Optimization Of The Conditions For Microbial Biosynthesis And Application Of S-equol

The non-natural product equol,which is a metabolite of soy isoflavone daidzein,has higher and broader physiological functions than that of daidzein,such as estrogen-like effects,antioxidant effects,and lipid-lowering effects.In the previous study,our lab members isolated one strict anaerobic bacterial strain,which is named Eggerthella sp.HAU-JLC44,the bacterium of which can convert the substrate daidzein to S-equol under anaerobic conditions.The invertase gene involved in the synthesis of S-equol from the bacterium mentioned previously,which include daidzein reductase(dgr),dihydrodaidzein reductase(dhdr),tetrahydrodaidzein reductase(thdr)and dihydroracemase(ddrc)were cloned.Subsequently,the four invertases were cloned into Escherichia coli,and a double plasmid co-expression system E.coli BL21/pET-30a-DHDR-TDHDR/pACYCduet-1DDRC-DGR was constructed.In order to decrease a large accumulation of the intermediate dihydrodaidzein,the preliminary transformation conditions in the test tube were optimized.In this study,both the culture conditions and the transformation conditions for the recombinant system were optimized by using the response surface analysis method.Afterwards,it was the first time to attempt to expand culture and aerobic bioconversion in shaking flask as well as fermentors.This study not only reduced the cost of the culture medium,but also significantly improved the bioconversion efficiency of the recombinant system under aerobic conditions.Finally,we separated and purified the product equol and added the purified equol to different foods to provide a reference for the development of foods containing equol.The main results of this study are as follows:(1)Use response surface methodology to optimize the medium composition and transformation conditions of recombinant system.The medium components determined by the single-factor experiment include glucose,tryptone and sodium chloride;the main factors affecting the bioconversion efficiency include induction temperature,inducer concentration,inoculum amount,initial pH value and concentration of the carbon source lactose.Plackett-Burman test was performed on the above eight factors,and four important influencing factors were screened out,namely carbon source,nitrogen source,induction temperature and IPTG concentration.The steepest climbing test design determined the optimal value range of each main factor,and the average conversion rate of equol can reach 89.02%under the optimized conditions.The Box-Behnken design-response surface method carries out a four-factor three-level test,aiming at the best value point to obtain the best bioconversion efficiency.Finally the optimized conditions were verified by the bioconversion test.The average bioconversion rate of substrate daidzein into S-equol was 94.15%.(2)Gradually expand the culture of recombinant bacterium at the shake flask levelThrough single-factor experiments on the four conditions of filling volume,induction time,bioconversion period and substrate addition volume,the best conditions are:30%filling volume,induction time 6 h,conversion time 72 h.The strain transforms 1.4 mM daidzein into S-equol with an average transformation rate of 96.06%.(3)Biotransformation in a 5 L fermentorExpand the culture of the recombinant strain in the fermentor,and the OD600 value of the recombinant bacterium reached 4.061,which is significantly higher than that of the unoptimized LB medium(OD600=2.345).The average bioconversion rate of the substrate daidzein to S-equol was 89.04%when the concentration of the substrate daidzein added was increased to 2.0 mM and the bioconversion period was 60 h.The bioconversion rate is currently the highest at present.(4)The purified S-equol was added to soybean powder to make soy milk tablets,or added to natto polysaccharide mucus to make a multifunctional oral liquid.Both the sensory quality and the stability of S-equol was preliminarily determined.