Blown Pack Research Articles

Spoilage potential of bacterial species from chilled vacuum-packed lamb

The objective of this study was to establish whether specific organisms play important roles in the spoilage rate of vacuum-packed (VP) lamb at low storage temperatures. The spoilage potential of representative organisms (n = 13) of the spoilage community of VP lamb were investigated through a series of shelf-life challenge trials. Each isolate was individually inoculated onto sterile (irradiated) and non-sterile (i.e., containing natural microbial community) VP lamb meat. Meat quality was assessed over time by measuring sensorial qualities, bacterial growth and pH. Among all test organisms, Clostridium spp. had the highest spoilage potential and had a major effect on the spoilage rate of VP lamb (based on sensory assessment). C. estertheticum caused premature ‘blown pack’ spoilage; however, the spoilage was delayed in a community setting. C. putrefaciens and C. algidicarnis caused premature spoilage of VP lamb independently and in a community setting. In contrast, all facultative anaerobes and Pseudomonas sp. tested were not capable of spoiling meat independently or within a community, expect for Carnobacterium divergens and Serratia spp., which spoiled meat prematurely when present in a community. Overall, these results highlight that Clostridium could be one of the main taxa driving the faster rate of quality loss of chilled VP lamb compared to beef. This research can help to inform opportunities for shelf-life extension by targeting organisms with ‘high’ spoilage potential, such as Clostridium.

An Evaluation of Sporicidal Treatments against Blown Pack Spoilage Associated Clostridium estertheticum and Clostridium gasigenes Spores

Blown pack spoilage (BPS) occurs when meat is cross-contaminated with Clostridium estertheticum or Clostridium gasigenes spores, often from the meat processing environment. This study tested the efficacy of four sporicidal disinfectants commonly used in beef processing plants against C. estertheticum and C. gasigenes spores in a suspension test. D-values were obtained under model ‘clean’ (sterile distilled water, SDW) and ‘dirty’ (3 g/L bovine serum albumin, BSA) conditions. Mean concentration (log10 CFU/mL) were calculated from direct counts. The levels of dipicolinic acid (DPA), indicating damage to the core of these spores, was also monitored using a terbium (Tb)-DPA assay for treatment 1 (peracetic acid as the active ingredient) in SDW and BSA. In SDW sporicidal treatment 3 (containing peroxymonosulphate) was the most effective against C. estertheticum spores but under ‘dirty’ (BSA) conditions sporicidal treatments 1 and 2 were more effective. A similar pattern was obtained with C. gasigenes with treatment 3 being the most effective in SDW but treatment 2 (sodium hypochlorite as the active ingredient) being more effective in BSA. The lower DPA concentrations obtained in SDW versus BSA demonstrated the protective effect of organic matter. It was concluded that meat processors should use a 5% formulation containing sodium hypochlorite, sodium hydroxide and alkylamine oxide to eliminate BPS Clostridial spores in the abattoir.

Detection of Psychrophilic Clostridium spp. in Fecal Samples from Cattle of Different Ages Sampled at the Slaughterhouse Level

Clostridium estertheticum and C. estertheticum-like spp. are obligate anaerobic psychrophiles causing "blown pack" spoilage of chilled vacuum-packed meat. The present study aimed at detecting and isolating these spoilage bacteria in fecal samples of cattle of different ages at the slaughterhouse level. One hundred two swab fecal samples were obtained and enriched anaerobically in prereduced peptone-yeast-glucose-starch (PYGS) medium for 3 weeks at 4°C and then screened for C. estertheticum and C. estertheticum-like spp. by using a 16S rRNA gene-based real-time PCR (RT-PCR) assay. The RT-PCR-positive samples were further enriched for 3 weeks in prereduced PYGS medium and then subjected to an ethanol (50%, v/v) and lysozyme (4 mg/mL) treatment. Isolation was carried out anaerobically on Columbia agar with 5% defibrinated sheep blood at 4°C for 3 weeks. Isolated strains were identified morphologically and by the 16S rRNA gene. Forty (39%) of 102 samples were RT-PCR positive. The frequency of positive samples was the following: 9 (45%) of 20 in calves (aged ≤160 days), 23 (43%) of 54 in young cattle (aged 161 to 1,000 days), and 8 (29%) of 28 in cows or bulls (aged >1,000 days). Six strains were isolated from 6 of 40 RT-PCR-positive samples. Of these, five were from the calves (n = 1) and young cattle (n = 4). The six isolates were identified as C. estertheticum (n = 1), Clostridium frigoriphilum (n = 1), and C. estertheticum-like spp. (n = 4). The present findings confirm that feces of cattle are an important source of psychrophilic Clostridium spp. The fecal carriage among livestock animals at slaughter is strongly correlated with the risk of carcass contamination. Therefore, the maintenance of slaughter hygiene is of central importance.

