Psychrotrophic Clostridium Research Articles

Influence of reduced levels or suppression of sodium nitrite on the outgrowth and toxinogenesis of psychrotrophic Clostridium botulinum Group II type B in cooked ham

Outgrowth and toxinogenesis of Clostridium botulinum Group II (non-proteolytic) type B were studied in cooked ham prepared with different NaNO2 (ranging from 0 to 80 mg/kg) and sodium chloride (NaCl, ranging from 12 to 19 g/kg) incorporation rates. Cured ground pork batters were inoculated with a cocktail of 3 strains of C. botulinum Group II type B at 3.5 log10 CFU/g, portioned and samples of 50 g were vacuum packed then cooked and cooled based on thermal processing employed by the meat processing industry. These cooked ham model samples were stored under reasonably foreseeable conditions of use and storage i.e. for 14 days at 4 °C, followed by a cold chain break for 1 h at 20 °C then up to 33 days at 8 °C. Storage times and temperatures were used to mimic those commonly encountered along the supply chain. Enumeration of C. botulinum and detection of the botulinum neurotoxin type B (BoNT/B) were performed in triplicate at different storage times. Under these experimental conditions, incorporation rates of NaNO2 ≥ 30 mg/kg prevented the outgrowth and toxinogenesis of C. botulinum Group II type B in the cooked ham model, regardless of the NaCl concentrations tested. In contrast, total removal of nitrite allowed outgrowth and toxin production during storage of the processed meat product. Results showed that the maximum ingoing amount of nitrite (i.e. 150 mg/kg) that may be added according to the EU legislation (Regulation (EC) No 1333/2008) can be reduced in cooked ham while still ensuring control of C. botulinum Group II type B. According to the multiple factors that could affect C. botulinum behavior in processing meat products, outgrowth and toxin production of C. botulinum should be evaluated on a case by case basis, depending on the recipe, manufacturing process, food matrix and storage conditions.

A mathematical model for predicting growth/no-growth of psychrotrophic C. botulinum in meat products with five variables

The objective of this study was to develop a mathematical model for predicting growth/no-growth of psychrotrophic Clostridium botulinum in pasteurised meat products packed in modified atmosphere for combinations of storage temperature, pH, NaCl, added sodium nitrite and sodium lactate.Data for developing and training the artificial neural network (ANN) were generated in meat products. A total of 249 growth experiments were carried out in three different meat products with different combinations of storage temperature, pH, NaCl, sodium nitrite and sodium lactate. The meat batter was inoculated with approx. 104 spores/g using a 4-strain cocktail of gas-producing C. botulinum. The meat products were sliced, packed in modified atmosphere (30% CO2/70% N2) and stored at 4 °C, 8 °C and 12 °C, respectively, for up to 8 weeks. The enumeration of C. botulinum was performed when the volume of the package had increased by 9% or more, or at the end of the storage period.Based on 10–20 replicates for each combination, the “frequency of growth” was calculated. An ANN with 5 input neurons, 3 hidden and a single output neuron was trained using the 5 hurdle values as inputs and the observed “frequency of growth” as target value.The inputs for the final model are the five variables: temperature, pH, added sodium nitrite, NaCl and sodium lactate within the ranges 4–12 °C, 5.4–6.4, 0–150 ppm, 1.2–2.4% and 0–3% respectively. As reference a logistic regression method was also applied and subsequently compared to the full neural network model. Based on RMSEC value of 0.104 and 0.144 for ANN and the logistic regression model respectively, the ANN was preferred. On a separate set of test data (n = 60) the ANN model was validated by comparing the predicted “probability of growth” with the observed growth. A bias of 0.0166 was obtained, indicative of a model that is slightly fail-safe.

