Vacuum-packaged Meat Research Articles

The pepR gene of Lactobacillus sakei is positively regulated by anaerobiosis at the transcriptional level.

Lactobacillus sakei is a lactic acid bacterium belonging to the natural flora of meat products. It constitutes the main flora of vacuum-packed meat and is largely used in western Europe as a starter for the manufacturing of fermented sausages. This species is able to grow both under aerobiosis and anaerobiosis. In many technological processes involving it, oxygen is scarce. The aim of this study was to identify the major proteins affected by growth under anaerobiosis. Using two-dimensional electrophoresis, we showed that one spot was 10-fold overexpressed when cells were grown under anaerobiosis. By N-terminal sequencing it was identified as a peptidase (PepR), and the pepR gene was cloned. Northern analysis revealed that pepR was expressed as a single 1.27-kb transcript induced under anaerobiosis. A mutant was constructed by single crossover in the pepR gene, and its growth and survival were not affected by anaerobiosis.

Effectiveness of some recent antimicrobial packaging concepts

A new type of active packaging is the combination of food-packaging materials with antimicrobial substances to control microbial surface contamination of foods. For both migrating and non-migrating antimicrobial materials, intensive contact between the food product and packaging material is required and therefore potential food applications include especially vacuum or skin-packaged products, e.g. vacuum-packaged meat, fish, poultry or cheese. Several antimicrobial compounds have been combined with different types of carriers (plastic and rubber articles, paper-based materials, textile fibrils and food-packaging materials). Until now, however, few antimicrobial concepts have found applications as a food-packaging material. Antimicrobial packaging materials cannot legally be used in the EU at the moment. The potential use would require amendments of several different legal texts involving areas such as food additives, food packaging, hygiene, etc. The main objective of this paper is to provide a state of the art about the different types of antimicrobial concepts, their experimental development and commercialization, and to present a case study summarizing the results of investigations on the feasibility of a low-density polyethylene (LDPE)-film containing triclosan to inhibit microbial growth on food surfaces and consequently prolong shelf-life or improve microbial food safety. In contrast with the strong antimicrobial effect in in-vitro simulated vacuumpackaged conditions against the psychrotrophic food pathogen L. monocytogenes, the 1000mg kg -1 containing triclosan film did not effectively reduce spoilage bacteria and growth of L. monocytogenes on re frigerated vacuum-packaged chicken breasts stored at 7 ° C.

Mechanisms of Pink Color Formation in Irradiated Precooked Turkey Breast Meat

ABSTRACT: Precooked turkey breast meat was aerobically packaged or vacuum‐packaged and irradiated at 0, 2.5, or 5.0 kGy. CIE color, reflectance, oxidation‐reduction potential (ORP), gas production, and lipid oxidation were determined at 0, 7, and 14 d. Irradiation increased redness of vacuum‐packaged meat, and the redness was distinct and stable under vacuum. Irradiation decreased ORP and produced carbon monoxide (CO). This indicated that the pink color was caused by the heme pigment‐CO complex formation. The reflectance of meat and the absorption spectra of myoglobin solution supported the assumption that denatured CO‐myoglobin is the pigment in irradiated precooked turkey breast.

Use of oxygen sensors for the non-destructive measurement of the oxygen content in modified atmosphere and vacuum packs of cooked chicken patties; impact of oxygen content on lipid oxidation

Samples were packaged under vacuum and modified atmosphere (70% N 2:30% CO 2) and stored at 4 °C, under 616 lux fluorescent lighting, for 25 days. Oxygen sensors attached to the inside of the lidding material in MAP packages or inserted into vacuum packages were capable of monitoring changes in oxygen levels. Lipid oxidation of samples was measured at regular intervals. Residual oxygen levels were typical of those found in MAP and vacuum-packaged meats at 0.9–1.1% and 0.11–0.15%, respectively. MAP samples contained the highest levels of oxygen and were significantly ( P<0.05) more oxidised than vacuum-packaged samples. A significant ( P<0.05) reduction in lipid oxidation was observed in vitamin E supplemented patties compared to unsupplemented samples. Vitamin E supplementation had an important effect in counteracting the influence of freely available oxygen on lipid oxidation. The ability to continually assess oxygen levels within packs containing cooked processed meats, through continual, non-destructive assessment using oxygen sensors, provides a far more realistic approach to determining realistic shelf-life stabilities of foods held in vacuum or modified food packing systems.

