Growth and bacteriocin production by lactic acid bacteria in vegetable broth and their effectiveness at reducing Listeria monocytogenes in vitro and in fresh-cut lettuce

The fresh-cut fruit and vegetable industry is searching for alternatives to replace chemical treatments with biopreservative approaches that ensure the safety of the product and fulfil consumer preferences for minimally processed foods. In this study, the use of bacteriocins produced by lactic acid bacteria has been tested as a substitute for chemical disinfection of fresh-cut iceberg lettuce. First, the ability of several non-plant origin bacteriocinogenic strains (nisin Z(+), plantaricin C(+), lacticin 481(+), coagulin(+) or pediocin PA-1(+)) to grow in a lettuce extract at 4 degrees C, 10 degrees C and 32 degrees C was tested. All strains were able to grow, but bacteriocin production was predominantly detected at 32 degrees C. Addition of bacteriocinogenic supernatants (nisin(+), coagulin(+) and a nisin-coagulin(+) cocktail) to tryptic-soy agar plates inoculated with Listeria monocytogenes reduced Listeria counts by approximately 1-1.5 log units compared with the control plates without bacteriocin, after 48 h of storage at 4 degrees C. The effect of washing with bacteriocin-containing solutions on survival and proliferation of Listeria monocytogenes was also evaluated in fresh-cut lettuce packaged in macro-perforated polypropylene bags and stored for 7 days at 4 degrees C. Washing fresh-cut lettuce with these solutions decreased the viability of Listeria monocytogenes by 1.2-1.6 log units immediately after treatment, but, during storage at 4 degrees C, bacteriocin treatments only exerted minimal control over the growth of the pathogen. Natural microbiota were little affected by bacteriocins during storage.     

Consequences of the development of nisin-resistant Listeria monocytogenes in fermented dairy products

Wild Listeria isolates representing serovars found in artisanal cheeses commercialized in Asturias (northern Spain) were assessed for their susceptibility to several bacteriocins. Pediocin PA-1 was the most active bacteriocin followed by enterocin AS-48, nisin, and plantaricin C. However, some Listeria monocytogenes and Listeria innocua strains were already highly resistant to PA-1. Among the wild L. monocytogenes populations, the frequency of development of nisin resistance ranged from 10(-6) up to 10(-3), depending on the strain. Highly stable mutants with increased nisin resistance (two- to fourfold) were isolated and tested for potential cross-resistance to lysozyme, EDTA, and various NaCl concentrations and pH values. All mutants were cross-resistant to lysozyme but sensitive to EDTA. In contrast, no clear correlation could be established between nisin resistance and an altered susceptibility to NaCl or pH changes. Nisin-resistant variants were able to survive and even to multiply in milk fermented by a nisin-producing Lactococcus, but the growth of the wild-type strain was inhibited. The different phenotypes evaluated in this study are indicative of the unpredictability of the consequences of the development of nisin resistance in a dairy environment. This resistance should be considered when making a risk assessment of the long-term use of nisin to control L. monocytogenes.     

Viability loss and morphology change of foodborne pathogens following exposure to hydrostatic pressures in the presence and absence of bacteriocins

Cell suspensions of three pathogens were exposed to hydrostatic pressure (HP), bacteriocin mixture (nisin and pediocin) or a combination of HP+bacteriocins and changes in colony forming units (cfu) and cell-morphology by scanning electron microscopy (SEM) were studied. Cell viability loss, as determined from the reduction in cfu before and after a treatment, occurred in Listeria monocytogenes by all three treatments and in Salmonella typhimurium and Escherichia coli O157:H7 by HP and HP+bacteriocin combination. Cell wall and cell membrane collapse and cell lysis was indicated in L. monocytogenes exposed to bacteriocin or HP+bacteriocin and in Salmonella and E. coli exposed to HP or HP+bacteriocin.     

