基于多组学技术研究植物乳杆菌增强红枣多酚生物利用度的分子调控机制

More than 90% of the dietary polyphenols ingested is absorbed by the degradation of intestinal probiotics, and then plays its biological effects. And different intestinal probiotics composition in individuals will cause the absorption differences in dietary polyphenols. Therefore, the in vitro biological conversion of dietary polyphenols by intestinal probiotics to enhance its bioavailability has become a research hotspot in the field of Food Science and Nutrition. We have screened Lactobacillus plantarum WCFS1 as the best strains enhancing the bioavailability of jujube polyphenols, but its molecular mechanism and regulatory mechanism are still unclear. In view of this, Lactobacillus plantarum WCFS1 was used to fermentate the whole jujube to fully degrade the polyphenols. The fermented jujube puree containing the polyphenol metabolites with high bioavailability was selected and used for the following study. The RNA-seq and the iTRAQ techniques were used to analyze the differential expression of genes and proteins associated with the polyphenol metabolism-related enzymes, in order to predict the genes related with the formatation of polyphenol metabolites with high bioavailability. Furthermore, the main regulatory factors were determined by constructing the gene knockout mutants and the recombinant bacteria. Combined with the differentiated polyphenol metabolites in the jujube puree and the fermented jujube puree by the non-target metabolomics, the molecular regulation mechanism of enhancing the bioavailability of jujube polyphenols by Lactobacillus plantarum was revealed. This will provide a basis for the further understanding of the interaction between probiotics and dietary polyphenols and the nutritional value of the fermented foods by probiotics.

人体摄入的膳食多酚90%以上是在肠道益生菌作用下被吸收利用，继而发挥其生物效应。而不同人体的肠道益生菌组成会造成膳食多酚的吸收差异。因此利用肠道益生菌体外生物转化膳食多酚，增强其生物利用度，已成为食品科学和营养学的研究热点。我们前期已筛选出植物乳杆菌WCFS1是增强红枣多酚生物利用度的高效菌株，但对其增强的分子机制和调控机制仍不清楚。针对此问题，本项目拟采用植物乳杆菌对红枣进行全果发酵，筛选含高生物利用度多酚代谢产物的发酵红枣浆，采用RNA-seq和iTRAQ技术分析与多酚代谢相关酶的基因和蛋白质的差异表达，预测调控高生物利用度多酚代谢产物形成的相关基因，并通过构建基因敲除突变株和重组菌，确定其主要调控因子，再结合非靶标代谢组学筛选的发酵前后差异多酚代谢物，进而揭示植物乳杆菌增强红枣多酚生物利用度的分子调控机制，这将为更深入认识益生菌与膳食多酚的相互作用和益生菌发酵食品的营养价值提供依据。

(对项目的背景、主要、重要结果、关键数据及其等做简单概述，800字以内，含标点符号)：人体摄入的膳食多酚90%以上是在肠道益生菌作用下被吸收利用，继而发挥其生物效应。而不同人体的肠道益生菌组成会造成膳食多酚的吸收差异。因此利用肠道益生菌体外生物转化膳食多酚，增强其生物利用度，已成为食品科学和营养学的研究热点。我们前期已筛选出植物乳杆菌是增强红枣多酚生物利用度的高效菌株，但对其增强的分子机制和调控机制仍不清楚。基于此，本项目围绕植物乳杆菌增强红枣多酚生物利用度的研究主题，开展了系列研究工作，取得了一定有价值的研究成果。建立了同时测定红枣中六种酚酸的气相色谱-质谱联用法，为红枣中游离态酚酸的测定提供了可靠的分析方法；对我国16种红枣中的酚酸和黄酮组成及其抗氧化活性进行了评价，发现佳县红枣和赞皇大枣是含有多酚及其抗氧化活性最高的红枣品种；红枣皮中含有大量具有高抗氧化活性的不溶性多酚，发现这些多酚是通过MAPK信号通路，抑制了NF-κB 的活化，从而大大下调了RAW264.7巨噬细胞中 iNOS和COX-2的蛋白表达水平，其抗炎活性高于红枣核多酚，可见红枣皮中不溶性多酚可作为抗炎剂的一个潜在资源；不同乳酸菌菌种发酵对红枣浆中游离态酚酸及其抗氧化活性影响很大，植物乳杆菌是单独发酵红枣浆的最佳乳酸菌菌种，其与嗜酸乳杆菌以１：１体积比复合发酵可提高红枣浆中游离态酚酸及其抗氧化活性，有利于红枣中不溶性多酚的生物转化，提高其多酚的生物利用度；在复合乳酸菌发酵前，先采用酵母菌发酵，将红枣浆中的糖类消化，再和乳酸菌共同发酵，所得发酵红枣汁的抗氧化活性增强，品质提高，贮藏期稳定；将红枣浆以3%的比例加入羊乳中，采用乳酸菌发酵生产酸羊乳，红枣浆的添加改善了酸羊乳的风味，提高了酸羊乳的质构特性及其抗氧化活性；采用代谢组学技术已明确植物乳杆菌转化红枣多酚的主要代谢产物是具有降脂减肥作用的槲皮素和具抗肿瘤作用的毛地黄黄酮。这些成果可为新型高营养红枣食品的研发提供科学依据，也可为高生物利用度多酚的高效生物合成提供新的途径。