of bavaricin A, a bacteriocin produced by Lactobacillus bavaricusMI401. Journal of Applied Bacteriology 75, 113–122.
Leal-Sánchez, M.V., Jiménez-Díaz, R., Maldonado-Barragán, A., Garrido-Fernández, A., Ruiz-Barba, J., 2002. Optimization of bacteriocin productionby batch fermentation of Lactobacillus plantarum LPCO10. Applied andEnvironmental Microbiology 68, 4465–4471.
Lee, S., Iwata, T., Oyagi, H., 1993. Effects of salts on conformational change ofbasic amphipathic peptides from β-structure to α-helix in the presence ofphospholipid liposomes and their channel-forming ability. Biochimica etBiophysica Acta 1151, 75–82.
Leistner, L., 1978. Hurdle effect and energy saving. In: Downey, W.K. (Ed.),Food Quality and Nutrition. Applied Science Publishers, London, UK,pp. 553–557.
Leistner, L., 1999. Combined methods for food preservation. In: ShafiurRahman, M. (Ed.), Handbook of Food Preservation. Marcel Dekker, NewYork, pp. 457–485.
Leistner, L., 2000. Basic aspects of food preservation by hurdle technology.International Journal of Food Microbiology 55, 181–186.
Leistner, L., Gorris, L.G.M., 1995. Food preservation by hurdle technology.Trends in Food Science & Technology 6, 41–46.
Leroy, F., De Vuyst, L., 1999. The presence of salt and a curing agent reducesbacteriocin production by Lactobacillus sakei CTC 494, a potential starter forfermented sausage. Applied and EnvironmentalMicrobiology 65, 5350–5356.
Leroy, F., De Vuyst, L., 2003. A combined model to predict the functionality ofthe bacteriocin-producing Lactobacillus sakei strain CTC 494. Applied andEnvironmental Microbiology 69, 1093–1099.
Leroy, F., De Vuyst, L., 2005. Simulation of the effect of sausage ingredients andtechnology on the functionality of the bacteriocin-producing Lactobacillussakei CTC 494 strain. International Journal of Food Microbiology 100,141–152.
Leroy, F., Verluyten, J., De Vuyst, L., 2006. Functional meat starter cultures forimproved sausage fermentation. International Journal of Food Microbiology106, 270–285.
Liang, Z., Mittal, G.S., Griffiths, M.W., 2002. Inactivation of SalmonellaTy-phimurium in orange juice containing antimicrobial agents by pulsed electricfield. Journal of Food Protection 65, 1081–1087.
Long, C., Phillips, C.A., 2003. The effect of sodium citrate, sodium lactate andnisin on the survival of Arcobacter butzleri NCTC 12481 on chicken. FoodMicrobiology 20, 495–502.
López-Pedemonte, T., Roig-Sagués, A.X., Trujillo, A.J., Capellas, M., Guamis,B., 2003. Inactivation of spores of Bacillus cereus in cheese by highhydrostatic pressure with the addition of nisin or lysozyme. Journal of DairyScience 86, 3075–3081.
Luchansky, J.B., Call, J.E., 2004. Evaluation of nisin-coated cellulose casingsfor the control of Listeria monocytogenes inoculated onto the surface ofcommercially prepared frankfurters. Journal of Food Protection 67,1017–1021.
Lüders, T., Birkemo, G.A., Fimland, G., Nissen-Meyer, J., Nes, I.F., 2003. Strongsynergy between a eukaryotic antimicrobial peptide and bacteriocins fromlactic acid bacteria. Applied and EnvironmentalMicrobiology 69, 1797–1799.
Lungu, B., Johnson, M.G., 2005. Fate of Listeria monocytogenes inoculatedonto the surface of model Turkey frankfurter pieces treated with zeincoatings containing nisin, sodium diacetate, and sodium lactate at 4 ºC.Journal of Food Protection 68, 855–859.
Mahadeo, M., Tatini, S.R., 1994. The potential of nisin to control Listeriamonocytogenes in poultry. Letters in Applied Microbiology 18, 323–326.
Maisnier-Patin, S., Tatini, S.R., Richard, J., 1995. Combined effect of nisin andmoderate heat on destruction of Listeria monocytogenes in milk. Lait 75,81–91.