Changes in Hygienic Quality of Vacuum-packed Pork during Storage

Napravnikova E., L. Vorlova, L. Malota: Changes in Hygienic Quality of Vacuumpacked Pork During Storage.Acta Vet. Brno 2002, 71: 255‐262. Hygienic quality of boned vacuum-packed pork was monitored during storage by sensory, chemical, and microbiological methods. Pork was vacuum-packed into polyamide /polyethylene (PA/PE) foil 72 h after slaughter. Initial temperature did not exceed 9° C. The meat was stored at 2.5 ± 0.5°C in the dark for 35 days and samples for laboratory examinations were collected at days 14, 21, 28, and 35 to assess appearance, colour, odour, and characteristics of released meat juice, to determine pH and concentrations of ammonia and D- and L-forms of lactic acid, and to enumerate lactacidogenic bacteria. Lactobacillus curvatus and Lactobacillus xylosus were identified as the causative agents of meat spoilage using the API system. The results were subjected to correlation analyses. The shelf life of boned vacuum-packed pork varied between 21 and 28 days, depending on initial pH. The results of correlation analyses indicated that only ripe pork with the optimum pH = 5.8 to 5.95 is suitable for vacuum-packing. In addition to the lactacidogenic bacteria count (LAC), the shelf life can be estimated also from ammonia concentration (LAC/ammonia r = 0.9584, P < 0.05), pH (LAC/pH r = -0.9317, P < 0.05) and the concentration of D-lactate (LAC/D-lactate r = 0.9867, P < 0.01). Pork, vacuum packing, lactobacilli, ammonia, pH, lactic acid Meat spoilage is a complex process in which microorganisms present in the muscular tissue due to secondary contamination during processing are involved, and which depends on ambient temperature. The storage of meat is associated with changes in quality resulting from microbial activity, shift of pH, production of toxic substances, and aberrant odour (Huis 1996). Most prone to spoilage are foods with a high protein content, such as meat, poultry, fish, and milk, which have a high dietetic value, neutral, or lightly acid pH, and a high water content providing favourable conditions for bacterial growth (Huis 1996). Bauer (1995) demonstrated that enzymes of the muscular tissue and bacterial enzymes are involved in chemical changes (production of biogenic amines, changes in colour and fat) occurring during storage. The most important factors influencing the development and growth of spoilage microorganisms on raw meat in cold storage include the initial relative number of psychrotrophic flora and its growth dynamics at low temperatures. Qualitative and quantitative composition of the microbial population responsible for meat spoilage depend on pH, water activity, storage atmosphere, and among-species relations (Gould 1995). Dominant in the microbial population of vacuum-packed meat are lactacidogenic bacteria which participate in the development of an unpleasant odour. Dainty (1996) detected indol and hydrogen sulphide by gas chromatography and identified them as the cause of this sensory defect. The microbial activity results in the production of typical metabolites including invariably the D and L forms of lactic acid. D-lactate is produced by bacteria exclusively and is regarded as an indicator of the growth of specific microbial flora (Sinell and L uke 1979; Schneider et al. 1983). Further metabolites produced by the

Use of restriction fragment length polymorphism analysis to differentiate strains of psychrophilic and psychrotropic clostridia associated with blown pack' spoilage of vacuum-packed meats.

Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP). Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing. DNA-RFLP resolved 19 of the 22 meat strains into 11 groups. Three meat strains were untypable using this method. All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes. With PCR-RFLP, the 22 meat strains produced eight distinct genotypes. 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence. PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used. Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species. Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms. The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir.

Carbon monoxide-heme pigment is responsible for the pink color in irradiated raw turkey breast meat

Turkey breast muscles were aerobically or vacuum packaged, irradiated at 0, 2.5, or 5.0 kGy using a Linear Accelerator (electron beam), and stored at 4°C. The CIE color values, reflectance scan, oxidation-reduction potential (ORP), production of gaseous compounds, and lipid oxidation of samples were determined at 0, 1, and 2 weeks of storage. Absorption spectra of sample drips were determined at 1 week of storage. Irradiation increased the a-value of both aerobically and vacuum-packaged turkey breast, but vacuum-packaged meat had stronger intensity than the aerobically packaged. The increased redness in vacuum-packaged meat was stable during the 2 weeks of storage. The production of CO in meat, which can bind to myoglobin as a sixth ligand, was proportional to irradiation dose. The ORP was decreased by irradiation, but was increased during storage. The ORP and lipid oxidation values were lower in vacuum-packaged than those in aerobically packaged turkey breast. Therefore, increased a-values in irradiated turkey breast should be caused by the decreased ORP and heme pigment-CO ligand formation. The absorption spectra of meat drip also showed that the peak wavelengths of irradiated turkey breast were similar to that of the CO-myoglobin.