Detection of Psychrophilic Clostridium spp. Causing “Blown Pack” Spoilage in Meat Juice Samples from Chilled Vacuum-Packed Beef and Lamb Meat Imported from Different Countries to Switzerland

"Blown pack" spoilage (BPS) of chilled vacuum-packed meat is mainly caused by anaerobic and psychrophilic Clostridium spp., including C. estertheticum, C. gasigenes, C. frigoriphilum, and C. frigidicarnis. Recently, its occurrence has been reported in several countries, especially in internationally traded meat. Therefore, this study aimed at detecting the occurrence of psychrophilic Clostridium spp. causing BPS in meat juice samples (MJS) from chilled vacuum-packed beef and lamb meat imported from other countries to Switzerland. One hundred fifty-four MJS (n = 78 from beef; n = 76 from lamb meat) were screened for psychrophilic Clostridium spp. by quantitative PCR, whereby MJS with a crossing point PCR cycle value <35 and >35 were considered positive and negative, respectively. Psychrophilic Clostridium spp. were detected in 10 MJS, of which 2 were from beef and 8 were from lamb meat. The two beef MJS originated from Spain and Lithuania, whereas the lamb MJS originated from New Zealand (six) and Australia (two). This is the first report of psychrophilic Clostridium spp. in MJS from chilled vacuum-packed beef and lamb meat imported from other countries to Switzerland and provides further evidence that the risk of BPS in lamb meat is higher than in beef.

Antimicrobial effect of different peroxyacetic acid and hydrogen peroxide formats against spores of Clostridium estertheticum

“Blown pack” spoilage is primarily caused by Clostridium estertheticum. The primary source of contamination is probably pelts, faeces and soil during opening cuts and de-hiding. Peroxyacetic acid (POAA) based fogs are commonly included in an abattoir's routine cleaning process. Hydrogen peroxide (H2O2) is a powerful oxidizing agent that penetrates microbe cell walls causing cell death. In this study, we compared the ability of H2O2 and OXYSAN ZS (POAA containing 1-hydroxyethylidine-1,1-diphosphonic acid as a stabilizer) in different formats to inactivate C. estertheticum spores. Hydrogen peroxide treatment using Phytagel™ gel as carrier was effective on fleece against both naturally contaminating microflora and C. estertheticum spores. This is the first time an antimicrobial treatment has been shown to inactivate C. estertheticum spores on such a complex and highly contaminated matrix. Both H2O2 and OXYSAN ZS treatments inactivated C. estertheticum spores on stainless steel indicating their potential use as an in-plant decontamination procedure or inclusion in routine in-process cleaning.

“Blown pack” Probabilistic Modeling for C. Algidicarnis and C. Estertheticum under the Effects of Storage Temperature, Vacuum Level and Package Shrink Temperature

A model for ‘blown pack’ probability (BPP) caused by spores of C.estertheticum DSM8809 and C.algidicarnis, was developed as a function of vacuum packaging variables: storage temperature(ST:-2, 2, 4 and 15°C), vacuum level(VL:6 and 9mBar) and heat shrink temperature(HST:83 and 87°C). Beef meat pieces, were inoculated with spore suspensions individually at 102spores/cm2, packed and daily monitored up to 90 days. The lower BPP, estimated by the log-logistic model, for C.algidicarnis was 0.8% at:-1.5°C/6mBar/87°C while for C.estertheticum was 99.13% at the same conditions. For both organisms, tested variables were unable to eliminate the risk of blown packaged spoilage, at 102spores/cm2 contamination level.

