西藏牦牛源枯草芽孢杆菌的分离培养及鉴定

Isolation,Culture and Identification of Bacillus Subtilis from Yak in Tibet

从西藏健康牦牛的新鲜粪便中分离、纯化枯草芽孢杆菌,并筛选出耐受性最好的菌株,并进行形态学观察、生化试验、PCR扩增、电泳检测、16s rRNA序列测定、构建进化树并进行同源性分析,对通过鉴定的枯草芽孢杆菌进行耐肠液、耐胆盐、耐高温等耐受性试验。结果显示:分离出疑似枯草芽孢杆菌4株,革兰氏染色为阳性;芽孢染色液染色菌体为蓝色,芽孢不着色。菌体呈椭圆到柱状,芽孢位于菌体中央或次极端。16s rRNA测序有4株枯草芽孢杆菌,与标准株序列同源性高达99.6%,分别命名为Yak-KC1,Yak-KC5,Yak-KC6,Yak-KC7,该4株菌对肠液、胆盐、高温均具有一定的耐受性,Yak-KC7的耐受性最佳,具有进一步开发为益生菌的潜能。

The study was to isolate and purify Bacillus subtilis from the fresh faeces of healthy yaks in Tibet,and to select the strains with the best tolerance.Morphological observation,biochemical test,PCR amplification,electrophoresis detection,16s rRNA sequence determination,construction of evolutionary tree and homology analysis were carried out on the isolated strains.Bacillus subtilis was tested for tolerance to intestinal fluid,bile salt and high temperature.The results showed that 4 suspected Bacillus subtilis strains were isolated,and gram staining was positive.The spores were blue in colour and did not stain.The thalli were from elliptic to columnar with spores in the center or subextreme of the thalli.Four strains of 16s rRNA sequencing were Bacillus subtris,with 99.6%homology with the standard strains,and were named as Yak-KC1,Yak-KC5,Yak-KC6 and Yak-KC7,respectively.These four strains have certain tolerance to intestinal fluid,bile salt and high temperature,and Yak-KC7 had the best tolerance,which had the potential to be further developed as probiotics.