新疆地区不同动物原奶微生物组及优势种群Lactococcus遗传结构的宿主特异性研究

Raw milk is known to harbour a complex microbial community. These microorganisms include bacteria of technological relevance such as the lactic acid bacteria (LAB), which can contribute to subsequent desirable fermentative reactions. However the presence of spoilage bacteria can have considerable negative effects on the quality of milk and dairy products while the presence of pathogens can have more severe repercussions. Lactococcus is an organism of substantial economic importance, used extensively in the production of fermented foods and widely held to have evolved from plant strains..The main purpose of this work is to select Lactococcus strains from dairy products from different environmental niches, xinjiang. Taxonomic identity and genetic diversity of strains isolated are determined by using universal 16S rRNA gene sequences and phenotypic characteristics. Accurate identification to the species level of the Lactococcus population is obtained by means of using species-specific primers and genomic fingerprintings (PFGE, rep-PCR, AFLP), and then we will apply rapid MLST genotyping method to determine the population genetic structure of Lactococcus from different environmental niches,I ncluding raw milk of different farm animals and other econiches. By PCR amplification of structural genes for antimicrobial bacteriocins, antibiotic resistance and virulence, we will detect antimicrobial activity and virulence traits of Lactococcus isolates and compare their occurrence in clonal clusters generated by MLST and genotypic fingerprints (PFGE, rep-PCR). In addition, we will use illumina high-throughput sequencing to comprehensively survey composition of the microbiota and the diversity of lactic acid bacteria (LAB) in a variety of raw milk of different farm animals in Xinjiang region..In this work, To conclude, our objectives are test whether the clonal clusters of Lactococcus lactis isolates from different origins exhibit genetic isolation and host-specifity and further assess the probiotic properties, functionality, and safety of the tested Lactococcus spp.

新疆地区少数民族众多，家养动物原奶自然发酵乳品大多以家庭作坊式的手工加工为主，开展各种动物原奶微生物组的解析对少数民族健康的影响非常必要。Lactococcus是原奶中最丰富、乳品工业中应用最广泛的乳酸菌，其野生型菌株开发潜力巨大。本项目拟广泛取样新疆南、北疆动物原奶，基于rRNA基因的高通量测序技术，比较空间上隔离的特定地理区域、不同动物原奶微生物菌群的结构组成，揭示动物原奶微生物组是否具有宿主动物特异性和地域性格局。深度分离筛选Lactococci菌株，分子指纹分型去重后对代表菌株采用MLSA、PFGE技术和耐药基因、毒力基因、细菌素基因多重PCR检测技术，结合高通量表型芯片方法，揭示乳球菌的群体遗传多态性与表型组学、耐药谱的相关性以及遗传结构分化的宿主特异性。项目的开展有助于实时评估不同动物原奶加工乳品对少数民族健康的潜在影响，为安全的野生型益生菌资源的开发和应用奠定理论参考依据。

采集获得新疆地区（伊犁、哈密、塔城和喀什）7种动物原乳（马乳、驴乳、山羊乳、绵羊乳、牦牛乳、驼乳、牛乳），共142份样品，通过高通量测序技术，比较不同地理区域、不同动物原奶微生物菌群的结构组成。通过乳酸菌分离培养法，深度分离筛选乳酸菌菌株，分子指纹分型去重后对代表菌株采用MLSA、PFGE技术和多重PCR检测技术，揭示乳酸菌的群体遗传多态性。并采用体外试验筛选潜在益生乳酸菌，进一步研究其发酵性能（凝乳时间、滴定酸度、感官评价、酸化能力、后酸化能力、双乙酰和乙醛等）。为发展区域特色乳品和益生菌产品奠定理论基础。. 测序结果显示，多样性丰富度为：驼奶>牦牛奶>马奶>牛奶>绵羊奶>山羊奶>驴奶；物种种类丰富度为：驼奶>绵羊奶>山羊奶>牦牛奶>马奶>驴奶。优势菌门皆为变形菌门和厚壁菌门；不同地区和不同动物类型来源的原奶中微生物群落结构相差甚远，并检测到环境细菌和致病菌。. 分离鉴定共得到842株乳酸菌，其中Lactobacillus 84株，Lactococcus 77株，Leuconostoc 35株，Enterococcus 69株，Pediococcus 72株，Streptococcus 21株，Bacillus 1株，Weissella 182株。马奶有297株，驼奶360株，驴奶153株，山羊奶32株。不同来源的原奶中乳酸菌的群落结构和物种丰度具有明显的差异性。. 体外实验筛选得8株潜在益生乳酸菌，其中Lactobacillus delbrueckii subsp.bulgar 2株、Lb. plantarum 4株、Lb. helveticus 2株。综合多种发酵性能指标分析， Lactobacillus delbrueckii subsp.bulgar T63-2和Lb. helveticus T62-46的生产利用价值高。