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Abstract:The objective of this study was to determine formation of antibacterial substances in supernatants of L. delbrueckii during cold storage and evaluate whether the application of this bacteria to raw ground beef would result in significant reductions of E. coli O157:H7 during refrigerated storage. Antibacterial activity of a newly isolated Lactobacillus delbrueckii MH 10 at refrigeration temperatures against food-borne pathogen Escherichia coli O157: H7 was studied. The size of inhibition zone depends on concentration of LAB cells. The cells (~10 9 CFU/ml) of L. delbrueckii produced significant amount of antibacterial substances mainly hydrogen peroxide ~ 35 µg/ml in sodium phosphate buffer (0.2 M, pH 6.5) and ~ 40 µg/ml in beef broth at 5°C during submerged cultivation without of growth. Submerged cocultivation of E. coli O157: H7 with lactobacilli in NB broth at 5°C reducing the total number of the pathogen more than 3 log for 5 days. The cell suspension intendent for treatment must contain 10 8-9 CFU/ ml of LAB. L. delbrueckii reduced initial amount 2x10 5 of E. coli O157: H7 in ground beef cocultivation up to 3 log for 3 days and become undetectable after 7 days. The application of L. delbrueckii bacteria does not cause any changes in sensory characteristics of ground beef by itself; moreover promote expanding of shelflife due to inhibition of psychrophilic spoilage microorganisms.

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It was found out that among rifampicin resistant mutants (Rif) of Lactobacillus delbrueckii subsp. lactis MDC 9617 the frequency of occurrence of active hydrogen peroxide producers is about five times higher than in Rif + strain. Aerobically growing Rif mutants show relatively higher kinetics of H 2 O 2 production and earlier entry into the stationary phase due to auto-inhibition caused by inhibitory concentrations of H 2 O 2 . As a result, they synthesize about the same amount of H 2 O 2 but with a smaller biomass. The concentration of H 2 O 2 , which causes auto-inhibition of aerobically growing cultures is approximately 2 times lower than the MIC of exogenous H 2 O 2 for anaerobically growing cultures. The higher sensitivity of aerobically growing cultures is rather due to the existing intracellular H 2 O 2 , which together with extracellular H 2 O 2 causes an additive/cumulative effect. Lack of auto-inhibition at 5 °C allows the mutants to accumulate 50-70% more peroxide than the parental strain. Reduction of the food-borne pathogen Escherichia coli O157:H7 by selected active Rif-4 mutant during co-cultivation in LAPTg at 5 °C was significantly higher than that of the parental strain. Due to high (~40%) co-aggregation ability of LAB with E. coli , the death of the pathogen detected at an early stage of co-cultivation in the absence of inhibitory concentrations of H 2 O 2 , may occur by direct release of H 2 O 2 onto the target cell. The ability of Rif mutants to produce higher amounts of H 2 O 2 with a smaller biomass enable them to extend the shelf-life of refrigerated food without risk of altering sensory characteristics of the latter.