Effects of the initial numbers of spores of Clostridium estertheticum on the development of blown pack spoilage of vacuum packaged beef of normal and intermediate pH

Effects of the initial numbers of spores of Clostridium estertheticum on the development of blown pack spoilage of vacuum packaged beef of normal and intermediate pH

Characterization of a potentially novel ‘blown pack’ spoilage bacterium isolated from bovine hide

To characterize a psychrotrophic bacterium, designated TC1, previously isolated from a cattle hide in Ireland, and to investigate the ability of this strain to cause 'blown pack' spoilage (BPS) of vacuum-packaged beef primals. TC1 was characterized using a combination of phenotypic, chemotaxonomic and genotypic analyses and was assessed for its ability to spoil vacuum-packaged beef at refrigerated temperatures. TC1 was Gram-positive and formed elliptical subterminal endospores. The strain was able to grow between 0 and 33 °C, with optimal growth between 23 and 24 °C. TC1 could be differentiated from its phylogenetically closest neighbour (Clostridium lituseburense DSM 797(T)) by 16S rRNA gene sequencing, pulsed-field gel electrophoresis and cellular fatty acid composition. TC1 spoiled (BPS) beef within 42 days when inoculated in cold-stored (1 °C) vacuum-packed beef. The phenotypic, chemotaxonomic and genotypic characterization indicated that TC1 may represent a potentially novel, cold-tolerant, gas-producing bacterium of considerable economic significance to the beef industry. This study reports and characterizes an emerging BPS bacterium, which should be considered in future activities designed to minimize the psychrophilic and psychrotrophic spoilage of vacuum-packaged beef.

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 heat shrink treatment and storage temperature on the time of onset of “blown pack” spoilage

This study determined the effects of (a) the short “heat shrink” treatment frequently applied to vacuum packed meats within normal commercial production, and (b) chill holding storage temperature, on the subsequent time to onset (TTO) of “blown pack” spoilage (BPS). Beef or lamb steaks were inoculated with 10 3 CFU/cm 2 of spore suspensions of five gas producing clostridia, vacuum packed (VP) and treated as follows: no heat, 50 °C/15 s, 70 °C/10 s or 90 °C/3 s. Samples were stored at −1.5, 1 or 4 °C and examined daily to determine TTO of BPS. For each strain, pack treatment and storage temperature had significant ( P < 0.05 and P < 0.001 respectively) effects on TTO of BPS, i.e. 90 °C/3 s < 70 °C/10 s < 50 °C/15 s ≤ “no heat”, and 4 °C < 1 °C < −1.5 °C. The study suggested that the meat industry could reduce the risks of BPS by avoiding higher temperature (90 °C/3 s or 70 °C/10 s) heat shrinking, and by storing VP meats at lower temperatures (e.g. −1.5 °C).

A CASE OF “BLOWN PACK” MEAT LINKED TO CLOSTRIDIUM ESTERTHETICUM IN IRELAND

ABSTRACT The objective of this study was to determine whether Clostridium estertheticum can be linked to the “blown pack” of vacuum‐packed beef and lamb. Vacuum‐packed chilled beef and lamb samples that had been spoiled by gross gas distension were obtained from two Irish meat processing plants. A sample of purged liquid from each sample was removed from the each meat pack and divided into 3 replicate samples. Both polymerase chain reaction amplification and conventional culturing methods was used to detect C. estertheticum in each purge sample. DNA sequencing was used to confirm its detection. C. estertheticum DNA was detected in beef but not detected in lamb. C. estertheticum was not isolated from the beef or lamb purge samples. This study represents the first confirmed link between C. estertheticum and the blown pack vacuum‐packed beef in Ireland.PRACTICAL APPLICATIONSBlown pack spoilage of vacuum packed meat is a significant problem in the meat industry and has the potential to cause considerable economic loss to meat processors. This study found, the first evidence of Clostridium estertheticum in vacuum packed meat in Ireland. This research will contribute to a better understanding of blown pack spoilage of vacuum packed meat caused by C. estertheticum.

