传统分离培养结合DGGE技术研究新疆传统发酵酸驼乳中乳酸菌的多样性  

Analysis of Lactic Acid Bacteria Diversity in Traditional Fermented Camel Milk Products in Xinjiang by the Culture-independent and Denaturing Gradient Gel Electrophoresis Methods

作　　者：樊哲新 李宝坤[1] 李开雄[1] 朱敏[1] 蒋琰洁 李浩然[1] 

机构地区：[1]石河子大学食品学院,新疆石河子832000

出　　处：《中国食品学报》2015年第4期208-217,共10页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金青年项目(31201395);国家自然科学基金地区项目(31460007;30960001);新疆生产建设兵团博士资金专项(2014BB005)

摘　　要：目的：分析新疆传统发酵酸驼乳中乳酸菌多样性,为我国传统乳制品微生物资源的利用提供基础数据。方法：采用传统分离培养和基于16S rDNA的PCR-DGGE方法,对12份酸驼乳样品中分离出的87株可培养乳酸菌进行形态、生理生化结合16S rDNA分子法鉴定菌株;对DGGE图谱上的15条16S rDNA目标条带切胶回收测序。结果：传统培养法显示样品中乳酸菌总数在106-108CFU/m L之间,其中植物乳杆菌为优势菌群,次优势菌群为乳酸片球菌;通过DGGE目标条带序列分析与相似性比对,植物乳杆菌、瑞士乳杆菌丰度最高,是优势菌群。此外,鉴定出干酪乳杆菌、屎肠球菌、乳明串珠菌、芽孢杆菌。DGGE图谱显示：样品混合菌群中有5条条带无对应的纯菌株,而纯菌株在混合菌群的图谱上都有相应的条带。结论：传统分离培养与PCR-DGGE在分析优势细菌种群上结果一致,均为植物乳杆菌;用传统分离培养未鉴定到屎肠球菌和芽孢杆菌。DGGE图谱能更全面地反映样品中的弱势菌群,体现样品细菌多样性的真实水平。Objective：To provide essential database for the utilization of microbial in the traditional fermented milk product of China, analysis the diversity of lactic acid bacteria（LAB）, which originated from Xinjiang traditional fermented camel milk product.Methods：Both culture-dependent and culture-independent methods, polymerase chain reaction-denaturing gradient gel electrophoresis（PCR-DGGE） based on the sequence of 16 S rDNA V6-V8 region gene, were applied to analysis the samples. 87 LAB strains were isolated and purified based on culture-dependent, the isolates were identified by physiology-biochemistry characteristics and the molecular method of 16 S rDNA. By the DGGE fingerprint technology, 15 target bands obtained from the gel were sequenced, identified and a phylogenetic tree was contructed.Results：The population of LAB varied from 106-108CFU/m L, The result showed that the dominant LAB was Lactobacillus plantarum and Pediococcus acidilactici followed by it through the traditional isolation method. The profile of DGGE revealed that the most dominant LAB population belonged to Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus Casei, Enterococcus faecium, Leuconostoc lactis, Bacillus were also identified in DGGE profiles. 5 bands of total bacterial DNA in DGGE profile of samples were not recovered by cultivation. Conversely, all species had the band of total bacterial DNA in DGGE fingerprint. Discussion Conclusion： In term of dominant bacteria, the two methods both showed that Lactobacillus plantarum was predominant group, However Enterococcus faecium, Bacillus were not recovered by cultivation. The DGGE fingerprint contained some minority of groups, this suggested that DGGE can be applied to analysis of bacterial effectively, which will reflect the true level of bacterial diversity.

关 键 词：酸驼乳 变性梯度凝胶电泳 多样性 传统分离培养 16S RDNA 

分 类 号：TS252.1[轻工技术与工程—农产品加工及贮藏工程]