The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria

The sensitivities of vegetative cells of strains ofListeria, Clostridium, Staphylococcus, Lactococcus, Lactobacillus, MicrococcusandPediococcus, and of spores ofClostridiumandBacillusto three broad spectrum bacteriocins (nisin A, nisin Z and pediocin) from lactic acid bacteria were determined by a critical dilution micro-assay. The minimal inhibitory concentrations (MIC) of partially purified bacteriocins, prepared by a pH-dependent adsorption/desorption process, were determined and expressed in arbitrary units ml⁻¹and in μ g ml⁻¹of pure bacteriocin. The MICs of bacteriocins varied considerably between species and even between strains of the same species, as clearly shown for nine strains ofListeria monocytogenes. When bacteriocin activity was expressed in μ g ml⁻¹, pediocin was more effective againstListeria monocytogenesthan nisin A or nisin Z. The latter bacteriocins, in concentrations between 23 and 69 μ g ml⁻¹, prevented outgrowth ofClostridiumandBacillusspores for at least 10 days. Although pediocin at 17 μ g ml⁻¹prevented outgrowth ofB. stearothermophilusandC. butyricumspores for up to 7 days, it apparently activated the germination ofB. subtilisspores.     

Purification, characterization and amino acid sequencing of divergicin M35: a novel class IIa bacteriocin produced by Carnobacterium divergens M35

Carnobacterium divergens M35, isolated from a commercial sample of frozen smoked mussels, produces a new bacteriocin, divergicin M35, a class IIa bacteriocin. Divergicin M35 is sensitive to pronase-E, alpha-chymotrypsin and proteinase K, but not to trypsin and withstands thermal treatments up to 121 degrees C for 30 min. Divergicin M35 was extracted from the culture supernatant of C. divergens M35 using an SP-Sepharose cation-exchange column, desalted and purified on a C18 Sep-Pack column and further purified by reverse phase-high pressure liquid chromatography. This procedure allowed the recovery of 10% of the bacteriocin present in the culture supernatant with purity higher than 99%. Divergicin M35 had a molecular mass of 4518.75 Da as determined by mass spectrometry, a pI value of 8.3 and positive net charge (+3). The amino acid sequence of divergicin M35 was found to consist of 43 amino acid with four cysteine residues (Cys10, 15, 25, 43) and showed 80.5% homology with divercin V41 (80.5%) and 80.0% with bavaricin MN. Divergicin M35 showed powerful antilisterial activity, especially against Listeria monocytogenes and was also active against carnobacteria but not against strains of Lactococcus, Lactobacillus, Enterococcus, Bifidobacteria and Escherichia. Divergicin M35 production began in late exponential phase and reached a maximum activity of 65,000 AU/ml in early stationary phase. Initial broth pH, Tween 80 and acetate did not affect C. divergens M35 growth or divergicin production. This bacteriocin may be a potential tool for inhibiting L. monocytogenes in seafood products that do not usually undergo an adequate heat treatment.     

Comparative Study on Antibacterial Effect of Pediocin ACCEL and Nisin Against Fluorescencestained Listeria monocytogenes BCRC 14845

ABSTRACT: Effects of pediocin ACCEL (Class IIa, pediocin-like bacteriocins) and nisin on carboxyfluorescein diacetate (cFDA) -stained Listeria monocytogenes BCRC 14845 was carried out. Decrease of fluorescence intensity in stained cells and increase in efflux were observed with the increase in bacteriocin concentrations up to 12.5 μg/mL. No further decreases in fluorescence intensity and cell numbers were obtained when the concentration of bacteriocins was over 12.5μg/mL. The leakage of efflux was further confirmed by the release of intracel-lular ultraviolet-absorbing substances. Comparing the leaking time of fluorescence, the intensity of cFDA in nisin-treated cells within 1 min incubation was almost comparable to that in pediocin-treated samples after 40 min incubation. Considering the leakage of cFDA, UV-absorbing substances, changes in cell numbers during incubation of cFDA-stained cells, and restoration ability, nisin seemed to reveal mainly bacteriostatic effect and partially bactericidal effect on L. monocytogenes and to cause easier-to-repair membrane damage. However, pediocin ACCEL appeared to have both bacteriostatic and bactericidal effects on L. monocytogenes and to cause more difficult-to-repair membrane damage.     

Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product

Screening for bacteriocin production of 500 strains of lactic acid bacteria (LAB) from various African fermented foods resulted in the detection of a bacteriocin producing Lactococcus lactis (BFE 1500) isolated from a dairy product called wara. The bacteriocin inhibited not only the closely related LAB, but also strains of Listeria monocytogenes, Listeria innocua, Clostridium butyricum, Clostridium perfringens, Bacillis cereus and Staphylococcus aureus. It was heat stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10), but highest activity was observed in the lower pH range. The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K, but not by other proteases. Growth kinetic assay indicated stronger growth inhibition by the bacteriocin produced by Lc. lactis BFE 1500 on L. monocytogenes WS 2250 and B. cereus DSM 2301 than with the nisin A producing strain DSM 20729. Polymerase chain reaction indicated the presence of the nisin operon in strain BFE 1500 and sequencing of its structural gene showed that Lc. lactis BFE 1500 produced the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The genetic determinants for bacteriocin production in strain BFE 1500 are located on a conjugative transposon. The ability of the bacteriocin produced by Lc. lactis BFE 1500 to inhibit a wide range of food-borne pathogens is of special interest for food safety, especially in the African environment with perennial problems of poor food hygiene.     

Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham,a traditional Thai fermented sausage

A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production. One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus. Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10). The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases. The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin. The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer. Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27. The ability of the bacteriocin produced by Lc. lactis WNC 20 may be useful in improving the food safety of the fermented product.     

Nisin-curvaticin 13 combinations for avoiding the regrowth of bacteriocin resistant cells of Listeria monocytogenes ATCC 15313

Nisin (25-100 IU/ml) and curvaticin 13 (160 AU/ml), a bacteriocin produced by Lactobacillus curvatus SB13, were shown to have a bactericidal effect against Listeria monocytogenes ATCC 15313 in TSB-YE broth (pH 6.5), but it was only transitory. Regrowth was not due to the loss of bacteriocin activity. Cells surviving nisin or curvaticin 13 were more resistant to the respective bacteriocin than the parental strain. Survivors to curvaticin 13 were resistant to the class IIa bacteriocins (camocin CP5, pediocin AcH) but remained sensitive to nisin. The frequencies of spontaneous nisin resistants decreased with increasing bacteriocin concentration and the presence of salts (NaCl, K2HPO4). The behaviour of nisin (1000 IU/ml) or curvaticin 13 (640 AU/ml) resistant variants (Nis1000, Curv645) was investigated in the presence of nisin (100 IU/ml) or curvaticin 13 (320 AU/ml) at 22 and 37 degrees C, and compared with that of the parental strain. The effectiveness of nisin was the same at both temperatures, whereas curvaticin 13 displayed a faster bactericidal action at 37 degrees C. Nis1000 cells were less sensitive to curvaticin 13 than the parental strain, whereas Curv640 cells were more sensitive to nisin than the parental strain. Simultaneous or sequential additions of nisin (50 IU/ml) and curvaticin 13 (160 AU/ml) were performed at 22 degrees C in broth inoculated with the parental strain. All combinations induced a greater inhibitory effect than the use of a single bacteriocin. Simultaneous addition of bacteriocins at t0 led to the absence of viable cells in the broth after 48 h.     

INABILITY OF HEAT STRESS TO AFFECT SENSITIVITY OF LISTERIA MONOCYTOGENES TO PEDIOCIN IN PORK

The effect of heat-shock (48C for 20 min) on sensitivity of Listeria monocytogenes Scott A to bacteriocin produced by Pediococcus acidilactici was evaluated. Exposure of Listeria to pediocin (68, 267 AU/ mL) resulted in lower number of survivors in the first 2 h of incubation in Trypticase soy broth at 37C compared with samples without pediocin, regardless of whether the cells were heat-shocked or not. Further incubation for 24 h resulted in resumption of growth by L. monocytogenes, with counts reaching the same level as samples without pediocin. Cold storage of Listeria in pork in the presence of pediocin (8,192 AU/g) resulted in a 2.0 log₁₀ reduction after 24 h, when compared with controls not exposed to pediocin, regardless of whether the samples were stored under air, vacuum, or a modified atmosphere. Prior heat-shocking did not have an effect on sensitivity of Listeria to pediocin during storage. Thus, storage atmosphere and exposure to heat shock, which are known to induce thermotolerance in L. monocytogenes, had no effect in sensitivity of this organism to pediocin .     