The effect of storage temperature and inoculum level on the time of onset of ‘blown pack’ spoilage

To examine the effect of storage temperature and inoculum level on the time of onset of 'blown pack' spoilage (BPS) caused by psychrotolerant bacteria in vacuum-packed (VP) meats. Gas-producing species and strains (n = 11), recovered in our laboratory or reported as associated with BPS, were inoculated onto beef or lamb meat pieces at final levels of <10, 10, 10(2) and 10(3) CFU cm(-2), VP and stored at -1.5, 1 or 4 degrees C. Six strains produced observable amounts of gas within 42 days and a further four strains produced gas within 100 days. BPS was observed earliest in VP meats inoculated with Clostridium estertheticum ssp. estertheticum at all inoculum levels/storage temperature combinations examined. Storage temperature and inoculum level significantly affected (P < 0.001 and P < 0.05 respectively) the onset of BPS in all cases. Controlling contamination levels and lowering the storage temperature delay the onset of BPS. The study demonstrates the positive effects of low contamination-low temperature as control interventions preventing/delaying BPS in VP chilled meats and identifies some of the contaminants most likely to cause BPS in chilled stored VP meat products.

Sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats, as determined by PCR amplification procedure

To determine possible preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats. Molecular methods based on the polymerase chain reaction (PCR) amplification of specific 16S rDNA fragments were used to detect the presence of Clostridium gasigenes, Clostridium estertheticum, Clostridium algidicarnis and Clostridium putrefaciens in a total of 357 samples collected from ten slaughter stock supply farms, slaughter stock, two lamb-processing plants, their environments, dressed carcasses and final vacuum-packed meat stored at -0.5 degrees C for 5(1/2) weeks. Clostridium gasigenes, C. estertheticum and C. algidicarnis/C. putrefaciens were commonly detected in farm, faeces, fleece and processing environmental samples collected at the slaughter floor operations prior to fleece removal, but all these micro-organisms were detected in only 4 out of 26 cooling floor and chiller environmental samples. One out of 42 boning room environmental samples tested positive for the presence of C. gasigenes and C. estertheticum, but 25 out of 42 of these samples were positive for C. algidicarnis/C. putrefaciens. Nearly all of the 31 faecal samples tested positive for the presence of C. gasigenes and C. estertheticum; however, only two of these samples were positive for C. algidicarnis and/or C. putrefaciens. Clostridial species that were subject to this investigation were frequently detected on chilled dressed carcasses. The major qualitative and quantitative differences between the results of PCR detection obtained with the primers specific for 'blown pack' -causing clostridia (C. gasigenes and C. estertheticum) and those obtained with primers specific for C. algidicarnis and C. putrefaciens suggest that the control of meat spoilage caused by different groups of meat clostridia is best approached individually for each group. This paper provides information significant for controlling meat spoilage-causing clostridia in the meat-processing plants.

The effect of peroxyacetic acid-based sanitizer, heat and ultrasonic waves on the survival of Clostridium estertheticum spores in vitro

To determine the effect of selected physical and chemical treatments on the survival of 'blown pack'-causing Clostridium estertheticum. The study investigated the survival of the spores of 'blown pack'-causing C. estertheticum following the four treatments, which include: heat alone, ultrasound followed by heat treatment, peroxyacetic acid (POAA)-based sanitizer followed by heat treatment and POAA sanitizer followed by heat treatment in the presence of 20% animal fat. No C. estertheticum survivors were recovered in spore preparations that underwent either of the two treatments with the sanitizer, resulting in the inactivation of 4 to 5 log CFU ml(-1) of spores. Similarly, no survivors were detected in spore preparations that were treated with the sanitizer for 5 min at room temperature without further heat treatment. When using heat alone and ultrasound followed by heat treatment, complete spore inactivation did not occur for spores heated at times and temperature combinations other than 240 s at 100 degrees C. POAA sanitizer used with or without heat is capable of in vitro inactivation of at least 4 log CFU ml(-1)C. estertheticum spores. The data generated in the study provide background information for controlling 'blown pack'-causing clostridia on dressed carcasses and in meat plant environment.

