1株解淀粉芽孢杆菌B7快速液态发酵优化工艺研究

Study on the Optimization Technology of Rapid Liquid Fermentation of a Strain of Bacillus amyloliquefaciens B7

本试验以1株饲用益生菌解淀粉芽孢杆菌B7(Bacillus amyloliquefaciens B7,B.amyloliquefaciens B7)为研究对象,为了增强其在液态条件下的发酵水平,提高其芽孢发酵工艺优化效率,拟采用测定芽孢吡啶二羧酸(dipicolonic acid,DPA)浓度的方法来代替传统的平板芽孢计数法,以探讨此测定方法在芽孢发酵优化工艺中的可行性。首先,通过单因素试验筛选出对菌株产孢有显著促进作用的因子,进一步通过正交优化试验确定蛋白胨、玉米粉、大米蛋白粉和Mn^(2+)组合的最优水平。结果表明,发酵液中的芽孢浓度与测定的DPA荧光强度呈线性相关,在蛋白胨20 g/L、玉米粉10 g/L、大米蛋白粉20 g/L、Mn^(2+)1.0 mmol/L时发酵48 h,检测到DPA荧光强度达到最大,其相应的芽孢浓度达到7.0×10^(9)CFU/mL。与实验室常用的LB培养基相比,芽孢浓度提高了3.3倍;与工业生产用培养基相比,芽孢浓度提高了2.2倍。本研究为芽孢杆菌发酵过程中芽孢浓度测定提供了一种快速方法,为工业菌株B.amyloliquefaciens B7的发酵提供了一种成分简单且高产芽孢的培养基。

In this study,a strain of probiotic Bacillus amyloliquefaciens B7(B.amyloliquefaciens B7)was used as the research object,in order to improve its level of fermentation under liquid conditions,and to improve the optimum efficiency of spore fermentation.The method of determination of dipicolonic acid(DPA)concentration was proposed to replace the traditional plate spores counting method to explore the feasibility of this method in the optimum process of spore fermentation.The significant factors affecting spore production were first screening through the single-factor experiment.Firstly,the factors that had significant promoting effect on spore production of strain were screened out by single factor test.The optimum combination of peptone,corn powder,rice protein powder and Mn^(2+)was determined by orthogonal experiment.The result showed that the spore concentration was linearly dependent on the fluorescence intensity of DPA in the method,and the optimal medium for higher spore concentration was peptone 20 g/L,corn flour 10 g/L,rice protein powder 20 g/L,Mn^(2+)1.0 mmol/L.After 48 h of fermentation,spore concentration reached 7.0×10^(9)CFU/mL.Compared with the LB medium and the industrial production medium used in practice,the spore concentration was increased by 3.3 and 2.2 times,respectively.This study provided a rapid method for the determination of spore concentration during fermentation of Bacillus,and a simpler medium with high spore yields was presented for industrial spore fermentation of B.amyloliquefaciens B7 based on the rapid quantification of spore concentration.