Screening For High Yield Broad-spectrum Antibacterial Peptide Strains And Optimization Of The Fermentation Conditions

The aim of this project was to screen strains of producing antibacterial peptide. Fermentation conditions of antibacterial peptide were studied in order to enhance the titer of antibacterial peptide and the biological features of coarse antibacterial peptide were also determined.We have Collected 156 soil samples from different regions in China, 1 strain which ha-ve antimicrobial activity had been isolated from these nutritious soil samples. after exclud-ing inhibitive effect of organic acid and organic Bases, through salting-out, dialysis and protease experiment, preliminary determined that the Antibiotics producted by S62-9 were polypeptide- antibacterial peptide. The strain S62-9 was identified. According to themorpho-logical features, physiological and biochemical features and 16SrDNA sequence analyse of the strain, strain S62-9 were identified as Brevibacillus laterosporus. Different factors were investigated such as the ingredients of medium, temperature, pH, inoculating amount and medium capacity on production of antibacterial peptide in order to optimize fermentation condition of S62-9. The optimal results were dextrin 1%, peptone 2%, CaCL2 0.15%, Tween-20 1.5%. Through shake-flask experiments, the best culture conditions were pH 7.0, 37℃, 250 rpm, 40 mL/300 mL, and 3% inoculation, for 22h.Cultured in this condition, the yield of antibacterial peptide increased 59.9% compared with Initial fermentative conditions.The biological features of coarse antibacterial peptide showed that the bacteriocin has good stability against temperature and pH. The antimicrobial activity changes little from pH 3 to pH 12. The antimicrobial activity only have 12% loss suffered under the temperature of 100℃forl0 min, and even under the temperature of 121℃for 30 min Still able to maintain 77% of the activity. It was resistant to protease K and Pepsin. It is not sensitive to proteinase K, processing by pepsin, activity decreased only 28%.The inhibition spectrums of the antibacterial peptide was very wide, it could inhibit both Gram- bacteria and some Gram+ bacteria, such as Staphylococcus aureus, acillus subilis, Bacillus cereus, Lactobacillus plantarum, Lysobacter, E.coli, Enterobacter deformation, Salmonella typhimurium and Shigella. In addition, it could also inhibit some fungi suchas Kluyveromyces yeast, Candida albicans, Aspergillus niger and Penicillium fungi.The antibacterial substance can be isolated from other groups by salting out with 60% (NH4)2SO4, 80℃heat treatment and Sephacryl S-100 analysis. Three paks can be obtained by Sephacryl S-100 hromatography and both peaks have antimicrobial activity. So the antibacterial peptide is consisted of two proteins with different MW. Only one protein with larger MW can be detected through SDS-PAGE, and its MW is Less than 14.4 kD. Another protein as a result of too small MW(<8 kD), failed to detect.