Influence of different shrinking temperatures and vacuum conditions on the ability of psychrotrophic Clostridium to cause ‘blown pack’ spoilage in chilled vacuum-packaged beef

This study determined the ability of psychrotrophic Clostridium strains isolated from vacuum-packaged beefs and abattoir environments to cause ‘blown-pack’ spoilage of vacuum-packaged beef stored at 2 and 15°C. The influence of shrinking temperatures (83, 84 and 87°C) and vacuum pressure (6 and 9mbar) on the occurrence of such spoilage as well as the effects of simulated transportation (500km) on the integrity of packages was determined. At 15°C and 2°C, twelve and six strains caused ‘blown-pack’ spoilage, respectively. The combination of vacuum pressure (9mbar) combined with shrinking temperature (87°C) retarded the occurrence of spoilage. The simulated transportation under the experimental conditions did not affect the integrity of packages. More studies that assess the factors that may contribute for the occurrence of ‘blown-pack’ spoilage should be performed to avoid the occurrence of such spoilage during its shelf-life.

Involvement of Clostridium gasigenes and C. algidicarnis in ‘blown pack' spoilage of Brazilian vacuum-packed beef

The objectives of this study were to isolate psychrotrophic clostridia from Brazilian vacuum-packed beef cuts (spoiled or not) and to identify the isolates by using 16S rRNA gene sequencing. Anaerobic psychrotrophic microorganisms were also enumerated and samples were collected to verify the incidence of psychrotrophic clostridia in the abattoir environment. Vacuum-packed beef cuts (n=8 grossly distended and n=5 non-spoiled) and environmental samples were obtained from a beef packing plant located in the state of São Paulo, Brazil. Each sample was divided in three subsamples (exudate, beef surface and beef core) that were analyzed for vegetative forms, total spore-forming, and sulfide reducing spore-forming, both activated by alcohol and heat. Biochemical profiles of the isolates were obtained using API20A, with further identification using 16S rRNA gene sequencing. The growth temperature and the pH range were also assessed. Populations of psychrotrophic anaerobic vegetative microorganisms of up to 10(10)CFU/(g, mL or 100 cm(2)) were found in 'blown pack' samples, while in non-spoiled samples populations of 10(5)CFU/(g, CFU/mL or CFU/100 cm(2)) was found. Overall, a higher population of total spores and sulfide reducing spores activated by heat in spoiled samples was found. Clostridium gasigenes (n=10) and C. algidicarnis (n=2) were identified using 16S rRNA gene sequencing. Among the ten C. gasigenes isolates, six were from spoiled samples (C1, C2 and C9), two were isolated from non-spoiled samples (C4 and C5) and two were isolated from the hide and the abattoir corridor/beef cut conveyor belt. C. algidicarnis was recovered from spoiled beef packs (C2). Although some samples (C3, C7, C10 and C14) presented signs of 'blown pack' spoilage, Clostridium was not recovered. C. algidicarnis (n=1) and C. gasigenes (n=9) isolates have shown a psychrotrophic behavior, grew in the range 6.2-8.2. This is the first report on the isolation of psychrotrophic Clostridium (C. gasigenes and C. algidicarnis) in Brazil. This study shows that psychrotrophic Clostridium may pose a risk for the stability of vacuum-packed beef produced in tropical countries during shelf-life and highlights the need of adopting control measures to reduce their incidence in abattoir and the occurrence of 'blown pack' spoilage.

Microbial deterioration of vacuum-packaged chilled beef cuts and techniques for microbiota detection and characterization: a review.

Gas production from microbial deterioration in vacuum-packs of chilled meat leads to pack distension, which is commonly referred as blown pack. This phenomenon is attributed to some psychrophilic and psychrotrophic Clostridium species, as well as Enterobacteria. The ability of these microorganisms to grow at refrigeration temperatures makes the control by the meat industry a challenge. This type of deterioration has been reported in many countries including some plants in the Midwestern and Southeastern regions of Brazil. In addition to causing economic losses, spoilage negatively impacts the commercial product brand, thereby impairing the meat industry. In the case of strict anaerobes species they are difficult to grow and isolate using culture methods in conventional microbiology laboratories. Furthermore, conventional culture methods are sometimes not capable of distinguishing species or genera. DNA-based molecular methods are alternative strategies for detecting viable and non-cultivable microorganisms and strict anaerobic microorganisms that are difficult to cultivate. Here, we review the microorganisms and mechanisms involved in the deterioration of vacuum-packaged chilled meat and address the use of molecular methods for detecting specific strict anaerobic microorganisms and microbial communities in meat samples.

