兔肠道大豆异黄酮还原菌株的分离鉴定及其转化特性

Isolation, identification and biotransforming property of an isoflavone reducing bacterium isolated from rabbit intestinal microflora

摘要:

【目的】从兔新鲜粪样中分离对大豆异黄酮黄豆苷原和染料木素具有转化作用的特定细菌菌株。【方法】在厌氧工作站内对獭兔新鲜粪样进行梯度稀释后涂板，挑取单菌落与底物黄豆苷原和染料木素分别厌氧混合培养，用高效液相色谱检测底物被转化情况。【结果】分离得到一株对大豆异黄酮黄豆苷原和染料木素均具有转化作用的革兰氏阳性严格厌氧细菌菌株AUH-JLR41 (KJ188150)。根据产物的高效液相保留时间、紫外吸收图谱和质谱分析结果，将菌株AUH-JLR41代谢底物黄豆苷原和染料木素生成的产物分别鉴定为二氢黄豆苷原和二氢染料木素。经手性高效液相系统检测，产物二氢黄豆苷原和二氢染料木素均呈现两个等面积物质峰，表明这两个产物的对映体过量率均为0。通过转化动态研究发现，菌株AUH-JLR41分别在底物黄豆苷原和染料木素加入48 h和72 h后将底物全部转化为产物，该菌株能转化底物黄豆苷原和染料木素的最大浓度均为0.6 mmol/L。经BLAST比对，菌株AUH-JLR41的16S rRNA基因序列与斯奈克氏菌属菌株Slackia equolifaciens DZE (EU377663)的相似性高达99.6%。【结论】兔肠道分离的斯奈克氏菌属菌株Slackia sp. AUH-JLR41在厌氧条件下能将大豆异黄酮黄豆苷原和染料木素分别还原为二氢黄豆苷原和二氢染料木素。

Abstract:

[Objective] To isolate specific bacteria capable of biotransforming isoflavones daidzein and genistein from fresh feces of rabbit. [Methods] In an anaerobic chamber, fresh fecal samples of rabbit were diluted before being spread on agar plate. Single colonies were picked randomly and incubated with daidzein or genistein. High performance liquid chromatography (HPLC) was used to detect whether the isolated bacteria were able to biotransform the substrate daidzein or genistein. [Results] A Gram-positive obligate anaerobic bacterium, which we named AUH-JLR41 (KJ188150), capable of biotransforming isoflavones daidzein and genistein was isolated. Based on the HPLC retention time, UV spectra and electrospray ionization mass spectrometry (ESI-MS) analysis, the metabolites of daidzein and genistein were identified as dihydrodaidzein (DHD) and dihydrogenistein (DHG), respectively. Chiral stationary-phase HPLC successfully separated the two peaks which had the same peak area, indicating that the enantiomeric excess of biosynthesized DHD and DHG was zero. Biotransformation kinetics showed that daidzein was reduced to DHD completely after 48 h incubation and genistein was reduced to DHG after 72 h incubation under anaerobic conditions. Moreover, the maximal concentrations of daidzein and genistein that strain AUH-JLR41 was able to convert were 0.6 mmol/L. BLAST search on the GenBank revealed that the 16S rRNA gene sequence for strain AUH-JLR41 had the highest similarity to that of Slackia equolifaciens DZE (EU377663), the similarity of which was 99.6%. [Conclusion] Bacterium strain AUH-JLR41 isolated from rabbit feces was able to reduce isoflavones daidzein and genistein to DHD and DHG, respectively, under anaerobic conditions.