Vacuum-packaged Meat Research Articles

Microbiological Safety of Retail Vacuum-Packed and Modified-Atmosphere-Packed Cooked Meats at End of Shelf Life

A study of retail modified-atmosphere-packed and vacuum-packed cooked ready-to-eat meats was undertaken from September through mid-November 2003 to determine the microbiological quality at the end of shelf life and to establish any risk factors in the production, storage, and display of this product. Examination of 2,981 samples using Microbiological Guidelines criteria revealed that 66% were of satisfactory or acceptable microbiology quality, 33% were of unsatisfactory quality mainly due to high aerobic colony counts and Enterobacteriaceae concentrations, and 1% were of unacceptable quality due to the presence of Listeria monocytogenes at 100 CFU/g or higher (27 samples; range of 102 to 106 CFU/g) and Campylobacter jejuni (1 sample), indicating a risk to health. All samples at the end of the shelf life had satisfactory (<20 CFU/g) and/or acceptable (<102 CFU/g) levels of Staphylococcus aureus and Clostridium perfringens, four samples (<1%) had unsatisfactory levels of Escherichia coli (range of 102 to 106 CFU/g) and 5.5% of the samples contained L. monocytogenes at <20 CFU/g (4.8%) or between 20 and 100 CFU/g (0.7%). More samples of chicken (45%; 224 of 495 samples), beef (43%; 160 of 371 samples), and turkey (41%; 219 of 523 samples) were of unsatisfactory or unacceptable quality compared with ham (23%; 317 of 1,351 samples) or pork (32%; 67 of 206 samples). Twelve different L. monocytogenes typing characters (serotype–amplified fragment length polymorphism type–phage type) were evaluated for isolates recovered from samples of unacceptable quality, and the 1/2-IX-NT type was recovered from almost half (48%) of these samples. Salmonella was not detected in any samples examined. Risk factors identified for cooked meats that were microbiologically contaminated more frequently included vacuum packaging, packaging on retail premises, slicing, temperature not monitored in display units, and no hazard analysis system in place. Results from this study also suggest that the shelf life assigned to some modified-atmosphere-packed and vacuum-packed meats may not be appropriate.

Intraspecific Diversity of Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus sakei, and Leuconostoc mesenteroides Associated with Vacuum-Packed Meat Product Spoilage Analyzed by Randomly Amplified Polymorphic DNA PCR

The intraspecific diversity of Leuconostoc mesenteroides, Lactobacillus curvatus, Lactobacillus sakei, and Lactobacillus plantarum was analyzed by randomly amplified polymorphic DNA (RAPD) PCR with universal primers M13 and T3. The study included 100 reference strains and 210 isolates recovered from two vacuum-packed Spanish meat products, fiambre de magro adobado and morcilla, previously identified by rDNA–restriction fragment length polymorphism profiles. The RAPDM13 profiles identified isolates at species level in L. plantarum and L. mesenteroides, while RAPD-T3 provided profiles in L. sakei. The combination of RAPD-M13 and RAPD-T3 fingerprints revealed a total of 17 profiles in L. mesenteroides, 6in L. sakei, 12 in L. plantarum, and6in L. curvatus. Of these, six profiles corresponding to L. mesenteroides and one corresponding to L. sakei were found in both products. The Shannon-Weaver diversity index (H′), calculated according to RAPD-M13 and RAPD-T3 profiles during storage, revealed that most profiles appeared only in single samplings in both products, indicating a high strain substitution rate during chilled storage of vacuum-packed meat products. When bloating appeared, only one profile corresponding to L. mesenteroides subsp. dextranicum was present throughout the storage period.

Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods

To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, 'morcilla' and 'fiambre de magro adobado' obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).

Inhibition of Listeria innocua and Brochothrix thermosphacta in vacuum-packaged meat by addition of bacteriocinogenic Lactobacillus curvatus CRL705 and its bacteriocins

