A Lactobacillus sake strain LKE5 and four strains of Carnobacterium piscicola were evaluated as biopreservation cultures to control the growth of Listeria monocytogenes on vacuum-packed, cold-smoked salmon stored at 5 degrees C. All five strains were antilisterial as live cultures in an agar diffusion assay. Cell-free supernatants of two strains of C. piscicola and L. sake LKE5 were also antilisterial because of the production of bacteriocins. The presence of high cell numbers of strains of C. piscicola had no influence on the sensory quality of cold-smoked salmon stored at 5 degrees C, but L. sake LKE5 caused strong sulfurous off-flavors and was rejected as a culture for biopreservation of cold-smoked salmon. A bacteriocin-producing strain of C. piscicola (A9b) initially caused a 7-day lag phase of L. monocytogenes, followed by a reduction in numbers of L. monocytogenes from 10(3) CFU/ml to below 10 CFU/ml after 32 days of incubation, coinciding with the detection of antilisterial compounds. The presence of a nonbacteriocin-producing strain of C. piscicola (A10a) prevented the growth of L. monocytogenes during the 32-day incubation. The growth of L. monocytogenes was strongly repressed on cold-smoked salmon in the presence of C. piscicola A9b and A 10a, respectively. The initial cell numbers of L. monocytogenes that were found on Oxford plates incubated at 25 degrees C reached low maximum cell counts of 10(4) and 2 x 10(3) after 14 and 20 days of storage in mixed culture with C. piscicola A9b and A10a.

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Aims: This study elucidates the mechanisms by which a nonbacteriocinogenic Carnobacterium piscicola inhibits growth of Listeria monocytogenes. Methods and Results: Listeria monocytogenes was exposed to live cultures of a bacteriocin-negative variant of C. piscicola A9b in co-culture, in a diffusion chamber system, and to a cell-free supernatant. Suppression of maximum cell density (0-3AE5 log units) of L. monocytogenes was proportional to initial levels of C. pisciola (10 3 -10 7 CFU ml )1 ). Cell-to-cell contact was not required to cause inhibition. The cell-free C. piscicola supernatant caused a decrease in L. monocytogenes maximum cell density, which was abolished by glucose addition but not by amino acid, vitamin or mineral addition. The fermentate also gave rise to a longer lag phase and a reduction in growth rate. These effects were independent of glucose and may have been caused by acetate production by C. piscicola. 2D gel-electrophoretic patterns of L. monocytogenes exposed to C. piscicola or to L. monocytogenes fermentate did not differ. Treatment with C. piscicola fermentate resulted in down-regulation (twofold) of genes involved in purine-or pyrimidine metabolism, and up-regulation (twofold) of genes from the regulon for vitamin B 12 biosynthesis and propanediol and ethanolamine utilization. Conclusions: A nonbacteriocinogenic C. piscicola reduced growth of L. monocytogenes partly by glucose depletion. Significance and Impact of the Study: Understanding the mechanism of microbial interaction enhances prediction of growth in mixed communities as well as use of bioprotective principles for food preservation.

Aims: To study the importance of bacteriocin production for the antilisterial effect of a bacteriocinogenic Carnobacterium piscicola strain A9b on growth of Listeria monocytogenes in broth and cold-smoked salmon systems. Methods and Results: Acriflavin treatment of strain A9b resulted in loss of bacteriocin production and of immunity to carnobacteriocin B2. Two plasmids present in the wild-type were lost in the variant that was also more sensitive to bavaricin and leucocin A than the wild-type indicating cross-resistance to class IIa bacteriocins. The growth rate of the bac ) mutant was higher than that of the wild-type at 5 and 37°C but not at 25 or 30°C. In salmon juice the maximum cell density of L. monocytogenes was suppressed 3 and 6 log by co-culture with C. piscicola A9b bac ) and bac + , respectively, as compared with the control. Sterile filtered cultures of C. piscicola A9b bac ) caused a limited suppression of the maximum cell density of L. monocytogenes similar to that observed when sterile buffer was added in equal amounts. Semi-purified carnobacteriocin B2 caused a 3AE5 log decline in viable cell count after 6 day of incubation in cold-smoked salmon juice at 5°C. High resistance level to carnobacteriocin B2 was observed for L. monocytogenes cells exposed to semi-purified and in situ produced carnobacteriocin B2. Conclusions:The presence of bacteriocin production in C. piscicola enhances its inhibition of L. monocytogenes Significance and Impact of the Study: Due to the emergence of resistance, a bacteriocin negative lactic acid bacteria may be more suited for practical use as a bioprotective agent against L. monocytogenes in ready-to-eat foods.

This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis r ) cells grown in broth at 4°C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis r cells. Wild-type cells grown in 100% CO 2 were two to five times longer than cells grown in air. Nisin (2.5 g/ml) did not decrease the viability of Nis r cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO 2 . There was a quantifiable synergistic action between nisin and CO 2 in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 g of nisin per ml increased from 3.1 to 12.5 g/ml over 35 days, but this increase was markedly delayed for cultures in CO 2 . This synergism between nisin and CO 2 was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO 2 and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO 2 atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4°C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30°C. Cells grown in the presence of 100% CO 2 and those grown at 4°C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO 2 is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.The ability of Listeria monocytogenes to resist environmental stresses has made this food-borne pathogen a major concern to the food industry. This pathogen is found on various foods and has been implicated in several large food-borne outbreaks worldwide (C. B. Dalton, C. Austin, J. Sobel, P. Hayes, B. Bibb, J. Mellen, and P. Griffin, Proc. 44th Annu. Epidemic Intelligence Serv. Conf., abstr. 19, 1995; 11). New preservation strategies have been developed to control the growth of L. monocytogenes in foods, including application of nisin (10,15). Nisin is an antimicrobial peptide which kills L. monocytogenes as well as many other gram-positive bacteria. It acts on the cytoplasmic membrane of sensitive cells by forming transient pores which allow efflux of small hydrophilic compounds like ATP, ADP, monovalent cations, and amino acids (1, 25). This pore formation leads to dissipation of the membrane potential and ionic gradient across the membrane and subsequently results in the destruction of energy metabolism and cell death (3, 18).The practical application of nisin as a food preservative can be compromised by the existence of L. monocytogenes strains which are naturally resistant to nisin (13, 17) and selection of nisin-resistant bacteria during exposure to progressively higher nisin concentrations (16,17). However, the use of a multiplehurdle system may reduce d...