Studies on the mode of action of diacetyl and carbon dioxide as inhibitors of meat spoilage bacteria

Carbon dioxide (CO2 and diacetyl (DA; 2, 3-butanedione) inhibit Gram-negative bacteria. Diacetyl inhibits the amino acid utilization of arginine. This inhibition occurs in the periplasmic space and CO2 may exert its influence in the periplasmic space as well. Therefore, this study investigated the effect of CO2 and diacetyl alone or combined on the Gram-negative spoilage organisms found in ground beef. The effect of diacety1 on decarboxylase activity of spoilage organisms found in fresh ground beef was determined using an automated system that provides real time information of microbial activity producing a dynamic curve similar to the growth curve. The influence of CO2 on pure cultures of Pseudomonas fragi, Escherichia coli O157:H7 (both associated with meat), and Escherichia coli K12 W3110 (reference organism) in AOAC synthetic broth was studied using two-dimensional electrophoresis (2-DE) to determine the effect of CO2 on cellular proteins. Pure cultures were grown in AOAC broth under 0 or 40% CO2 for 7 and 12 days, respectively, at 5°C. Cellular proteins were extracted and the method of 2-DE was performed. No difference was observed in proteins synthesized by the organisms. Testing the effect of diacetyl and CO2, alone or combined, on refrigerated ground beef showed that 10% CO2 alone was more inhibitory to the spoilage organisms than the highest concentration of diacetyl (100µg/g). The combination of 8.6 µg/g diacetyl and 10% CO2 was more effective in extending the shelf-life of refrigerated ground beef than other combinations tested. Automated recording of the metabolic activity in refrigerated ground beef confirmed that the natural spoilage microflora increased in untreated meat and sharply decreased in diacetyl-treated meat. The effect of diacetyl was indicated by inhibition of amino acid decarboxylase activity and change in microbiota. These findings confirm the usefulness of the BIOSYS 32 in objective assessment of microbial activity in fresh meats alone and in the presence of antimicrobial agents. From studies with ground beef, it appears that CO2 and diacetyl do not share the same inhibitory sites (periplasmic space) in the cell, however, if they do, the target sites may be limited. In broth, CO2 inhibition of protein synthesis was evident, but was dependent upon time of incubation and number of cells