Identification And Screening Of Bacteriocin-producing Lactic Acid Bacteria And Fermentation Condition Of Bacteriocin

The aim of this project was to screen strains of producing broad-spectrum Bacteriocin. Fermentation conditions of Bacteriocin were studied in order to enhance the titer of Bacteriocin and the biological features of coarse Bacteriocin were studied. A Bacteriocin-producing Lactic Acid Bacteria R260 strain was screened from plant-material. The morphological, physiological and biochemical features of R260 strain were as follows, the bacterial cell was gram-positive, straight rot-shaped, single or pairs and the spor wasn't found. VP reaction, nitrate reaction, gelatin liquefy and catalase reaction were negative. Glucose, fructose, sucrose and maltose were fermented to organic acids. R260 strain was identified as a Lactobacillus Plantarum, according to its morphological and physiological properties. The supernatant of R260 strain can inhibit indicator strains strongly excluding inhibitive effect of H2O2 and organic acid, but the inhibitive activity decreased sharply after treatment with trypsin and pepsin. The results confirmed that this inhibitory material was a kind of protein, could be classed as Bacteriocin. We investigated different factors such as different medium, the ingredients of medium, temperature, time, seed age, pH and Tween80 on production of Bacteriocin in order to optimize the fermentation condition. R260 strain can grow in all of four types of media including M17S,CM,MRS and APT, but it grow well and the highest value of titer of Bateriocin in MRS. The optimum carbon source, nitrogen source and growth factor was sucrose, soybean peptone and Fe2+ respectively and the optimal concentration was 2%,1.0% and 0.02% respectively by orthogonal experiment. The study on condition of fermentation showed that the optimum initial pH for producing Bacteriocin is 6.5 , it produced the highest amount of Bacteriocin at 30℃.The optimum inoculating amount and seed age was 3% and 12 hours respectively. The relationship of the growth, pH and Bacteriocin yield showed that R260 strain grew very well in MRS medium. The pH almost changed little at the first 4h, but decreased apparently after 4h. R260 strain produced Bacteriocin at 7h, the amounts of Bacteriocin reached the highest level after 20h and its activity decreased sharply after 22h. The biological features of coarse Bacteriocin showed that the optimum pH was 5.0. The Bacteriocin was steady in acid conditions. It was still active after boiled in 100℃ for 20mins, so Bacteriocin has good thermal stability. The inhibition spectrum of Bacteriocin is very wide, it can inhibit both positive bacteria and some negative bacteria, but not effect on yeast and mold. It is sensitive to pepsin and trypsin but insensitive to hydrogen peroxidase. Bacteriocin of L. Plantarum R260 inhibited B.thuringiensis from growing rather than killing it.