Isolation And Identification Of The Equol-producing Bacterial Strain, From Vegetarian Intestinal Tract

Soybeans contain a lot of soybean isoflavone and It has been accepted by many peoples that Soybean isoflavones can prevent and improve various diseases. In recent years a large number of studies show that many biological activity of soybean isoflavone is equol to achieve, but the source of equol was scarce, at present even if the females in the international phenol only through chemical method synthesis, due to the large intermediate step synthesis involves a large amount of by-product, not only reduces the conversion efficiency and increases the difficulty of equol isolation and purification, chemical catalyst price plus the use of expensive, resulting in the equol standard international market sale price expensive, Sigma,3990yuan/5mg, the Domestic Company2000/5mg, this may be one reason why the human intervention less.The project in vegetarian and non vegetarian urine samples, detection of high yield of equol for subjects and collect the fecal bacteria culture, the separation and purification technology and UV method, in order to equol production as the evaluation index, comparative observation by equol producing bacteria, isolation and culture of equol producing strain. The main insight into the types and isolation and culture conditions of vegetarian by equol producing ability and producing bacteria, then through single factor and orthogonal test the effect of culture conditions on equol producing ability. For species identification of single strains isolated by physiological and biochemical tests and16S rRNA sequence analysis, and the optimum culture conditions for improving the ability of transformation into equol. Through in-depth study of vegetarian species and separation of equol producing bacteria culture conditions, laid a foundation for the realization of in vitro equol high biotransformation, for new bacterial flora and new technology platform for the new strain, built to do on the humble. The results of this study not only has theoretical significance, value added to enhance the soybean and soybean products in the economy, but also the practical value and significance to develop equol sources.In order to Hefei Lan Xu Biological Technology Co., Ltd. equol standard as samples, for the standard of qualitative and quantitative analysis by UV spectrophotometry, the measured maximum absorption wavelength of204nm. In order to vegetarians and non vegetarians urine samples of urine, bleaching conditions on the single factor and orthogonal experiments and the purification process through, to equol production as the evaluation index, the conditions of decolorization decolorization time40-60min. bleaching temperature70℃, the dosage of activated carbon3G. desorption agent concentration80%, desorption agent55mL. volume of sample8mL. adsorption time5h purification conditions measured average content of50ml in urine of equol concentration was28.6020μg/mL, and detection of three kind of life condition of vegetarian and non vegetarian, under the morning urinary equol contents, the health care food> soybean isoflavone; normal life> fasting soybean products, and vegetarian average producing equol were higher than non vegetarians3-5times, in the consumption of soy isoflavones vegetarians produce equol was10.64μg/mL,3.49μg/mL non vegetarian. The high yield of equol vegetarian as fecal tested by colony, clarity and equol production index by single factor experiments, the optimal dilution of10-4are most suitable for subsequent experiments, respectively, medium from Mark Kang Kai, MRS, IRIE, BHI of Bifidobacterium adolescentis five, after the roll tube method, anaerobic tube method flat plate method, three kinds of training methods, respectively, were cultured and screened, finally obtains under anaerobic conditions with10-4diluent to streak plate inoculated to mark Kang Kai and BHI medium conclusion, produce equol intestinal bacteria was the best. In order to equol yield as indexes, optimum cultivation conditions based middle equol bacteria by single factor and orthogonal experiments of Makanke and BHI, pH7.0in wheat Kang Kai36h of culture, medium, substrate concentration was4.25mg/mL under the conditions of continuous culture of N times, get a single colony, and was named LJ-G1, after gram staining and scanning electron microscopy showed that LJ-G1was gram negative, rod-shaped bacteria is a flagellum; BHI culture medium with glucose as carbon source, at37℃,26h continuous culture N training PH7.0anaerobic environment, obtain more single colony, and named LJ-Q2, Gram staining and scanning electron microscope after that, LJ-Q2is gram positive cocci bacteria, spherical, bacteria without flagella round. Through physiological and biochemical tests and16S rDNA sequencing results, refer to the "common bacterial system identification manual" that LJ-G1strain belongs to the river born Escherichia coli (Enterobacter.amnigenus); LJ-Q2strains with amino acids (Acidaminococcus Rogosa) and Staphylococcus aureus are very similTo sum up, the research content in the completion of the project, the process to achieve the desired objectives achieved the following results of innovation. Separating success from vegetarian intestinal bacteria are cultured in high yield of equol bacteria, will be the first. Identification of high yielding equol bacteria, the discovery of new species and provide basic data for application of a strain. The results of this study not only has theoretical significance, value added to enhance the soybean and soybean products in the economy, but also the practical value and significance to develop equol sources.