Acid-adapted Listeria monocytogenes displays enhanced tolerance against the lantibiotics nisin and lacticin 3147.

Log-phase Listeria monocytogenes cells become tolerant to a variety of environmental stresses following acid adaptation at pH 5.5. We demonstrated that adapted cells also exhibit increased tolerance to nisin and, to a lesser extent, lacticin 3147. At nisin concentrations of 100 and 200 IU/ml the survival of acid-adapted cells was approximately 10-fold greater than nonadapted cells. However, acid adaptation had only a moderate effect on the tolerance of L. monocytogenes to lacticin 3147, a phenomenon that possibly reflects the distinct mode of action of this bacteriocin. Analysis of the fatty acid composition of the bacterial membrane indicated that straight-chain fatty acids C14:0 and C16:0 were significantly increased in acid-adapted cells while levels of C18:0 decreased. The results indicate that stress mechanisms that are induced in mildly acidic conditions provide protection against the antimicrobial action of bacteriocins. This increased resistance of acid-adapted L. monocytogenes could cause increased survival of this pathogen in food products in which nisin or other bacteriocins are used as preservatives.     

COMBINATION WITH PLANT EXTRACTS IMPROVES THE INHIBITORY ACTION OF DIVERGICIN M35 AGAINST LISTERIA MONOCYTOGENES

ABSTRACT

The susceptibility of 11 strains of Listeria monocytogenes to divergicin M35, a bacteriocin produced by Carnobacterium divergens strain M35, and to aqueous extracts of garlic, onion, oregano, red chili and black pepper at 30 and 10C, was evaluated using a microdilution assay. The susceptibility of divergicin‐resistant strains to combinations of these agents was also evaluated. Three strains were resistant to divergicin M35 (>500 µg/mL) at 30C but were more susceptible at 10C. Garlic gave the most inhibitory plant extract, followed by onion, while oregano, red chili and black pepper extracts were less active at both temperatures. Garlic extract and divergicin M35 combined or with other extracts increased inhibitory activity against the divergicin‐resistant strains. The garlic/divergicin combination was the most effective at inhibiting these strains and was bactericidal at both temperatures. Log‐phase cells were the most susceptible to the garlic/divergicin combination. Stationary‐phase cells were much more resistant at both incubation temperatures. Furthermore, the effect of the garlic/divergicin combination at inhibiting divergicin‐resistant L. monocytogenes in a food system was also studied using cold‐smoked salmon as a food model. Results indicated that this combination could efficiently reduce the viability of L. monocytogenes in smoked salmon stored at 10C.

PRACTICAL APPLICATIONS

There is increasing popularity worldwide for chemical preservative‐free, ready‐to‐eat and minimally processed seafood with low salt, fat and sugar content. Bacteriocins produced from lactic acid bacteria can have a potential application to prolong the shelf life of cold‐smoked salmon. Also, plant and spice extracts have been shown to contain antibacterial substances with potential for application in foods. Thus, this research explores the combination of divergicin M35, a bacteriocin produced by Carnobacterium divergens strain M35, and aqueous extracts of garlic, onion, oregano, red chili and black pepper to inhibit Listeria monocytogenes and to prolong the shelf life of cold‐smoked salmon.     

A rapid turbidometric microplate bioassay for accurate quantification of lactic acid bacteria bacteriocins

A 1 day turbidometric microplate bioassay (TMB) was developed for the rapid, accurate and precise quantification of lactic acid bacteria (LAB) bacteriocins (nisin Z and pediocin PA-1). Parameters such as the concentration of the indicator strains and the incubation time were optimized for each bacteriocin. A high correlation coefficient (r²=0.992±0.004) was obtained for the exponential regression in the nisin Z concentration range of 20–120 ng/ml with 1×10⁷ CFU indicator strain (Pediococcus acidilactici UL5) and an incubation time of 3 h. Using these parameters, the detection limit was estimated at 80 ng/ml (3.2 IU/ml), compared to 300 ng/ml for the agar diffusion assay (ADA). High precision (<7%) and accuracy (10%) were obtained for all nisin Z concentrations tested. Similar results were obtained with pediocin PA-1 with r²=0.993±0.005, a precision (8.2%) and an accuracy lower than 15%.     