Protein expression under uracil privation in Lactobacillus sakei.

Lactobacillus sakei is a lactic acid bacterium belonging to the natural flora of fresh and vacuum-packed meat, and is used as the starter for manufacturing fermented sausages. This species is now being studied at the genetic level. We investigated uracil prototrophy of strain 23K in order to validate the proteomic approach to study metabolism regulations. Cells grown without uracil had lower growth rates than with uracil. Protein analysis by 2D gel electrophoresis showed that at least three polypeptides were specifically induced in the absence of uracil. Two of these polypeptides were identified as orotate phosphoribosyl transferase, catalyzing the fifth step of pyrimidine biosynthesis, and PyrR, the transcriptional regulator of the pyr operon, respectively.

Industrial application of an antilisterial strain of Lactobacillus sakei as a protective culture and its effect on the sensory acceptability of cooked, sliced, vacuum-packaged meats

The application of a protective lactic acid bacterium (LAB) during the commercial production of cooked meat products is described. The LAB, a strain of Lactobacillus sakei, was previously isolated from cooked ham and inhibited growth of Listeria monocytogenes and Escherichia coli O157:H7 in this product. L. sakei was applied to the cooked products at a concentration of 10 5–10 6 cfu/g immediately before slicing and vacuum-packaging using a hand-operated spraying bottle. The LAB strain inhibited growth of 10 3 cfu/g of a cocktail of three rifampicin resistant mutant L. monocytogenes strains both at 8°C and 4°C. Consumer acceptance tests of cooked ham and of servelat sausage, a Norwegian non-fermented cooked meat sausage, showed that control and inoculated products were equally acceptable. The products were still acceptable after storage for 28 days at 4°C and, after opening the packages, for a further 5 days at 4°C. The findings presented here confirm that the L. sakei strain is suitable for use as a protective culture and may technically easily be implemented in the commercial production of cooked meat products.

The spoilage microflora of cured, cooked turkey breasts prepared commercially with or without smoking

Lactobacillus sakei subsp. carnosus was predominant in the spoilage flora of sliced, vacuum-packed, smoked, oven-cooked turkey breast fillets which developed mild, sour spoilage flavors after 4 weeks storage at 4°C. In contrast, Leuconostoc mesenteroides subsp. mesenteroides predominated in the spoilage flora of sliced, vacuum-packed, unsmoked, boiled turkey breast fillets from the same plant which were also stored at 4°C. The spoilage flora of the unsmoked breasts grew faster than that of the smoked breasts and was more diverse. Lactobacillus sakei, Weissella viridescens and an atypical group of leuconostoc-like bacteria were also members of the unsmoked turkey breasts flora. Consequently, the unsmoked breasts spoiled after 2 weeks at 4°C: the packs swelled and the meat developed strong sour odors and flavors and abundant slime. Except for the unidentified leuconostocs, which apparently survived boiling of the unsmoked turkey, all the spoilage organisms contaminated the meats during the slicing and vacuum packaging operations. From their biochemical reactions and cellular fatty acid profiles, the atypical leuconostocs were more closely related to Leuconostoc carnosum than W. viridescens. Carnobacteria and Brochothrix thermosphacta were present in relatively large numbers on the raw turkey, but were not numerous in the spoilage flora of the cooked, vacuum-packed meat products.

Specific microflora of packed meet – a review

Meat is an attractive medium for microorganism multiplication due to its composition. The microflora of unpacked meat or of meat packed in wrapping plastic foil is significantly different from the microflora of vacuum-packed or controlled-atmosphere packed meat. Dominant microorganisms of unpacked meat are some species of the genus Pseudomonas sp., namely Pseudomonas fragi, Shewanella putrefaciens, Psychrobacter and Acinetobacter, which largely contribute to meat spoilage. From vacuum-packed or controlled-atmosphere packed meat were isolated mainly Brochotrix thermosphacta, heterofermentative lactobacilli (L. cellobiosus), Carnobacterium sp. and Leuconostoc sp. Characteristic microorganisms of vacuum-packed meat were Leuconostoc carnosum, Leuconostoc gelidum and Carnobacterium divergens. Quantitative proportions of the groups of microorganisms in packed meat are related to meat pH value, plastic foil permeability for oxygen and type of packed product.

