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The objectives of this study were to determine the microbial ecology of “blown-pack” fresh beef obtained from a federally inspected facility and to use biopreservation to prevent spoilage of vacuum packaged chilled beef. Organic acids and alcohols in the purge obtained from the “blown” beef packages were detected using HPLC. PCR analysis indicated that Clostridium spp. were present in the purge of the commercial sample. Biochemical tests, RFLP and 16S rDNA sequencing were used to identify organisms isolated from the meat. Out of 66 isolated strains, 26 isolates were strict anaerobes and RFLP indicated that all were clonal isolates. Based on sequence analysis, the isolate was identified as Clostridium putrefaciens. The isolate caused “blown-pack” spoilage and produced butyric and propionic acids when inoculated onto fresh meat and Carnobacterium maltaromaticum UAL307 prevented the production gas and obvious signs of spoilage. / Food Science and Technology

Clostridium sporogenes PA 3679 spores were inoculated into a meat-vegetable mixture before extrusion, cooking, and vacuum packaging into "stewsticks" to simulate Clostridium botulinum growth. The experiment was a 3 x 5 x 2 x 3 factorial which determined the influence of pH, water activity, initial spore load, and storage period on spore survival. Spore levels decreased throughout storage for all treatments. Spore levels decreased linearly (P = 0.02) as water activity increased, in samples that were heated to kill vegetative cells and activate spores. Other significant interactions of heat-treated samples were observed with inoculum level (P < 0.01) and storage time (P < 0.01). Spore levels in stored products were also significantly affected by water activity* inoculum level (P = 0.03), pH * time, water activity* time (P = 0.01), inoculum level * time (P < 0.01), and water activity * inoculum levels * time (P < 0.01). The interaction between pH * water activity * time tended towards significance (P = 0.06). Most probable number estimates in nonheated samples accounted for naturally occurring viable cells and spores, and added spores and were significantly affected by the main effects of inoculum level (P < 0.01) and time (P < 0.01). The two-way interactions of water activity * inoculum level (P = 0.04), pH * inoculum level (P < 0.01),water activity * time (P < 0.01), and three-way interaction of pH * inoculum level * time (P = 0.03) were significant. Spore levels approached 102, or less (compared to an inoculum level of 106 spores per gram) due to the effects of many treatments. Some stewstick packages were observed to become "gassy" or "loose" during storage. Subsequently the stewstick packages were used to isolate microorganisms that were able to grow at water activities of 0.96-0.86, in glycerol-adjusted Rogosa agar, and were acid tolerant to pH 4.4-4.2. One produced gas in pure culture, and some produced indole. These bacteria were not destroyed by heating to 74°C for at least 30 minutes, and lowered the pH in the stewstick during storage. In conclusion, in all stewstick samples, regardless of pH or Aw, inoculated clostridial spore levels decreased during storage, apparently because spores germinated and vegetative cells subsequently died. Thus, if stewsticks are cooked to 74°C throughout, have a Aw ≤ 0.86 and pH ≤ 4.8, they appear to be safe.