导　　师： 陈宗道;Azlin Mustapha

学科专业： 083201

授予学位： 博士

作　　者： ;

机构地区： 西南大学

摘　　要： 牦牛奶酪乳酸菌是我国重要而且独特的生物资源,有极其重要的收集、整理、保存、研究的价值。本研究从西藏、云南、新疆三个地区采集17份牦牛奶酪样品,分离纯化出了其中的乳酸菌,研究了乳酸菌的表型、基因型和益生特性。 表型特性的研究即用传统的菌落形态、革兰氏染色显微形态、生理生化、在不同环境/(温度、pH值、盐度/)中的生长特生,碳水化合物发酵方式等实验项目,将乳酸菌划分到属,进而初步判断出菌种,此外,还分析了产酸活性、利用柠檬酸盐等表型特性,以获得乳酸菌的基本数据, 基因型特性的研究包括16S rRNA序列分析和随机扩增多态性DNA技术。本研究建立了属特异性PCR→16S rRNA序列分析→种特异性PCR的快速鉴定模式,构建了系统进化树,以分析乳酸菌菌株的遗传亲缘关系。建立了适用于乳酸菌的RAPD反应体系和程序,用于分离鉴定出的乳酸菌,用NTsys2.10e软件对其图谱做聚类分析,与16S rRNA序列分析的结果相比较,分析菌株之间的遗传亲缘关系,分析菌株聚类方式与地域之间的相关性。 益生特性的研究包括乳酸菌菌株的产细菌素、产胞外多糖、产叶酸、降胆固醇的益生特性,并研究了菌株对抗生素的抗性、体内环境的耐受性、黏附活性,筛选出有突出益生功能,抗逆性较好的菌株,有希望作为益生菌加以开发应用。 主要的研究结果如下： /(1/)西藏、云南、新疆三个地区的样品平均pH值分别为4.31±0.39；4.69±0.29：4.52±0.22,地区之间不存在显著性差异/(P=0.176>0.05/),三个地区菌落计数平均值分别为4.17±0.43；4.30±0.68；3.9±0.59log CFU//g,也不存在显著性差异/(P=0.468>0.05/) /(2/)从17份样品中分离出39株乳酸菌,通过表型方法初步判断出菌种。35株菌为杆菌,被分为7个菌种：L.buchneri2株/(Y15.Y16/),L.casei16株/(T7.Y19,X21,X22,X23,X26,X27,X28,X29,X30,X31,X32 There are various kinds of lactic acid bacteria existing in Chinese yak milk cheeses, which is an extremely precious natural microbiology resource. It is a research work of great values that collecting, organizing, preserving and investigating those lactic acid bacteria. This study collected17yak milk cheese samples from Tibet, Yunnan and Xinjiang province, isolated lactic acid bacteria, studied their phenotypic, genotypic and probiotic characterization. The phenotypic characterization study included species identification of isolated lactic acid bacteria with classic taxonomy methods, such as colony morphology, Gram stain and microscopic morphology, primary biochemical and physiological properties, growth capability in different conditions /(temperature, pH value, and salt concentration/), carbohydrates fermentation patterns, acid producing activity, capability of utilizing citrate and so on. Isolated lactic acid bacteria were assigned to genus level, and preliminary species level. Phenotypic experiment results provided a comprehensive data of lactic acid bacteria. The capability of producing acid and utilizing citrate directly demonstrated lactic acid bacteria's role in fermentation. The genotypic characterization study included16S rRNA sequenc analysis and randomly amplified polymorphic DNA /(RAPD/). This study established a rapid identification mode:genus specific PCR→16S rRNA sequence analysis→species specific PCR. Isolated lactic acid bacteria were identified to species level with this mode, phylogenetic tree was constructed based on16S rDNA sequences to confirm species assignment and demonstrate strains'genetic relationship. This study also established a stable RAPD reaction system and amplification program for lactic acid bacteria. Isolated lactic acid bacteria were subjected to this RAPD analysis, NTsys2.10e software was used to perform cluster analysis of strains'RAPD fingerprinting patterns. The cluster result were compared with that of16S rRNA sequence analysis to figure out genetic relati

关 键 词： 乳酸菌 牦牛奶酪 表型特性 基因型特性 益生特性

领　　域： [轻工技术与工程] [轻工技术与工程]