To evaluate the inhibition effectiveness of Lactobacillus curvatus CRL705 used as a bioprotective culture and of its bacteriocins, lactocin 705 and lactocin AL705, against Listeria innocua, Brochothrix thermosphacta and indigenous lactic acid bacteria (LAB) in vacuum-packaged meat stored at 2 degrees C. The live culture of Lact. curvatus CRL705 as well as synthetic lactocin 705 and purified lactocin AL705 were shown to be similarly effective in preventing the growth of B. thermosphacta and L. innocua in meat discs in contrast to control samples in which these micro-organisms grew rapidly, their numbers increasing by 3.0- and 2.1-log cycles respectively. In addition, indigenous LAB population showed a lower growth rate in the presence of lactocin 705. Bacteriocin activity was detected in the meat discs during 36 days at 2 degrees C irrespective of the biopreservation strategy applied. Changes in pH were not significantly different in meat discs treated with the protective culture when compared with control samples. Lactobacillus curvatus CRL705 and the produced bacteriocins, lactocin 705 and lactocin AL 705, were effective in inhibiting L. innocua and B. thermosphacta. The use of the bioprotective culture in refrigerated vacuum-packaged fresh meat would be more feasible from an economic and legal point of view. Establishment of biopreservation as a method to ensure the microbiological safety of vacuum-packaged fresh meat at 2 degrees C.

Changes of bacterial diversity and main flora in chilled pork during storage using PCR-DGGE

This study was designed to explore the bacterial diversity and the main flora in chilled pork by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Longissimus muscle was removed from pork carcasses at 24 h postmortem. The muscle was tray- and vacuum-packaged at 4 °C for 2, 4, 7 days to extract the bacteria total DNA, respectively. The results indicated that the bacterial diversity of chilled pork decreased with storage time regardless of packaging method. Nine types of bacteria were identified, including Arthrobacter sp., Enterococcus sp., Staphylococcus sp., Moraxella sp., Pseudomonas sp., Lactobacillus sp., Aeromonas sp., Acinetobacter sp., Brochothrix thermosphacta. For tray-packaged pork, Pseudomonas sp. and B. thermosphacta were the dominant micro-organisms. The differences in the species found were related with the presence of Lactobacillus sp. in vacuum-packaged meat. The results of the present study might be useful to study the changes of the contaminating bacteria and their characteristics in chilled pork.

Contamination of some vacuum-packaged meat productswithListeria monocytogenes

In this study, the contamination of vacuum-packaged processed meat products by L. monocytogenes was investigated in the samples collected from retail markets in Van, Turkey. Of the 100 samples studied, 44 (44%) were positive for Listeria spp., while 14 samples (14%) of Listeria spp. isolates were identified to be contaminated by L. monocytogenes. Listeria spp. and L. monocytogenes were detected in 60% and 20% of Turkish fermented sausage samples, 48% and 12% of sausage samples, 40% and 16% of salami samples, and 28% and 8% of pastirma samples, respectively. Because of high level of L. monocytogenes incidence in examined vacuum-packaged meat products, it is essential to make sure that all necessary sanitary requirements be met to avoid any contamination. It is concluded that inadequte cooking practices or raw consumption of these contaminated products may cause potential risks for public health.

Use of a spectrophotometric bioassay for determination of microbial sensitivity to manuka honey

The antimicrobial activity of manuka honey has been well documented (Molan, 1992a,b,c, 1997) [Molan, P.C., 1992. The antibacterial activity of honey. 1: the nature of the antibacterial activity. Bee World 73 (1) 5-28; Molan, P.C., 1992. The antibacterial activity of honey. 2: variation in the potency of the antibacterial activity. Bee World 73 (2) 59-76; Molan, P.C., 1992. Medicinal uses for honey. Beekeepers Quarterly 26; Molan, P.C., 1997. Finding New Zealand honeys with outstanding antibacterial and antifungal activity. New Zealand Beekeeper 4 (10) 20-26]. The current bioassays for determining this antimicrobial effect employ a well diffusion (Ahn and Stiles, 1990) [Ahn, C., Stiles, M.E., 1990. Antibacterial activity of lactic acid bacteria isolated from vacuum-packed meats. Journal of Applied Bacteriology 69, 302-310], (Weston et al., 1999) [Weston, R.J., Mitchell, K.R., Allen, K.L., 1999. Antibacterial phenolic components of New Zealand manuka honey. J. Food Chem. 64, 295-301] or disc diffusion (Taormina et al., 2001) [Taormina, Peter J., Niemira, Brendan A., Beuchat, Larry R., 2001. Inhibitory activity of honey against food borne pathogens as influenced by the presence of hydrogen peroxide and level of antioxidant power. Int. J. Food Microbiol. 69, 217-225] assay using zones of inhibition as indicators of bacterial susceptibility. The development of a 24-h spectrophotometric assay employing 96-well microtiter plates, that is more sensitive and more amenable to statistical analysis than the assays currently employed, was undertaken. This simple and rapid assay permits extensive kinetic studies even in the presence of low honey concentrations, and is capable of detecting inhibitory levels below those recorded for well or disc diffusion assays. In this paper, we compare the assay to both well and disc diffusion assays. The results we obtained for the spectrophotometric method MIC values show that this method has greater sensitivity than the standard well and disc diffusion assays. In addition, inter- and intra-assay variance for this method was investigated, demonstrating the methods reproducibility and repeatability.

