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

Spoilage 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.     

The spoilage characteristics of Brochothrix thermosphacta and two psychrotolerant Enterobacteriacae in vacuum packed lamb and the comparison between high and low pH cuts

The spoilage potential of Brochothrix thermosphacta, Serratia proteamaculans and Rahnella aquatilis was investigated in vacuum packaged high (5.9 to 6.4) and low (5.4 to 5.8) pH lamb. Vacuum packaged fore shank (m. extensor carpi radialis) and striploins (m. longissimus dorsi) (n = 306) inoculated with ~ 100 CFU of individual bacteria were stored for twelve weeks at temperatures − 1.5, 0, 2 and 7 °C. Spoilage characteristics and bacterial numbers were recorded and analysed in comparison to un-inoculated control samples. All three bacterial species were shown to grow in vacuum packaged lamb of pH values between 5.4 and 6.4, when stored at chilled temperatures (− 1.5 to 7 °C) for up to 84 days. B. thermosphacta and S. proteamaculans caused spoilage to the meat under these conditions whilst R. aquatilis spoiled high pH meat at 7 °C. These results go against previous beef models stipulating that Brochothrix and Enterobacteriacae species cannot grow on or cause spoilage of low pH meat in the absence of oxygen.     

Detection of cold-tolerant clostridia other than Clostridium estertheticum in raw vacuum-packed chill-stored meat

Samples from raw chill-stored vacuum-packed beef, lamb and venison or the meat processing environment, associated with a spoilage problem, but negative for Clostridium estertheticum using a specific real-time PCR test, were examined for other Clostridium spp. using direct 16S rDNA PCR-RFLP and sequencing. Of 291 samples tested by PCR, presence of clostridia was indicated in 123 and there was sufficient PCR product in 35 to be further investigated. Presence of Clostridium spp. was confirmed by RFLP and sequencing in 25/35 samples (11 of 14 incidents). Species detected in spoiled meat were (incidents): Clostridium tagluense-like (4), Clostridium putrefaciens (2), Clostridium algidicarnis (3), Clostridium frigoris/estertheticum-like (3) and Clostridium. gasigenes (2). More than one species was detected in some incidents. All of the above species have previously been associated with spoiled meat apart from the Cl. tagluense-like species. Clostridia were also confirmed in 4/7 samples from the environment, with two Cl. frigoris/estertheticum-like and two mesophilic species of Clostridium. Our study showed that, cold-tolerant Clostridium species other than Cl. estertheticum are occasionally associated with spoiled vacuum-packed meat, particularly lamb. Further studies are required to confirm the exact identity of the Cl. tagluense-like species and its role in meat spoilage.     

Factors affecting microbial spoilage and shelf-life of chilled vacuum-packed lamb transported to distant markets: A review

Vacuum-packaging and stringent control of storage temperatures enable the export of meat to distant markets, supplying a chilled product that can favourably compete with local fresh meats. To save fuel and reduce emissions, the speed of ships travelling to international markets has decreased resulting in requirement for the shelf-life of chilled lamb to be extended beyond the recognised time of 60–70 days. Growth of microorganisms and ability to cause spoilage of vacuum-packed lamb are dependent on many factors, including the type and initial concentration of spoilage bacteria, meat pH, water activity, availability of substrates, oxygen availability and, most importantly, storage time and temperature of the packaged product. This paper reviews the existing knowledge of the spoilage bacteria affecting vacuum-packed lamb, discusses the impact of these bacteria on product quality, shelf-life and spoilage, and concludes that under specified conditions the shelf-life of chilled lamb can be extended to beyond 70 days.     

Possible involvement of psychrotolerant Enterobacteriaceae in blown pack spoilage of vacuum-packaged raw meats

Recent investigations of blown pack spoilage in New Zealand chilled vacuum-packaged meats have found moderate to high numbers of Enterobacteriaceae in the spoilage flora, but no clostridia, such as C. estertheticum and C. gasigenes, that are usually associated with blown pack spoilage. This study showed that pyschrotolerant Enterobacteriaceae produced gas in a lamb homogenate model under anaerobic conditions and that these organisms could cause blown pack spoilage of vacuum-packaged chilled meats. Significant gas production was observed with the majority of the psychrotolerant Enterobacteriaceae strains tested including presumptive species of Enterobacter, Serratia, Hafnia and Rahnella. However, no gas was produced in lamb homogenates inoculated with presumptive species of Ewingella americana or Yersinia enterocolitica. Gas production was also confirmed in vacuum-packaged lamb shoulders stored at 4 degrees C for 21 days after being inoculated with individual representative Enterobacteriaceae isolates. Biochemical characterisation proved to be more useful than genotype-based typing of 16S rRNA genes for discriminating different psychrotolerant Enterobacteriaceae from naturally contaminated meat microflora.     