Efficacy of nisin in combination with protective cultures against Listeria monocytogenes Scott A in tofu.

Nisin can be used as a biopreservative to control growth of Listeria monocytogenes in various minimally processed foods. Tofu is an example of a non-fermented soybean product, which may allow growth of Listeria at refrigeration temperatures and in which nisin may be applied to prevent multiplication of Listeria. The efficacy of nisin against Listeria may be compromised by the emergence of spontaneous nisin-resistant mutants. Exposure of L. monocytogenes Scott A to nisin in a culture medium or in a food product results in an initial reduction of Listeria population which is followed by regrowth of survivors to nisin during further incubation. In vitro studies using Standard I Nutrient broth showed that Enterococcus faecium BFE 900-6a and Lactobacillus sakei Lb 706-1a used as protective cultures in combination with nisin were able to suppress proliferation of Listeria cells not killed by nisin at 10 degrees C. Growth and bacteriocin production of these two strains and a third protective culture, Lactococcus lactis BFE 902 was also observed in soymilk and tofu at 10 degrees C. Inoculation studies with tofu prepared with nisin and protective cultures showed that lower amounts of nisin are required for an effective inhibition of L. monocytogenes Scott A when either E. faecium BFE 900-6a or Lc. lactis BFE 902 are used in addition. The combination of nisin with these bacteriocinogenic lactic acid bacteria (LAB) resulted in a complete suppression of listerial growth in homemade tofu stored at 10 degrees C for 1 week. Lb. sakei Lb 706-1a was less effective and did not prevent a slight increase of L. monocytogenes Scott A numbers during storage.     

片球菌素的纯化及稳定性研究

片球菌素是一类具有良好热稳定性的未修饰的小分子蛋白质。其理化性质稳定,能抑制多种革兰氏阳性菌,对单核细胞增多症李氏杆菌抑制作用最为显著。文中着重总结了近年来在片球菌素稳定性及分离纯化方法领域的研究进展,同时也对其研究过程中存在的问题进行了探讨。     

Detection of pediocin PA-1-producing pediococci by rapid molecular biology techniques

Five bacteriocinogenic lactic acid bacteria strains (347, X13, Z102, A172 and P20) independently isolated from fermented sausages were identified asPediococcus acidilacticiby carbohydrate fermentation patterns and other biochemical characteristics. This fact, together with their activity againstListeria monocytogenes, suggested that they could be pediocin PA-1 producers. Rapid molecular biology techniques were used to detect the pediocin PA-1 operon in these strains. PCR, dot-blot and Southern hybridization, and DNA sequencing results confirmed that all of them had the genetic determinants for pediocin PA-1 biosynthesis encoded in a 9.4 kb plasmid. The bacteriocin produced byP. acidilactici347 has been purified by a procedure that included ammonium sulphate precipitation and cation exchange, hydrophobic-interaction and reverse-phase chromatography. The amino acid sequence of this bacteriocin was identical to pediocin PA-1, confirming the expression of the pediocin PA-1 genes.     

乳酸片球菌素PA-1在大肠杆菌中的表达与纯化

为了实现该细菌素的外源表达,本实验首先利用聚合酶链式反应从乳酸片球菌PAF中扩增出乳酸片球菌素PA-1的结构和免疫基因,然后克隆到表达载体pGEX-6p-1,构建了N端含有GST-His-DDDDK标签的重组质粒pGEX/his-pedAB,然后转化进入大肠杆菌Rosetta（DE3）感受态细胞,经异丙基硫代半乳糖苷诱导,重组乳酸片球菌素PA-1在大肠杆菌胞内成功表达。表达的融合蛋白先经过镍亲合层析柱纯化,然后注入谷胱甘肽S-转移酶亲和色谱柱用肠激酶处理,释放出成熟的乳酸片球菌素PA-1。利用高效液相色谱和质谱技术检测乳酸片球菌素PA-1纯度。以单核细胞增生李斯特氏菌CMCC54004为指示菌,利用琼脂扩散法检验乳酸片球菌素PA-1活性。结果表明,携带GST-His-DDDDK标签的融合蛋白无活性,标签切除后其抑菌活性恢复,且其纯度达90%以上。     

