Screening Of Intestinal Bacteria And Their Enzyme Systems That Hydrolyze Flavonoid Glycoside

ObjectiveTo obtain a single strain and efficient enzyme from the intestinal bacteria that can hydrolyze the flavone C-glycoside to corresponding aglycones and provide a convenient,efficient and environment friendly method to solve the hydrolysis problem of flavone C-glycosides,so as to improve their pharmaceutical value.MethodsAnaerobic cultivation was used to isolate intestinal flora with the capacity of hydrolyzing flavone 8-C-glycosides(orientin,vitexin),flavone 6-C-glycosides(isoorientin,isovitexin)and isoflavone C-glycoside(puerarin)to corresponding aglycones.The single strain was separated by plate streaking and identified by traditional bacterial identification and 16S rDNA technique.The crude enzyme was separated and purified by precipitation with ammonium sulfate,Sephadex gel filtration chromatography and ion exchange chromatography,and its composition were identified by LC-MS-MS and molecular biological methods.The enzyme production conditions were optimized by investigating the relationship between the composition of the medium and the transformation activity.ResultsIn this study,two bacterial communities with the capacity of hydrolyzing flavone C-glycosides to the corresponding flavonoid were screened from the intestinal flora of 10 healthy people.They were identified by morphological identification and 16S rDNA identification as Enterococcus faecalis(HCG-W-12-1)and Enterococcus gallinarum(HCG-P-5-8).They could hydrolyze orientin,vitexin and isovitexin to their corresponding aglycones,but didn’t work for isoorientin and puerarin.An enzyme system that hydrolyzed flavone 8-C-glycosides was prepared from the fermentation of strain HCG-W-12-1,and it consisted of about 43 proteins.The content of carbon source in the medium was an important condition to induce the production of living proteins.The activity of hydrolyzing the flavone 8-C-glycoside was significantly enhanced under the condition of low carbon sources in the medium.The composition of the enzyme system is significantly different from that of the normal carbon medium.ConclusionsTwo mixed flora,two single strains and a crude enzyme which selectively hydrolyzes flavone 8-C-glycosides and flavone 6-C-glycosides were screened and identified from human intestinal flora,respectively.The study first found the protein induced phenomenon with the low carbon source in the medium,and optimized the enzyme production conditions of the strain.This study laid a foundation for further searching for the protein that can efficiently hydrolyze flavone C-glycosides.