Abstract
Lactic acid bacteria (LAB) isolated from various foods are important due to their potential to inhibit microorganisms, including drug-resistant bacteria. The objectives of this investigation were to isolate and identify antibacterial substances producing LAB from Hentak, a traditional fermented fish product of Manipur (North-East India), and to optimize the production of antagonistic substances present in cell free neutralized supernatant (CFNS) against enteric bacterial pathogens using the 'one factor at a time' (OFAT) method. Out of 10 LAB, the most potent bacterium producing antibacterial substances was isolated and identified as Lactobacillus pentosus strain LAP1 based upon morphological, biochemical and molecular characterization. MRS (de Man, Ragosa and Sharpe) medium was determined to provide better bactericidal activity (AU/ml) than other tested media against the indicator enteric bacteria, including Staphylococcus epidermidis MTTC 3615, Micrococcus luteus MTCC 106, Shigella flexneri MTCC 1457, Yersinia enterocolitica MTCC 840 and Proteus vulgaris MTCC 1771. The culture conditions (pH: 5, temperature: 30 degrees C and inoculum volume: 1 %) and medium components (carbon source: lactose and nitrogen source: ammonium chloride) were observed to be the most influential parameters of significant antagonistic activity of CFNS against the enteric pathogens. MRS medium supplemented with Tween20 effectively stimulated the yield of antibacterial substances. The CFNS of strain LAP1 exhibited sensitivity to proteolytic enzyme (pepsin) treatment and heat treatment (60 degrees C for 60 min, 100 degrees C for 30 min and 121 degrees C for 15 min) and lost its inhibitory properties. The CFNS was active at an acidic (pH 3.0) to neutral pH (pH 7.0) but lost its antagonistic properties at an alkaline pH. The CFNS obtained from strain LAP1 scavenges the DPPH (1,1-diphenyl-2 picrylhydrazyl) significantly in a concentration-dependent manner within the range of 8.8 +/- 0.12-57.35 +/- 0.1 %. The OFAT-based approach revealed the baseline for statistical optimization, the scale-up process and efficient production of CFNS by L. pentosus strain LAP1, which could be used as a potential antibacterial and free radical scavenging agent.