Purification and characterisation of acidocin D20079, a bacteriocin produced by Lactobacillus acidophilus DSM 20079

Bacteriocins are natural antimicrobial agents produced by food fermentative bacteria. Lactobacillus acidophilus DSM 20079 produces a small bacteriocin, with a molecular mass of 6.6 kDa, designated acidocin D20079. This antimicrobial peptide was extremely heat-stable (30 min at 121 °C) and was active over a wide pH range. It was found to be sensitive to proteolytic enzymes (trypsin, ficin, pepsin, papain, and proteinase K). Acidocin D20079 has a narrow inhibitory spectrum restricted to the genus Lactobacillus which includes L. sakei NCDO 2714, an organism known to cause anaerobic spoilage of vacuum-packaged meat. Maximum production of acidocin D20079 in MRS broth was detected at pH 6.0, and the peptide was purified by ammonium sulphate precipitation followed by sequential cation exchange and hydrophobic interaction chromatography. Purified acidocin D20079 spontaneously formed spherulite crystals during dialysis. As the N-terminus was found to be blocked for sequencing, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used to determine a partial sequence, and the molecular mass of the bacteriocin in the formed crystals (6.6 kDa). Estimates of the molecular weight of the partially purified peptide, using tricine-SDS-PAGE, in which bacteriocin activity was confirmed by overlayer techniques were in accordance with this value.

Presence of acylated homoserine lactones (AHLs) and AHL-producing bacteria in meat and potential role of AHL in spoilage of meat.

Quorum-sensing (QS) signals (N-acyl homoserine lactones [AHLs]) were extracted and detected from five commercially produced vacuum-packed meat samples. Ninety-six AHL-producing bacteria were isolated, and 92 were identified as Enterobacteriaceae. Hafnia alvei was the most commonly identified AHL-producing bacterium. Thin-layer chromatographic profiles of supernatants from six H. alvei isolates and of extracts from spoiling meat revealed that the major AHL species had an R(f) value and shape similar to N-3-oxo-hexanoyl homoserine lactone (OHHL). Liquid chromatography-mass spectrometry (MS) (high-resolution MS) analysis confirmed the presence of OHHL in pure cultures of H. alvei. Vacuum-packed meat spoiled at the same rate when inoculated with the H. alvei wild type compared to a corresponding AHL-lacking mutant. Addition of specific QS inhibitors to the AHL-producing H. alvei inoculated in meat or to naturally contaminated meat did not influence the spoilage of vacuum-packed meat. An extracellular protein of approximately 20 kDa produced by the H. alvei wild-type was not produced by the AHL-negative mutant but was restored in the mutant when complemented by OHHL, thus indicating that AHLs do have a regulatory role in H. alvei. Coinoculation of H. alvei wild-type with an AHL-deficient Serratia proteamaculans B5a, in which protease secretion is QS regulated, caused spoilage of liquid milk. By contrast, coinoculation of AHL-negative strains of H. alvei and S. proteamaculans B5a did not cause spoilage. In conclusion, AHL and AHL-producing bacteria are present in vacuum-packed meat during storage and spoilage, but AHL does not appear to influence the spoilage of this particular type of conserved meat. Our data indicate that AHL-producing H. alvei may induce food quality-relevant phenotypes in other bacterial species in the same environment. H. alvei may thus influence spoilage of food products in which Enterobacteriaceae participate in the spoilage process.

