莲子抗性淀粉对肠道菌群乳酸发酵产丁酸调控机制的研究

The content of butyrate in the intestine is associated with the butyrate-producing bacteria in cecum and colon, and these bacteria are regulated by the microbiota-accessible carbohydrates (such as resistant starch) which can pass through the digestive tract without undergoing metabolic transformation and pass into the large intestine. It is a fact that resistant starch can be fermented by gut microbiota to produce butyrate, but its regulatory mechanism of the production of butyrate by gut microbiota is not yet clear. Our previous research found that lactate conversion to butyrate fermented by gut microbiota in mice was promoted by lotus seed resistant starch (LRS3) through modulating the growth of starch-utilizing bacteria and butyrate-producing bacteria. The results provided a new idea for investigating the production of butyrate from the carbohydrate fermentation in the gut. Therefore, the different structural characteristics of LRS3 were prepared by the autoclaving treatment. The crystallization kinetics of LRS3 was used to quantitatively express its structural properties, and then the relationship between its crystallization kinetic properties and the structural properties was constructed. In addition, the effect of the different structural properties of LRS3 on the gut microbiota and metabolites in mice was investigated, then the regulatory effect of the structural properties of LRS3 on lactate conversion to butyrate fermented by gut microbiota was elucidated. Moreover, the fermentation system with the specific structure of LRS3 and [3-13C] lactic acid in vitro was established. The acetyl CoA pathway of lactate conversion to butyrate fermented by gut microbiota was detected using the isotope-labelled method. Finally, the regulatory mechanism of LRS3 on lactate conversion to butyrate fermented by gut microbiota was revealed. The results from this project will provide a scientific evidence for the development of a new prebiotics with high production of butyrate.

肠道内丁酸含量与盲肠和结肠内丁酸产生菌有关，而该肠道菌群又受能够进入大肠的可利用碳水化合物（如：抗性淀粉）所调控。抗性淀粉经肠道菌群发酵产丁酸已获证实，但其调控肠道菌群产丁酸机制尚未明确。我们发现，莲子抗性淀粉通过调控小鼠肠道内淀粉利用菌和丁酸产生菌的生长，促进乳酸发酵产丁酸，这为碳水化合物代谢产丁酸的研究提供了新思路。因此，本课题通过压热处理获得不同结构特性莲子抗性淀粉，采用结晶动力学方程定量表征其结构特性，构建结晶动力学特性与结构特性之间的关系；研究不同结构特性莲子抗性淀粉对小鼠肠道菌群和代谢产物的影响，阐明莲子抗性淀粉结构对肠道菌群乳酸发酵产丁酸的调控作用；在此基础上，建立特异结构莲子抗性淀粉和[3-13C]乳酸体外发酵体系，采用同位素示踪法，探明肠道菌群乳酸发酵产丁酸的乙酰CoA途径，揭示莲子抗性淀粉对肠道菌群乳酸发酵产丁酸的调控机制。该研究为开发高产丁酸的新型益生元提供科学依据。

莲子淀粉属直链淀粉含量高的特异性淀粉，极易老化生成3型抗性淀粉。抗性淀粉在健康人体小肠内不能被消化吸收但能在大肠中发酵或部分发酵代谢，产生短链脂肪酸等。肠道内丁酸含量与肠道内丁酸产生菌有关，而该肠道菌群又受抗性淀粉所调控。因此，本课题通过莲子抗性淀粉结构特性、结晶动力学和肠道菌群调节作用的研究，建立特异结构莲子抗性淀粉和[3-13C]乳酸钠体外发酵体系，揭示莲子抗性淀粉对肠道菌群乳酸发酵产丁酸的调控机制。研究结果如下：.水分含量显著影响莲子抗性淀粉的结构特性，70％和80％的水分含量有利于抗性淀粉的形成。莲子抗性淀粉在4、25及4/25℃下的回生均为一次成核，但晶核的扩散速率为4℃>4/25℃>25℃。体外粪便菌群研究发现，莲子抗性淀粉的结构与菌群生长具有相关性。结合生产效率及菌群调节功能，确定LRS-70%为特异结构莲子抗性淀粉。.莲子抗性淀粉可显著影响小鼠的肠道菌群结构，降低具有致病能力的菌群数量，增加短链脂肪酸产生菌的数量。小鼠粪便中丁酸含量随着喂养时间的延长而增加，而乳酸含量随着喂养时间的延长呈现先增大后减小，且丙酸含量变化规律与乳酸一致，这表明莲子抗性淀粉促进了肠道菌群乳酸发酵产丁酸。大鼠肠道菌群研究与小鼠肠道菌群研究规律一致，但非靶向代谢组学未能筛选出差异的短链脂肪酸，这可能与筛选的方式及短链脂肪酸在代谢产物中所占的比例有关。.莲子抗性淀粉和[3-13C]乳酸钠对生长对数期以及稳定期菌群的影响结果表明，莲子抗性淀粉可以促进大鼠粪便菌群中双歧杆菌等益生菌的增殖，同时抑制志贺氏菌等有害菌的生长。经过菌群的发酵，抗性淀粉结构发生了明显的变化，菌群首先利用抗性淀粉的无定形区以及亚结晶区。结合同位素示踪分析，构建了基于（丁酰CoA：乙酸）CoA转移酶为主要途径的莲子抗性淀粉调控肠道菌群乳酸发酵产丁酸的乙酰CoA途径。该研究为开发高产丁酸的新型益生元提供科学依据。