乳酸菌发酵对荔枝果肉多糖结构特征与益生作用的影响及其机制

Lactic acid bacteria (LAB) fermentation is an important way to improve the nutrition of litchi pulp. Our previous study revealed that LAB fermentation could change the physicochemical properties of the polysaccharides of litchi pulp, and increase its stimulation of probiotic proliferation in vitro. However, the mechanism about the change of structure and prebiotic activities of litchi polysaccharide with LAB fermentation is not clear. The polysaccharides from fresh and fermented litchi pulp with LAB will be used as the materials for this project. To clarify the effect of fermentation treatment on the prebiotic activities of litchi polysaccharide, the prebiotic activities of polysaccharides from both fresh and fermented litchi pulp will be studied in vitro and in vivo. Furthermore, the primary structure and advanced structure, as well as the prebiotic activities in vitro of different polysaccharide fractions from fresh and fermented litchi pulp will be analyzed to explore polysaccharides’ structural changes and its structure-activity relationship. Moreover, the respective primary active polysaccharide fraction from both fresh and fermented litchi pulp will be used to study the differences in glycolysis metabolism of probiotics by analysis of carbohydrate enzymes. The purpose of this project is to reveal the effect of LAB fermentation treatment on prebiotic activities of litchi pulp polysaccharides and its mechanism. These results will enrich the theoretical basis of the nutritional properties of foods improved with lactic acid bacteria fermentation and provide scientific basis for the intensive processing of litchi functional products.

乳酸菌发酵是提高荔枝营养特性的重要手段。项目组前期研究发现乳酸菌发酵可以改变荔枝果肉多糖的理化性质，提高其体外促益生菌增殖作用，但对于荔枝果肉多糖如何通过乳酸菌发酵处理改变其结构而影响其益生活性的作用机制尚不清楚。本项目拟通过体内动物实验和体外益生活性评价实验，比较新鲜和发酵后荔枝果肉多糖的体内外益生活性变化；解析发酵前后荔枝多糖不同级分的一级结构和高级结构差异，结合体外益生活性评价，探明发酵前后荔枝果肉多糖的结构变化规律及其影响益生活性的构效关系，并确定益生活性主级分；在此基础上，围绕碳水化合物酶，探究益生菌对发酵前后各多糖主活性级分的酵解利用差异，阐明发酵处理对荔枝果肉多糖益生作用的影响机制。研究结果将丰富乳酸菌发酵处理提高食品营养特性的理论基础，为荔枝营养功能食品精深加工提供理论依据。

乳酸菌发酵是一种常见的荔枝加工方式，荔枝果肉富含活性多糖，乳酸菌发酵对荔枝果肉多糖的结构和生物活性的影响并不清楚。本项目考察乳酸菌发酵不同时间点（0-72h）荔枝果肉多糖结构和益生活性的差异，发现益生活性最好的是发酵6h的荔枝果肉多糖（LP-6）；通过体内动物实验比较发酵前荔枝果肉多糖（LP-0）和发酵后荔枝果肉多糖（LP-6）对肠道菌群及肠粘膜屏障的影响差异，发现2种多糖在体内均能促进肠道益生菌含量，增加结肠ZO-1、MUC-2、Lgr-5等表达，且LP-6具有更好的效果；进一步采用体外模拟消化酵解模型探究两者的消化酵解差异，发现两种多糖经胃液消化后结构发生轻微降解，在肠液中无显著变化，经粪便菌群发酵后都发生一定程度的降解，但不能被微生物完全利用，LP-6的保留率为35.59%，LP-0的保留率为42.87%；采用DEAE-Sepharose Fast Flow阴离子交换柱分别纯化发酵前后荔枝果肉多糖，每个多糖均有4个级分，采用益生活性跟踪筛选出各自的主活性级分，LP-0-4和LP-6-3，解析其结构特征：LP-0-4的Mw为47.8kDa，主要由1→3,6-α-D-Galp-、→3-α-Galp-1→、1→5-α-Araf, 1-α-L-Araf组成；LP-6-3的Mw为45.3kDa，主要由1→3,6-α-D-Galp-、1→5-α-Araf, 1-α-L-Araf组成；进一步以LP-0-4为原料，从体内探究其对肠道菌群的调节作用机制，发现荔枝果肉多糖级分能通过调节肠道菌群组成和丰度，促进短链脂肪酸等代谢产物，以及短链脂肪酸受体GPR41、GPR43的mRNA和蛋白表达，增强阿拉伯糖苷酶、半乳糖苷酶和葡萄糖苷酶等碳水化合物酶活，发挥调节肠道菌群的作用。