Isolation,Partial Purification and Characterisation of Bacteriocins from Fermented Foods
Bacteriocins are antibacterial proteins produced by bacteria that can kill or inhibit the growth of other bacteria. Many lactic acid bacteria produce a high diversity of different bacteriocins. Many bacteriocins have been characterized biochemically or genetically and though there is a basic understanding of their structure, function, biosynthesis and mode of action, many aspects of these compounds are still unknown. The project aims at isolation, partial purification and characterization of bacteriocins from fermented vegetables. The fermented extracts of carrot, cucumber and beetroot were sources for isolation of bacteriocinogenic LAB. Test organisms employed were the major food borne pathogens, Staphylococcus aureus, E.coli and Bacillus subtilis. Media used was MRS)and nutrient agar. .Bacteriocin producing Lactic acid bacteria (LAB) were isolated from fermented carrot. Well diffusion assay was carried out with the cell free supernatant of the isolated bacteriocin producer strain (neutralized and treated with catalase to eliminate the effect of hydrogen peroxide) and it presented a broad inhibitory spectrum against the indicator strains tested such as E. coli ATCC29522,S. aureus ATCC29523, and Bacillus cereus .This gave evidence that the antimicrobial activity is due to the production of bacteriocins. Partial purification of bacteriocin was performed using ammonium sulphate precipitation. Protein precipitated out at 80% ammonium sulphate saturation.WDA assay using the partially purified bacteriocin confirmed the inhibitory activity of the bacteriocin against the test organisms used in ranges of AU /ml of .10,000 AU/ml.To confirm that the inhibitory effect was not because of the presence of salts a control well with ammonium sulphate at 80% saturation was also maintained which did not demonstrate an inhibition zone. As for the effect of incubation temperature, the bacterial proliferation were most significant when cultured at 30ºC and the amount of bacteriocin production when compared to the other incubation temperatures (20, 25, 35 and 40ºC) suggested that ambient growth temperature played an important role on the production of bacteriocin. Th effect of pH on the bacteriocin activity was checked and it was found that maximum activity was observed at (1200 AU/mL) at pH 6.5.Increased production of bacteriocin was noticed even at low pH, i.e., 4 -4.5 (1000 AU/mL at both pH). The inference can be made that the optimal production of the bacteriocin isolated from LAB was favored by lowered pH as reported for the bacteriocins of Lactobacillus plantarum or Lactobacillus caseii. As the bacteriocin isolated expressed favourable temperature ranges and wide pH tolerance further characterisation of the strain and bacteriocin will impart both probiotic and preservative potentials.