Maldonado, A., Ruiz-Barba, J.L., Floriano, B., Jimenez-Diaz, R., 2002. Thelocus responsible for production of plantaricin S, a class IIb bacteriocinproduced by Lactobacillus plantarum LPCO10, is widely distributed amongwild-type Lact. plantarum strains isolated from olive fermentations.International Journal of Food Microbiology 77, 117–124.
Maldonado, A., Ruiz-Barba, J.L., Jimenez-Diaz, R., 2004. Production ofplantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presenceof different types of gram-positive bacteria. Archives of Microbiology 181,8–16.
Mansour, M., Linder, M., Millière, J.-B., Lefebvre, G., 1998. Combined effectsof nisin, lactic acid and potassium sorbate on Bacillus licheniformis sporesin milk. Le Lait 7, 117–128.
Mansour, M., Amri, D., Bouttefroy, A., Linder, M., Milliere, J.B., 1999.Inhibition of Bacillus licheniformis spore growth in milk by nisin,monolaurin, and pH combinations. Journal of Applied Microbiology 86,311–324.
68 A. Gálvez et al. / International Journal of Food Microbiology 120 (2007) 51–70
Martínez, B., Bravo, D., Rodriguez, A., 2005. Consequences of the developmentof nisin-resistant Listeria monocytogenes in fermented dairy products.Journal of Food Protection 68, 2383–2388.
Martínez, B., Zomer, A.L., Rodríguez, A., Kuipers, O.P., 2006. Cell wallsynthesis inhibition by the antimicrobial peptide Lcn972 is sensed by thelactococcal two-component system KinD/LlrD. FOODMICRO 2006. Foodsafety and biotechnology: diversity and global impact. The 20th Interna-tional ICFMH Symposium, Bologna (Italy), August 29-September 2, p. 87.Book of Abstracts.
Masschalck, B., Van Houdt, R., Michiels, C.W., 2001. High pressure increasesbactericidal activity and spectrum of lactoferrin, lactoferricin and nisin.International Journal of Food Microbiology 64, 325–332.
Matijašić, B.B., Rajšp, M.K., Perko, B., Rogelj, I., 2007. Inhibition of Clos-tridium tyrobutyricum in cheese by Lactobacillus gasseri. InternationalDairy Journal 17, 157–166.
Mattila, K., Saris, P., Tyopponen, S., 2003. Survival of Listeria monocytogeneson sliced cooked sausage after treatment with pediocin AcH. InternationalJournal of Food Microbiology 89, 281–286.
Mauriello, G., Ercolini, D., La Storia, A., Casaburi, A., Villani, F., 2004.Development of polythene films for food packaging activated with anantilisterial bacteriocin from Lactobacillus curvatus 32Y. Journal of AppliedMicrobiology 97, 314–322.
Mauriello, G., De Luca, E., La Storia, A., Villani, F., Ercolini, D., 2005.Antimicrobial activity of a nisin-activated plastic film for food packaging.Letters in Applied Microbiology 41, 464–469.
Mazzotta, A.S., Montville, T.J., 1997. Nisin induces changes in membrane fattyacid composition of Listeria monocytogenes nisin-resistant strains at 108 °Cand 30 °C. Journal of Applied Microbiology 82, 32–38.
Ming, X., Daeschel, M.A., 1993. Nisin resistance of foodborne bacteria and thespecific resistance responses of Listeria monocytogenes Scott A. Journal ofFood Protection 56, 944–948.
Ming, X., Weber, G.H., Ayres, J.W., Sandine, W.E., 1997. Bacteriocins appliedto food packaging materials to inhibit Listeria monocytogenes on meats.Journal of Food Science 62, 413–415.
Modi, K.D., Chikindas, M.L., Montville, T.J., 2000. Sensitivity of nisin-resistantListeria monocytogenes to heat and the synergistic action of heat and nisin.Letters in Applied Microbiology 30, 249–253.
Møretrø, T., Naterstad, K., Wang, E., Aasen, I.M., Chaillou, S., Zagorec, M.,Axelsson, L., 2005. Sakacin P non-producing Lactobacillus sakei strainscontain homologues of the sakacin P gene cluster. Research in Microbiology156, 949–960.