가열방법과 포장조건이 칠면조육 패티의 저장중 지방산화와 콜레스테롤 산화물에 미치는 효과

가열방법과 포장방법이 저장기간 동안 칠면조육 패티의 지방산화(TBARS)와 콜레스테롤 산화물 함량에 미치는 효과를 조사하기 위하여, 칠면조 다리육을 분쇄하여 패티를 제조하였다. 칠면조육 패티는 5가지 가열방법(oven cooking, pan frying, deep frying, boiling, microwaving)으로 가열한 후 각각 함기포장과 진공포장한 후 저장하면서 시험에 공시하였다. 지방산패도는 함기포장과 진공포장 모두 저장초기에는 boiling 방법이 유의적으로(P < 0.05) 높게 나타났으나, 저장 말기에는 microwaving 방법이 가장 높은 지방산패도를 나타내었다(P < 0.05). 총 콜레스테롤 산화물의 량은 전 저장기간동안 boiling 방법이 가장 낮은 함량을 나타내었고(P < 0.05), microwaving 방법이 가장 높은 함량을 나타내었다(P < 0.05).각각의 콜레스테롤 산화물의 함량을 보면,7<TEX>$\alpha$</TEX>-hydroxycholesterol은 pan frying 방법이 가장 낮은 함량을 나타내었고(P < 0.05), microwaving 방법이 가장 높은 함량을 나타내었다(P < 0.05). <TEX>$\lpha$</TEX>-epoxide의 량은 boiling 방법이 가장 낮은 함량을 나타내었으며, pan frying 방법에서 가장 높게 나타났다(P < 0.05). <TEX>$\beta$</TEX>-epoxide 함량은 pan frying 방법이 가장 낮은 함량을 나타내었으며(P < 0.05), deep frying 방법과 microwaving 방법에서 가장 높은 함량을 나타내었다(P < 0.05). 또한 7-ketocholesterol의 함량은 microwaving 방법에서 가장 높게 나타났다(P < 0.05). Turkey thigh meats with skin were ground twice through a 3-mm plate and patties were prepared. Patties were cooked using 5 different methods(oven cooking, pan frying, oil deep frying, boiling, and microwaving) to an internal temperature of 85<TEX>${\sim}90^{\circ}C$</TEX> and packaged in either oxygen permeable PVC zipperbags or oxygen impermeable PVDC bags. The samples were analyzed for thiobarbituric acid reactive substances(TBARS) and cholesterol oxidation products(COPs) after storage at <TEX>$4^{\circ}C$</TEX>. The TBARS of cooked meat increased during the storage regardless of cooking methods, vacuum packaged thigh meat produced less TBARS and COPs than the aerobically packaged samples. At the beginning of storage, aerobically packaged meat cooked by boiling method produced higher TBARS than that of others. However, the TBARS of microwaved meat with vacuum packaging increased rapidly after 3 days of storage at <TEX>$4^{\circ}C$</TEX>. The amount of total COPs in cooked thigh meat increased linearly with storage time. The level of total COPs in aerobically packaged cooked meat was higher than the vacuum-packaged meat. Microwave produced higher level of total COPs in meat than other cooking methods during storage.

Simultaneous detection of Carnobacterium and Leuconostoc in meat products by multiplex PCR.

To develop a multiplex PCR approach for simultaneous detection of Leuconostoc and Carnobacterium and its validation in meat products. Two multiplex PCR assays were developed using newly designed 16S rDNA-directed primers adapted to the current taxonomic situation of genera Leuconostoc and Carnobacterium that allow: (i) simultaneous detection of both genera, and members of the nonmotile species of genus Carnobacterium and (ii) identification in a single assay of the nonmotile species C. divergens, C. maltaromicum and C. gallinarum. Sensitivity values of 10(3) and 10(4) CFU g(-1) were determined for multiplex PCR detection of Carnobacterium and Leuconostoc, respectively, following artificially inoculated meat trials. In addition, both multiplex PCR assays were validated in 14 naturally contaminated samples covering nine types of meat products. Results obtained by colony identification were confirmed by PCR detection. The methods described in this study provide a rapid and reliable tool for PCR detection of Carnobacterium and Leuconostoc, in meat products, and for colony identification. This multiplex PCR approach will help in the analysis of the spoilage microbiota of refrigerated vacuum-packaged meat product in order to determine the appropriate preservation method.

Double-packaging is effective in reducing lipid oxidation and off-odor volatiles of irradiated raw turkey meat

