一株凝结芽孢杆菌的分离筛选及产孢条件优化

Isolation and optimization on spore-forming conditions of Bacillus coagulans

摘要:

【背景】凝结芽孢杆菌除了具有一般乳酸菌的益生功能外，还具较强的耐酸、耐胆盐、耐高温、易贮存等生物特性。【目的】从泡菜中筛选一株性能优良的凝结芽孢杆菌用于微生态制剂的制备，并对其产孢率进行优化，为该菌株的进一步工业化生产提供参考依据。【方法】采用选择性培养基通过特定的培养条件，筛选到一株抑菌效果良好的产酸芽孢杆菌，并对其进行特异性引物的鉴定、16S rRNA基因序列分析及生理生化实验。通过单因素及正交试验对菌株的产芽孢条件进行优化。【结果】筛选得到一株凝结芽孢杆菌BC01，该菌株对大肠杆菌(Escherichia coli CVCC 1527)、鼠伤寒沙门氏菌(Salmonella typhimurium CVCC 2228)、产气荚膜梭菌(Clostridium perfringens CVCC 46)、猪霍乱沙门氏菌(Salmonella choleraesuis CVCC 503)等均有较强的抑制作用；模拟胃液处理120 min存活率达到94%；0.3%的胆盐存活率达到84.3%。单因素及正交试验优化后的最适培养基配方：糖蜜10.0 g/L，酵母浸出粉20.0 g/L，NaCl 5.0 g/L，K2HPO4?5.0?g/L，MnSO4 10.0 mg/L；最适培养条件：接种量4%，温度45 °C，初始pH 7.0，转速200?r/min，培养时间36 h。在该优化条件下，其活菌数最高达到6.7×109 CFU/mL，产孢率达到89.2%。【结论】筛选得到一株可用于微生态制剂的菌株——凝结芽孢杆菌BC01，对其产孢率进行了优化，为工业化生产奠定了基础。

Abstract:

[Background] In addition to the general function of lactic acid bacteria, Bacillus coagulans have strong biological properties of resistance to acid, bile salts, heat and easy storage. [Objective] Screen a strain of Bacillus coagulans from pickle to apply for the preparation of probiotics and optimize its sporulation rate as a guide for further industrial production. [Methods] Bacillus with good antibacterial effect screened by specific culture conditions, are identified by specific primers, 16S rRNA gene sequence analysis and physiological & biochemical experiments. The single-factor and orthogonal experiments are used to optimize the spore-producing conditions. [Results] Bacillus coagulans BC01 with a strong inhibitory effect to Escherichia coli CVCC 1527, Salmonella typhimurium CVCC 2228, Clostridium perfringens CVCC 46 and Salmonella choleraesuis CVCC 503 was screened. Its survival rate under 120 min of gastric acid treatment is up to 94%. 84.3% after 6 h in the 0.3% bile salt solution. The best medium is obtained by single factor and orthogonal experiment optimization. The optimized formula includes, molasses 10.0 g/L, yeast leaching powder 20.0 g/L, NaCl 5.0 g/L, K2HPO4 5.0 g/L and MnSO4 10.0 mg/L. The optimum culture conditions are as follows, inoculation quantity 4%, temperature 45 °C, initial pH 7.0, rotational speed 200 r/min, culture time 36 h. Under the conditions, the number of viable cells up to 6.7×109 CFU/mL and the rate of sporulation 89.2%. [Conclusion] Bacillus coagulans BC01 for microecological preparation was screened. The sporulation rate of Bacillus coagulans BC01 was optimized. It will lay down a foundation for industrial scale production of Bacillus coagulans.