诺丽自然发酵过程中微生物群落解析及关键菌株对主要活性成分形成的影响

Fermented Noni（Morinda citrifolia Linn）juice is a popular healthy drink, which is made by close natural fermentation and frequently suffers serious microbial contamination and quality instability. In this project, we will investigate the culturable microorganisms and their dynamical changes in the fermented noni juice using traditional method, and also analyze the microbiota structure and evolution in the fermented noni juice by using high-throughput sequencing and quantitative PCR. Then we will select the predominant microbes in the fermented noni juice and the key strain or strains to enhance significantly the production of the main functional components in the fermented juice, such as polysaccharides, iridoids, scopoletin. With the key strain(s) as the model, we will study the metabolics of the fermented juice using CPMG 1H-NMR and analyze the evolution of the main functional components during noni fermentation, and then elucidate the effects of the key strain(s) on functional components production. This project will supply a theoretical guide and technical support for controlled noni fermentation by inoculating the key strains and therefore solve the bottle neck for fermented noni juice industry, and supply a new approach to enhance the functional properties of fermented noni juice and to develop healthy products with some specific functions, and also lay the foundations for further research on the metabolic regulation of noni juice fermentation.

诺丽发酵汁（诺丽酵素）是一种广受欢迎的保健饮品，一般采用密闭自然发酵而成。生产过程中普遍存在微生物污染严重、产品品质不稳定等难题。本项目首先以传统手段分析诺丽发酵过程中可培养微生物群落结构及其变化，再以高通量测序和定量PCR综合分析全部微生物群落结构和优势菌种及其变化，然后对优势菌种逐一筛选，获得能显著促进主要活性成分（多糖、环烯醚萜类、东莨菪素等）形成的关键菌株或菌株组合。在此基础上，以关键菌株/组合为研究模型，对接种关键菌株后的诺丽发酵汁进行基于核磁共振分析（CPMG 1H-NMR）的代谢组学分析，解析主要活性成分的变化规律，揭示关键菌株对主要活性成分形成的影响。这一方面可为建立“人工接种、有控制地诺丽发酵技术”、解决行业生产技术瓶颈提供理论指导和技术支撑，同时可为提高诺丽发酵产品的保健功效和开发特定保健功能的新产品提供新的思路，并可为进一步研究诺丽发酵的代谢调控奠定基础。

诺丽发酵汁是一种广受欢迎的保健饮品，一般采用密闭自然发酵而成。高通量测序分析发现，诺丽果自然发酵过程中，细菌群落中有26个门，418个属，260个种。真菌群落中有6个门，77个属，116个种。前期（3 d~30 d）丰度值较高的细菌为醋酸杆菌属、葡糖杆菌属和不动杆菌属，占比90%以上；后期（40 d~60 d）丰度值较高的为醋杆菌属、葡糖杆菌属和葡糖醋杆菌属，三者占比95%以上。真菌的优势菌为毛霉属，含量始终在90%以上。宏基因组学技术分析表明，主要微生物为：未鉴定醋酸杆菌、醋化醋杆菌、弗托氏葡糖杆菌、未鉴定葡萄糖杆菌，占比分别为56.90%、18.78%、17.31%和8.04%。转录组学分析表明，诺丽果的核心微生物群为泛菌属、葡糖杆菌属、伯克霍尔德菌、不动杆菌和假单胞菌，分别占6 1.3%、23.9%、5.9%、3.7%和2.9%；自然发酵过程中变形菌门的贡献度最高，达96%以上。在种水平，发酵初期以Acetobacter sp.为主，发酵后期以Acetobacter aceti和Gluconobacter sp.为活性微生物。从诺丽自然发酵汁中共分离出88个菌株，其中Gluconobacter japonicus N11和Acetobacter pasteurianus N4发酵汁抗氧化性能表现较好。Acetobacter syzygii N5发酵汁中东莨菪素含量最高，比自然发酵汁样品提升了45.6%。选择从不同发酵阶段诺丽自然发酵汁中分离的发酵性能优良的醋酸菌Acetobacter lambici C20-14、乳酸菌Lactobacillus plantarum A33，分别接种于诺丽汁进行发酵。发酵至15d时，东莨菪素、去乙酰车叶草苷酸含量及抗氧化能力趋于稳定。将分离鉴定的新种诺丽醋杆菌Acetobacter nonis AN02接种发酵，可以显著降低异味物质己酸、辛酸和丁酸的含量。本研究可为建立诺丽果接种发酵技术、解决行业生产技术瓶颈提供理论指导和技术撑，同时可为诺丽发酵产品的功效提升和风味改善奠定基础。