Morgan, S.M., Galvin, M., Kelly, J., Ross, R.P., Hill, C., 1999. Development ofa lacticin 3147-enriched whey powder with inhibitory activity againstfoodborne pathogens. Journal of Food Protection 62, 1011–1016.
Morgan, S.M., Ross, R.P., Beresford, T., Hill, C., 2000. Combinationof hydrostatic pressure and lacticin 3147 causes increased killing ofStaphylococcus and Listeria. Journal of Applied Microbiology 88,414–420.
Moschetti, G., Blaiotta, G., Villani, F., Coppola, S., 2001. Nisin-producingorganisms during traditional ‘Fior di latte’ cheese-making monitored bymultiplex-PCR and PFGE analyses. International Journal of Food Microbi-ology 63, 109–116.
Mulet-Powell, N., Lacoste-Armynot, A.M., Vinas, M., Simeon De Buochberg,M., 1998. Interactions between pairs of bacteriocins from lactic bacteria.Journal of Food Protection 61, 1210–1212.
Muñoz, A., Maqueda, M., Gálvez, A., Martínez-Bueno, M., Rodriguez, A.,Valdivia, E., 2004. Biocontrol of psychrotrophic enterotoxigenic Bacilluscereus in a non fat hard type cheese by an enterococcal strain-producingenterocin AS-48. Journal of Food Protection 67, 1517–1521.
Murano, E., Hugas, M., Garriga, M., Monfort, J.M., 2002. New mildtechnologies in meat processing: high pressure as a model Technology.Meat Science 62, 359–371.
Natrajan, N., Sheldon, B.W., 2000. Efficacy of nisin-coated polymer films toinactivate Salmonella typhimurium on fresh broiler skin. Journal of FoodProtection 63, 1189–1196.
Nattress, F.M., Baker, L.P., 2003. Effects of treatment with lysozyme and nisinon the microflora and sensory properties of commercial pork. InternationalJournal of Food Microbiology 85, 259–267.
Nes, I.F., Johnsborg, O., 2004. Exploration of antimicrobial potential in LAB bygenomics. Current Opinion in Biotechnology 15, 100–104.
Neysens, P., De Vuyst, L., 2005. Carbon dioxide stimulates the production ofamylovorin L by Lactobacillus amylovorus DCE 471, while enhancedaeration causes biphasic kinetics of growth and bacteriocin production.International Journal of Food Microbiology 105, 191–202.
Neysens, P., Messens, W., De Vuyst, L., 2003. Effect of sodium chloride ongrowth and bacteriocin production by Lactobacillus amylovorus DCE 471.International Journal of Food Microbiology 88, 29–39.
Nilsson, L., Huss, H.H., Gram, L., 1997. Inhibition of Listeria monocytogeneson cold-smoked salmon by nisin and carbon dioxide atmosphere.International Journal of Food Microbiology 38, 217–227.
Nilsson, L., Chen, Y., Chikindas, M.L., Huss, H.H., Gram, L., Montville, T.J.,2000. Carbon dioxide and nisin act synergistically on Listeria mono-cytogenes. Applied and Environmental Microbiology 66, 769–774.
Nykänen, A., Weckman, K., Lapvetelainen, A., 2000. Synergistic inhibition ofListeria monocytogenes on cold-smoked rainbow trout by nisin and sodiumlactate. International Journal of Food Microbiology 61, 63–72.
Olasupo, N.A., Fitzgerald, D.J., Narrad, A., Gasson, M.J., 2004. Inhibition of Ba-cillus subtilis and Listeria innocua by nisin in combination with some naturallyoccurring organic compounds. Journal of Food Protection 67, 596–600.
O'Mahony, T., Rekhif, N., Cavadini, C., Fitzgerald, G.F., 2001. The applicationof a fermented food ingredient containing ‘variacin’, a novel antimicrobialproduced by Kocuria varians, to control the growth of Bacillus cereus inchilled dairy products. Journal of Applied Microbiology 90, 106–114.
O'Sullivan, L., Ross, R.P., Hill, C., 2002. Potential of bacteriocin-producinglactic acid bacteria for improvements in food safety and quality. Biochimie84, 593–604.
