西藏开菲尔粒中Lactobacillus kefiri的分离与鉴定  

Isolation and identification of Lactobacillus kefiri from Tibetan kefir grains

作　　者：贾宇声 王兴兴[1] 潘迎捷[1,2,3] 王永杰 

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海水产品加工及贮藏工程技术研究中心,上海201306 [3]农业部水产品贮藏保鲜质量安全风险评估实验室,上海201306

出　　处：《上海海洋大学学报》2018年第1期141-148,共8页Journal of Shanghai Ocean University

基　　金：上海高校特聘教授东方学者岗位计划基金(20101222);上海市科学技术委员会科技支撑项目(10540503000);上海市科委工程中心建设(11DZ2280300)

摘　　要：Lb.kefiranofaciens是西藏开菲尔粒中的优势菌株,约占乳酸菌总数的95%。通过分离纯化,构建克隆文库并结合16S rRNA基因测序、变性梯度凝胶电泳(DGGE)、菌落PCR和显微镜观察等手段分析了开菲尔粒中的非优势乳酸菌Lb.kefiri。扫描电镜显示,其细胞形态呈短杆状(0.8~1.5×0.6~0.7μm),比Lb.kefiranofaciens短,且细菌聚集生长的趋势更为明显。在所构建的克隆文库中,25个菌落经测序分析,发现有1条序列属于Lb.kefiri。以开菲尔粒为样品,通过分离纯化,应用菌落PCR方法验证的22个单菌落中,有5个单菌落是Lb.kefiri。此外,对开菲尔粒进行PCR-DGGE检测,发现4个开菲尔粒样品中都有Lb.kefiri的存在。这些结果都证明了Lb.kefiri在开菲尔粒中稳定存在且所占比例相对较小,是一种非优势乳酸菌。Our previous work suggests that Lb.kefiranofaciens is the dominant bacterial species in Tibetan kefir grains and comprises approximately 95% of all lactic acid bacteria.In this research,bacterial isolation and purification,16 S rRNA gene cloning and sequencing,denaturing gradient gel electrophoresis（DGGE）,colony PCR and scanning electron microscope observation were performed to understand non-dominant lactic acid bacteria in kefir grains.Scanning electron microscope observation revealed that the cells of Lb.kefiri are short rod（0.8-1.5 × 0.6-0.7 μm）,which were shorter than Lb.kefiranofaciens.Lb.kefiri cells tended to form aggregates in pure culture condition.After sequencing analysis,1 of 25 clones was related to Lb.kefiri.For 22 colonies isolated from kefir grains,5 colonies are Lb.kefiri.In addition,DGGE results showed that Lb.kefiri exists in all kefir grain samples.These results demonstrate that Lb.kefiri exists in kefir grain and remains stable and is non-dominant.

关 键 词：西藏开菲尔粒 Lb.kefiri 扫描电镜 克隆文库 分离培养 DGGE 细胞形态观察 

分 类 号：TS201.3[轻工技术与工程—食品科学] S917.1[轻工技术与工程—食品科学与工程]