Construction Of Producing Strains Of The Intestinal Pathogenic Bacteria Antimicrobial Proteins And The Preliminary Study Of Its Fermentation Conditions

As a kind of green and healthy microecologics,Bacillus subtilis could balance the intestinal flora and promote the nutrient absorption and enhance immunity.It is one of the ideal antibiotics alternative,and has been used widely in livestock breeding.The aim of the study was to construct the antibacterial protein gene in Bacillus subtilis and to obtain the LW1 industry producing strain.In this study the optimal culture conditions of Bacillus subtilis LW1 was got,which provided a reference for subsequent in-depth study of its antibacteria function and industrial production.In this study a genetic engineering strain named Bacillus subtilis LW1 was constructed,which could express the intestinal pathogenic bacteria antimicrobial protein.The functions of intestinal probiotics and the antibacterial protein were explored by establishing a poison attack model.Mice were immunized with intestinal probiotics,LW1and antibacterial protein.Antibacterial activity was tested by taking an intestinal bacteria inhibition experiment.The results showed that the number of E.coli in feces and intestines were all reduced in varying degrees.The medium compositions and fermentation conditions of LW1 were optimized in this study.Single factor experiment and orthogonal experiment were used to ascertain the optimal medium formula.The optimal result was:sucrose 1.5%,yeast extract 0.5%and MgCl₂ 0.6%.The optimal fermentation conditions were:temperature 35oC,initial pH 7.2,inoculation quality 10%,volume 20 mL/250mL.5 L fermentor was used to explore the basic fermentation conditions.The best industrial nitrogen source was soybean meal determined by single factor experiment.The finally optimal components of fermentation medium were:1.5%carbon source?sucrose/cornstarch 1:1?,1.5%nitrogen source?yeast extract/soybean powder 1:1?and 0.9%inorganic salts?MgCl₂/K₂HPO₄/KH₂PO₄ 1:1:1?,Set different fermentation parameters,and the results indicated that at 30oC³5oC,initial pH 7.0⁷.2 and 300 r/min were the optimal conditions.