Isolation And Characterizing Of Daidzein-Degrading Bacteria From The Intestinal Tract Of Pig

Isoflavones are flavonoids present in various plants, particularly in soybean germ. They are classified as phytoestrogens because their structures resemble that of estrogen and they have a weak affinity for the estrogen receptor. Epidemiologic and experimental studies showed that they had preventive effects on breast cancer, prostate cancer, cardio-vascular disease, osteoporosis and menopausal symptoms. Isoflavones exist as glycosides in some plants, mainly as daidzin and genistin. In the intestine, daidzin is converted to daidzein byβ-glucosidase. Then, daidzein is converted via dihydrodaidzein to O-desmethylangolensin (O-DMA) or equol by enzymes derived from intestinal bacteria Equol has a stronger estrogenic activity than daidzein and O-DMA. Equol producers (humans have capablity of producing equol from daidzein) have a lower risk of developing breast and prostate cancers than non-equol producers. To get a better understanding of the bio-degradation of daidzein, and widen the application of daidzein in the swine production, a study was initiated aimed to: (1) characterize the changes of feces bacteria community with the supplement of daidzein in vitro.,(2) establish a practical method to detect the level of daidzein and equol at the same time, (3) isolate several novel members of intestinal bacteria can convert daidzein to equol, (4) find out the possible factors may affect the efficiency of the equol-producing bacteria.In the first study, PCR and denaturing gradient gel electrophoresis (DGGE) were used to monitor the development of feces bacteria community of Suzhong pig over time with the supplement of daidzein in the BHI medium. The V6-V8 region of 16S rDNA of fecal bacteria were amplified by polymerase chain reaction(PCR) with bacterial universal primers 968f/1401r. DGGE profiles of the PCR amplicons were done similarity analysis by Molecular Analyst. Results revealed: some predominant bands disappeared and few new bands got richness over time in the BHI medium with daidzein. The similarity index of the three repetitions got 100% on hour 48. The community achieved stable since hour 72. This shows marked selective effect of daidzein on the composition of fecal bacteria community in vitro.The second study established a high performance liquid chromatographic (HPLC) method to determine the level of daidzein and equol at the same time. Waters C18 column (4.6mm i.d.x 150mm, 4μm) was used with a mixture of acetonitrile-methanol-water (20:30:50, V/V) as the mobile phase and a detector set at 260nm for daidzein and 205nm for equol. The calibration curve was linear(r~2= 0.9898) in the range of 0.05～10μg/ml of daidzein and was also linear(r~2= 0.9965) in the range of 0.05～10μg/ml of equol.In the third study, daidzein-degrading bacteria were anaerobically isolated from pig feces by selective medium in this study. HPLC was used to analyse daidzein and its metabolites, equol. In this study, we got two strains from 36 purified strains can convert daidzein to equol, and named them as zx-5 and zx-7. The production of equol was over 0.05ug/ml in pure culture, and the equol production in pure culture is higher than the mixed bacteria. This is the first report about single strain of equol-producing bacteria has been isolated from pig feces.In the forth study the strains zx-5 and zx-7 were identified by morphological, biochemical and molecular biological methods. Strains zx-5 and zx-7 are obligate anaerobe and gram positive bacterium with spore. They can utilize glucose, maltose and galactose, and produce large amount of gas. The results of the 16S rRNA BLAST revealed that strains zx-5 and zx-7 have a closest relative with Swine manure bacterium RT-1A and rumen bacterium Clostridium bifermentans strain HT2, the similarity over 99% respectively. As the biochemical traits of these two strains resemble the traits of Clostridium bifermentans, they can be demonstrated belong to Clostridium bifermentans.The last study investigated the equol-producing ability of the strains zx-5 and zx-7. Add components which got from the carbon-utilization experiment to simulate the chemical environment of gut lumen, with the purpose to find out possible factors may affect the equol production. After generating in the BHI-daidzein medium, the equol-producing ability of these two strains can be improved dramatically. Addition of cysteine can improve the daidzein disappearance in the medium, and maltose can improve the equol production significantly.