优良枯草芽胞杆菌的筛选及对小鼠安全性评价

Study on Screening Bacillus subtilis and Evaluation of Its Safety in Mice

为进一步鉴定、分析和评价分离于鸡粪便的13株芽胞杆菌,根据枯草芽胞杆菌16SrRNA序列设计特异性引物,PCR扩增13株分离于鸡粪便的芽胞杆菌,将扩增片段测序并分析同源性;将鉴定的枯草芽胞杆菌进行耐酸、耐胆盐、耐高温等试验,同时用不同剂量菌液分别以不同方式感染小鼠,定期称重、观察各脏器病理变化和计算主要脏器系数。结果显示,Q04、Q05、Q09与GenBank公布的枯草芽胞杆菌16S rRNA序列同源性为99%,且Q04在pH2.5,胆盐浓度2g/L以及100℃时生长良好;感染小鼠全部健康存活,体重均有增加;小鼠脏器无病变,脏器系数无显著差异(P>0.05)。由结果可知,分离鉴定菌株Q04、Q05、Q09为枯草芽胞杆菌,其中Q04具有更好的生物学特性,并且对小鼠安全无毒,可作为微生态制剂的候选菌株。

In order to identify,analyze and evaluate 13 Bacillus strains isolated from chicken feces,the primers were designed according to the 16 S rRNA sequences of Bacillus subtilis .The 13 Bacillus strains were amplified by PCR,and the amplified fragments were sequenced and analyzed for homologies.The identification of Bacillus subti-lis was conducted by acid,bile and high temperature resistance tests.The mice were infected with different doses of bacteria with various ways,and weighed regularly.The pathological lesions of various organs were observed and the main organ coefficients were calculated.The results showed that the homologies of Bacillus subtilis 16 S rRNA sequences of Q04,Q05,Q09 were 99% compared with that in GenBank.The Q04 grew well in pH2.5,bile salt concentration of 2 g/L and 100 ℃.The infected mice all survived healthily,their weights increased.There were no pathological lesions in organs,and the organ coefficients had no significant differences （P〉0.05）.In conclusion, the strains Q04,Q05 and Q09 were isolated and identified as Bacillus subtilis ,of which Q04 has better biological properties.The strains Q04,Q05 and Q09 were safe to mice,and can be used as candidate strains of probiotics.