The effect of sage, sodium erythorbate and a mixture of sage and sodium erythorbate on the quality of turkey meatballs stored under vacuum and modified atmosphere conditions

1. The combined effect of sage (S), sodium erythorbate (SE), a mixture of sage and sodium erythorbate (MIX) and vacuum packaging (VP) and modified atmosphere packaging (MAP) on the quality of cooked turkey meatballs stored at 4°C was investigated. The physicochemical properties (colour, MDA, AV, pH, water activity), microbiological quality characteristics (counts of mesophilic and psychrotrophic bacteria, fungi, coliforms and Clostridium sp.) and flavour attributes of meatballs were determined. 2. The values of the colour parameters L*, a* and b* were affected by the additives and packaging method. The colour of meatballs was better protected by sodium erythorbate than by sage or a mixture of sage and sodium erythorbate. The additives effectively stabilised lipids against oxidation and slowed down hydrolytic changes in turkey meatballs. Sage and a mixture of sage and sodium erythorbate showed stronger antioxidant properties than sodium erythorbate added alone. Products with additives were characterised by better sensory quality than control samples. Sage and MIX prevented the growth of mesophilic and psychrotrophic bacteria. All additives inhibited the growth of coliforms. 3. MAP was more effective than VP in maintaining the microbial and sensory quality stability of cooked turkey meatballs. However, VP appears to be a better method as regards the maintaining of lipid stability in turkey meatballs.

Psychrophilic and psychrotrophic clostridia: sporulation and germination processes and their role in the spoilage of chilled, vacuum‐packaged beef, lamb and venison

SummarySpoilage of beef, lamb and venison by psychrophilic and psychrotrophic clostridial species renders meat unacceptable resulting in financial losses and reduced consumer confidence. A number of clostridial strains, including Clostridium algidicarnis, Clostridium algidixylanolyticum, Clostridium estertheticum, Clostridium frigidicarnis and Clostridium gasigenes, have been implicated in red meat spoilage. Unlike other spoilers, these clostridia are able to grow in anaerobic conditions and at chilled temperatures (some at −1.5 °C the optimal storage temperature for chilled red meat). The spoilage they cause is characterised by softening of the meat, production of large amounts of drip (exudates), offensive odours and in the case of C. estertheticum and C. gasigenes production of gas. Spoilage occurs following the introduction of clostridial spores into vacuum packages during processing. Germination of spores is necessary for the growth of vegetative cells, which cause spoilage. Current mitigation strategies focus on good management practice within meat processing plants. However, this is not always sufficient to prevent spoilage. This review summarises the issues associated with meat spoilage because of psychrotolerant clostridia and discusses areas that require further study.

STUDY OF “BLOWN PACK” SPOILAGE OF CHILLED VACUUM PACKED BEEF

Blown pack spoilage of vacuum packed chilled beef was characterised by chemical (GCMS) analysis, microscope evaluation and microbiological analysis. Large amounts of butyric acid were found; the alteration was probably caused by psychrotrophic clostridia.

Influence of the preparation, storage temperature, modified atmosphere and vacuum packaging on shelf-life of sliced, chilled yellow pepper and pre-cooked sweet corn

Sliced peppers can be stored at 4.5 °C without chilling injury. At 8 °C 7 days shelf-life was achieved in active MAP, at 4.5 °C 7 days also in passive MAP. At 8 °C the development of a slight off-flavour was observed from day 7 th in the passive MAP. Washing in chlorinated water was more efficient for the reduction of Listeria monocytogenes than flash washing in warm water, which reduced the spoilage efficiently. The growth of Listeria monocytogenes was limited to the first 3 days of chilled storage at 4.5–8 °C in all samples and a slight decrease was observed up to day 10, indicating the likelihood of a competitive inhibition effect of the spoilage microbiota. In case of storage at 1.5 °C the sensory properties of blanched vacuum packed corn on the cob were acceptable for more than 30 days. The shelf-life is limited by safety considerations, toxin formation of the psychrotrophic Clostridium botulinum due to the higher temperatures in the distribution chain might occur. Efficient pre-cooling delays the sp...

