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“Blown-pack” Spoilage of Beef
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- Author / Creator
- Ho, Linda
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“Blown-pack” spoilage of beef by psychrotrophic Clostridium spp. occurs sporadically and resistance of the endospores to most interventions makes them very difficult to control. Current interventions used to control spoilage and pathogenic bacteria, such as acid washes and heat treatments, do not inactivate the endospores and do not control “blown-pack” spoilage. Research was done to identify bacteria isolated from “blown-pack” spoiled beef. The heat and pressure resistance of psychrotrophic Clostridium spp. isolated from “blown-pack” spoiled beef was determined and the impact of antimicrobials on heat and pressure resistance was assessed. Presence of small acid soluble (SASPs) proteins was detected because of their role in heat resistance. Genomes of psychrotrophic Clostridium spp. were sequenced to confirm identity and to confirm the presence of genes that encode for SASPs. Psychrophilic and psychrotrophic Clostridium spp. were isolated from the meat and purge of a vacuum packaged beef loin that had evidence of “blown-pack” spoilage. During isolation of bacteria, the meat samples and cultures were kept under anaerobic conditions, and organisms were cultivated on rich media under anaerobic conditions at 7°C. Presumptive identification of anaerobic organisms by species-specific PCR and sequencing of the 16S rDNA confirmed them to be Clostridium estertheticum and Clostridium putrefaciens. HPLC analysis of volatile compounds in the purge detected the presence of volatiles. The strain of Cl. estertheticum isolated was only able to grow at temperatures below 10°C whereas the strain identified as Cl. putrefaciens was able to grow at 7 to 21°C. When Clostridium endospores were heated to 90°C under aerobic conditions, no survivors were detected; however, when endospores were heated under anaerobic conditions they survived heating at 70°C for 8 min. Endospores of a strain of Cl. estertheticum were able to survive heating to 90°C for 4 min under anaerobic conditions. Small acid soluble proteins, which are critical for heat resistance of endospores, were detected in endospores of cold-tolerant Clostridium. Combination of high hydrostatic pressure (HHP) at 400 MPa and heat (4, 40 and 70°C) increased inactivation of Clostridium endospores; however, additives altered the efficacy of HHP. When endospores were subjected to HHP in meat with the antimicrobial preparation Micocin X, endospores survived 15 min of pressure at 4°C, whereas without the Micocin X, endospores of Clostridium estertheticum BP09-13 and ATCC 51377 were inactivated at 8 min. Similar results were observed when the supernatant of a strain of Carnobacterium maltaromaticum or APT were added to the meat prior to pressure treatment. To determine if the presence of peptidoglycan could account for the increased survival, peptidoglycan from Bacillus spp. was added to saline containing endospores and samples were subjected to pressure treatment. In this case, the addition of peptidoglycan increased the inactivation of the endospores; thus peptidoglycan is not responsible for the protective effect observed with the addition of Micocin X. The genomes of the 3 isolates from this study were sequenced. This was the first time genomes of Clostridium spp. isolated from “blown-pack” spoiled beef there sequenced. Comparison of the genomes of the two strains of psychrophilic Cl. estertheticum and the strain of psychrotrophic Cl. putrefaciens revealed that the Cl. estertheticum had a larger genome than Cl. putrefaciens. The strain that was presumptively identified by 16S rDNA sequencing as Cl. putrefaciens was closely aligned to Clostridium algidicarnis and was thus renamed. Annotation of the genes indicated that nearly half of the genes present were hypothetical for all three genomes analyzed. Analysis of the genomes of the strains of Cl. estertheticum revealed 16 single nucleotide polymorphisms in transport proteins. The presence of genes that encode small acid soluble proteins was confirmed in Cl. estertheticum and Cl. algidicarnis. Current interventions used in industry are insufficient to prevent the occurrence of “blown-pack” spoilage in vacuum packaged beef, therefore the organisms responsible for “blown-pack” spoilage continue to be a problem for beef producers. Combinations of pressure and antimicrobial preparations did not increase activation, but instead increased survival of the Clostridium spp. Despite unsuccessful control of Clostridium spp. in meat, genomic information could identify key weaknesses in the life cycle or structure of the endospore to target. Further research and analysis of the genome could reveal the answers required to prevent the sporadic occurrence of “blown-pack” spoilage of vacuum packaged meats.
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- Graduation date
- Fall 2016
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.