PCR-based 16S ribosomal DNA detection technique for Clostridium estertheticum causing spoilage in vacuum-packed chill-stored beef

Chill stored vacuum-packaged meat is sometimes spoiled by psychrotrophic or psychrophilic clostridia. Clostridium estertheticum was first isolated from vacuum-packed beef from southern Africa, but has recently been found in beef originating from northern Europe. This organism is difficult to isolate using conventional methods, and two PCR-based methods have been devised for use in measures to control the bacterium in the abattoir and to study its ecology. In the first method, primers were designed having a high annealing temperature of 65°C to increase specificity, producing a PCR product of 567 bp from the 16S rDNA. Two species of Enterobacteriaceae found in meat cross-reacted in this test, and so it was necessary to use a second step, digesting the PCR product with two restriction enzymes. Subsequently a further set of primers was designed, producing a PCR product of 641 bp, and using an annealing temperature of 60°C. The second procedure was more specific and did not require subsequent restriction analysis of the PCR product. The two sets of primers appeared to have similar sensitivity, detecting 10–100 cells of C. estertheticum in broth, meat or meat purge (drip). A semiquantitative method is described for estimating numbers of the target bacterium.

Clostridium frigidicarnis sp. nov., a psychrotolerant bacterium associated with 'blown pack' spoilage of vacuum-packed meats.

Two strains of a psychrotolerant Clostridium, isolated from vacuum-packed, temperature-abused beef, were characterized using a multiphasic approach. The strains were Gram-positive motile rods producing elliptical subterminal spores during early stationary growth phase. The strains were psychrotolerant. At pH 7.0, they grew between 3.8 and 40.5 degrees C; their optimum growth temperature was 30.0-38.5 degrees C. At 30 degrees C, the pH range for growth was between 4.7 and 9.5; the optimum pH for growth was 6.4-7.2. The organisms were proteolytic and saccharolytic, lecithinase-positive and hydrolysed gelatin. The fermentation products formed in peptone/yeast extract/glucose/starch broth were acetate, ethanol, butyrate, isovalerate, butanol, isobutyrate, oxalacetate, lactate, hydrogen and carbon dioxide. The DNA G + C compositions of the two meat strains were 27.3 and 28.4 mol%. Phylogenetic analyses indicated that the strains belong to Cluster I of the genus Clostridium (sensu Collins et al., 1994). The new strains differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of phenotypic and genotypic characterization data, the strains were assigned to a new species for which the name Clostridium frigidicarnis is proposed; strain SPL77AT (= DSM 12271T) is the type strain.

Characterisation of ropy slime-producing Lactobacillus sakei using repetitive element sequence-based PCR

Eighteen previously characterised Lactobacillus sakei strains exhibiting varying slime production capabilities in vacuum-packaged meat products were analysed using repetitive element sequence-based PCR (rep-PCR). The single primers BOXA1R and RW3A and the primer pair REP1R-Dt and REP2R-Dt were evaluated for their applicability in L. sakei genotyping. The five different patterns produced by RW3A were the least useful, with the discriminatory power equal to ribotyping. BOXA1R and REP–primer pair both produced six different banding patterns and the combination of these results yielded seven different rep-types. Rep-PCR was concluded to have approximately the same discriminatory power as randomly amplified polymorphic DNA (RAPD) analysis, but was inferior to pulsed-field gel electrophoresis (PFGE). However, if the results of rep-PCR and RAPD were combined, the discrimination was comparable to PFGE, with the exception that within Ribogroup I non-slime-producing strains were indistinguishable from weak slime producers. It was concluded that the combination of the two PCR-based typing techniques, rep-PCR and RAPD, would be a valuable tool in large scale contamination studies at meat processing plants, since results can be obtained rapidly with fewer isolates needing further analysis by PFGE.

