The Screening,Identification And Fermentation Optimization Of Probiotics Strains Bacillus With Anti-Vibrio Effects

The vibrio screening and inspection procedures of prawns were established.The vibrio of prawns from Fuzhou(Fuqing,Lianjiang,Changle)regional were separated and identificated.20 strains of vibrio including 3 kinds of vibrio were separated.They included 2 strains of Vibrio hollis,4 strains of Moderate aeromonas and 14 strains of Vibrio furnissii.The detection rate of vibrio was 13.33%.The results in a certain showed that the prawns of Fuzhou regional were contaminated by Vibrio furnissii seriously.Vibrio furnissii Vf-6 isolated was choosed as the indicator bacteria used for screening Vibrio bacteria inhibition.According to the characteristics of pathogenic vibrio,four kinds of storage methods were compared to determine the optimum preservation method for pathogenic vibrio.The results showed that the sequence in the storage time from long to short was liquid wax preservation at room temperature>slant cant preservation at room temperature>liquid wax preservation at 4 °C>slant cant preservation at 4?.Therefore the preservation method of liquid paraffin at room temperature showed easy operation and good bacteria.biological characteristics stability was suitable for short-term storage of Vf-6..Through the method of bacteriostatic circle,preliminary screening with plate and secondary screening with shake flask were carried.Bs-5 was screened from 20 strains of bacillus preserved in laboratory,which has good antibacterial effect on Vibrio Furnissii Vf-6.By morphological characteristics,physiological and biochemical test and 16SrDNA molecular identification method the taxonomic position of Bs-5 strain was determined,Bs-5 strains was identified as Bacillus subtilis.The studies of bacteriostatic spectrum of B.subtilis Bs-5 showed that the antimicrobial metabolites have certain inhibition on Staphylococcus aureus,Micrococcus luteus and Vibrio furnissii Vf-6.It also has a strong inhibitory effect on trichoderma and pyricularia oryzae.But it has no bacteriostasis on Escherichia colilThe studies of physical and chemical properties of B.subtilis Bs-5 showed that antimicrobial metabolites showed good thermal stability,optimum pH 7.0 and good stability of protease and organic solvent.Different metal ions showed different effects on the antibacterial activity of metabolites.The fermentation of bacteria metabolism was optimized by single factor optimization,orthogonal experimental design and BP neural network optimization,the optimal fermentation medium compositions were sucrose concentration 5.4 g/L,flour 25.5 g/L,ammonium chloride 0.68 g/L,ZnSO4·7H2O 0.025 g/L.The optimum fermentation conditions were inoculated quantity 2%,shake flask temperature 35?,shake flask speed 180 r/min,the initial pH 6.5,loading fluid amount to 45 mL(250 mL conical flask).In the end the metabolites of bacteriostatic circle diameter reached 32.93 mm and showed 99.58%more than that of initial 16.50 mm.