Isolation And Characterization Of The Equol-Producing Bacteria From Human Feces

Soybean isoflavones are phytoestrogens present in plant. Daidzein and genistein, the main ingredient of glycosides, have extensive biological activity. Epidemiological studies suggest that soybean isoflavones have biological activity of preventing breast cancer, prostate cancer, cardiovascular disease, osteoporosis, climacteric symptoms remission and so on. In recent years, with the development of research, researchers find that equol produced by metabolizing daidzein has higher biological acitivity compared with soybean isoflavones, more stable nature, more easily absorbed by the human, and slower clearance rate in plasma. So presumably biological function of soybean isoflavones may attribute to equol in some extent. But only30%-50%of the population can metabolize daidzein to equol. Effects of equol production factors include intestinal physiological environment, host genetic and dietary factors. Because equol only is produced by intestinal microbial specificity, and the biological function of equol and the applicability of the crowd is better than that of soybean isoflavones, therefore isolating equol-producing bacteria will have very broad application prospects for preparation pf equol with microbial technology. The research of soybean isoflavones degradation bacteria focus on human and mouse at home and abroad and bacteriums of metabolizing daidzein are also only a few. In this study equol producers were screened from human urine. Daidzein-degrading bacteria were anaerobically isolated from human feces by selective mediun. Two strains metabolizing daidzein to equol were isolated and identified, and the strains properties are made certain exploration.In the paper equol was determinated in human urine with high performance liquid chromatography (HPLC). Hypersil ODS2-C18(250*4.6mm,5μm) was used at25℃, and mobile phase was acetonitrile:methanol:water=20:30:50(V/V) with flow rate of1mL/min. Detection wavelength by ultraviolet spectrophotometer was at205nm. On the detection methods of verification, the results showed:equol concentration is0.1-25μg/mL, A and C showed a good linear relation, the linear regression equation was:A=103170C-10178(R2=0.999). The relative standard deviation(RSD) of equol in instrument precision experiment was0.8%. The RSD of equol in stability experiment was1.24%. The RSD of equol in method precision experiment was1.32%. The samples average recovery rate was99.72%, and the RSD was0.13%. Four persons were equol producers in twelve subjects.With BHI culture medium adding200μmol/L daidzein as substrates in the isolation of producing equol, in the anaerobic condition the feces of equol producers were isolated and purified, and56strains were gotten. The content of equol in the culture medium was detected with HPLC. There were two strain produced equol after repeated purification.The concentration of equol of the two strains HY-1and HY-2were0.26and0.36μg/mL respectively. The bacteria physiological and biochemical identification and16S rDNA sequence analysis showed that:the strain HY-1was facultative anaerobes, judged for the family enterococcus, the strain HY-2is strictly anaerobic bacterium, judged for the family Slackia. The16S rDNA sequence of the strain HY-1had a closest relative with Enterococcus faecium sequence, the homology over99%. The16S rDNA sequence of the strain HY-2had a closest relative with Strain HE8sequence, the homology more than99%. The two strains of bacteria in GenBank of NCBI were JQ768054and JQ796697.The properties of the two strains were studied respectively.The strain HY-1entered rapid growth period within10h after inoculation of bacteria, and entered decline period after18h. The strain HY-2entered rapid growth period within10h after inoculation of bacteria, and entered decline period after14h. The pH value of strain HY-1decreased faster at0-6h in BHI medium, and reached a minimum value of6.27at6h, after a rebound. The pH value picked up6.51after48h culturing. The pH value of strain HY-2decreased slowe at0-8h in BHI medium, and reached a minimum value of6.72at8h, after a rebound. The pH value picked up6.83after48h culturing. The strains HY-1and HY-2grew well in the temperature range of30～40℃, belonging to mesophilic microorganisms, where30～35℃the optimum growth temperature range, too high or too low temperature is not conducive to the growth of bacteria. Different carbon sources had different effect on the strains HY-1and HY-2producing equol. Glucose, maltose, lactose, rhamnose, arabia glucose and mannose can promote the strain HY-1to produce equol. Xylose and sorbitol can inhibited equol production. Xylose, glucose, rhamnose, arabia glucose, sorbitol obviously can promote the strains HY-2to produce equol, maltose and lactose inhibited equol production.