Soybean Antibacterial Liquid Preparation With Fermentation Process Optimization And Isolation

In recent years, with the generation of antibiotic resistance, people have been paying increasingly attention to the research of looking for new antibacterial method. With soybean protein as raw material, through the microorganism fermentation to prepare a liquid containing antibacterial activity peptides. Filedred strains for fermentation, research the impact of add quantity of fermentation medium, fermentation process parameters on the antibacterial activity of fermented liquid.Through orthogonal experiment to optimize the process. Then research on extraction and purification of the antimicrobial components from the fermented liquid.First determine the composition of soybean isolated protein, fermented with aspergilus Niger, bacillus subtilis, plant lactobacillus and bacillus Natto that are commonly used for fermentation. Compare the antibacterial activity for optimize strains. Selected common mold, gram-positive bacteria, gram-negative bacteria and yeast as indicator bacteria, determine the broad spectrum antimicrobial resistance. The results showed that the material soybean protein had almost 90% protein content which was a good source of nitrogen for fermentation strains and nutrient source. In strains filtration experiment, aspergilus Niger showed no bacteriostasis, bacillus subtilis and plant lactobacillus fermented liquid had antibacterial resistance, bacillus natto indicated largest bacteriostatic diameter. So choose bacillus natto as experiment fermentation strain. The femented liquid of bacillus natto had certain degree of inhibition zone to gram-positive bacteria, gram-negative bacteria, fungi and yeast that verified its broad spectrum antimicrobial resistance.Through single factor experimental, research on different culture medium and fermentation conditions on the bacteriostatic and influence of peptide content in fermented liquid and contrast diffence of the two trends. The results showed that with additive amount of soybean protein 6%, glucose 0.4% and NaCl 0.05%, the fermented liquid showed best bacteriostatic effect. Meanwhile fermented time 56h, liquid amont 100mL/250mL, temperature 37℃, strain quantity 12%, pH 7.0 were best fementation condition. In the meantime, effect on polypeptide content has a general trend.With method of salting-out, ammonium sulfate precipitation was to extract the antibacterial ingredient in fermented liquid. Though different impact of ammonium sulfate on liquid extract bacteriostatic to determine the best ammonium sulfate saturation. Reseach on the influence of temperature, pH, protease on extrac bacteriostasis to provided the basis for thought fermented liquid storage conditions and stability. With method of SDS-PAGE, determine the protein molecular weight range in fermented liquid and extract. Sephadex G-75 gel column chromatography isolated and purified different molecular weight and with Fourier infrared spectrum, analyzed the changes of raw material soybean protein before and after fermentation. The results showed that 70% was the best ammonium sulfate saturation. Temperature below 60℃ the antibacterial activity was relatively stable, pH extracted a smaller influence on bacteriostasis, but it effected a bit in overly acidic or basic treatment. The antibacterial activity significantly reduced in treatment by trypsin. Extract relative molecular quality basically below 31KDa. Sephadex G-75 gel column chromatography isolated two molecular weight polypeptides composition, bacteriostatic experiment showed that smaller molecular weight component had antibacterial activity. Part C-N bond ruptured after fermentation, free amino content increased and the fermentation process of macromolecular protein by enzyme hydrolysis to small molecular polypeptids.