Preliminary Study On The Xylooligosaccharides Utilization Mechanism Of Bifidobacterium Animalis Subsp.lactis JNFE03

Xylo-oligosaccharides(XOS)are formed by xylose molecules with β-(1,4)glycosidic bonds.A large number of in vitro studies and in vivo studies have proved the probiotic function of XOS,but these studies are limited to global studies.The specific metabolic regulation mechanism of bifidobacterium utilizing XOS is still unclear.In this study,we analyzed the XOS utilization ability of different bifidobacteria and then took B.animalis subsp.lactis JNFE03 strain as the research object.The metabolic pathways of B.animalis subsp.lactis JNFE03 using XOS were analyzed through cell physiology analysis and transcriptomic analysis.The regulatory mechanism of the key transcription factor Xos R was elucidated.Firstly,based on the phylogenetic evolution analysis of the XOS-degrading enzyme system in bifidobacteria,the in vitro culture experiments were used to analyze the XOS utilization ability of B.adolescentis,B.longum subsp.long,B.pseudocatenulatum,B.animalis subsp.lactis,B.pseudolongum and B.bifidum.A strain named B.animalis subsp.lactis JNFE03 that efficiently utilizes XOS was screened.When the strain was fermented and cultured in the XOS carbon source system for 24 hours,the OD value of the system was 1.01,and the contents of xylotetraose and xylotriose decreased by 79.7% and 59.2%,respectively;the content of unsaturated fatty acids in the cell membrane and the fluidity of the cell membrane increased.When the strain was fermented and cultured in the XOS carbon source system for 36 h,the activity of β-xylosidase increased to 4.75 U/m L,and the total content of short-chain fatty acids increased to 1793.51 μg/m L.On this basis,the differentially expressed genes of B.animalis subsp.lactis JNFE03 on different carbon source mediums were determined by RNA-seq sequencing.Through GO enrichment analysis,the differentially expressed genes were mainly concentrated in ATP binding,membrane composition and cytoplasm.Through KEGG enrichment analysis,differentially expressed genes were mainly enriched in amino acid metabolism,translation and membrane transport.Based on the bioinformatics database,the XOS utilization pathway of B.animalis subsp.lactis JNFE03 was described.The study found that B.animalis subsp.lactis JNFE03 mainly uptook XOS through the ABC transport system and MFS transport system and uptook protein through the Opp transport system,DPP transport system and LIV transport system.Finally,the regulatory mechanism of the Lac I family transcription factor xos R obtained in transcriptomics results was explored,which has a helix-angle-helix(HTH)structure DNA binding domain.The promoter PI of xos R was verified by promoter fluorescence intensity determination,and the Xos R transcription factor was solublely expressed in E.Coli BL21(DE3),with a molecular weight of about 44.3 k Da,which can bind to BIFANG＿00034 target fragments at a concentration of 20 nmol/L.Through circular dichroism analysis and molecular docking simulation,it was found that Xos R protein can bind to XOS.After that,the Xos R protein cannot bind to the original target,thus promoting the expression of structural genes.