Spoilage characteristics of Brochothrix thermosphacta and campestris in chilled vacuum packaged lamb,and their detection and identification by real time PCR

The spoilage potential of Brochothrix campestris and Brochothrix thermosphacta was investigated in vacuum-packed lamb. Striploins (n = 338) were inoculated and stored for twelve weeks at temperatures − 1.5, 0, 2 and 7 °C. Growth around 5–6 log₁₀ CFU/cm² was recorded after six weeks at 0, 2 and 7 °C, and ~ 3 log₁₀ CFU/cm² after nine weeks at − 1.5 °C. B. campestris was shown to cause spoilage by nine weeks at temperatures above 0 °C by the presence of green drip and unacceptable odours. Molecular based assays for the detection and differentiation of B. thermosphacta and B. campestris were developed and validated. A TaqMan assay was designed to target a unique single-nucleotide polymorphism in the Brochothrix 16 s rRNA gene with a sensitivity of < 7 CFU per reaction. Secondly a specific PCR was designed for B. campestris targeting the structural genes, brcA and brcB. These testing regimes offer a rapid and cost effective method for the detection and screening of Brochothrix species in meat products and processing environments.     

Shelf life extension of whole Norway lobster Nephrops norvegicus using modified atmosphere packaging

Once a nuisance by-catch, today the Norway lobster (Nephrops norvegicus) is a valuable UK fisheries commodity. Unfortunately, the species is very susceptible to quality deterioration post harvest as it quickly develops black spots and also spoils rapidly due to bacterial growth. Treatment with chemicals can stop the blackening and carefully monitored cold storage can result in a sensory shelf life of up to 6.5 days. The high susceptibility to spoilage greatly restricts the extent to which N. norvegicus can be distributed to retailers and displayed for sale. The application of modified atmosphere (MA) could be extremely beneficial, allowing the chilled product to stay fresh for a long period of time, thus ensuring higher sales. In the present study, we identified a gas mix for the MA packaging (MAP) of whole N. norvegicus lobster into 200 g retail packs. Our results show that a shelf life extension to 13 days can be achieved when retail packs are stored in MAP at 1 °C. Effectiveness of the MAP was evaluated by using a newly developed QIM for MA-packaged whole N. norvegicus and also by analyzing bacterial plate counts. Changes in the microflora and effects of different storage temperatures on the quality of the MA packs are also presented. The main specific spoilage organism (SSO) of modified atmosphere packaged Norway lobster is Photobacterium phosphoreum.     

Development and validation of a real-time PCR assay specific for Clostridium estertheticum and C. estertheticum-like psychrotolerant bacteria

A new real-time PCR assay was developed targeted to the psychrotolerant spoilage bacteria, Clostridum estertheticum, a causative agent of ‘blown-pack’ spoilage of vacuum packaged meats during chilled storage. Further, a robust validation of the sensitivity and specificity in different meat processing related matrices was carried out. Results show that real-time PCR is a valid method for the detection of C. estertheticum spores as long as consideration is given to the matrix being tested and the sensitivity of detection required. For meat, hide, blood/drip and environmental swabs it was possible to detect low numbers of C. estertheticum spores (approx 3 spores per ml) by direct real-time PCR (without pre-enrichment of the samples). For faeces and soil matrices, a cold temperature enrichment step was required prior to DNA extraction and real-time PCR analysis to increase the ability to detect samples containing C. estertheticum spores; this was particularly important when the samples contained low numbers of spores (less than 3 spores per ml). For matrices with high levels of PCR inhibitors such as soil, it was necessary to dilute the extracted DNA sample 100 fold especially for detection of high levels of contamination (greater than 10³ per ml) otherwise a pre-enrichment was required.     

Effects of dietary rosemary extract on lamb spoilage under retail display conditions

A dietary rosemary extract (RE) was tested to extend the shelf life of raw lamb meat. Lambs were supplemented with 0.6 mg kg⁻¹ RE during fattening (from 13 to 25 kg live weight). Meat spoilage (total viable counts, psycrophilic bacteria, Enterobacteriaceae, molds and yeasts), TBARS, CIE L*a*b* color and the sensory traits of lamb cuts were analyzed on days 0, 7, 14 or 21 under retail display conditions (70/30 O₂/CO₂ atmosphere, 2 °C temperature and 1600 lx lighting). Supplementation of the lamb diet with RE was effective (P < 0.05) in prolonging the time chilled-packed lamb cuts could be kept under retail display conditions. Dietary rosemary clearly inhibited lipid oxidation and rancidity, and was moderately efficient in preventing sensory deterioration and microbial spoilage. Although the results concerning meat preservation were limited, the dietary use of rosemary extracts in lambs seems to be promising as a nutritional strategy for improving meat quality.     

