PCR-DGGE法分析西藏传统发酵乳制品中乳酸菌的多样性  

Investigating the Diversity of Lactic Acid Bacteria in Tibetan Traditional Fermented Dairy Products by PCR-DGGE

作　　者：蒋厚阳[1] 陈芝兰[2] 赵国华[1,3] 杨吉霞[1,3] 

机构地区：[1]西南大学食品科学学院,重庆400715 [2]西藏大学农牧学院生物技术中心,西藏林芝860000 [3]重庆市农产品加工技术重点实验室,重庆400715

出　　处：《食品科学》2014年第1期167-173,共7页Food Science

基　　金：中央高校基本科研业务费专项(XDJK2009C039);国家自然科学基金项目(31060020)

摘　　要：目的:运用聚合酶链式反应和变性梯度凝胶电泳(polymerase chain reaction-denatured gradient gel electrophoresis,PCR-DGGE)技术分析西藏传统发酵乳制品中乳酸菌的生物多样性。方法:从西藏8个牧区采集19份样品,提取样品总DNA,用巢式和降落PCR扩增16S rRNA的V3区段,对扩增产物做变性梯度凝胶电泳,用NTsys 2.10e软件分析条带的相似性,切胶回收条带并测序,鉴定菌种并构建系统进化树、分析优势菌种。结果:19份样品中的乳酸菌菌群组成包括Lactobacillus paracasei、Lactobacillus helveticus、Lactobacillus fermentum、Lactobacillus crispatus、Lactobacillus delbrueckii、Lactobacillus buchneri、Lactococcus raffinolactis、Leuconostoc mesenteroide、Lactobacillus plantarum、Pediococcus pentosaceus、Lactococcus lactis、Streptococcus thermophilus。综合样品和牧区的乳酸菌分布情况,确定Lactobacillus delbrueckii为优势菌种。结论:PCR-DGGE技术能够有效分析西藏地区发酵乳制品中乳酸菌的多样性。Nineteen samples of Tibetan fermented dairy products were collected from eight pasturing areas and a polymerasechain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） method was established to investigate the biodiversity oflactic acid bacteria in Tibetan fermented dairy products. After total DNA was extracted from dairy samples by a commercialkit （Tiangen DP326）, the V3 region of the 16S rRNA gene was amplified by nested PCR and touchdown PCR, and the PCRamplified products were subjected to DGGE electrophoresis. All bands were excised from the gel and sequenced, and thespecies of strains were identified and a phylogenetic tree was constructed. NTsys 2.10e software was used to analyze thesimilarity of band profiles. Results showed that the microbial flora of lactic acid bacteria in Tibetan fermented dairy productswas composed of Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus crispatus,Lactobacillus delbrueckii, Lactobacillus buchneri, Lactococcus raffinolactis, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, Lactococcus lactis and Streptococcus thermophilus. Therefore the conclusion is drawnthat Lactobacillus delbrueckii is the dominant species in most of the samples, and it simultaneously distributes in all eightpasturing areas. This study has successfully established a PCR-DGGE method to investigate the biodiversity of lactic acidbacteria in Tibetan fermented dairy products.

关 键 词：乳酸菌 生物多样性 聚合酶链式反应和变性梯度凝胶电泳 系统进化树 优势菌种 

分 类 号：TS252.54[轻工技术与工程—农产品加工及贮藏工程]