Parente, E., Giglio, M.A., Riccardi, A., Clementi, F., 1998. The combined effectof nisin, leucocin F10, pH, NaCl and EDTA on the survival of Listeriamonocytogenes in broth. International Journal of Food Microbiology 40,65–75.
Patterson, M., 2000. High pressure treatments of foods. In: Robinson, R.K.,Batt, C.A., Patel, P.D. (Eds.), Encyclopedia of food microbiology. AcademicPress, San Diego, pp. 1059–1065.
Peláez, C., Requena, T., 2005. Exploiting the potential of bacteria in the cheeseecosystem. International Dairy Journal 15, 831–844.
Periago, P.M., Palop, A., Fernandez, P.S., 2001. Combined effect of nisin,carvacrol and thymol on the viability of Bacillus cereus heat-treatedvegetative cells. Food Science and Technology International 7, 487–492.
Pol, I.E., Smid, E.J., 1999. Combined action of nisin and carvacrol on Bacilluscereus and Listeria monocytogenes. Letters in Applied Microbiology 29,166–170.
Pol, I.E., Mastwijk, H.C., Bartels, P.V., Smid, E.J., 2000. Pulsed-electric fieldtreatment enhances the bactericidal action of nisin against Bacillus cereus.Applied and Environmental Microbiology 66, 428–430.
Pol, I.E., Mastwijk, H.C., Slump, R.A., Popa, M.E., Smith, E.J., 2001a.Influence of food matrix on inactivation of Bacillus cereus by combinationsof nisin, pulsed electric field treatment, and carvacrol. Journal of FoodProtection 64, 1012–1018.
Pol, I.E., van Arendonk, W.G., Mastwijk, H.C., Krommer, J., Smid, E.,Moezelaar, R., 2001b. Sensitivities of germinating spores and carvacrol-adapted vegetative cells and spores of Bacillus cereus to nisin and pulsed-electric-field treatment. Applied and Environmental Microbiology 67,1693–1699.
Ponce, E., Pla, R., Sendra, E., Guamis, B., Mor-Mur, M., 1998. Combined effectof nisin and high hydrostatic pressure on destruction of Listeria innocua andEscherichia coli in liquid whole egg. International Journal of FoodMicrobiology 43, 15–19.
Ray, B., 2002. High hydrostatic pressure: microbial inactivation and foodpreservation. In: Britton, G. (Ed.), The Encyclopedia of EnvironmentalMicrobiology. Wiley, New York, pp. 1552–1563.
Rayman, K., Aris, B., Hurst, A., 1981. Nisin: possible alternative or adjuct tonitrite in the preservation of meats. Applied and Environmental Microbi-ology 41, 375–380.
69A. Gálvez et al. / International Journal of Food Microbiology 120 (2007) 51–70
Rayman, K., Malik, N., Hurst, A., 1983. Failure of nisin to inhibit outgrowthof lostridium botulinum in a model cured meat system. Applied andEnvironmental Microbiology 46, 1450–1452.
Rekhif, N., Atrih, A., Lefebvre, G., 1994. Selection and properties compositionof spontaneous variants of Listeria monocytogenes ATCC 15313 resistant todifferent bacteriocins produced by lactic acid bacteria strains. CurrentMicrobiology 28, 237–241.
Roberts, C.M., Hoover, D.G., 1996. Sensitivity of Bacillus coagulans spores tocombinations of high hydrostatic pressure, heat, acidity and nisin. Journal ofApplied Bacteriology 81, 363–368.
Robertson, A., Tirado, C., Lobstein, T., Jermini,M., Knai, C., Jensen, J.H., Ferro-Luzzi, A., James,W.P.T. (Eds.), 2004. Food and health in Europe: a newbasisfor action. WHO Regional Publications, European Series, vol. 96. Geneva.
Rodríguez, J.M., Martinez, M.I., Horn, N., Dodd, H.M., 2003. Heterologousproduction of bacteriocins by lactic acid bacteria. International Journal ofFood Microbiology 80, 101–116.
Ross, R.P., Sporns, P., Dodd, H.M., Gasson, M.J., Mellon, F.A., McMullen, L.M.,2003. Involvement of dehydroalanine and dehydrobutyrine in the addition ofglutathione to nisin. Journal of Agriculture and Food Chemistry 51,3174–3178.
