人体肠道菌群体外生物转化大黄过程优化及成分变化研究

目的 探究人体肠道菌群体外生物转化大黄过程条件及成分变化。方法 采集3名健康儿童（1～3岁）新鲜粪便制备菌群悬液；以活菌数保持最多为依据进行初始培养基的选择；按照初始培养基中原有的0.5%碳源和0.5%氮源的添加量，进行不同碳源/氮源组合，依据活菌计数结果判断最优组合；通过指纹图谱技术筛选菌群体外培养条件，包括大黄添加方式（大黄粉或大黄水浸液）、转化时间（24或48 h）、氧需求（有氧或无氧）；基于LC-MS代谢组学进行转化前后成分分析，采用SIMCA软件（V16.0.2）进行主成分分析（PCA），差异代谢物分析采用MetaboAnalyst 5.0。结果 菌落计数和指纹图谱结果显示，蔗糖/大豆胨为改良YCFA培养基最适组合，粉状大黄为添加方式，培养基与大黄粉最适添加体积质量比为32∶1，有氧培养24 h为最优生物转化条件。代谢组学结果表明，主要代谢产物共计763种，数目较多的代谢物类别分别为脂质和类脂质分子（244种）、苯丙烷和聚酮化合物（138种）、有机杂环化合物（101种）、有机酸及其衍生物（92种）和苯类（59种）；转化前后代谢物PCA图能良好区分，确定18种关键差异代谢物，山柰酚-3-O-刺槐二糖苷和薯蓣皂苷上调最明显。结论 采用指纹图谱技术联合代谢组学方法揭示了大黄在菌群生物转化前后存在的成分差异，通过菌群生物转化可提高大黄活性成分的溶出和转变。

Study on optimization of Rhei Radix et Rhizoma biotransformation process and changes of its ingredients in vitro by human gut microbiota

Objective To explore the conditions and composition changes in the process of Rhei Radix et Rhizoma biotransformation in vitro by human gut microbiota. Methods Fresh feces from three healthy children (1-3 years old) were collected to prepare bacterial flora suspension. The initial medium was selected based on the maximum number of viable bacteria. According to the amount of 0.5% carbon source and 0.5% nitrogen source added in the initial medium, different carbon source/nitrogen source combinations were performed, and the optimal combination was determined according to the viable count results. The out-ofpopulation culture conditions of bacteria were screened by fingerprint technology, including Rhei Radix et Rhizoma addition method (Rhei Radix et Rhizoma powder or Rhei Radix et Rhizoma water immersion), transformation time (24 or 48 h), oxygen requirement (aerobic or anaerobic). Component analysis before and after transformation was performed based on LC-MS metabolomics, principal component analysis (PCA) was performed using SIMCA software (V16.0.2), and MetaboAnalyst 5.0 was used for differential metabolite analysis. Results The results of colony count and fingerprinting technology showed that the optimal combination of sucrose/soy peptone as the modified YCFA medium, and powdered Rhei Radix et Rhizoma as the addition method, the optimal addition ratio (v/w) of the medium and Rhei Radix et Rhizoma powder was 32:1. Culturing in oxygen for 24 h was the optimal conditions for biotransformation. The results of metabolomics showed that there were 763 main metabolites in total, the most abundant metabolite groups were lipids and lipid-like molecules (244 species), phenylpropanes and polyketides (138 species), organic heterocyclic compounds (101 species), organic acids and their derivatives (92 species), and benzenes (59 species). The PCA diagrams of metabolites before and after biotransformation were well distinguished, also, 18 key different metabolites were identified. Biorobin and dioscin were up-regulated most obviously. Conclusion The compositional differences of Rhei Radix et Rhizoma before and after biotransformation were revealed by fingerprint technology and metabolomics. The dissolution and transformation of Rhei Radix et Rhizoma active components could be improved by biotransformation of the flora.