BACTERIOCIN PRODUCED BY Lactococcus lactis CRL 1584 ISOLATED FROM A Rana catesbeiana HATCHERY

PASTERIS, S.E.; ROIG BABOT, G.; GUIDOLI, M.G.; BUHLER, M.I.; NADER-MACIAS, M.E.

Sevilla. España

Conferencia; II INTERNATIONAL CONFERENCE ON ENVIRONMENTAL, INDUSTRIAL AND APPLIED MICROBIOLOGY. BIOMICROWORLD 2007; 2007

Rana catesbeiana hatcheries are developed all around the world by their meat value and by-products (skin, liver, and gut) requirements. Bullfrog production is an intensive process where animals are more susceptible to some bacterial infectious diseases, such as red leg syndrome (RLS). Farmers usually sacrifice infected animals or treat them with antibiotics. Because of the problem of antibiotic resistance and the subsequent reluctance to use them, the application of probiotics in aquaculture is becoming increasingly popular. Probiotics generally include lactic acid bacteria (LAB) which could participate in the control of pathogenic microorganisms by producing organic acids, hydrogen peroxide or bacteriocins. In previous studies, it has been demonstrated that a strain of Lactococcus lactis CRL 1584 isolated from balanced feed in a R. catesbeiana hatchery has some beneficial properties that indicates their potential use in a probiotic product. The aim of this work was to characterize the lactococcin, a bacteriocin-like metabolite produced by this microorganism. The producing strain was identified by phenotypic assays. L. lactis CRL 1584 was grown in LAPTg medium for 24h at 37°C. The indicator strains used were Proteus vulgaris, P. mirabilis, Pseudomonas aeruginosa, Citrobacter freundii, Staphylococcus epidermidis (RLS-associated pathogens) and also S. aureus, Listeria monocytogenes Scott A and L. inocua (meat spoilage bacteria). The pathogenic strains were grown in nutritive or BHI broth media for 24h at 37°C. Antimicrobial activity was determined by the agar well-diffusion method by using different initial concentrations of the indicator strains. Culture supernatants of L. lactis were able to inhibit the growth of enterobacteriaceae, and Staphylococci by acidity. One vancomicin resistant Enterococcus faecium strain, Ps. aeruginosa strains, L. monocytogenes Scott A, and L. inocua were inhibited by organic acids, a bacteriocin-like metabolite and low amounts of hydrogen peroxide. The action of proteolytic enzymes on cell-free supernatants indicates a proteinaceous nature of the inhibitory metabolite. Lipase and amylase exerted a low inhibitory effect, indicating that both, lipidic and glusidic fractions are also involved in the antagonistic activity. The bacteriocin named Lactococcin CRL 1584 was produced following the microbial growth with a maximum of 2,100 AU/mL at the end of the exponential growth phase. It was completely stable at -20°C for 3 months and at 80, 100, and 115°C for 30 min in untreated supernatants. Is resistant to 80°C for 10 min when supernatants were neutralized, indicating a possible protective synergistic effect with the organic acids produced. The activity was also stable from pH 2 to 7 for 3 days at 8°C, and from pH 2 to 5.5 for 2 days when supernatants were stored at 25°C. Neither hexadecane nor ethyl acetate affected the bacteriocin activity. 10% and 20% chloroform produced an inhibition of 21.4 and 42.9%, respectively, while a 100% inactivation was caused by ethanol. This is the first report about a bacteriocin-producing L. lactis strain isolated from a R. catesbeiana hatchery, that inhibits the growth of LAB selected by beneficial properties, as Lactobacillus plantarum, Pediococcus pentosaceus, E. faecium isolated from the same ecological niche, which are able to inhibit pathogens also by production of organic acids and/or hydrogen peroxide. For the formulation of a combined probiotic including the selected LAB and L. lactis 1584, the resistant strains to Lactococcin should be obtained. The bacteriocin producer L. lactis shows some beneficial properties to be used as probiotic for raniculture, and also as a biopreservative to control meat spoilage bacteria. This strain is also able to control Lactococcus garvieae, a fish pathogenic LAB, that encourage the potential application of this strain in fish aquaculture. Keywords: Probiotics; Aquaculture; Bacteriocin.