Ross, R.P., Stanton, C., Hill, C., Fitzgerald, G.F., Coffey, A., 2000. Novelcultures for cheese improvement. Trends in Food Science & Technology 11,96–104.
Ross, R.P., Morgan, S., Hill, C., 2002. Preservation and fermentation: past,present and future. International Journal of Food Microbiology 79, 3–16.
Ross, A.I.V., Griffiths, M.W., Mittal, G.S., Deeth, H.C., 2003. Combiningnonthermal technologies to control foodborne microorganisms. InternationalJournal of Food Microbiology 89, 125–138.
Ryan, M.P., Ross, R.P., Hill, C., 2001. Strategy for manipulation of cheese florausing combinations of lacticin 3147-producing and-resistant cultures.Applied and Environmental Microbiology 67, 2699–2704.
SanMartín, F., Harte, F.M., Lelieveld, H., Barbosa-Canovas, G.V., Swanson, BarryG., 2001. Inactivation effect of an 18-T pulsed magnetic field combined withother technologies onEscherichia coli. Innovative Food Science and EmergingTechnologies 2, 273–277.
Santi, L., Cerrutti, P., Pilosof, A.M., de Huergo, M.S., 2003. Optimization of theconditions for electroporation and the addition nisin for Pseudomonasaeruginosa inhibition. Revista Argentina de Microbiología 35, 198–204.
Scannell, A.G.M., Hill, C., Buckley, D.J., Arendt, E.K., 1997. Determination ofthe influence of organic acids and nisin on shelf-life and microbiologicalsafety aspects of fresh pork sausage. Journal of Applied Microbiology 83,407–412.
Scannell, A.G.M., Hill, C., Ross, R.P., Marx, S., Hartmeier, W., Arendt, E.K.,2000a. Development of bioactive food packaging materials using immobi-lised bacteriocins Lacticin 3147 and Nisaplins. International Journal of FoodMicrobiology 60, 241–249.
Scannell, A.G., Ross, R.P., Hill, C., Arendt, E.K., 2000b. An effective lacticinbiopreservative in fresh pork sausage. Journal of Food Protection 63,370–375.
Schillinger, U., Geisen, R., Holzapfel, W.H., 1996. Potential of antagonisticmicroorganisms and bacteriocins for the biological preservation of foods.Trends in Food Science & Technology 71, 58–64.
Schved, F., Henis, Y., Juven, B.J., 1994. Response of spheroplasts and chelator-permeabilized cells of Gram-negative bacteria to the action of the bacteriocinspediocin SJ-1 and nisin. International Journal of Food Microbiology 21,305–314.
Schved, F., Pierson, M.D., Juven, B.J., 1996. Sensitization of E. coli to nisin bymaltol and ethyl maltol. Letters in Applied Microbiology 22, 189–191.
Shearer, A.E.H., Dunne, C.P., Sikes, A., Hoover, D.G., 2000. Bacterial sporeinhibition and inactivation in foods by pressure, chemical preservatives, andmild heat. Journal of Food Protection 63, 1503–1510.
Sip, A., Grajek, W., Boyaval, P., 1998. Enhancement of bacteriocin productionby Carnobacterium divergens AS7 in the presence of a bacteriocin-sensitivestrain Carnobacterium piscicola. International Journal of Food Microbiol-ogy 42, 63–69.
Siragusa, G.R., Cutter, C.N., Willett, J.L., 1999. Incorporation of bacteriocin inplastic retains activity and inhibits surface growth of bacteria on meat. FoodMicrobiology 61, 229–235.
Smelt, J.P.P.M., 1998. Recent advances in the microbiology of high pressureprocessing. Trends in Food Science & Technology 9, 152–158.
Sobrino-Lopez, A., Martin Belloso, O., 2006. Enhancing inactivation of Sta-phylococcus aureus in skim milk by combining high-intensity pulsedelectric fields and nisin. Journal of Food Protection 69, 345–353.
Stergiou, V.A., Thomas, L.V., Adams, M.R., 2006. Interactions of nisin withglutathione in a model protein system and meat. Journal of Food Protection69, 951–956.