Influence of Oxygen Transmission Rate of Packaging Film on Outgrowth of Anaerobic Bacterial Spores

ABSTRACT Modified atmosphere packaging (MAP) has been applied to many food products, particularly respiring horticultural products. For non-respiring products, such as meat and seafood, reduced oxygen environments have been shown to preserve product quality. Recent efforts by seafood manufacturers to employ reduced oxygen packaging (ROP) for fresh fish have been halted by the Food and Drug Administration's import alert # 16–125, which specifies a minimum film permeability of 10,000 cc/m2/day for hermetically sealed fresh seafood. The purpose of this work was to determine the impact of package area and factors related to oxygen transmission rate (OTR) on anaerobic spore outgrowth. Studies were conducted to determine effects of temperature and relative humidity on OTR of commercially available packaging films (Clysar 60 HGPF, Clysar 75 HGPF, AET PST2-060 and Cryovac BDF1000). Three films with wide OTR range were selected to study effects of package area on outgrowth of Clostridium sporogenes spores. Films were converted into packages with different areas (0.083 and 0.325 m2). Inoculated petri dishes were packed in nitrogen flushed bags and incubated at 10 to 35°C. Oxygen concentrations were measured in growth media and headspace. Oxidation reduction potentials (ORP) of media were measured before and after incubation. It was found that package area, within limits of practicality, was insufficient to modify spore outgrowth behavior. However, results suggest an interesting hypothesis that packaging film OTR is related to a key window of time in which outgrowth is more likely to occur, which may be exploited during distribution. Results should be confirmed using psychrotrophic Clostridium botulinum.

Microbiological Quality of Rabbit Meat

World rabbit meat production is estimated to be over 1 million tons, and Spain is the third largest producer. Although rabbit meat is marketed and consumed worldwide, information on microbiological quality is very scarce. Here, we report indicator organisms, spoilage flora, sensory quality, and some physicochemical traits of 24 h postmortem chilled rabbit carcasses and prepackaged rabbit meat stored chilled in air for 0 to 3 days at the retail level. The mean total bacterial count (4.01±0.48 log CFU/g) for carcasses dressed at a small abattoir by a manual process was significantly lower (P < 0.05) than that (4.96±0.90 log CFU/g) for carcasses dressed at a large abattoir in automated slaughter lines. Both groups of carcasses had mean pH values of 5.98. The dominant contaminants on carcasses from the small abattoir were Pseudomonas, lactic acid bacteria, and yeasts. These microorganisms and Brochothrix thermosphacta were dominant on carcasses from the large abattoir. On prepacked hind legs (pH 6.26 ± 0.18) stored at −1 to + 1° C (supermarket 1), mean aerobic mesophilic count was 5.87±1.03 log CFU/g, and the major microbial groups were Pseudomonas, yeasts, lactic acid bacteria, and B. thermosphacta. On prepacked whole carcasses (pH 6.37 ± 0.18) displayed at −1 to + 5° C (supermarket 2), mean aerobic mesophilic count was 6.60±1.18 and the same microbial groups were dominant. Relative Escherichia coli incidence was supermarket 2 > large abattoir > supermarket 1 > small abattoir. Overall, low numbers of coliforms, Enterobacteriaceae, psychrotrophic clostridia, coagulase-positive staphylococci, and molds were found. Sensory scores, pH values, and L-lactic acid content differentiated fresh carcasses from retail samples. Data obtained suggest that the microflora of chilled rabbit meat are different from those found on the meat of other animals.

Co-incubation of Clostridium botulinum with protective cultures

Potential neurotoxin production by psychrotrophic Clostridium botulinum is the significant food safety risk in refrigerated foods of extended durability. Protective cultures (PC), nisin-producing Lactococcus lactis (6.3 × 10 5 cfu/ml) and pediocin A-producing Pediococcus pentosaceus (10 7 cfu/ml), were co-incubated with C. botulinum type B (6.2 × 10 2 cfu/ml) in tryptose, peptone, glucose and yeast extract broth for 31 days at 5 °C, 14 days at 10 °C and for 10 days at 15 °C. Bacterial populations, pH, botulinal toxin and bacteriocin production were measured. At 5 °C, the populations of all species did not change during incubation. At 10 and 15 °C, the PCs increased by 1–3log. At 15 °C, C. botulinum increased by 1.5–3log and was not inhibited by the PCs. At 10 °C, this pathogen was not detected after 10 days of co-incubation. The reduction in C. botulinum population coincided with the nisin level produced by L. lactis exceeding 100 IU/ml, but it was not rapid enough to prevent botulinal toxin formation.

