The Isolation, Identification Of Lactic Acid Bacteria Producing Bacteriocin And The Optimization Of Medium

Lactic acid bacteria(LAB),a kind of probiotics,play an essential role in food fermentation processes.It can inhibit or kill various food-borne pathogenic and spoilage bacteria, so it is beneficial to human health. It can inhibit or kill various food-borne pathogenic and spoilage bacteria, the antimicrobial effect may be due to the production of organic acids, hydrogen peroxide and diacetyl, as well as bacteriocins, a family of antimicrobial peptides, synthesized by the ribosome. These substances are proteinaceous, antimicrobial compounds produced by bacteria usually only inhibit closely related species. There has been a recent explosion in the interest of bacteriocins as natural bio-preservatives in food systems and they have been considered to be the most effective alternative to antibiotics. Therefore,the aim of this study here is to screen out a lactic acid bacteria which produced high level bacteriocin and optimiz the fermentation medium for bacteriocin produced by LAB using response surface methodology. we can get the highest antibacterial activity of bacteriocin through this method.The lactic acid bacteria producing bacteriocin were screened from various materials by the agar well diffusion method, using Escherichia coli1490å’ŒStaphylococcus aureus2241 as the indicator. Ten strains showed the higher antibacterial activity, among which the Num.8 strain (isolated from ensilage) named 15d was a lactic acid bacteria producing bacteriocin. The 16SrDNA fragment of strain 15d was amplified through general PCR with the template of its total DNA and bacteria 16SrDNA universal primers. The 16SrDNA sequence was analyzed and compared to that of other Lactobacillus strains held in GenBank DNA database using the BLAST algorithm. In this study, biochemical experiment of strain 15d was also carried out. The experiments of cataloes, starch hydrolysis, yield of hydrogen sulphide, V-P, litmus milk patronization and gelatin liquefaction showed negative. And the tests of glucose, gluconate, acid producing by litmus milk and litmus milk reduction were positive. Strain 15d was finally characterized as Lactobacillus casei. The experiment showed that the natural plasmid did not exist in the Lactobacillus casei by using the modified alkaline lysis method. Response Surface Methodology (RSM) was applied to optimize the liquid medium of MRS for bacteriocin produced by Lactobacillus casei. Two level factorial design of PB was used to evaluate the influence of the related factors. Then the concentrations of the main factors were further optimized by using RSM. The results showed the optimized composition.A bacteriocin-producing lactic acid bacteria Lactobacillus casei was isolated from ensilage in this study. The produced bacteriocin could inhibit the gram-negative bacteria Escherichia coli1490 which is not inhibited by most bacteriocins. The property of this bacteriocin makes it possible that adding the bacteriocin into the chicken feeds and piglet feeds. In addition, the analysis of the possible mechanisms that the nutrients changed the bacteriocin producing capacity from the perspective of molecular can provide ideas for the future researches.