Studies On Some Properties Of Bacteriocins Produced By Lactobacillus Species Isolated From Agro-Based Waste

摘要

Lactic acid producing bacteria (LAB) were isolated from vegetable waste. These organisms were studied for bacteriocin production. Among the isolated cultures Lactobacillus lactis and Lactobacillus plantarum were potent producers of bacteriocins. Bacteriocin produced by these Lactobacillus species had a large spectrum of inhibition against food spoilage microorganisms and various related strains of lactic acid bacteria. The bacteriocin inhibited the growth of Escherichia coli but it showed least activity against Candida albicans. The antibacterial activity appeared to be pronounced between late logarithmic phase and early stationary phase. The bacteriocins were found to be heat stable. Supplementation with lactose, peptone and yeast extract enhanced the production of bacteriocin. Introduction Bacteriocins are highly specific antibacterial proteins produced by strains of bacteria active mainly against some other strains of same or related SPP (Gaur et al 2004). The bacteriocins produced by Lactic acid bacteria (LAB) are potent bio-preservative agents and the applications of these in food are currently the subject of extensive research. The search for new bacteriocins with a wider spectrum of activity and compatibility with different food system is being studied by some investigators.Fruits and vegetable waste provide a good source for isolating LAB having antagonistic and probiotic properties. However, work on bacteriocinogenic LAB from agro-based waste has been limited. Therefore the present studies are carried out to examine LAB population isolated from damaged and spoiled vegetable wastes collected from local market yard. Bacteriocins produced by these organisms are characterized with respect to inhibition spectrum, effect of physical and chemical parameters and effect of cultural conditions on baccteriocin production. Materials and Methods Vegetable waste containing spoiled cabbage and cucumber was collected from local market yard. It was then crushed, homogenized with kitchen blender, and inoculated in the deMan Ragosa and Sharpe (MRS) broth for enrichment of resident LAB. The tubes were incubated at 37°C for 24 hours.Screening for bacteriocin producersThe enriched broth was serially diluted prior to being pour plated on MRS agar. The plates were incubated at 37°C for 16 hours till colonies appeared. The plates were then overlaid with MRS soft agar inoculated with indicator organisms isolated from raw milk. All the plates were incubated at 37°C for 24 hours. Colonies showing zone of inhibition were considered as potential bacteriocin producers.Identification of bacteriocin producersThe bacterial strains that were selected as potential bacteriocin producers were subjected to morphological, cultural and biochemical characterization according to Bergey's Manual of Determinative Bacteriology (1997). Bacteriocin assayThe strains that were selected as potential bacteriocin producers were grown in MRS broth at 37°C for 48 hours. Cells were separated by centrifugation at 5000 rpm for 10 minutes. The pH of the cell free supernatant was adjusted to 5.5 with sterile 0.2 N NaOH. Bacteriocin activity in the supernatant was then tested by agar well diffusion assay (Geis et al 1983). The antagonistic effects of the culture supernatants of bacteriocin producing LAB were tested on various Gram Positive and Gram Negative organisms listed in Table - 2. MRS agar was used for lactic strains, nutrient agar for gram positive and gram negative organisms and Saboraud Dextrose Agar was used for Candida albicans. All cultures were grown aerobically at 37°C for 48 hours. Inhibition zones around the wells were measured. The anti-microbial activity of the bacteriocin is defined as the reciprocal of the highest dilution showing inhibition of the indicator lawn and is expressed in activity units per ml (Nakamura et al 1983).