Inhibition of Nonproteolytic, Psychrotrophic Clostridia and Anaerobic Sporeformers by Sodium Diacetate and Sodium Lactate in Cook-in-Bag Turkey Breast ,

A nonproteolytic, psychrotrophic Clostridium isolate, designated strain OMFRI1, was recovered from cook-in-bag turkey breasts (CIBTB) that displayed an intense pink discoloration and an off-odor following extended refrigerated storage. The viability of strain OMFRI1 in CIBTB containing sodium diacetate (at 0, 0.25, and 0.5%) and/or sodium lactate (at 0, 1.25, and 2.5%) was subsequently evaluated. Raw CIBTB batter was inoculated with 9 to 30 spores of strain OMFRI1 per g, vacuum packaged, cooked to an instantaneous internal temperature of 71.1°C, chilled, and incubated at 4°C for up to 22 weeks. In the absence of food-grade antimicrobial agents, spoilage (i.e., an off-odor) occurred within 6 weeks, and anaerobic plate counts reached 6.6 log10 CFU/g. The CIBTB containing sodium diacetate (0.25%) and that containing sodium lactate (1.25%) required 12 weeks for spoilage to occur and for anaerobic plate counts to reach 7.0 and 6.0 log10 CFU/g, respectively. When sodium diacetate (0.25%) and sodium lactate (1.25%) were used in combination, no off-odor was detected and anaerobic plate counts did not exceed 2.3 log10 CFU/g over 22 weeks of storage at 4°C. In related experiments, sodium diacetate (at 0, 0.25, and 0.5%), sodium lactate (at 0, 1.25, and 2.5%), and combinations of both ingredients were evaluated in uninoculated CIBTB incubated at 25°C for up to 22 days. In the absence of antimicrobial agents and in CIBTB containing sodium diacetate (0.5%), spoilage occurred within 8 days and anaerobic plate counts reached 6.8 and 6.6 log10 CFU/g, respectively. Samples of CIBTB containing sodium lactate (2.5%) showed signs of spoilage within 22 days, and anaerobic plate counts for these samples ranged from ≤1.0 to 6.3 log10 CFU/g. In CIBTB containing both sodium lactate (2.5%) and sodium diacetate (0.25%), spoilage was not evident and anaerobic plate counts were ≤1.0 log10 CFU/g within 22 days. These data validate the efficacy of sodium lactate and sodium diacetate in extending the shelf life of CIBTB.

Numbers and types of microorganisms in vacuum-packed cold-smoked freshwater fish at the retail level

Fifty-four packages (each one belonging to a different lot) of vacuum-packed cold-smoked salmon (30) and trout (24) produced by six Spanish smokehouses were obtained at retail level after 3 weeks storage at 2±1 °C. Sensorial, chemical, physicochemical and microbiological characteristics were examined. Overall, pH, a w, salt content in water phase, aerobic plate counts at 30 and 25 °C, levels of Enterobacteriaceae, lactic acid bacteria (LAB), fungi and presumptive aeromonads and staphylococci are in agreement with available data on lightly preserved fish products. Psychrotrophic clostridia ranged between 1.71 and 2.21 log CFU/g. Levels of ethanol were highly variable and not significantly related ( p>0.05) to sensory scores or to microbial numbers. Salmonella, Escherichia coli and Listeria monocytogenes were not detected in any sample. Listeriae other than L. monocytogenes were isolated from three packages. Levels of Staphylococcus aureus lower than 4 log CFU/g were also found in three packages. Among 377 bacteria randomly isolated from aerobic 25 °C plate counts, LAB predominated, with Carnobacterium ( C. piscicola) and Lactobacillus (eight species) being the genera most frequently found. The second and third major groups were Enterobacteriaceae and Micrococcaceae, respectively. Proteus vulgaris, P. mirabilis and Serratia liquefaciens were dominant among Enterobacteriaceae and coagulase-negative staphylococci among Micrococcaceae. Minor microbial groups such as aerobic Gram-negative bacilli ( Acinetobacter, Moraxella and Pseudomonas), Brochothrix, Aeromonas, Bacillus and Vibrio constituted less than 17% of the total flora.

