Purification and characterization of a broad spectrum bacteriocin produced by a selected Lactococcus lactis strain 63 isolated from Indian dairy products.

Lactococcus lactis subsp. lactis strain 63 was isolated from Indian dairy products, produced a bacteriocin with broad spectrum inhibition against several of food pathogens like Listeria monocytogenes and Bacillus cereus as well as Gram negative bacteria viz., E. coli, Yersinia, Citrobacter, Proteus, Enterobacter, Klebsiella and Serratia strains. Bacteriocin production was higher in GM-17 and MRS as compared to TYGE broth and enriched skim milk broth and reached the maximum level during the early stationary phase. The bacteriocin was purified by performing ammonium sulfate precipitation. The bacteriocin was able to survive 90 °C/10 min but not 100 °C/10 min. Complete inactivation of bacteriocin was observed after autoclaving. The bacteriocin maintained its activity over a wide range of pH (3-9). The antimicrobial compound produced by the isolate 63, was sensitive to papain, pepsin, trypsin and amylase but was resistant to detergents like SDS and urea. Tween 20, Tween-80 as well as Triton X-100 enhanced its activity. Since the treatment with proteolytic enzymes resulted in loss of activity, this shows that the proteinaceous nature of the antimicrobial substance. Tentative molecular weight of the bacteriocin was found to be between 3.5 and 5 kDa by Tricine SDS-PAGE. Finally, we confirmed the presence of gene for nisin, and the sequence thus obtained, was identical to the sequences previously described for nisin Z. Lactococcus lactis subsp. lactis 63 or its bacteriocin, which has a wide inhibitory spectrum, has the potential for use as a starter or protective culture in the manufacture of fermented products.