自然发酵酸马奶细菌多样性及其基因动态变化研究

发布时间：2018-05-26 08:24

本文选题：酸马奶 + PacBio　；参考：《内蒙古农业大学》2017年博士论文

【摘要】：作为一种发酵乳饮料,酸马奶因具有丰富的营养成分和重要的经济价值,而深受中亚和东欧人喜爱。虽然酸马奶具有独特的微生物群落结构,但目前对其核心菌群及微生物群落动态变化和功能基因研究不够深入。本研究首先采用PacBio SMRT测序技术在分类学地位"种"水平上对其核心细菌类群进行了甄别;继而以5个牧民家庭为采样点,分不同时间点在酸马奶制作过程中进行了样品的动态采集,利用宏基因组策略对酸马奶中细菌微生物多样性和功能基因动态变化进行了研究。通过本研究的实施可以全面、深入了解微生物群落结构动态变化对酸马奶品质形成的功能和相互作用机制,为酸马奶的品质改良和工业化生产提供原始数据和理论指导。主要研究结论如下:(1)酸马奶的核心细菌类群主要由隶属于Proteobacteria和Firmicutes 2个细菌门的 Lactobacillus helveticus、Lactobacillus kefiranofaciens、Lactobacillus gallinarum 和 Acetobater pasteurianus4个细菌种组成。纳入本研究的11个酸马奶样品共发现25个核心OTUs,其相对含量占所有质控后合格序列数的 43.64%,且其中 24 个 OTUs为Lactobacillus helveticus 由此可见,虽然有的样品中可能含有一些较为独特的种系型,但酸马奶共有大量的核心细菌菌群。(2)在酸马奶制备过程中,细菌群落扮演了重要角色。为了探究酸马奶发酵过程中的细菌群落结构和生物多样性的动态变化,本研究采用Pacbio SMRT三代测序技术对从5个采样点收集的22个样品进行了 16s rRNA基因的测序。结果表明,酸马奶样品中共确定了 148个细菌种,归属于82个属、8个门;细菌群落结构的差异归因于分离地的不同;D组样品细菌组成与其他组相比差异最显著;细菌群落组成的动态变化结果显示,在酸马奶发酵0-9h期间,瑞士乳杆菌(Lactobacillus helveticus)数量逐渐增加,在发酵9h达到峰值,然后降低,而粪肠球菌(Enterococcus faecalis),耐久肠球菌(Enterococcus durans)和酪黄肠球菌(Enterococcus casseliflavus)在整个发酵过程中均呈现增加的趋势,于24h达到最大值。(3)在基因水平,发酵不同时间点的样品都主要由COG功能大类L(复制、重组与修复)、E(氨基酸运输代谢)、J(翻译、核糖体结构与生物合成)、G(碳水化合物转运与代谢)组成。功能大类F(核苷酸转运和代谢)、M(细胞壁/膜/包膜生物合成)、V(防御机制)、Q(次级代谢产物的生物合成、运输和分解代谢)、L(复制、重组和修复)和U(胞内运输、分泌和囊泡运输)彼此之间存在较显著的正相关关系。除此之外,COG功能大类与酸马奶中的营养成分之间也存在较为密切的关系。基于KEGG数据库的注释结果发现,在发酵的前9个小时样品共享了 92.7%的KO,这期间KO号种类上并没有明显的增加。随着发酵时间的延长,酸马奶样品中微生物的基因丰度和代谢通路的分布会产生一定的变化。总体而言,微生物基因相对含量在刚接种进去发酵剂时(B2)与鲜乳样品(B1)之间存在较大差异。随着发酵时间延长,菌群功能基因变化趋缓。
[Abstract]:As a fermented milk drink, acid horse milk is very popular in Central Asia and Eastern Europe because of its rich nutritional components and important economic value. Although acid Mais milk has a unique microbial community structure, the research on its core microbial community and microbial community dynamic changes and functional basis is not thorough. First of all, PacBio SM was used in this study. The RT sequencing technology identifies the core bacterial groups at the level of taxonomic status, and then takes 5 herdsmen families as sampling points and carries out dynamic collection of samples during the process of acid Mais milk production at different points of time. The dynamic changes of microbial diversity and functional genes in acid horse milk are carried out by macrogenome strategy. Study. Through the implementation of this study, we can thoroughly understand the function and interaction mechanism of the dynamic changes of microbial community structure to the quality of acid horse milk, and provide the original data and theoretical guidance for the quality improvement and industrial production of acid Mais milk. The main conclusions are as follows: (1) the core bacterial groups of the acid mare's milk are mainly subordinate to them. The composition of Lactobacillus helveticus, Lactobacillus kefiranofaciens, Lactobacillus gallinarum and Acetobater pasteurianus4 bacteria of 2 bacterial gates of Proteobacteria and Firmicutes. A total of 25 core OTUs were found in the 11 acid milk samples of this study, and their relative content accounted for 43.64% of the number of qualified sequences after all quality control. 24 of them OTUs is Lactobacillus helveticus, although some of the samples may contain some more unique species, but the acid horse milk has a large number of core bacterial flora. (2) the bacterial community plays an important role in the process of acid horse milk preparation, in order to explore the bacterial community structure and birth in the fermentation process of acid horse milk. In this study, the Pacbio SMRT three generation sequencing technology was used to sequence the 16S rRNA gene of 22 samples collected from 5 sampling points. The results showed that the acid horse milk samples identified 148 species of bacteria, belonging to 82 genera and 8 gates, and the differences in the structure of bacterial communities were attributed to the different separation sites; the sample of D group was fine. The microbial composition was the most significant difference compared with the other groups, and the dynamic changes of the bacterial community composition showed that the number of Lactobacillus helveticus increased gradually during the fermentation of 0-9h milk, and the peak value of the fermentation 9h, and then decreased, and the Enterococcus faecalis (Enterococcus faecalis), the Enterococcus durans (Enterococcus durans) and the casein. Enterococcus (Enterococcus casseliflavus) showed an increasing trend during the whole fermentation process and reached the maximum at 24h. (3) at the gene level, the samples at different time points were mainly composed of COG functions, L (replication, recombination and repair), E (amino acid transport and metabolism), J (translation, ribosome structure and biosynthesis), G (carbohydrate transport). Composition. Functional major F (nucleotide transshipment and metabolism), M (cell wall / membrane / capsule biosynthesis), V (defense mechanism), Q (biosynthesis of secondary metabolites, transport and catabolism), L (replication, recombination and repair) and U (intracellular transport, secretion and vesicle transport) have significant positive correlation with each other. In addition, COG work The KEGG database based on the KEGG database found that the samples shared 92.7% of the KO in the first 9 hours of the fermentation, and there was no significant increase in the number of the samples during the period of the fermentation. On the whole, the relative content of microorganism has a great difference between the fresh milk sample (B2) and the fresh milk sample (B1). With the prolongation of the fermentation time, the change of the functional gene of the bacteria group slows down.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS252.1

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