Application of reuterin produced by Lactobacillus reuteri 12002 for meat decontamination and preservation.

Lactobacillus reuteri strain 12002 was used for reuterin production in the two-step fermentation process. A batch culture fermentation was used to produce a maximum biomass of L. reuteri. Then cells were harvested, resuspended in a glycerol-water solution, and anaerobically incubated to produce reuterin. The lyophilized supernatants (approximately 4000 activity units (AU) of reuterin per ml) were diluted in distilled water for decontamination and preservation trials. The MIC values of reuterin for Escherichia coli O157:H7 and Listeria monocytogenes were 4 and 8 AU/ml, respectively. In meat decontamination experiments, the surface of cooked pork was inoculated with either L. monocytogenes or E. coli O157:H7 at a level of approximately log10 5 CFU/cm2, incubated for 30 min at 7 degrees C, and decontaminated by exposure to reuterin (500 AU/ml). The bactericidal effect of reuterin was analyzed 15 s and 24 h after exposure at 7 degrees C. After 15 s of exposure to reuterin, viable numbers decreased by 0.45 and 0.3 log10 CFU/cm2 for E. coli O157:H7 and L. monocytogenes, respectively. After 24 h the numbers decreased by 2.7 log10 CFU/cm2 for E. coli O157:H7 and by 0.63 log10 CFU/cm2 for L. monocytogenes. In the same experiment, the combined effect of reuterin and lactic acid was also investigated. Adding lactic acid (5%, vol/vol) to reuterin significantly enhanced (P < or = 0.05) the efficacy of reuterin. No additional effect (P < or = 0.05) was found when ethanol (40%) was added to the mixture of reuterin and lactic acid. To evaluate the preservative effect of reuterin during meat storage, reuterin was added to raw ground pork contaminated with E. coli O157:H7 or L. monocytogenes. Reuterin at a concentration of 100 AU/g resulted in a 5.0-log10 reduction of the viability of E. coli O157:H7 after 1 day of storage at 7 degrees C. Reuterin at a concentration of 250 AU/g reduced the number of the viable cells of L. monocytogenes by log10 3.0 cycles after 1 week of storage at 7 degrees C.

The effect of Reuterin on Listeria monocytogenes and Escherichia coli O157:H7 in milk and cottage cheese.

A broad-spectrum reuterin produced during anaerobic fermentation of glycerol by Lactobacillus reuteri strain 12002 was found to be inhibitory and bactericidal for Listeria monocytogenes and Escherichia coli O157:H7. Lyophilized reuterin was prepared by a two-step fermentation process. A batch fermentation in a 15-liter fermentor was applied to produce a maximum biomass of L. reuteri using a modified MRS broth at pH 4.3. Further, harvested cells were used to ferment glycerol (250 mM) under anaerobic conditions. The sensitivity to reuterin of 10 strains of Listeria spp., including 6 strains of L. monocytogenes, and 6 strains of E. coli, including one enterotoxigenic E. coli strains and two enterohemorrhagic E. coli strains, was estimated. Strains of L. monocytogenes were more resistant to reuterin than E. coli strains. In cottage cheese, pH 5.4, L. monocytogenes increased by 0.4 log while E. coli O157:H7 decreased by 0.5 log in 21 days at 7 degrees C; addition of reuterin (50 to 250 units per g) to the cottage cheese reduced the viability of both organisms. The inactivation rate was more pronounced (P < or = 0.05) with E. coli O157:H7 than L. monocytogenes and it was dependent on reuterin concentration. The rate of E. coli O157:H7 population reduction reached to 2, 3, and 6 log cycles by day 7 for reuterin concentrations of 50, 100, and 150 units per g of creamed cottage cheese, respectively. While, 100, 150, and 250 units of reuterin per g caused reductions in L. monocytogenes counts by 2, 5, and greater than 5 log cycles, respectively. In UHT skim milk with 150 units of reuterin per ml, stored at 7 degrees C, the decline in the numbers of L. monocytogenes cells was higher than that in cottage cheese. Milk fat in the range of 0.5 to 3% did not affect the reuterin activity (P < or = 0.05). Addition of 3% salt enhanced the lethal effect of reuterin and diminished the initial population of L. monocytogenes by 4.5 log cycles in three days at 7 degrees C.

Growth and survival kinetics of Yersinia enterocolitica IP 383 0:9 as affected by equimolar concentrations of undissociated short-chain organic acids.

The influence of different organic acids (lactic, acetic, formic and propionic acids) at equimolar concentrations of undissociated acid with pH range of 3.9, 5.8, on the aerobic and anaerobic growth and survival kinetics of the virulent strain of Y. enterocolitica IP 383 0:9, was determined in tryptone soy broth at 4 degrees C. Growth and survival data were analyzed and fitted by a modification of the Whiting and Cygnarowicz-Provost model, using the Minpack software library. Initial generation times, initial specific growth rates, lag time and dead rate were subsequently calculated from the model parameters. The results demonstrate that the inhibitory effects of the acids were divided into two categories dependent upon pH. At high pH (5.8) the order of inhibition was formic acid > acetic acid > propionic acid > lactic acid, whereas at lower pH it became formic acid > lactic acid > acetic acid > propionic acid. The inhibitory effect of lactic acid is enhanced under anaerobic condition. Nevertheless, when the organism was cultured anaerobically, it was shown to be more tolerant to formic and acetic acids. Moreover, these variables (type of organic acid, pH and atmosphere) did not lead to the loss of the virulence plasmid in growing and surviving cells. The mechanism of inhibitory effect for each of the acids are also discussed.

Kinetics of interactions of lactic acid, pH and atmosphere on the growth and survival of Yersinia enterocolitica IP 383 O:9 at 4 degrees C.

The influence of different lactic acid concentrations (0.1, 0.3, 0.5, 0.7, 0.9, 1.1% v/v), within pH range of 3.9 to 5.8 on the aerobic and anaerobic growth and survival kinetics of the virulent strain of Y. enterocolitica IP 383 O:9, was determined in Tryptone Soy Broth at 4 degrees C. Growth and survival data were analyzed and fitted by a modification of the Whiting and Cygnarowicz-Provost model, using the Minpack software library. Initial generation times, initial specific growth rates, lag times and death rates were subsequently calculated from the model parameters. The stability of the virulence plasmid in growing and surviving cells was examined using crystal violet binding, low-calcium response and congo red uptake. The results demonstrate the dependancy of the growth and survival kinetics on the interaction between the three variables. The effect of lactic acid on Y. enterocolitica is greater under anaerobic than aerobic conditions. Nevertheless, the organism was found to be more tolerant of low pH conditions under anaerobic atmosphere than under an aerobic atmosphere in the absence of lactic acid. The interaction between the variables did not lead to loss of the virulence plasmid in growing or non-growing cells.