兼性肠球菌Enterococcus hirae AUH-HM195对黄豆苷原的开环转化

C-ring cleavage of isoflavone daidzein by a newly-isolated facultative Enterococcus hirae AUH-HM195 from Crossoptilon mantchuricum feces

【目的】从褐马鸡粪样中分离对大豆异黄酮黄豆苷原具有转化作用的功能微生物菌株。【方法】在厌氧工作站内对褐马鸡新鲜粪样进行梯度稀释后涂板,从板上挑取单菌落与底物黄豆苷原厌氧混合培养,用高效液相色谱检测底物被转化情况。【结果】分离出一株对黄豆苷原具开环转化作用的革兰氏阳性兼性好氧菌株AUH-HM195(EU919863),经BLAST比对,该菌株的16S rDNA基因全序与肠球菌属菌株Enterococcus hirae(DSM20160)的相似性为100%。根据保留时间、代谢产物最大紫外吸图谱以及核磁共振氢谱和碳谱等分析结果,将菌株Enterococcu shirae AUH-HM195降解黄豆苷原的代谢产物确定为去氧甲基安哥拉紫檀素。纯化后的代谢产物在手性柱上出现两个物质峰,其对映体过量率为66.9%。【结论】细菌菌株Enterococcus hirae AUH-HM195为国内外首次报道的对黄豆苷原具有开环转化作用的肠球菌属菌株,同时也是第一个报道的对大豆异黄酮具有转化作用的兼性微生物菌株。

[ Objective] To isolate specific bacteria capable of biotransforming isoflavone daidzein. [ Methods] Fresh crossoptilon mantchuricum feces was diluted serially from 10^-1 to 10^-8 in Brain Heart Infusion （BHI）liquid medium in the anaerobic chamber at 37℃. Single colony was isolated and incubated in BHI liquid medium containing 0. 1 mmol/L daidzein under anaerobic conditions. The culture medium was detected by HPLC method after being incubated for 3 days. [ Results] A grampositive facultative anaerobic bacterium designated strain AUH-HM195 capable of cleaving the C-ring of isoflavone daidzein was isolated. Basic Local Alignment Search Tool （BLAST） search on the GenBank revealed that the full-length of 16S rDNA gene sequence for strain AUH-HM195 has 100% similarity to that of Enterococcus hirae （DSM20160）. Based on HPLC retention time, UV spectrum, I H and 13C NMR analysis, the metabolite of daidzein by strain Enterococcus hirae AUH-HM195 was identified as O-desmethylangolensin. Two peaks were observed when purified O-desmethylangolensin was eluted on a chiral column. The enantiomeric excess （ % e.e. ） of O-desmethylangolensin was 66.9%. [ Conclusion] Bacterial strain Enterococcus hirae AUH- HM195 is the first reported Enterococcus bacterium with C-ring cleavage activity. It is also the first reported facultative bacterium capable of metabolizing isoflavones.