Studies on Plasmidassociated Bacteriocin Production by Lactobacilli

Abstract

One hundred and twenty five lactobacilli isolates were collected from different food and clinical samples including milk, yogurt, fermented fruits, fermented vegetables, fermented pulses, fermented meat, dental carries and infant faeces. All the isolates were identified by their morphological, colonial characters and catalase reaction. The isolates were screened against each other for bacteriocin production by agar-well diffusion assay and overlay method. Twenty one lactobacilli isolates produced an inhibitory substance effective against some of the other isolates tested. All the bacteriocin producing isolates were immune to the inhibitory effect of their own bacteriocin. Of all twenty one, five best produces (LM-06, LM-07, LM-05, LY-06 and LC-09) were selected for further studies. The inhibitory substance produced by all five isolates were identified as lipoproteinic in nature as their activity was completely abolished after treatment with proteolytic and lipolytic enzymes while they retained full activity after treatment with catalyze and lysozyme. The stability of proteinaceous inhibitors to pH and temperature were studied. LC-09 appeared to be a noble bacteriocin as it resisted 100°C for approximately 3 hours. The bacteriocin produced by LM-06 was the strongest of all as it inhibited a majority of the other isolates against which tested and hence LM-06 was retained for detailed investigations. The isolates was identified as L. casei on the basis of biochemical reactions and its bacteriocin was designated as lactocin LM-06. An increase in lactocin LM-06 preparation inhibited the growth of L. monocytogenes an important food-borne pathogen. The bacteriocin bioactivity was lost after one month at room temperature while was stable for more than two years at -20°C. To get the direct evidence of plasmid association of lactocin LM-06 production, arcidine orange and ethidium bromide as curing agent were tried. The curing of Bac+ phenotype was achieved by 120 µg/ml of ethidium bromide while acridine orange was not effective in this respect. Beside ethidium bromide, elevated temperature(42°C) mediated curing of LM-06 also revealed plasmid association of the bacteriocinogenic determinants in lactocin LM-06 production. The restriction analysis of the isolated plasmid pNRJ revealed a plasmid of approximately 60kb responsible for the production. Attempts to purify the protein LM-06 resulted in an increase in lactocin LM-06 titer and its present yield. The approximate molecular weight of the protein determined by natie gel electrophoresis appeared to be 15 KDa.