Study On High Cell Density Culture And Preservation Of Bacillus Subtilis CS27

Bacillus subtilis is one of the15probiotics strains that have been approved by ministry of agriculture, more and more probiotics strains have been developed into industrial microorganisms. Its preparations are environmental protection product with non-toxic and non-pollution, which have broad prospects for development, have been widely used in animal husbandry, aquaculture and feed industry, have showed great economical, social benefits and ecological benefits. The objective of this topic is to develop microbial agents of Bacillus subtilis CS27for forage. Orthogonal experiments and Response Surface Analysis were used to optimize the liquid fermentation medium of CS27and initially optimize the CS27fermentation conditions of5L sartorius stedim biotech.All of these researches laid a solid foundation for the industrial production of CS27microbial agents.In addition,the solid preservation conditions of CS27microbial agents were studied. The results are as follows:(1) The liquid fermentation medium and technological conditions of Bacillus subtilis CS27were optimized by single factor experiments and orthogonal experiments. The results showed that optimum medium formula was molasses1.5%, soybean powder1.5%, NH4C10.5%,NaCl1.0%,MgSO4·7H2O0.1%,sodium citrate0.1%.The favorable fermentation condition was determined:fermentation temperature32℃,initial pH value of medium7.0, inoculation amount10%, liquid volume in flask50mL.(2) A strain of Bacillus subtilis CS27was isolated from the mud of pound, and the Plackett-Burman design was used to study the8factors influencing the growth of Bacillus subtilis CS27. the results shows that soybean powder, ammonium chloride, the initial pH were the main factors. Response surface methodology was also used to research the optimization for the3main factors, and the results show that the concentration of Bacillus subtilis CS27was significantly increased and the cell biomass increased froml.34x109cfu/mL to1.55×109cfu/mL, which was increased by20%. (3) Bacillus subtilis CS27was cultured by using a5L fermentor and the conditions for culture of the Bacillus subtilis CS27were obtained.The optimal conditions for the Bacillus subtilis CS27were studied and the results were:fermentor180r/min, pH6.5, dissolved oxygen10%and cultivating time21h.Under the optimized conditions, he Bacillus subtilis CS27could reach aboutl.45×1010cfu/mL.(4) The result of solid microbial agents preservation showed that the best method of preservation is zeolite adsorption when capacity was1:7.Three months later, hightest bacterial survival rate was up to70.28%.