Isolation Of An Isoflavone Reducing Bacterium From Of Pregnant Horse Feces And It's Effect On Intestinal Bacterial Diversity In Rats

The subjective of this paper is to isolate specific bacteria capable of biotransforming daidzein from fresh feces of pregnant horse,to optimize the of fermentation medium for bacteria by single factor experiment and response surface methodology,and to study the effect of the isolated bacteria on bacterial flora diversity in the rat intestinal and the metabolism of soy isoflavones.Isolation and identification of an isoflavone reducing bacterium isolated from fresh feces of pregnant horse.The Hungate anaerobic roller tube was used as anaerobic culture.Single colonies were picked randomly and incubated with daidzein.High performance liquid chromatography was used to detect whether the isolated bacteria were able to biotransform the substrate daidzein.A strain capable of reducing daidzein was selected and was identified by 16S r DNA sequence analysis and phylogenetic tree.The morphological characteristics and physiological and biochemical responses of the strain were determined.A Facultative anaerobic bacteria,which were named HXBM408(MF992210),capable of biotransforming isoflavones daidzein to dihydrodaidzein was isolated.The isolated strain belonged to Gram positive.Basic Local Alignment Search Tool(BLAST)search of HXBM408's 16S r DNA sequence on Gen Bank suggested that the isolated stain has 99%similarity to that of Pediococcus acidilactici strain DSM(NR042057).The morphological and physiological biochemical characteristics of HXBM408 showed highly similarity to Pediococcus.Based on the above characteristics,the strain was identified Pediococcus acidilactici.Optimization of fermentation medium components for bacterium using response surface methodology.On the basic of BHI medium,the main factors(carbon source,nitrogen source,growth factor and inorganic salt)and the appropriate range of concentration were determined by single factor test.The best composition in the medium were analyzed by the Box-Behnken central composite experiment and response surface methodology.The significant factor were determined by the single factor experiment,include glucose,peptone,VB₁ and KH₂PO₄,Response surface analysis was determined that the strain medium to supplement with 10.22 g/L glucose,6.00 g/L peptone,0.49 g/LVB₁ and 0.82 g/L KH₂PO₄ on the basic of BHI medium.Validation experiments showed that the DHD concentration was 0.27?g/m L,which was close to the predictive value 0.28?g/m L.The effect of bacteria on bacterial flora diversity of rat intestinal and the metabolism of soybean isoflavones.Twenty female SD rats with initial body weight of(160.00±10.00)g were randomly divided into experimental group and control group.The two groups of rats were treated with antibiotics(Azithromycin)continuously for 7 days.After,rats in control group were given sterilized water and daidzein by gavage for 7 days.The rats in experimental group were given HXBM408 solution and daidzein by gavage for 7 days.The rat feces,intestinal digesta and plasma were collected.The concentration of daidzein and its metabolites in feces and plasma of rats were measured by HPLC.The bacterial diversity of feces and intestinal digesta were determined by Ion S5^TMXL technique.The content of daidzein metabolite equol in rat feces was significantly higher than that in the control group(P<0.05)at the seventh day and the fourteenth day.But the content of daidzein and its metabolites in feces was not significantly different(P>0.05)with the increase of experimental time.There was no significant difference in the plasma daidzein and its metabolites concentration in rat(P>0.05).On the 7th day,the estimation of fecal microbial abundance in the experimental group was significantly lower than that in the control group(P<0.05),but with the prolongation of time,the difference was not significant(P>0.05).In the intestinal digesta of rats,the proteobacteria of experimental group was significantly lower than that of the control group(P<0.05).On the 7th day,the relative abundance of Lachnospiraceae,Streptococcaceae and Streptococcus in the faces of experimental group rats was significantly higher than the control group(P<0.05),but the difference was disappeared as the extension of time(P>0.05).In the intestinal digesta of rats,the relative abundance of Bacteroidaceae and Bacteroides in experimental group was significantly higher than that of the control group(P<0.01),however,the relative abundance of Streptococcaceae and Streptococcus in experimental group were significantly lower than of the control group(P<0.05).Bacterium strain HXBM408 isolated from pregnant horse feces was able to reduce daidzein to dihydrodaidzein.The optimal ingredients of medium for the bacterium were 38 g/L BHI,10.22 g/L glucose,6.00 g/L peptone,0.49 g/L VB₁ and 0.82 g/L KH₂PO₄.The bacterium can increase the degradation ability of soybean isoflavones short during period after ingestion,and regulate the intestinal flora.