Spoilage of Vacuum-Packaged Refrigerated Beef by Clostridium

Abstract

A motile, gram-positive, spore forming, anaerobic, psychro-trophic bacterial species, probably from the genus Clostridium, was involved in spoilage of vacuum-packaged refrigerated fresh beef. The spoilage was associated with accumulation of large quantities of foul smelling gas and purge in the bag and loss of color and texture of the meat. Attempts to grow the organism in several laboratory media were not yet successful; however, inoculation of purge from a spoiled sample into a fresh beef, vacuum-packaging and refrigeration storage facilitated growth of this species and produced characteristic spoilage of beef. Vacuum-packaging and refrigeration have become popular to extend shelf-life of fresh meat. The predominant bacteria associated with spoilage of vacuum-packaged refrigerated meat are several species of lactobacilli, Leuconostoc and Enterobacteriaceae, Brocothrix ther-mosphacta and Alteromonas putrefaciens (2,4,5,10,12,15). At normal pH of meat (pH 5.5 to 5.8) growth of B. ther-mosphacta, A. putrefaciens and several Enterobacteriaceae species are restricted (4,5,7,8), and as the storage temperature nears 0°C, the growth of psychrotrophic lactobacilli predominates (3,5,12). The predominance of psychrotro-phic lactobacilli in these products can also be due to production of some antimicrobial compounds that restrict growth of associative bacteria (4,5,15). In contrast, B. th-ermosphacta and several psychrotrophic gram-negative facultative bacteria may grow in high pH beef (pH above 5.8) and cause spoilage (4-6,14,18). The spoilage of vacuum-packaged refrigerated beef from the growth of lactobacilli, Leuconostoc spp. and B. thermosphacta is associated with sour, acid, and cheesy odor from the production of short chain fatty acids and other organic acids (2-4). In contrast, the gram-negative psychrotrophs, including A. putrefaciens, produce low molecular weight sulfur compounds and amines, give offensive odor and cause greening of meat (5,6,13,14,18). A specific Lactobacillus strain produced hydrogen sulfide with greening of vacuum-packaged refrigerated meat (16). Spoilage of vacuum-packaged meat by Clostridium spp. at relatively higher storage temperature has been reported (9). At present we are investigating the microbiological causes of extensive spoilage of vacuum-packaged fresh beef produced by a large processor. The spoilage is associated with accumulation of large amounts of foul smelling gas and fluid and extensive proteolysis. Our preliminary studies suggested that the causative organism is a Clostridium spp. MATERIALS AND METHODS Examination of commercial spoiled beef samples. Spoiled samples of top round, chuck roll, tri tip, and strip loin with accumulation of large quantities of gas were obtained under refrigeration from the beef processor. The samples were examined for color and texture changes and fluid accumulation (purge). The bags were opened, gas was examined for odor, and the purges were used for pH and bacteriological examination and inoculation in fresh beef. Bacteriological examination. The purges from spoiled samples were examined directly under a phase contrast microscope to visualize the predominant microflora and also by gram-staining, and staining for spores (17) and flagella (1). The materials were serially diluted and pour plated for aerobic plate counts (plate count agar, 30°C for 2 d), psychrotrophic counts (plate count agar, 10°C for 7 d) and psychrotrophic lactic acid bacteria counts (APT agar, pH adjusted to 5.0 with lactic acid, 10°C for 7 d). For the isolation of the suspected Clostridium species dilutions of the purges were pour plated in thioglycollate agar (fluid thiogly-collate broth + 1.5% agar), brain heart infusion agar supplemented with either 0.1% Na-thioglycollate or 0.1% Na-thiogly-collate + 0.05% L-cysteine, lactose egg-yolk-milk agar and trypticase peptone glucose yeast extract agar (broth +1.5% agar) (17). The plates were incubated either under vacuum or in 50:50 N 2 +C0 2 at 1 to 3°C for two to three weeks and examined for colonies. Cells from the representative colonies were purified and examined by phase contrast microscopy and gram-staining. Inoculation of fresh beef. Fresh beef steaks with pH between 5.5 to 5.6 were trimmed and cut aseptically in 100 to 125 g pieces from semitendinosus muscle of cows slaughtered in our facility. Each piece was placed in an oxygen impermeable sterile plastic bag, inoculated with 1 ml of purge from a spoiled beef, vacuum sealed (about 5mm Hg) and stored at 1 to 3°C in the dark. Controls were beef without the inoculum of purge. At intervals the samples were examined for gas accumulation, changes in color, texture and odor and the predominant microflora (by phase