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.     

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.     

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.     

Effects on the development of blown pack spoilage of the initial numbers of Clostridium estertheticum spores and Leuconostoc mesenteroides on vacuum packed beef

Beef steaks were inoculated with Clostridium estertheticum spores and Leuconostoc mesenteroides cells at all combinations of numbers of 0, 10, 100 or 1000/cm(2) for each organism. After vacuum packaging the steaks were stored at 4, 1, or -1.5°C. Pack volumes were determined by water displacement at suitable intervals. Irrespective of L. mesenteroides numbers, for packs containing meat inoculated with 0, 10, 100 or 1000 spores/cm(2), 60, 16, 3 and 1 of 60 packs did not swell. The times of onset of swelling were twice as long at -1.5 as at 4°C, but they were not affected by the inoculated numbers of L. mesenteroides. Rates of pack swelling increased with increasing storage temperature and number of spores, but decreased with increasing numbers of inoculated L. mesenteroides. Lactic acid bacteria can apparently prevent development of blown pack spoilage of vacuum packs containing meat contaminated with low numbers of C. estertheticum.     

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 103 CFU/cm2 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).     

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

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

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.     

Gas‐producing and spoilage potential of Enterobacteriaceae and lactic acid bacteria isolated from chilled vacuum‐packaged beef

This study aimed to enumerate and identify lactic acid bacteria and Enterobacteriaceae from spoiled and nonspoiled chilled vacuum‐packaged beef and determine their potential to cause blown pack spoilage. These microbial groups were also enumerated in nonspoiled samples and detected in abattoir samples. The potential of isolates to cause ‘blown pack’ spoilage of vacuum‐packaged beef stored at chilled temperature (4 °C) and abuse temperature (15 °C) was investigated. Populations of lactic acid bacteria in exudate of spoiled and nonspoiled samples were not significantly different (P > 0.05), whereas the number of lactic acid bacteria on the surface was significantly higher (P < 0.05) in spoiled samples as compared to nonspoiled samples. The population of Enterobacteriaceae species in exudate and on the surface of samples were significantly higher (P < 0.05) in spoiled packs in comparison with nonspoiled packs. Results of the deterioration potential showed that ‘blown pack’ spoilage was noticeable after 7 days at 15 °C and after 6 weeks at 4 °C for samples inoculated with Hafnia alvei.     

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.     

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 10(3)per ml) otherwise a pre-enrichment was required.     

Reduction of spoilage of chilled vacuum-packed lamb by psychrotolerant clostridia

Methods for the reduction of spoilage, of lamb, by psychrotolerant clostridia were investigated including exposure to air, hot and cold water spray washing and tyndallisation. Initially vegetative cells of psychrotolerant clostridia associated with spoilage of chilled vacuum-packed meat were exposed to aerobic cooked meat medium at room temperature (21 °C) to determine how long they remained viable. Survival of strains varied from 2 h to 3 days. Vegetative cells of Clostridium estertheticum subsp. estertheticum survived 7 days at 10 °C with little reduction in viable numbers. This ruled out exposure to air as a practical method for reducing spoilage. Trials were also carried out on chilled vacuum-packed lamb inoculated with spores of Cl. estertheticum subsp. estertheticum. The time until inoculated packs reached the loss of vacuum stage varied from 38 to 53 days. Hot and cold water washing extended the shelf life by 12 to 13 days in comparison to untreated packs.     

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.     

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.     

Isolation of lactic acid bacteria with inhibitory activity against pathogens and spoilage organisms associated with fresh meat

The use of lactic acid bacteria (LAB) as protective cultures in vacuum-packed chill-stored meat has potential application for assuring and improving food quality, safety and market access. In a study to identify candidate strains suitable for evaluation in a meat model, agar-based methods were employed to screen 181 chilled meat and meat process-related LAB for strains inhibitory to pathogens and spoilage organisms of importance to the meat industry. Six meat-derived strains, including Lactobacillus sakei and Lactococcus lactis, were found to be inhibitory to one or more of the target strains Listeria monocytogenes, Brochothrix thermosphacta, Campylobacter jejuni and Clostridium estertheticum. The inhibitory agents appeared to be either cell-associated or molecules released extracellularly with bacteriocin-like properties. Variations detected in the antimicrobial activity of LAB associated with changes to test parameters such as substrate composition underlined the importance of further in situ evaluation of the inhibitory strains in stored meat trials.     

