肠道菌群和发酵食品微生物的分析、分离中液相色谱技术的应用基础研究

The microbes in intestinal microflora and fermented foods are closely related to nutrition and health. Our preliminary study found that liquid chromatography can be a rapid and sensitive technique for the separation, analysis and preparation of microbial intact cell.. This project addresses the shortcomings of the methods to analyze intestinal microflora and microbes of fermented food, such as too much interference, limited detection sensitivity and accuracy, as well as microbial cells not being separated and recovered for further analysis. Using standardized samples like rat and rabbit droppings, yogurt and Koji, we will firstly analyze the binding mechanism of microbial cells with impurities, and then study the effects of liquid chromatographic methods to remove impurities, separate and recover different microbial cells, so as to improve the sensitivity and accuracy of PCR-DGGE method, as well as the recovery rate of the target strains. Finally, we will analyze the distribution of functional groups on cell surface of pure microbe by its adsorption and de-adsorption to liquid chromatographic resins and Caco-2 cell monolayer, so as to understand the adsorption and colonization to intestinal wall, fermentation tank and food raw materials, and the surface properties variation of these strains.. The project is expected to lay a base for the application of liquid chromatography in the analysis and the separation of food microbes, realize an innovation of the methodology for the analysis, separation and preparation of intestinal microflora and microecology of fermented foods.

肠道菌群和发酵食品微生物都与国民营养和健康密切相关，本项目组前期研究发现液相色谱可成为细菌完整细胞分离、分析和制备的快速、灵敏的技术手段。.本项目针对分子生物学技术等方法分析肠道菌群和食品微生物时，存在的干扰成分多、检测灵敏度和准确性受限以及菌体细胞无法分离回收、分析等问题，拟用标准化的鼠和兔粪便、酸奶和酿酒红曲等发酵食品为样品，首先分析其中的微生物与杂质成分的结合机理，然后研究液相色谱方法对杂质成分的去除、不同微生物细胞的分离以及回收的效果，从而提高PCR-DGGE等方法的灵敏度和准确性，以及目标菌株的菌体回收率。最后通过液相色谱、Caco-2细胞单层分析纯菌细胞表面的荷电、疏水等基团的分布，了解这些菌株在肠壁、发酵池和食品原料的定植、吸附以及表面特性变异等。.项目可望奠定液相色谱技术在食品微生物分析、分离中的应用基础，实现肠道菌群、发酵微生态微生物的分析、分离和制备的一个方法创新。

肠道菌群和发酵食品微生物与国民营养和健康密切相关，本项目针对原有分子生物学技术等方法分析肠道菌群和食品微生物时，存在的干扰成分多、检测灵敏度和准确性受限以及菌体细胞无法分离回收、分析等问题，研究探讨了液相色谱技术在解决这些问题方面的应用基础。. 实验首先用标准化的大鼠、小鼠粪便、酸奶和酿酒红曲等发酵食品为样品，分析确定肠道菌群、红曲微生物与杂质成分之间通过离子相互作用（静电引力）、疏水作用而相互结合。然后以红曲为例，通过红曲高效液相样品制备条件的优化、离子交换色谱条件的优化以及红曲中微生物的分离和鉴定，确认了液相色谱方法对杂质成分的去除、不同微生物细胞的分离以及回收的效果，以及提高PCR-DGGE方法的灵敏度和准确性、目标菌株的菌体回收率。最后通过液相色谱、静态吸附曲线和动态吸附曲线分析了纯菌细胞表面的荷电、疏水等基团的分布，了解这些菌株在肠壁、发酵池和食品原料的定植、吸附以及表面特性变异等。. 项目奠定了液相色谱技术在食品微生物分析、分离中的应用基础，实现了肠道菌群、发酵微生态微生物的分析、分离和制备的一个方法创新。