Spoilage Of Vacuum-packed Meats Research Articles

Unraveling the Genotypic and Phenotypic Diversity of the Psychrophilic Clostridium estertheticum Complex, a Meat Spoilage Agent.

The spoilage of vacuum-packed meat by Clostridium estertheticum complex (CEC), which is accompanied by or without production of copious amounts of gas, has been linked to the acetone–butyrate–ethanol fermentation, but the mechanism behind the variable gas production has not been fully elucidated. The reconstruction and comparison of intra- and interspecies metabolic pathways linked to meat spoilage at the genomic level can unravel the genetic basis for the variable phenotype. However, this is hindered by unavailability of CEC genomes, which in addition, has hampered the determination of genetic diversity and its drivers within CEC. Therefore, the current study aimed at determining the diversity of CEC through comprehensive comparative genomics. Fifty CEC genomes from 11 CEC species were compared. Recombination and gene gain/loss events were identified as important sources of natural variation within CEC, with the latter being pronounced in genomospecies2 that has lost genes related to flagellar assembly and signaling. Pan-genome analysis revealed variations in carbohydrate metabolic and hydrogenases genes within the complex. Variable inter- and intraspecies gas production in meat by C. estertheticum and Clostridium tagluense were associated with the distribution of the [NiFe]-hydrogenase hyp gene cluster whose absence or presence was associated with occurrence or lack of pack distention, respectively. Through comparative genomics, we have shown CEC species exhibit high genetic diversity that can be partly attributed to recombination and gene gain/loss events. We have also shown genetic basis for variable gas production in meat can be attributed to the presence/absence of the hyp gene cluster.

Comparative genomics of Clostridium species associated with vacuum-packed meat spoilage

Bacterial species belonging to the genus Clostridium have been recognized as causative agents of blown pack spoilage (BPS) in vacuum packed meat products. Whole-genome sequencing of six New Zealand psychrotolerant clostridia isolates derived from three meat production animal types and their environments was performed to examine their roles in BPS. Comparative genome analyses have provided insight into the genomic diversity and physiology of these bacteria and divides clostridia into two separate species clusters. BPS-associated clostridia encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) that enable them to utilize the intramuscular carbohydrate stores and facilitate sporulation. In total, 516 glycoside hydrolases (GHs), 93 carbohydrate esterases (CEs), 21 polysaccharide lyases (PLs), 434 glycosyl transferases (GTs) and 211 carbohydrate-binding protein modules (CBM) with predicted activities involved in the breakdown and transport of carbohydrates were identified. Clostridia genomes have different patterns of CAZyme families and vary greatly in the number of genes within each CAZy category, suggesting some level of functional redundancy. These results suggest that BPS-associated clostridia occupy similar environmental niches but apply different carbohydrate metabolism strategies to be able to co-exist and cause meat spoilage.

Characterization of the bacterial spoilage flora in marinated pork products

To investigate the microbiota in marinated, vacuum-packed pork and to characterize isolated bacteria with regard to their spoilage potential. Laboratory marinated pork meat and commercial products from three Norwegian producers were examined. Lactic acid bacteria dominated in all products at the expiration date. The flora in marinated products was similar only for products from the same plant. Strains of Lactobacillus algidus, Lactobacillus sakei, Lactobacillus curvatus, Carnobacterium divergens, Carnobacterium maltaromaticum, Leuconostoc mesenteroides, Leuconostoc carnosum and Leuconostoc sp. were isolated and tested for their spoilage potential. Samples inoculated with Lact. algidus or Leuc. mesenteroides were rated as most unpleasant by randomly selected people. A sensory panel scored samples with Lact. algidus highest for sour and intense odour. Lactobacillus algidus was found in products from two out of three production plants. Culture-independent DNA isolation confirmed that cultivation on Blood agar at 20 degrees C yielded a representative picture of the total flora in marinated flintsteak. Lactobacillus algidus may be an important, but underestimated, spoilage organism that needs to be focused on more when spoilage of vacuum-packed meat is considered. Routine microbial testing may have to be revised in order to detect spoilage LAB that are unable to grow under currently used conditions.

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.

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.

Clostridium frigidicarnis sp. nov., a psychrotolerant bacterium associated with 'blown pack' spoilage of vacuum-packed meats.

Two strains of a psychrotolerant Clostridium, isolated from vacuum-packed, temperature-abused beef, were characterized using a multiphasic approach. The strains were Gram-positive motile rods producing elliptical subterminal spores during early stationary growth phase. The strains were psychrotolerant. At pH 7.0, they grew between 3.8 and 40.5 degrees C; their optimum growth temperature was 30.0-38.5 degrees C. At 30 degrees C, the pH range for growth was between 4.7 and 9.5; the optimum pH for growth was 6.4-7.2. The organisms were proteolytic and saccharolytic, lecithinase-positive and hydrolysed gelatin. The fermentation products formed in peptone/yeast extract/glucose/starch broth were acetate, ethanol, butyrate, isovalerate, butanol, isobutyrate, oxalacetate, lactate, hydrogen and carbon dioxide. The DNA G + C compositions of the two meat strains were 27.3 and 28.4 mol%. Phylogenetic analyses indicated that the strains belong to Cluster I of the genus Clostridium (sensu Collins et al., 1994). The new strains differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of phenotypic and genotypic characterization data, the strains were assigned to a new species for which the name Clostridium frigidicarnis is proposed; strain SPL77AT (= DSM 12271T) is the type strain.