Mechanism Analysis And Screening Method Development Of Bifidobacterium Longum Occupying Of The Abundance Dominance Within The Genus In The Human Gut

Bifidobacterium is a representative of human intestinal probiotics,which has a high abundance in the human intestinal tract,and has been widely used as a probiotic preparation in food and medicine.However,the intake of Bifidobacteria as exogenous bacteria may cause the problem that the abundance decreases with time and cannot exert the beneficial effect for a long time.It has been reported that the abundance of each Bifidobacteria species in the intestinal tract varies with different diets,ages individualized characteristics due to changes in status.The reasons for the differences in the abundance of different species within the Bifidobacterium genus are currently unclear,thus further restricting the selection of Bifidobacteria strains that occupy a dominant abundance in the gut.Bifidobacterium longum（B.longum）has been found to have an abundance advantage at different ages.Therefore,this topic intends to explore the carbon and nitrogen source utilization ability of different species of Bifidobacteria,cell adhesion ability and adaptability to simulate the colonic environment,and find out that the abundance of B.longum is higher than that of other Bifidobacteria in the genus.The key indicators of the genus were verified by in vitro fermentation experiments to explain the dominant mechanism of B.longum,and to develop a screening method for Bifidobacterium strains with an abundance advantage in the human intestinal tract.Below are key research findings:（1）The mechanism by which B.longum dominates the abundance of Bifidobacteria in the human intestine may lie in its wide spectrum of carbon and nitrogen sources,especially the growth rate（generation time）and growth amount(OD₆₀₀)was higher than the other five species of Bifidobacterium,and B.longum had a stronger ability to adapt to the simulated colon environment.Specifically,the carbon source utilization results show that B.longum can utilize12 of the 17 common carbon sources,and the utilization ability of 10 of them is significantly higher than that of other Bifidobacteria.For example,in the D-xylose medium,the generation time of B.longum was significantly higher than that of Bifidobacterium animalis（B.animalis）,Bifidobacterium breve（B.breve）and Bifidobacterium pseudocatenulatum（B.pseudocatenulatum）,and the OD₆₀₀ was significantly higher than that of the other five species of Bifidobacteria.The results of nitrogen source utilization showed that the OD₆₀₀ of B.longum in 9 kinds of nitrogen source medium was significantly higher than that of B.bifidum.There was no significant difference with the reference strain Lactobacillus rhamnosus GG strain（L.rhamnosus GG）.The results of bile salt resistance showed that the generation time of B.longum in the simulated colonic environment medium with 0.4%bile salt was significantly lower than that of B.animalis,B.breve and Bifidobacterium adolescentis,（B.adolescentis）,which was no significant difference with L.rhamnosus GG and Bacillus coagulans 90（B.coagulans 90）.B.longum has a good ability to adapt to the environment of bile salts.（2）In order to verify the reason why B.longum occupies the dominant abundance within the genus,the following verifications were carried out through in vitro fermentation experiments of mouse and human feces:（1）Bifidobacteria,which occupy an abundance advantage,have a strong ability to utilize carbon and nitrogen sources;（2）After B.longum is inoculated in the in vitro fermentation medium,its proliferation is higher than that of other inoculated Bifidobacteria;（3）Under the condition of not occupying an abundance advantage,B.longum can regain the abundance advantage through efficient use of carbon sources.The specific content is as follows:（1）To verify whether in the intestinal tract of mice or human beings,there is a close relationship between the ability to utilize carbon and nitrogen sources and the abundance of Bifidobacteria.Bifidobacterium pseudolongum（B.pseudolongum）was the highest abundance of Bifidobacteria in the intestinal tract of C57BL/6J mice,of which rlative abundance was99.52%,and other Bifidobacteria accounted for 0.48%.For the other 3species of Bifidobacteria,the carbon and nitrogen source utilization experiment were carried out.The results showed that the generation time or OD₆₀₀ of B.pseudolongum with the highest abundance in 5 different carbon source media was significantly better than that of other isolated Bifidobacteria.It is also stronger than other isolated Bifidobacteria in the utilization of 6 kinds of nitrogen sources.（2）In order to verify that B.longum can still occupy the abundance advantage after entering the intestinal flora as exogenous bacteria,B.longum and other types of Bifidobacteria were introduced into the in vitro fermentation medium of mouse and human feces respectively.The results showed that the proliferation of B.longum was the highest after additional addition of different species of Bifidobacteria.It shows that after B.longum is introduced into the feces system as an exogenous bacterium,its ability to adapt to the environment is still stronger than other Bifidobacteria.（3）In order to verify that under the condition of not occupying the abundance advantage,B.longum can regain the abundance advantage through efficient use of carbon sources.Different species of Bifidobacterium were inoculated into the medium with D-xylose as the sole carbon source for in vitro fermentation.The proliferation of B.longum was found to be the highest.The results of flora analysis showed that in the experimental group with additional addition of other Bifidobacteria,B.longum could still occupy an abundance advantage through its strong D-xylose utilization ability,indicating that D-xylose can specifically increases the abundance of B.longum.This may be related to the higher xylulose kinase gene（xyl B）or xylose isomerase gene（xyl A）contained in B.longum.（3）Correlation analysis was performed on the data of all in vitro experiments of the 6species of Bifidobacteria and their abundance in the human intestinal tract,and 27 indicators significantly correlated with the abundance were obtained.A method for screening Bifidobacteria strains with the potential to dominate the abundance in the human gut was developed and validated according to the indicators.The indicators includes 4 items of time（D-xylose,fructo-oligosaccharide,glucose and inulin）,OD₆₀₀ 13 items（D-xylose,fructo-oligosaccharide,glucose,inulin,sucrose,galacto-oligosaccharide,lactose,D-galactose,control nitrogen source,yeast extract powder FM818/FM888,yeast peptone and yeast extract）,a total of 8 carbon source utilization capabilities（D-xylose,fructo-oligosaccharides,sucrose,galacto-oligosaccharides,lactose,D-galactose,D-maltose and trehalose）,2 items of adhesion ability（single plant and competitive adhesion ability）.In a word,this project explained that the mechanism of B.longum occupying the advantage of abundance in the genus lies in its excellent carbon source utilization ability,especially the utilization ability of D-xylose and fructooligosaccharides is stronger than that of B.longum.The verification experiment shows that the abundance advantage of Bifidobacteria is closely related to its strong carbon and nitrogen source utilization ability,and the Bifidobacteria that occupy the abundance advantage must have a strong carbon and nitrogen source utilization ability.B.longum can still regain the abundance advantage through extensive carbon resource utilization ability.Based on the above experimental results,this study developed a method for screening Bifidobacterium species with the potential to dominate the abundance in the human intestine,which provides a theoretical basis for screening the strains that dominate the abundance in the human intestine.