Irradiated vacuum-packed lamb meat stored under refrigeration: Microbiology,physicochemical stability and sensory acceptance

Reducing spoilage and indicator bacteria is important for microbiological stability in meat and meat products. The objective was to evaluate the effect of different doses of gamma radiation on the shelf-life of lamb meat, vacuum-packed and stored under refrigeration, by assessing the microbiological safety, physicochemical stability and sensory quality. Lamb loin cuts (Longissimus dorsi) were irradiated with 1.5 kGy and 3.0 kGy. The samples, including control, were stored at 1 ± 1 °C during 56 days. Samples were analyzed on zero, 14, 28, 42 and 56 days by their microbiological and physicochemical characteristics. Sensory quality was carried out on day zero. The results showed a reduction (p < 0.05) in the microbial load of the irradiated samples. The acceptance of lamb loins was not affected (p > 0.05) by the radiation doses. Thus gamma irradiation at 3.0 kGy was effective in reducing the content of microorganisms, without harming the physicochemical characteristics evaluated.     

Effects of sodium acetate dip treatment and vacuum-packaging on chemical,microbiological, textural and sensory changes of Pearlspot (Etroplus suratensis) during chill storage

The effects of sodium acetate dip treatment, followed by vacuum-packaging, on the shelf life of beheaded, scaled and gutted Pearlspot (Etroplus suratensis) during chill storage were examined. Sodium acetate (2%, w/v) solution was used for the dip treatment. Pouches (size: 15 × 22 cm) made of 12μ-polyester laminated with 300 gauge low-density polyethylene were used for packing fish. After packing, all the packs were iced with flake ice in the ratio (1:1) fish: ice in an insulated box and were kept in a cold room maintained at 0–2 °C. The control and the treated packs were analysed periodically for chemical (pH, TBA, TMA, TVB-N), microbiological (total viable count), textural and sensory characteristics. Changes in Staphylococcus aureus, Enterobacteriacea and Feacal streptococci were determined for fresh fish and for fish samples at the time of sensory rejection. Air packed samples were found to have a shelf life of about 8 days; vacuum-packed samples were found to be acceptable up to 10 days, whereas sodium acetate-treated vacuum packed samples were found to be acceptable up to 15 days. Thus, vacuum-packaging, in combination with sodium acetate, was found to delay the spoilage, thereby significantly extending the shelf life of Pearlspot at refrigeration temperatures.     

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

To understand how the initial numbers of Clostridium estertheticum spores on, and the concentration of glucose in meat affect the development of blown pack spoilage, beef of pH ≤5.6 and of pH ≥5.8 was inoculated with the spores at various numbers, vacuum‐packaged and stored at 2 °C. For beef of pH ≤5.6, the volumes of packs inoculated with ≤10 spores did not change; and packs inoculated with ≥30 spores started swelling after 35 days, and the rate of volume increase increased with increasing number of inoculated spores. For beef of pH ≥5.8, packs inoculated 0, three or ten spores slackened, and packs inoculated with ≥30 spores became swollen at the end of storage, but to a much lesser degree than the corresponding packs of beef of pH ≤5.6. Glucose was reduced by 21 mm and depleted in the rinse fluids from swollen packs of beef of pH ≤5.6 and of pH ≥5.8, respectively.     

Protective action of Lactobacillus curvatus CRL705 on vacuum-packaged raw beef. Effect on sensory and structural characteristics

Lactobacillus curvatus CRL705 was examined for its effectiveness as protective culture in the biopreservation of vacuum-packaged fresh beef stored during 60 days at 2 °C. For this purpose, L. curvatus CRL705, producer of lactocin 705 and lactocin AL705, was inoculated on the meat surface (10⁶ cfu g⁻¹). This microorganism became the dominating population throughout the storage period controlling the growth of Brochothrix thermosphacta and spoilage lactic acid bacteria naturally present on the meat. When the microstructural characteristics of the meat were evaluated using light microscopy, beef samples inoculated with the bioprotective culture showed a 10 days delay for the appearance of tissue degradation signs. Sensory analysis demonstrated that beef samples treated with L. curvatus CRL705 only developed an “acid” off-flavor after 60 days of refrigerated storage, and no undesirable off-odors were found. Therefore, inoculation with this bacteriocinogenic strain would provide an additional hurdle to improve storage life of refrigerated vacuum-packaged beef without affecting its sensory and structural characteristics.     