Influence of Peroxyacetic Acid–Based Carcass Rinse on the Onset of “Blown Pack” Spoilage in Artificially Inoculated Vacuum-Packed Chilled Beef

“Blown pack” spoilage is an increasingly reported spoilage condition of vacuum-packed chilled meats. This spoilage condition is primarily caused by a psychrophilic obligately anaerobic microorganism, Clostridium estertheticum. The present study investigated whether peroxyacetic acid (POAA)–based carcass rinse can delay the onset of gas production in chilled vacuum-packed beef artificially inoculated with C. estertheticum spores. The variables studied were (i) two prepackaging meat rinses (water and POAA-based rinse); (ii) three levels of C. estertheticum spores (0, 4, and 40 spores per cm2); and (iii) three postpackaging storage temperatures (−1.5, 0, and 2°C). Treatment with POAA-based rinse marginally delayed the onset of pack blowing in packs carrying high numbers of C. estertheticum spores but not in packs carrying low levels of inoculum or in uninoculated controls. The presence of as few as 4 spores per cm2 of meat surface effectively decreased by two-thirds the nominal shelf life of vacuum-packed chilled beef. Increasing the inoculum by 10-fold to 40 spores per cm2 resulted in the additional acceleration of the onset of pack blowing. The onset of gas production was significantly delayed by storing the packaged product at −1.5°C rather than at 0°C. The results of this study indicate that the POAA-based rinse tested will not eliminate the spoilage threat posed by clostridial blown pack spoilage spores present on meat surfaces. POAA-based rinse can be used alone to achieve some extension of shelf life of beef cuts heavily contaminated with C. estertheticum spores. Alternatively, the rinse may offer an opportunity for a more substantial extension of shelf life of contaminated cuts when used with additional hurdles.

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.

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.

The abattoir source of culturable psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled venison.

To identify the abattoir source(s) of culturable psychrophilic clostridia causing 'blown pack' spoilage of vacuum-packed chilled meats. Psychrophilic and psychrotolerant clostridia were isolated from hides, faeces and tonsils of deer slaughter stock, and from a meat plant environment. The isolates were differentiated using restriction fragment length polymorphism analysis of the 16S rDNA gene (PCR-RFLP) and 16S-23S rDNA internal transcribed spacer (ITS) analysis. PCR-RFLP group I clostridia were found to have restriction patterns indistinguishable from the patterns of 'blown pack'-causing Clostridium gasigenes DB1A(T) and R26. Gas production in packs inoculated with vegetative cells of PCR-RFLP group I clostridia was first evident after 14 days at 2 degrees C. The prevalence of these clostridia was similar in hide and faecal samples from slaughter animals, but these micro-organisms were absent from tonsils and the meat plant environment. Banding patterns of PCR-RFLP group II clostridia showed some cross-similarity with patterns of the 'blown pack'-causing micro-organism Cl. estertheticum DSM 8809(T) and Cl. estertheticum-like meat strains. The majority of clostridia in PCR-RFLP group II were found in the faeces of slaughter animals. Isolates representing PCR-RFLP group II did not, however, produce gas in vacuum packs stored at 2 degrees C for 84 days. The data suggest that soil particles attached to hide or present in faeces are the most probable primary reservoir from which 'blown pack' clostridia are introduced onto carcasses. Therefore, dressing procedure hygiene remains paramount in order to control the spread of psychrophilic Clostridium spp. in a meat plant. The paper provides information critical for controlling 'blown pack' spoilage in meat processing plants. It reports on the use of molecular techniques for determination of abattoir sources of 'blown pack'-causing clostridia.