Production optimization of broad spectrum bacteriocin of three strains of Lactococcus lactis isolated from homemade buttermilk

Three potent strains of lactic acid bacteria (LAB) with bacteriocin producing abilities have been isolated from homemade buttermilk. They can produce proteinaceous bacteriocins in the medium which are resistant to heat (121 °C for 15 min) and can tolerate range of pH from 3.0 to 11.0. They were found to be Gram positive, non-motile, non-endospore forming, coccoid structures. The strains LABW1 and LABW3 were present in chains while LABW4 were present singly or in pairs. By considering the 16S rDNA sequence homologies and phylogenetic analysis LABW1 and LABW3 were identified as Lactococcus lactis subsp. hordniae where as LABW4 was identified as Lactococcus lactis subsp. lactis. Maximum extracellular bacteriocin production by the isolates takes place in MRS medium (pH 5.0-7.0) at 28 °C and it showed strong correlation with bacterial growth. They started producing bacteriocin at early exponential phase (400 AU/ml) and reached its peak at the early stationary phase (12800 AU/ml for LABW1 and LABW3 and 25600 AU/ml for LABW4) which remain unchanged up to late stationary phase. The bacteriocin of the isolates had an antibacterial spectrum both against Gram positive as well as Gram negative pathogenic bacterial strains. The inhibition of Gram negative bacteria by bacteriocin of LAB is an unusual phenomenon. It exhibits bacteriocidal action on Staphylococcus aureus MTCC96 and Pseudomonas aeruginosa MTCC741. Prominent morphological aberrations, cell shrinkages and pore formations on cell wall of pathogenic bacterial cells upon bacteriocin treatment as evidenced from scanning electron microscope (SEM).