Evaluation of stored lamb bio-preserved using a three-strain cocktail of Lactobacillus sakei

Commercially prepared lamb was stored at −1.5 °C after inoculation with a combination of three Lactobacillus sakei strains previously shown to inhibit spoilage and pathogenic bacteria of importance to the meat industry. Between 6 and 14 weeks storage samples were evaluated for growth of inoculated strains, production of fermentation end-products and sensory acceptance of the cooked product. All three L. sakei strains flourished during storage, formed consistently dominant populations and were associated with lower surface pH and increased levels of lactic and acetic acids. Inoculated samples were determined to be as equally acceptable for smell, acidity, rancidity and overall liking as un-inoculated controls.     

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 degrees 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 degrees C rather than at 0 degrees 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.     

Influence of heat shrink treatments on the onset of clostridial “blown pack” spoilage of vacuum packed chilled meat

Storage trials were conducted to determine if short duration high temperature treatments applied in commercial heat shrinking of vacuum barrier packs affect the onset of clostridial blown pack spoilage. Spore suspensions of six significant gas producers were used: Clostridium estertheticum NCIMB 12511, and five local psychrotolerant clostridial isolates. Beef striploins, pH 5.5 and 6.0, were cut into steaks and placed into vacuum pouches. Duplicate pouches were inoculated with each test spore suspension for each meat pH/heat treatment/storage temperature combination. After vacuum packaging and heat shrinking, packs were stored at −1.5, 1 or 4°C. Based on time to first-phase gas production (small bubbles in drip), the test strains were considered statistically as representing two groups. With both groups, post-packaging heat treatment had a significant effect on time to gas production. Mean times, pooled for the three storage temperatures, to gas production with Group A were 49, 39, 36 and 35 days for the no heat, 70, 80 and 90°C treatments, respectively. With Group B these times were 77, 42, 39 and 36 days, respectively. Post-packaging heat shrink treatments of vacuum packaged meat accelerate the onset of Clostridium spp. mediated blown pack spoilage during chilled storage. Possible mechanisms for accelerated onset of blown pack spoilage following heat shrinking of vacuum packs are discussed.     

BOTULINAL TOXIN PRODUCTION IN VACUUM AND CARBON DIOXIDE PACKAGED MEAT DURING CHILLED STORAGE AT 2 AND 4C

This study was undertaken to determine if carbon dioxide packaging of meat afforded a food safety advantage over vacuum packaging with respect to botulinal toxin production during chilled storage. A cocktail of washed spores from five toxigenic clostridial strains – four reference Clostridium botulinum strains [types A, B (2 strains) and E] and a C. butyricum type E strain – was inoculated onto lamb chumps. Of these strains, two were psychrotolerant. The inoculated chumps were individually carbon dioxide packaged and duplicate packs were placed into storage at 10, 8, 6, 4 and 2C. All storage regimens included a weekly defrost cycle when meat surface temperatures increased by up to 6 to 7C during a 2 to 2.5 h period. After 84 days storage, packs were assessed for the presence of botulinal toxin using the mouse bioassay procedure. All packs contained botulinal toxin. To compare toxin production in vacuum and carbon dioxide packs at chill temperatures, the challenge trials were repeated for 4 and 2C storage. Packs were examined at regular intervals for toxin presence. Both pack types contained toxin after 21 and 48 days storage at 4 and 2C, respectively. In the unlikely, but not impossible, event that raw meat would be contaminated with psychrotolerant toxincapable clostridial spores, product safety, with respect to botulinal toxin presence after prolonged chilled storage, requires storage temperatures to be maintained below 2C for both vacuum and carbon dioxide packaged product.     

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.     

Effect on lamb meat quality of including thyme (Thymus zygis ssp. gracilis) leaves in ewes’ diet

The aim of this study was to investigate the effect of including thyme leaves (TL) in the diet of pregnant sheep on the sensorial characteristics, bacterial spoilage and oxidative stability of lamb meat stored in modified atmosphere (70% O₂:30% CO₂). For this, thirty-six sheep were randomly assigned to three groups: control (basal diet), T₁ (3.7% thyme leaves), T₂ (7.5% thyme leaves). Meat spoilage (TV, PSY, MY, ENT, and LA), TBARS, CIELAB coordinates, metmyoglobin and the sensory characteristics of fresh lamb meat were analyzed on days 0, 7, 14 and 21. The presence of antioxidant compounds in the diet containing TL delayed (P < 0.05) colour deterioration, lipid oxidation and bacterial counts, while at the same time imparting a better appearance to the fresh lamb meat. In general, this effect was more pronounced at the higher level of TL (7.5%). High Pearson’s correlation coefficients were found between the sensory attributes, CIELAB coordinates and TBARS.     

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 10¹⁰ CFU/(g, mL or 100 cm²) were found in ‘blown pack’ samples, while in non-spoiled samples populations of 10⁵ CFU/(g, CFU/mL or CFU/100cm²) 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.