Stevens, K.A., Sheldon, B.W., Klapes, N.A., Klaenhammer, T.R., 1991. Nisintreatment for inactivation of Salmonella species and other gram-negativebacteria. Applied and Environmental Microbiology 57, 3613–3615.
Stewart, C.M., Dunne, C.P., Sikes, A., Hoover, D.G., 2000. Sensitivity of sporesof Bacillus subtilis and Clostridium sporogenes PA3679 to combinations ofhigh hydrostatic pressure and other processing parameters. Innovative FoodScience and Emerging Technologies 1, 49–56.
Szabo, E.A., Cahill, M.E., 1998. The combined effects of modified atmosphere,temperature, nisin and ALTA™ 2341 on the growth of Listeriamonocytogenes. International Journal of Food Microbiology 43, 21–31.
Szabo, E.A., Cahill, M.E., 1999. Nisin and ALTATM 2341 inhibit the growth ofListeria monocytogenes on smoked salmon packaged under vacuum or100% CO2. Letters in Applied Microbiology 28, 373–377.
Taylor, J.I., Somer, E.B., Kruger, L.A., 1985. Antibotulinal effectiveness ofnisin-nitrite combinations in culture medium and chicken frankfurteremulsions. Journal of Food Protection 48, 234–249.
Terebiznik, M.R., Jagus, R.J., Cerrutti, P., De Huergo, M., Pilosof, A.M., 2000.Combined effect of nisin and pulsed electric fields on the inactivation ofEscherichia coli. Journal of Food Protection 63, 741–746.
Terebiznik, M.R., Jagus, R.J., Cerrutti, P., De Huergo, M., Pilosof, A.M., 2002.Inactivation of Escherichia coli by a combination of nisin, pulsed electricfields, and water activity reduction by sodium chloride. Journal of FoodProtection 65, 1253–1258.
ter Steeg, P.F., Hellemons, J.C., Kok, A.E., 1999. Synergistic actions of nisin,sublethal injury pressure, and reduced temperature on bacteria and yeast.Applied and Environmental Microbiology 65, 4148–4154.
Thomas, L.V., Wimpenny, J.W.T., 1996. Investigation of the effect of combinedvariations in temperature, pH, and NaCl concentration on nisin inhibition ofListeria monocytogenes and Staphylococcus aureus. Applied and Environ-mental Microbiology 62, 2006–2012.
Thomas, L.V., Davies, E.A., Delves-Broughton, J., Wimpenny, J.W.T., 1998.Synergistic effect of sucrose fatty acid ester on nisin inhibition of Gram-positive bacteria. Journal of Applied Microbiology 85, 1013–1022.
Todorov, S., Onno, B., Sorokine, O., Chobert, J.M., Ivanova, I., Dousset, X.,1999. Detection and characterization of a novel antibacterial substanceproduced by Lactobacillus plantarum ST 31 isolated from sourdough.International Journal of Food Microbiology 48, 167–177.
Trujillo, A.J., Capella, M., Saldo, J., Gervilla, R., Guamis, B., 2002.Applications of high-hydrostatic pressure on milk and dairy products: areview. Innovative Food Science and Emerging Technologies 3, 295–307.
Työppönen, S., Petäjä, E., Mattila-Sandholm, T., 2003. Bioprotectives andprobiotics for dry sausages. International Journal of Food Microbiology 83,233–244.
Ueckert, J.E., ter Steeg, P.F., Coote, P.J., 1998. Synergistic antibacterial action ofheat in combination with nisin and magainin II amide. Journal of AppliedMicrobiology 85, 487–494.
Uhart, M., Ravishankar, S., Maks, N.D., 2004. Control of Listeria mono-cytogenes with combined antimicrobials on beef franks stored at 4 degreesC. Journal of Food Protection 67, 2296–2301.
Ukuku, D.O., Fett, W.F., 2004. Effect of nisin in combination with EDTA,sodium lactate, and potassium sorbate for reducing Salmonella on wholeand fresh-cut cantaloupe. Journal of Food Protection 67, 2143–2150.
Ulmer, H.M., Heinz, V., Gänzle, M.G., Knorr, D., Vogel, R.F., 2002. Effects ofpulsed electric fields on inactivation and metabolic activity of Lactobacillusplantarum in model beer. Journal of Applied Microbiology 93, 326–335.