A bacteriocin from Carnobacterium piscicola for the control of Listeria monocytogenes in vacuum-packaged meat

The purpose of this study was to determine the inhibitory capacity of a bacteriocin-like substance produced by Carnobacterium piscicola L103 against Listeria monocytogenes , when inoculated into vacuum-packaged meat. The bacteriocin was partially purified by ammonium sulfate precipitation and gel filtration on Sephadex G-25, followed by lyophilization. Beef steaks were inoculated with the bacteriocin and a sensitive strain of L. monocytogenes . The vacuum-packaged meat was stored at 4°C and microbial analyses were carried out every 7 days, over 21 days. Results showed that in the control steaks, without bacteriocin, counts of L. monocytogenes stayed at the initial inoculation level during the complete storage period. The naturally growing lactic acid bacteria (LAB) reached log 7·4 cfu cm−2after 14 days of storage and no inhibitory effect on the growth of the LAB was observed in the presence of the bacteriocin. The pH of the meat dropped from 5·7 to 5·0 in the inoculated as well as in the control samples, while colour and odour remained acceptable up to 14 days of storage. On the meat inoculated with bacteriocin, L. monocytogenes was completely inhibited after 14 days of storage at 4°C. It was concluded that the use of the inhibitory substance from C. piscicola L103 could be an important contribution to meat safety, when inoculated into vacuum-packaged meat.

The Effectiveness of Triclosan-Incorporated Plastic against Bacteria on Beef Surfaces

Triclosan is a nonionic, broad-spectrum, antimicrobial agent that has been incorporated into a variety of personal hygiene products, including hand soaps, deodorants, shower gels, mouthwashes, and toothpastes. In this study, plastic containing 1,500 ppm of triclosan was evaluated in plate overlay assays and meat experiments as a means of reducing populations of bacteria. Plate overlay assays indicated that the triclosan-incorporated plastic (TIP) inhibited the following organisms: Brochothrix thermosphacta ATCC 11509, Salmonella Typhimurium ATCC 14028, Staphylococcus aureus ATCC 12598, Bacillus subtilis ATCC 6051, Shigella flexneri ATCC 12022, Escherichia coli ATCC 25922, and several strains of E. coli O157:H7. In meat experiment 1, irradiated, lean beef surfaces inoculated with B. thermosphacta, Salmonella Typhimurium, E. coli O157:H7, or B. subtilis were covered with TIP, vacuum packaged, and stored for 24 h at 4°C. Of the organisms tested, only populations of B. thermosphacta were slightly reduced. In meat experiment 2, prerigor beef surfaces were inoculated with E. coli O157: H7, Salmonella Typhimurium, or B. thermosphacta incubated at 4°C for 24 h, wrapped in TIP or control plastic, vacuum packaged, and stored at 4°C for up to 14 days. There was a slight reduction in the population of the organisms after initial application with TIP. However, bacterial populations following long-term, refrigerated (4°C), vacuum-packaged storage up to 14 days were not statistically (P≤ 0.05) or numerically different than controls. In meat experiment 3, even TIP-wrapped, vacuum-packaged beef samples that were temperature abused at 12°C did not exhibit significant (P ≤ 0.05) or sustainable reductions after 14 days of 4°C storage. Another study indicated that populations of E. coli O157:H7 or B. thermosphacta added directly to TIP were not affected after 2 h of refrigerated storage or that the antimicrobial activity could be extracted from the plastic. Additional experiments suggest that presence of fatty acids or adipose may diminish the antimicrobial activity of TIP on meat surfaces. This study demonstrates that while antimicrobial activity is detected against bacterial cultures in antimicrobial plate assays, plastic containing 1,500 ppm of triclosan does not effectively reduce bacterial populations on refrigerated, vacuum-packaged meat surfaces.

A model based on absorbance data on the growth rate of Listeria monocytogenes and including the effects of pH, NaCl, Na-lactate and Na-acetate

A mathematical model was developed for predicting the growth of L. monocytogenes at 9°C in the presence of 70 ppm sodium nitrite, and at different levels of pH (5.5–6.5), sodium chloride (1.0–4.0%), sodium lactate (0–0.5%) and sodium acetate (0–0.6%). Collection of the growth data was done using absorbance measurements in broth cultures and the absorbance measurement was evaluated. The model was compared to the Food MicroModel, and against the growth of L. monocytogenes in a vacuum-packed meat product stored at 9°C. A linear relationship was obtained, for the absorbance data on different dilutions of the inoculum, in the absorbance interval studied. There was also a linear relationship between the values of the maximum specific growth rates derived from the absorbance and the ones derived from viable count measurements; and corrections were made accordingly. The statistical evaluation showed that all the main factors, i.e. pH, sodium chloride, sodium lactate and sodium acetate were statistically significant for the growth rate of L. monocytogenes. Comparison to the Food MicroModel (FMM) showed a slight underprediction for the developed model (bias=0.84). The predictions were, on average, within 20% of the FMM predictions ( n=10). Validation against the observed growth of L. monocytogenes inoculated into an emulsion type of sausage ( n=4) also showed a slight underprediction by the model. The predictions were, on average, 16% below the observed values in the sausage (Bias 0.84, Accuracy 1.26).

