酶协同微生物发酵燕麦多酚的释放和转化机制研究

Microbial fermentation contributes to the release and transformation of functional active ingredients of cereals. It is an efficient and environmentally friendly method for functional food production, but the scientific mechanism needs to be urgently researched. This research focuses on building enzyme synergistic microbial fermentation system, to realize the efficient release and transformation of phenolic from oats. The cellular mechanism of phenolic release from oats will be studied through the micrographic morphology. Expression and regulation of key enzymes will be discovered by combining western blotting with RT-qPCR technique to reveal the enzyme catalytic mechanism of phenolic release from fermented oats. Means of chemistry and metabolomics are used to parse the structural differences of phenolic during fermentation. Based on the UHPLC/Q-TOF/MS technology, changes of the components of free, conjugated and bound phenolic fractions from fermented oats will be analyzed to illuminate the transformation pathway of phenolic. Finally, the functional active components of phenolic from bio-transformed oats will be studied using molecular target high-throughput screening technology. Moreover, multispectral spectroscopy techniques will be used to investigate the mechanism of active phenolic components from oats on hypolipidemic and lipid-lowering effect. This project is helpful to clarify the release and biotransformation mechanism and the key active compositions of phenolic in the fermented oats, and to provide new approaches for producing high-end functional food of oats, as well as enrich the theory of microbial metabolism and biotransformation with grain materials.

微生物发酵有助于谷物功能活性成分的释放与转化，是一种高效的环境生态友好型的功能性食品生产方式，但其科学机制亟待深入研究。本项目以燕麦为研究对象构建酶协同微生物发酵体系，实现燕麦多酚的高效释放和转化；通过显微影像形态学技术，研究燕麦多酚释放的细胞机制，结合Western blotting和RT-qPCR技术探索关键酶表达与调控规律，揭示燕麦多酚的酶催化释放机理；采用传统天然产物化学与代谢组学相结合手段，解析发酵前后燕麦多酚结构差异，基于UHPLC/Q-TOF/MS组学分析发酵过程中游离态、缀合态和结合态多酚组成变化规律，阐释燕麦酚类物质的转化途径；采用分子靶点高通量筛选技术研究燕麦多酚生物转化功能活性成分，结合多光谱学技术探索燕麦多酚活性物质的降糖降脂功效作用机理。本项目有助于明晰燕麦多酚释放转化机理和核心活性成分，为制造燕麦高端食品提供新的思路途径，为充实谷物微生物代谢转化理论提供基础。

燕麦作为全麦谷物食品已被广泛应用，但普通燕麦制品存在功能多酚释放和消化吸收困难问题。本项目采用微生物发酵技术，解决燕麦多酚高效释放与转化的关键问题，提高燕麦的生物活性及其营养附加值。. 本项目研究基于不同区域燕麦多酚成分，构建了HPLC指纹图谱；筛选获得适合与燕麦生长代谢的益生菌株（Monascus anka GIM 3.592、Bacillus subtilis 784、Saccharomyces cerevisiae GIM 2.139），构建了红曲菌单发酵、酶协同红曲菌发酵、微生物共发酵等多个微生物发酵燕麦体系，探索了分阶段、分批次的微生物发酵调控模式，实现了酶协同微生物发酵燕麦多酚的高效释放转化，燕麦总多酚含量最高可达35.075 GAE/g DW，相比未发酵提高了约70倍。SEM显微影像观察发现微生物发酵过程中燕麦表层包括淀粉颗粒和纤维外壳结构形态发生明显变化，SDS-PAGE和水解酶活性考察微生物发酵过程中多酚释放与酶含量的关联性，解析了微生物利用燕麦底物生长代谢水解酶系，首先破坏燕麦表面纤维网格结构，然后解离非水溶性多酚与大分子物质结合的酯键，进而促进多酚释放的生物途径。不同燕麦体系中，UHPLC-Q-TOF-MS鉴定了519种和384种代谢物，筛选得到121种多酚类化合物，其中对香豆酸、芥子酸、香草酸、咖啡酸、阿魏酸等存在一定的上调或下调。HPLC考察发现酶协同微生物发酵过程中燕麦游离态（槲皮素，芥子酸，咖啡酸，芦丁，绿原酸，阿魏酸，p-香豆酸，香草酸）和结合态（阿魏酸，芥子酸）多酚类化合物组成呈现动态变化；进一步揭示了阿魏酸、对香豆酸、咖啡酸和绿原酸之间的微生物发酵转化途径。微生物发酵燕麦多酚的DPPH、ABTS+抗氧化活性和Fe3+还原能力显著增强，并且与多酚组分具有一定的相关性；体外酶抑制实验评估微生物发酵燕麦多酚组分具有一定的降脂活性，紫外吸收光谱和荧光光谱探究了燕麦多酚与胰脂肪酶的相互作用机制；模拟探索了微生物发酵燕麦多酚体外降糖活性与作用机理，发现了小肠是燕麦多酚消化和作用的主要场所。. 本项目充实了谷物微生物发酵代谢转化理论，为制造谷类（燕麦）高端食品提供新的思路和途径。