Screening And Identification And Optimization Of Spore Production Conditions And Antifungal Protein Production Conditions Of Anti-calf-diarrhea Probiotics Strains

Research was done to screening and identification Basillus which could strongly inhibit Escherichia coli from healthy bovine intestine feces. With Escherichia coli as indicative bacteria, Basillus strains that could strongly inhibit Escherichia coli were prescreened and secondary screened with significance analysis to secondary screening result by SPSS software. Identification of these strains were done by morphological observation, physiological biochemical test and 16S rDNA sequence analysis. Growth curves of these strains were drawn by diluted viable count method. The fitting was done to obtain growth kinetic parameters of these strains with modified Gompertz equation and modified Logistic equation as models by SPSS software. 4 strains Basillus which could strongly inhibit Escherichia coli from healthy bovine intestine feces were obtained. The 4 strains were identified to Bacillus amyloliquefaciens. R squared of growth curves fitted by SPSS software was all above 0.97. 4 strains Bacillus as probiotics were obtained. Growth kinetic parameters of these strains could be predicted accurately by SPSS software.Research was done to improve ratio of spore production of strain Bacillus amyloliquefaciens BN-10, which could strongly inhibit Escherichia coli, screened from healthy bovine intestine feces. With single factor testing method, carbon source, nitrogen source and inorganic salts, which were suitable to spore production of strain BN-10 as probiotics, were chosen. With range analysis and general linear model analysis using SPSS software to results of orthogonal designs, fermentation medium and spore production conditions, which were suitable to spore production of strain BN-10, were determined. The ratio of spore production was 98.70% and the total bacterial counts was 2.26×10⁸cfu/mL when sucrose content was 1.5%, peptone content was 1.0%, MnSO₄ content was 0.05%, CaCl₂ content was 0.01%, medium initial pH 6.0, seed age was 12h, inoculation volume was 10%, media volume was 30mL/250mL, rotation speed was 200r·min^-1, cultured at 37℃for 72h.Research was done to optimize antifungal protein production conditions of strain Bacillus amyloliquefaciens BN-10 which could produce antifungal protein that could inhibit Escherichia coli. With single factor testing method, kinds of inorganic salt, nitrogen source and carbon source on the production of antifungal protein were chosen. With range analysis and general linear model analysis using SPSS software to results of orthogonal designs, it was determined that antifungal protein production conditions including fermentation medium and fermentation conditions which were most suitable to antifungal protein production of strain BN-10. The most suitable antifungal protein production conditions were that sucrose content was 2%, soybean cake powder was 5%, ZnSO₄ content was 0.01%, KCl content was 0.02%, medium initial pH 6.0, seed age was 12h, inoculation volume was 2%, media volume was 30mL/250mL, rotation speed was 200r·min^-1, culture for 24h at 37℃. The most suitable conditions for antifungal protein production were obtained. And the yield of antifungal protein was improved obviously.