Control of Nonproteolytic Clostridium botulinum Types B and E in Crab Analogs by Combinations of Heat Pasteurization and Water Phase Salt

Water phase sodium chloride (WPS) levels of 1.8 to 3.0% in combination with heat pasteurization for 15 min at temperatures of 75, 80, 85, and 90°C were evaluated as methods for the inactivation or inhibition of nonproteolytic, psychrotrophic Clostridium botulinum types B and E in crab analogs (imitation crab legs) subsequently stored at 10 and 25°C. Samples inoculated with 102 type B or E spores per g prior to pasteurization remained nontoxic for 120 days at 10°C and for 15 days at 25°C. With 104 type E spores per g and 80°C pasteurization, ≥2.4 and 2.7% WPS was required for inhibition at 10 and 25°C storage, respectively. Pasteurization at 85°C decreased the inhibitory level of WPS to 2.1% at 10°C and to 2.4% at 25°C. When the inoculum was 104 type B spores per g, samples with 2.7% WPS were toxic after 80 days of storage at 10°C. Samples inoculated with 103 type B spores per g and processed at 85°C remained nontoxic for 15 days at 25°C with a WPS of ≥2.4%. When pasteurization was carried out before inoculation and packaging, 1.8% WPS prevented toxin production by 102 and 104 type E spores per g for 30 days at 10°C, and this time period increased as the WPS concentrations increased. Three percent WPS prevented toxin production by 104 type E spores per g in vacuum-packaged analogs stored 110 days at 10°C. Pasteurization processes used in these experiments, however, do not inactivate the heat-resistant proteolytic types of Clostridium botulinum. Therefore, the most important factor controlling the growth of this bacterium is continuous refrigeration below 3.0°C or frozen storage of the finished product.

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.

Psychrotrophic Clostridia Causing Spoilage in Cooked Meat and Poultry Products

Certain types of commercially produced noncured turkey breast and roast beef are precooked in situ, stored at 4°C or below, and typically given use by dates of greater than 50 days. While of rare, sporadic occurrence, an unpleasant spoilage characterized by strong H2S odor and gas production has been observed in these products. This spoilage is due to the growth of psychrotrophic anaerobic sporeformers. Isolates from roast beef resemble Clostridium laramie while isolates from uncured turkey have been designated C. ctm for cooked turkey meat. The turkey breast isolates were characterized by temperature growth ranges, carbohydrate fermentations, and other biochemical reactions. Growth of all isolates was inhibited in broth media by 3.0% NaCl, 100 ppm nitrite, 2.0% sodium lactate, or 0.2% sodium diacetate. Inoculated studies were performed with three isolates in cooked turkey product. All three isolates grew and spoiled product at 10 and 3.3°C, and one isolate grew at 0.5 and −3°C. Some differences in growth were observed with the lactate and diacetate treatments in turkey meat among the three isolates. One isolate appeared to utilize the lactate, two were inhibited. Overall, 0.1% diacetate consistently delayed growth, although to different degrees, for all isolates.

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.

A PCR survey of psychrotrophic Clostridium botulinum-like isolates for the presence of BoNT genes.

Isolates (259) of psychrotrophic Clostridium spp. associated with either blown pack spoilage (five isolates) or slaughter stock (254 isolates) were screened for the presence of botulinum neurotoxin (BoNT) genes using degenerate PCR primers capable of amplifying A, B, E, F and G BoNT genes. No BoNT gene amplification products were detected using DNA templates from the 259 psychrotrophic isolates, including 249 isolates that showed the same 16S rRNA gene Restriction Fragment Length Polymorphism (RFLP) patterns as authentic Cl. botulinum type B. It is concluded that although the growth of such micro-organisms in vacuum-packed chilled meat leads to product spoilage, it does not prejudice product safety.

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.