本研究利用实验室筛选的高活性益生菌株Bacillus subtilis OKF-004进行液态发酵培养,通过单因素实验和正交试验探究了最适形成芽孢的液态发酵培养基组分与培养条件,并将所得菌液通过鼓风干燥制备成菌剂,优化确定了制备菌剂的工艺。以包含2.5%玉米淀粉、3%豆粕、2%葡萄糖的培养基在初始pH为6.5、装液量30 mL(250 mL锥形瓶中)、温度32℃、接种量7%的条件下培养时,枯草芽孢杆菌最适合形成芽孢,芽孢数最高可达到1.1×1010 CFU/mL;离心后将菌泥与浓度5%的保护剂甘油以1:7 g/mL的比例复溶制成菌悬液,以虾头粉末作为烘干载体,将载体与菌悬液以2:3 g/mL的比例混合后,于50℃鼓风烘干6 h,所得菌剂存活率最高,可达140.91%。本文研究的液态发酵培养、鼓风干燥的固态微生物菌剂制备方法具有过程简便、成本低廉、节省能源的特点,适合工业化生产。
In this study,a high-activity probiotic strain Bacillus subtilis OKF-004 which was screened by the laboratory was used for liquid fermentation culture. The single-factor experiments and orthogonal experiments were used to explore the optimal liquid fermentation medium components and culture conditions for spore formation. The obtained bacterial liquid was prepared by blast drying to prepare solid microbial agent,and the process for preparing the bacterial agent was optimized. It was most suitable for the formation of spores when the Bacillus subtilis was cultured in a medium containing 2.5% corn starch,3% soybean meal,and 2% glucose at initial pH of 6.5,liquid volume of 30 mL in 250 mL erlenmeyer flask,32℃,and inoculum size of 7%. The highest number of spores could reach 1.1×1010 CFU/mL. The bacterial sludge was reconstituted with a concentration of 5% of the protective agent glycerin at a ratio of 1:7 g/mL to prepare a bacterial suspension,and the shrimp head was used as a drying carrier,and the carrier and the bacterial suspension were mixed at a ratio of 2:3 g/mL.After drying at 50℃ for 6 h,the obtained bacterial agent had the best survival rate of 140.91%. The liquid fermentation culture and the solid microbial agent preparation method are simple in preparation,low in cost and suitable for industrial production.