The effects of double packaging on lipid oxidation, color, and volatile production were determined to establish a modified packaging method to improve quality changes in irradiated raw turkey meat. Sliced raw turkey breast and thigh meats were aerobically, vacuum- or double (vacuum and aerobic)-packaged, electron beam irradiated at 2.5 kGy, and then stored under refrigerated temperature. For the double-packaged samples, the outer vacuum bags were removed after 5, 7, or 9 d of refrigerated storage. 2-Thiobarbituric acid-reactive substances (TBARS) values, volatile compounds, and color values of the samples were determined after 10 d of storage. Irradiation and aerobic packaging promoted production of aldehydes (propanal and hexanal) related to lipid oxidation in turkey breast and thigh meats. Vacuum-packaged irradiated samples retained S-volatile compounds (methanethiol, dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide), mainly responsible for the irradiation off-odor, during storage. Exposure of double-packaged irradiated turkey meats to aerobic conditions by removing outer vacuum bags a few days before the test was effective in controlling both lipid oxidation-dependent (aldehydes) and radiolytic off-odor (S-compounds) volatiles. The a* values of raw turkey breast and thigh meats increased by irradiation regardless of packaging conditions. The a* value of double-packaged meats was lower than that of the vacuum-packaged meats but was not significant. Thus, the use of double-packaging alone was not enough to reduce the pink color of irradiated raw turkey meat. When lipid oxidation and irradiation off-odor should be minimized without any additional additives, however, double packaging is an excellent method to be used for turkey meats.

Rapid detection of meat spoilage by measuring volatile organic compounds by using proton transfer reaction mass spectrometry.

The evolution of the microbial spoilage population for air- and vacuum-packaged meat (beef and pork) stored at 4 degrees C was investigated over 11 days. We monitored the viable counts (mesophilic total aerobic bacteria, Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and Enterococcus spp.) by the microbiological standard technique and by measuring the emission of volatile organic compounds (VOCs) with the recently developed proton transfer reaction mass spectrometry system. Storage time, packaging type, and meat type had statistically significant (P < 0.05) effects on the development of the bacterial numbers. The concentrations of many of the measured VOCs, e.g., sulfur compounds, largely increased over the storage time. We also observed a large difference in the emissions between vacuum- and air-packaged meat. We found statistically significant strong correlations (up to 99%) between some of the VOCs and the bacterial contamination. The concentrations of these VOCs increased linearly with the bacterial numbers. This study is a first step toward replacing the time-consuming plate counting by fast headspace air measurements, where the bacterial spoilage can be determined within minutes instead of days.

PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats.

To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause 'blown pack' spoilage of vacuum-packed meat. Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of 'blown pack' causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of 'blown pack' causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT. The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g(-1) for non-enriched, and 102 CFU g(-1) for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of 'blown pack' spoilage in two packs, and Cl. estertheticum as the causative agent in the third. The developed method can be used for rapid detection of 'blown pack' causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. The paper reports practical procedures that can be used for rapid confirmation of the causative agents of clostridial 'blown pack' spoilage in commercial spoiled packs, or for detection of psychrophilic clostridia in epidemiological trace back of 'blown pack' spoilage incidents in meat processing plants.

Screening of lactic acid bacteria from Brazilian meats for bacteriocin formation

Twenty samples of vacuum-packaged meat products from Brazil were screened for the presence of bacteriocin-producing lactic acid bacteria, using an agar overlay method. Three bacteriocinogenic isolates were obtained and identified as Enterococcus sp. 18 (from bacon), Leuconostoc sp. 20 (from ham) and Lactobacillus sakei 29 (from hot home made “lingüiça”). Leuconostoc sp. 20 and Lactobacillus sakei 29 presented antilisterial activity, inhibiting all the strains of Listeria monocytogenes tested. However, the strains did not inhibit Brochothrix thermosphacta, Enterobacter sp., Salmonella sp. or Escherichia coli.

Positive effects of growth at suboptimal temperature and high salt concentration on long-term survival of Lactobacillus sakei

Lactobacillus sakei is a lactic acid bacterium commonly found on fresh meat and represents the predominant flora of vacuum-packed meat. In the present article, we studied the behavior of L. sakei in a chemically defined medium under various growth conditions relative to temperature or NaCl concentration. Growth occurred at each temperature, but growth rate and final cell density decreased at low temperature and survival was enhanced. In the presence of NaCl, we also observed a longer doubling time and a lower final cell density together with an enhanced long-term survival. When both conditions were combined, the long-term survival was greatly increased to about 28 weeks. Differences in cell morphology were observed under electron microscopy when cells were grown at low temperature in the presence of salt. We speculate that these are due to modifications in membrane structure. These results show that L. sakei is able to adapt to these environmental conditions and that slow growth is clearly associated with enhanced long-term survival.