Psychrotrophic clostridia mediated gas and botulinal toxin production in vacuum-packed chilled meat.

A cocktail of washed spores from six psychrotrophic Clostridium strains isolated from blown vacuum-packed meats was inoculated onto lamb chumps. A second washed spore cocktail of four toxigenic reference Cl. botulinum strains, types A, B (two strains) and E, and a Cl. butyricum type E strain, was similarly inoculated onto lamb chumps. All inoculated lamb chumps were individually vacuum-packed and placed into storage at various temperatures typical of good to grossly abusive chilled storage (-1 degree C to 15 degrees C). All packs were observed for gas production (pack-'blowing') over a 12 week storage period. On gas production, or after 12 weeks of storage, packs were examined by mouse bioassay for botulinum toxin production. The packs inoculated with the meat isolate cocktail showed evidence of gas production earlier than packs inoculated with reference strains. No botulinum toxin was recovered from the meat isolate inoculated packs, while botulinal toxin was detected in reference strain inoculated packs down to a nominal storage temperature of 2 degrees C.

Efficacy of heat and ethanol spore treatments for the isolation of psychrotrophic Clostridium spp. associated with the spoilage of chilled vacuum-packed meats

Psychrotrophic Clostridium spp. associated with chilled meat spoilage are difficult to isolate and culture. In this study the kinetics of heat and ethanol spore inactivation was determined as a first step towards optimising the recovery of psychrotrophic clostridial spores from meat and environmental samples. To determine heat inactivation, spores of nine isolates associated with spoiled chilled or frozen meat and a psychrotrophic reference strain Clostridium algidicarnis NCFB 2931, suspended in phosphate buffer, were exposed to temperatures between 75°C and 95°C for 0 to 120 min using a submerged tube procedure. Survivors after various temperature-time combinations were enumerated on Peptone Yeast Extract Glucose Starch (PYGS) agar containing lysozyme. D-values and z-values for each spore suspension were determined from their respective survival curves. To determine ethanol inactivation, similar phosphate buffer spore suspensions were mixed with equal volumes of absolute ethanol, incubated at 20°C and survivors enumerated on lysozyme-containing PYGS agar after 0 to 300 min. Based on spore heat inactivation, the 10 isolates could be grouped as having either heat-sensitive or heat-resistant spores. For heat-sensitive spore types, 60 min ethanol treatment gave maximum spore recovery whereas for heat-resistant spore types, heat treatment at 80°C for 10 min gave the best recovery. When the spore heat-resistance type is unknown, as would be the case when attempting an isolation from spoiled product, both an ethanol treatment and a separate heat treatment should be used, to ensure maximum spore recovery.

Characteristics and genetic determinants of bacteriocin activities produced by Carnobacterium piscicola CP5 isolated from cheese.

Carnobacterium piscicola CP5, isolated from a French mold-ripened soft cheese, produced a bacteriocin activity named carnocin CP5, which inhibited Carnobacterium, Enterococcus and Listeria spp. strains, and among the Lactobacillus spp. only Lactobacillus delbrueckii spp. [24]. The activity was purified by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography followed by reverse-phase high-performance liquid chromatography (RP-HPLC). This latter step separated two peaks with anti-listerial activity (CP51 and CP52). Carnocin CP51 was partially sequenced, and the N-terminal part revealed the presence of the "pediocin-like consensus" sequence-Tyr-Gly-Asn-Gly-Val-. Then, a degenerated 24-mer oligonucleotide probe was constructed from the N-terminal sequence and used to detect the structural gene. It was localized on a plasmid of about 40 kb. Cloning of restriction fragments of this one, followed by DNA sequencing, revealed the presence of the second anti-Listeria bacteriocin gene (CP52). By comparing sequences in data banks and confirming results with PCR reactions, carnocin CP51 shared homologies with carnobacteriocin BM1, and carnocin CP52 was similar to carnobacteriocin B2, both produced by C. piscicola LV17 [2]. However, carnobacteriocin A from C. piscicola LV17 gene was lacking in C. piscicola CP5, and the two microorganisms have been isolated from different ecological environments: C. piscicola CP5 and C. piscicola LV17 were isolated from soft cheese and vacuum-packed meat respectively. This fact could allow different application perspectives for C. piscicola CP5.