传统发酵乳制作过程中微生物的群落结构及功能动态变化研究

Traditionally fermented milk have a long history and are regarded as safe food. They are not only nutritionally rich, but are of important economical value. It is known that these dairy products harbor unique microbial communities. However, until now, there have only been few in-depth studies focusing on the dynamics and functions of these microbial communities during the fermentation process. In this project, samples will be taken from three selected local families of a pasturing area of the Chinese minority group, where traditional fermented products will be made. During the fermentation process, samples will be taken at different time points for further analysis. Firstly, the changes of the physical and chemical indicators including the sample composition, as well as the quantity and quality of the fermentation metabolites produced during the process will be determined. Secondly, the total microbial genomic DNA and RNA will be extracted from these samples. The sample-specific multiple barcode pyrosequencing technique will be applied to analyze the sequences of the total microbial 16S rRNA (for bacteria) and ITS (for fungi) in order to characterize the overall microbial community structure. Thirdly, the active microbial functioning will be investigated by comparatively analyzing the meta-transcriptomes of representative samples collected at different fermentation time points. Finally, all the obtained data will be collectively analyzed to reveal the dynamics and the regulation of the microbial communities during traditional fermentation processes. The project will fully and comprehensively reveal how the changes of the microbial communities are related to the quality of the fermentation products, and hence, further elucidate the mechanism, the functioning and the interaction of the microbiota with the products during fermentation. This study will provide novel data and will act as theoretical guidance for improving both the quality of traditionally fermented products and the process of their industrial production.

传统发酵乳历史悠久，食用安全，不仅具有丰富的营养价值，还有重要的经济价值。具有独特的微生物群落结构，但目前对发酵过程中微生物群落动态变化和功能研究不够深入。本项目拟以我国3个少数民族地区制作传统发酵乳的牧民家庭为采样点。在发酵乳制作过程中，分不同时间点动态采集样品，以这些样品为研究对象。首先，测定自然发酵过程中样品理化指标，发酵代谢产物组分和含量；其次，提取其中微生物基因组总DNA和RNA，采用样本特异性多重条形码焦磷酸测序技术测定其中微生物（细菌和真菌）的16S rRNA 和ITS基因序列,进行群落结构分析；之后，通过代表性样品不同发酵阶段微生物宏转录组学分析，研究微生物功能及互作。最终将数据进行联合分析，揭示发酵乳制作过程中微生物群落结构动态变化规律；全面、深入了解其中微生物群落结构动态变化对发酵乳品质形成的功能和相互作用机制，为传统发酵乳的品质改良和工业化生产提供原始数据和理论指导。

本项目从我国新疆、西藏、内蒙古以及俄罗斯卡尔梅克和布里亚特等地区采集自然发酵乳样品57份，从内蒙古锡林郭勒地区5个采样点，在酸马奶发酵过程中动态采集样品35份。应用二代测序技术完成了酸马乳、酸牛乳和酸牦牛乳中微生物多样性和群落结构的研究。然后选择酸马奶为重点研究对象，分析了样品发酵过程中的营养成分（脂肪、蛋白质，氨基酸、维生素、乳糖等）和理化指标（酸类、pH、滴定酸度）和代谢产物（风味物质，酸类）组分和含量的动态变化。同时，应用三代测序技术进行了酸马奶发酵过程中微生物多样性、群落结构、互作关系和宏基因组功能特性的深入研究。结果表明：乳酸菌和酵母菌是发酵中的优势微生物种群；乳源对发酵乳中微生物群落结构具有决定性影响；不同地域特点对传统发酵乳中微生物多样性和群落结构具有重要影响；不同发酵乳制作工艺对发酵乳中微生物多样性也具有重要作用。本项目的完成为传统发酵乳中微生物多样性的深刻了解提供了重要指导，同时为传统工艺的改良提供了重要的理论依据。通过三年的实施本研究发表论文9篇，其中SCI论文7篇；出版英文专著1部（其中本研究内容作为1章进行了撰写）申请发明专利1项，培养研究生3名，其中博士研究生1名，硕士研究生2名。