Effect of Packaging‐Film Thicknesses on Thermal Inactivation of Salmonella and Listeria innocua in Fully Cooked Chicken Breast Meat

ABSTRACT: The study was conducted to determine the effect of packaging‐film thicknesses on thermal inactivation of Salmonella and Listeria innocua in cooked chicken breast meat. The meat was inoculated to contain 107 CFU/g of Salmonella or L. innocua and vacuum‐packaged in a thickness of 0.0762‐ or 0.2032‐mm film. The packaged meat was heat‐treated in a water bath at 68°C for 10 to 120 s and then cooled in an ice‐water bath. The survivors of Salmonella or L. innocua were enumerated. This study revealed that packaging‐film thicknesses affected thermal inactivation of Salmonella and L. innocua. The results are useful for surface pasteurization of fully cooked and vacuum‐packaged meat and poultry products.

Molecular differentiation of clostridia associated with ‘blown pack’ spoilage of vacuum-packed meats using internal transcribed spacer polymorphism analysis

The 16S–23S rDNA internal transcribed spacer (ITS) polymorphism analysis was assessed for its suitability in rapid discrimination between species of psychrophilic and psychrotolerant clostridia associated with ‘blown pack’ spoilage of vacuum-packed meats. DNA isolated from 10 reference and 20 meat strains of psychrophilic and psychrotolerant clostridia were used as templates in PCR amplification with primers complementary to conserved regions of the 3′ end of the 16S rRNA and 5′ end of the 23S rRNA genes directly flanking the spacer. The majority of strains showed multi-band ITS patterns when products of spacer amplification were visualised on an agarose gel. With the majority of meat strains, PCR amplification generated single banding pattern for a single clostridial species. However, meat strains of Cl. algidicarnis produced four different ITS banding patterns. With reference strains of psychrophilic and psychrotolerant clostridia, variation in spacer length was also observed between nonproteolytic Cl. botulinum type B (17B), E (Beluga) and F (202F). On the other hand, the number and size of the ITS amplification products could not be used for a differentiation of Cl. laramiense ATCC 51254 T from Cl. estertheticum DSM 8809 T, Cl. putrefaciens DSM 1291 T from Cl. algidicarnis NCFB 2931 T, or Cl. frigidicarnis strains from nonproteolytic Cl. botulinum type B (17B). The presence of interstrain, and lack of interspecies, ITS polymorphism observed in the present study with some clostridial species may preclude the use of 16S–23S rDNA spacer amplification for species-level discrimination and identification, respectively, of psychrophilic and psychrotolerant clostridia associated with meat spoilage. However, where interstrain, intraspecies heterogeneity of ITS amplification products exists, ITS analysis could be useful for tracing back psychrophilic and psychrotolerant clostridia responsible for meat spoilage to their meat plant sources.

Leuconostoc carnosum 4010 has the potential for use as a protective culture for vacuum-packed meats: culture isolation, bacteriocin identification, and meat application experiments

A new culture, Leuconostoc carnosum 4010, for biopreservation of vacuum-packed meats is described. The culture originated from bacteriocin-producing lactic acid bacteria (LAB) naturally present in vacuum-packed meat products. Approximately, 72,000 colonies were isolated from 48 different vacuum-packed meat products and examined for antibacterial activity. Bacteriocin-producing colonies were isolated from 46% of the packages examined. Leuc. carnosum was the predominant bacteriocin-producing strain and Leuc. carnosum 4010 was selected for further experiments because it showed strong antilisterial activity without producing any undesirable flavour components in meat products. For identification of the bacteriocins produced, partial purification was carried out by ammonium sulphate precipitation, dialysis, and cation exchange chromatography. SDS-PAGE analysis revealed two bands with inhibitory activity corresponding to molecular sizes of 4.6 and 5.3 kDa. N-terminal amino acid sequencing showed that Leuc. carnosum 4010 produced two bacteriocins highly similar or identical to leucocin A and leucocin C. Application experiments showed that the addition of 10 7 cfu/g Leuc. carnosum 4010 to a vacuum-packaged meat sausage immediately reduced the number of viable Listeria monocytogenes cells to a level below the detection limit and no increase of L. monocytogenes was observed during storage at 5 °C for 21 days. The results presented demonstrate that Leuc. carnosum 4010 is suitable as a new protective culture for cold-stored, cooked, sliced, and vacuum-packed meat products.

Detection of the spoiling of meat using PTR–MS

Using the recently developed proton transfer reaction–mass spectrometer (PTR–MS) method, we investigated the spoiling of air- and vacuum-packed meat (beef, pork, and poultry) that was stored at 4°C for up to 13 days. We measured and identified partly the emitted volatile organic compounds (VOCs) as a function of storage time and found a large increase in these emissions after a few days of storage. Also a large difference in the spoiling behavior between vacuum- and air-packed meat was observed.