70 A. Gálvez et al. / International Journal of Food Microbiology 120 (2007) 51–70
Urso, R., Rantsiou, K., Cantoni, C., Comi, G., Cocolin, L., 2006a. Technologicalcharacterization of a bacteriocin-producing Lactobacillus sakei and its use infermented sausages production. International Journal of Food Microbiology110, 232–239.
Urso, R., Rantsiou, K., Cantoni, C., Comi, G., Cocolin, L., 2006b. Sequencingand expression analysis of the sakacin P bacteriocin produced by a Lacto-bacillus sakei strain isolated from naturally fermented sausages. AppliedMicrobiology and Biotechnology 71, 480–485.
Vaara, M., 1992. Agents that increase the permeability of the outer membrane.Microbiological Reviews 56, 395–411.
Verluyten, J., Schrijvers, V., Leroy, F., De Vuyst, L., 2002. Modelling thebehaviour of the potential meat starter culture Lactobacillus curvatus LTH1174 as influenced by different environmental factors important forEuropean sausage fermentations. Proceedings of the 18th InternationalICFMH Symposium FOOD MICRO 2002, pp. 167–172. Lillehammer,Norway, August 18–23.
Verluyten, J., Messens, W., De Vuyst, L., 2003. The curing agent sodium nitrite,used in the production of fermented sausages, is less inhibiting to thebacteriocin-producing meat starter culture Lactobacillus curvatus LTH 1174under anaerobic conditions. Applied and Environmental Microbiology 69,3833–3839.
Verluyten, J., Messens, W., De Vuyst, L., 2004a. Sodium chloride reducesproduction of curvacin A, a bacteriocin produced by Lactobacillus curvatusstrain LTH 1174, originating from fermented sausage. Applied andEnvironmental Microbiology 70, 2271–2278.
Verluyten, J., Leroy, F., De Vuyst, L., 2004b. Effect of different spices used inproduction of fermented sausages on growth of and curvacin A productionby Lactobacillus curvatus LTH 1174. Applied and EnvironmentalMicrobiology 70, 4807–4813.
Vignolo, G., Fadda, S., de Kairuz, M.N., de R. Holgado, A.P., Oliver, G., 1998.Effects of curing additives on the control of Listeria monocytogenes bylactocin 705 in meat slurry. Food Microbiology 15, 259–264.
Wandling, L.R., Sheldon, B.W., Foegeding, P.M., 1999. Nisin in milk sensitizesBacillus spores to heat and prevents recovery of survivors. Journal of FoodProtection 62, 492–498.
WHO, 2002. Food safety strategic planning meeting: report of a WHO strategicplanning meeting, WHO headquarters, Geneva, Switzerland, 20–22 February2001 (http://whqlibdoc.who.int/hq/2001/WHO_SDE_PHE_FOS_01.2.pdf).World Health Organization, Geneva.
Wouters, P., Álvarez, I., Raso, J., 2001. Critical factors determining inactivationkinetics by pulsed electric field food processing. Trends in Food Science &Technology 12, 112–121.
Yamazaki, K., Yamamoto, T., Kawai, Y., Inoue, N., 2004. Enhancement ofantilisterial activity of essential oil constituents by nisin and diglycerol fattyacid ester. Food Microbiology 21, 283–289.
Yang, R.G., Johnson, M.G., Ray, B., 1992. Novel method to extract largeamounts of bacteriocins from lactic acid bacteria. Applied and Environ-mental Microbiology 58, 3355–3359.
Young, L.L., Reverie, R.D., Cole, A.B., 1988. Fresh red meats: A place to applymodified atmospheres. Food Technology 42, 64–66, 68–69.
Yuste, J., Fung, D.Y., 2004. Inactivation of Salmonella typhimurium andEscherichia coli O157:H7 in apple juice by a combination of nisin andcinnamon. Journal of Food Protection 67, 371–377.
Zapico, P., Medina, M., Gaya, P., Nuñez, M., 1998. Synergistic effect of nisinand the lactoperoxidase system on Listeria monocytogenes in skim milk.International Journal of Food Microbiology 40, 35–42.