Destruction of Listeria monocytogenes in sturgeon (Acipenser transmontanus) caviar by a combination of nisin with chemical antimicrobials or moderate heat

The objective of this study was to investigate the effect of nisin in combination with heat or antimicrobial chemical treatments (such as lactic acid, chlorous acid, and sodium hypochlorite) on the inhibition of Listeria monocytogenes and total mesophiles in sturgeon (Acipenser transmontanus) caviar. The effects of nisin (250, 500, 750, and 1,000 IU/ml), lactic acid (1, 2, and 3%), chlorous acid (134 and 268 ppm), sodium hypochlorite (150 and 300 ppm), and heat at 60 degrees C for 3 min were evaluated for a five-strain mixture of L. monocytogenes and total mesophiles in sturgeon caviar containing 3.5% salt. Selected combinations of these antimicrobial treatments were also tested. Injured and viable L. monocytogenes cells were recovered using an overlay method. Treating caviar with > or =500 IU/ml nisin initially reduced L. monocytogenes by 2 to 2.5 log units. Chlorous acid (268 ppm) reduced L. monocytogenes from 7.7 log units to undetectable (<0.48 log units) after 4 days of storage at 4 degrees C. However, there were no synergistic effects observed for combinations of nisin (500 or 750 IU/ml) plus either lactic acid or chlorous acid. Lactic acid caused a slight reduction (approximately 1 log unit) in the microbial load during a 6-day period at 4 degrees C. Sodium hypochlorite was ineffective at the levels tested. Mild heating (60 degrees C for 3 min) with nisin synergistically reduced viable counts of L. monocytogenes and total mesophiles. No L. monocytogenes cells (<0.48 log units) were recovered from caviar treated with heat and nisin (750 IU/ml) after a storage period of 28 days at 4 degrees C.     

Pediocin PA-1, a wide-spectrum bacteriocin from lactic acid bacteria

Pediocin PA-1 is a broad-spectrum lactic acid bacteria bacteriocin that shows a particularly strong activity against Listeria monocytogenes, a foodborne pathogen of special concern among the food industries. This antimicrobial peptide is the most extensively studied class Ila (or pediocin family) bacteriocin, and it has been sufficiently well characterized to be used as a food biopreservative. This review focuses on the progress that have been made in the elucidation of its structure, mode of action, and biosynthesis, and includes an overview of its applications in food systems. The aspects that need further research are also addressed. In the future, protein engineering, genetic engineering and/or chemical synthesis may lead to the development of new antimicrobial peptides with improved properties, based on some features of the pediocin PA-1 molecule.     

Technological characterization of bacteriocin producing Lactococcus lactis strains employed to control Listeria monocytogenes in cottage cheese

In recent years, there has been a particular focus on the application of antimicrobial compounds produced by lactic acid bacteria (LAB) as natural preservatives to control the growth of spoilage and pathogenic bacteria in food. Bacteriocins are antimicrobial peptides which can be added to foods in concentrated forms as food preservatives, e.g. additives, or they can be produced in situ by starters or protective cultures. In this study, twenty Lactococcus lactis bacteriocin producers previously isolated from Italian fermented foods were subjected to a variety of physical and biochemical tests in order to identify those with the greatest potential as starter cultures in cheese production. Of these, four strains isolated from cheese (one nisin Z producer, one nisin A producer and two lacticin 481 producers) which fulfilled the desired technological criteria were assessed for their ability to control Listeria monocytogenes. The subsequent application of these bacteriocinogenic strains as starter cultures in Cottage cheese established that the nisin A producing Lact. lactis 40FEL3, and to a lesser extent the lacticin 481 producers 32FL1 and 32FL3, successfully controlled the growth of the pathogen. This is the first study to directly compare the ability of nisin A, nisin Z and lacticin 481 producing strains to control listerial growth during the manufacture and storage of Cottage cheese.