Spoilage-related microbiota associated with chilled beef stored in air or vacuum pack

In order to study the spoilage-related microbiota of beef at species level, a combination of culture-independent and culture-dependent methods was used to analyse nine different beef samples stored at 4 °C in air or in vacuum pack. Plate counts on selective agars after 0, 7 and 20 days of storage showed that vacuum packaging reduced the viable counts of Brochothrix thermosphacta, Pseudomonas spp. and Enterobacteriaceae, whereas the growth of lactic acid bacteria (LAB) was unaffected. Storage in vacuum pack mainly affected viable counts and not necessarily the species diversity of microbial populations on meat. Such populations were studied by PCR-DGGE of DNA directly extracted from meat and from bulk cells from culture media, followed by sequencing of DGGE fragments. Pseudomonas spp., Carnobacterium divergens, B. thermosphacta, Rahnella spp. and Serratia grimesii, or close relatives were detected in the meat at time zero. The use of the culture-independent method highlighted the occurrence of species that were not detected by plating. Photobacterium spp. occurred in most meat samples stored in air or in vacuum pack, which indicates this organism probably has a role in spoilage. In contrast, culture-dependent analysis allowed detection of bacterial species that were not found in DNA extracted directly from meat. This was the case for several species of Serratia or Rhanella among the enterobacteria, and Leuconostoc spp. among the LAB. Besides advancing our knowledge of the species involved in the spoilage of vacuum-packaged meat, this study shows the benefits of combining culture-based and direct approaches to enhance understanding of populations of spoilage bacteria.     

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.     

Effect of modified atmosphere packaging on structural and physical changes in buffalo meat

The effect of modified atmosphere packaging of buffalo meat on the structural parameters viz., fibre diameter, sarcomere length and myofibrillar fragmentation index and physical parameters viz., pH, drip loss and colour scores were studied. The buffalo meat was packed under aerobic, vacuum and modified atmosphere (80% oxygen+20% carbon dioxide) and stored at 4±1°C upto 21 days. The results obtained revealed that vacuum-packed buffalo meat had the lowest fibre diameter and myofibrillar fragmentation index and the highest sarcomere length, vacuum thus appears to enhance ageing. Buffalo meat packed in modified atmosphere had a low drip loss and a desirable colour. The modified atmosphere packed and vacuum-packed buffalo meat was acceptable for upto 14 days at 4±1°C.     

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.     

Vacuum skin pack of beef--a consumer friendly alternative

The aim of this study was to assess how beef quality traits are affected by skin packaging compared with vacuum and high-oxygen modified atmosphere packaging (MAP; 80% O(2) and 20% CO(2)). Both M. longissimus dorsi from 6 young bulls were cut and aged in vacuum for 7 days and then cut into 3.5-cm steaks, thereafter either frozen directly day 7 or stored in skin pack, vacuum pack or MAP for an additional 7 or 14 days and then frozen. Warner-Bratzler shear force, loss and colour were measured and sensory analysis was performed. The results showed no differences in shear force, sensory analysis and total loss between steaks packed in skin pack or vacuum, but skin packed steaks had lower purge loss. MAP steaks had lower sensory scores than the other treatments at days 14 and 21. Raw MAP-steaks were bright red, but showed signs of premature browning when cooked.     

Spoilage of vacuum-packed beef by a clostridium sp

An unusual spoilage condition of chill-stored normal pH vacuum-packed beef characterised by gross distension (blowing) of the packs and off-odours perceived on initial opening of the packs as ‘sulphurous’ and subsequently as ‘fruity’, ‘solvent-like’ and ‘strong cheese’ has been chemically defined. Hydrogen and carbon dioxide were the major components of the headspace gases. Other components included hydrogen sulphide, methanethiol, dimethyl sulphide, dimethyl di- and trisulphides, methylthioacetate, l-butanol, acetic and butanoic acids, and l-butyl esters of a range of C_1-C₆ fatty acids. Published sensory data are consistent with components of this mixture being responsible for the off-odours detected. When grown in pure culture on beef under identical storage conditions, a Clostridium sp isolated from one of the blown packs produced the same chemical and sensory spoilage characteristics.