Genetic And Phenotypic Diversity Of Bifidobacterium Longum And Their Relationship With The Immunoregulatory Function

Bifidobacterium is one of the predominant microbe in human gut,which could interact with their host.Some specific strains have been reported to inhibit enteroinvasive pathogens,inhibit colorectal cancer,and alleviate IBD and constipation.B.longum is the most widely existing Bifidobacteria in infant and adult.This study researched the diversity of B.longum in three aspect: comparative genomics,biological properties and in vivo function(alleviating colitis).The main results were as follows:(1)The microbiota and composition of Bifidobacterium of people of different ages or ethnics or genders or living in different regions in China was analyzed by high-throughput sequencing.The results of multivariate statistical analysis showed that,the gut microbiota of people of different ages or ethnics or living in different regions were different.By Random Forest analysis,we found that Bifidobacterium was one of the most varied genera among different people.The relative abundance of Bifidobacterium was significant higher in samples of Tibetan or child.The compositions of gut microbiota of different gender were similar.The analysis of composition of Bifidobacterium indicated that B.longum was the most abundant species with an average relative abundance of 34.30%.Region,age and ethnics compel significant effect on bifidobacterium composition,in which Tibetan had a higher relative abundance of B.kashiwanohense,Zhuang people had a higher relative abundance of B.dentium,and child had a higher relative abundance of B.pseudocatenulatum and B.breve.We sequenced the whole genomes of 45 B.longum strains isolated from people with different ages or living in different regions,and comparative genomic analysis was performed.Aligned with standard strain,we defined that there are two strains belonged to B.longum subsp.infantis,one strain belonged to B.longum subsp.suis and the other strains belonged to B.longum subsp.longum.Phylogenetic analysis based on whole genome sequences showed that the B.longum strains were not clustered according to the ages,region,ethnics or gender of their host.(2)The sequence alignment based on whole genome showed that there were 7 main variable regions in the genomes of B.longum(A-G),in which harbored genes related to sugar transportation and metabolism and EPS synthesis gene cluster.The analysis of genes related to sugar metabolism showed that,the composition of glycosyl hydrolases(GHs)genes was different between the two subspecies,but on strain level,there were no difference between strains isolated from people or different ages,ethnics,genders or regions;The composition of glycosyl transferases(GTs)genes was relatively conservative on both subspecies and strain level.Child originated strains possess relative slower growth rate(with a delayed period of 6-8 h)in MRS broth,and the growth rate of strains from elders were higher(with a delayed period of 4-6 h);the result of colony count showed that the CFU of child originated strains were significantly smaller than elder originated strains(8.89±0.32 log10 CFU/mL and 9.22±0.28 log10 CFU/mL).Carbohydrate fermentation tests showed that there were two strains that could not utilize arabinose but could utilize D-glucuronic acid,indicating that these two strains belonged to B.longum subsp.infantis,which coincided with the result of genome alignment.Furthermore,we found that most child originated strains could ferment D-mannose(4 in 18),and most adult originated strains could not ferment D-mannose(21 in 27).Acetic acid was the only short chain fatty acid in the culture supernatant of B.longum.The results of EPS determination showed that,the ability to produce capsular polysaccharide of child originated strains was worse than adult originated strains.Analysis of composition of EPS cluster showed that,that the numbers of GT genes in the EPS cluster of strains with lower capsular exopolysaccharide production(under 10 mg/g biomass)were significantly less than that of the higher ones,moreover,and there is a correlation between production of capsular exopolysaccharide and the number of GT genes in the EPS cluster.(3)The effect of different B.longum on colitis mice were analyzed.CCFM688?CCFM756?CCFM760?HAN42-10?M2-06?GX13-2?HAN30-6?YS108R administration could significantly reduce DAI value after DSS induced.CCFM688?CCFM756?CCFM760?HAN42-10?HUB37-A12?M2-06?HAN30-6?HAN4-25?YS108R administration could significantly reduce the level of IL-6 which was increased with DSS challenge.The expression level of tight junction protein and histological analysis showed that,CCFM688?CCFM756?CCFM760?HAN30-6?HAN42-10?M2-06?YS108R?HUB37-A12 could protect mice from colonic damage during DSS challenge;However,in HEN27-6?HUB2-25?M2-C administration groups,the mucosal structures were almost completely destroyed.Among strains with better effect on colitis,except for YS108 R,which produce ropy capsular polysaccharide,the other strains were all with poor ability to produce capsular polysaccharide;in contrast,strains with worse effect on colitis most have higher production of capsular polysaccharide.Therefore,capsular polysaccharides with different properties may affect the alleviative ability of B.longum on colits.(4)Cell-surface components were extracted and were tested for the immunomodulatory activity by cell model.The results showed that the culture supernatant exert no effect on LPS stimulated HT-29 cell.Only ropy capsular polysaccharide produced by YS108 R exert anti-inflammation effect on LPS stimulated HT-29 cell.Capsular polysaccharide produced by other strains exert no significant anti-inflammation effect.S-layer proteins produced by all strains could reduce the expression of IL-8.The effect of the mixture of S-layer protein and capsular polysaccharide on LPS induced HT29 cell showed that,anti-inflammation effect of S-layer protein were weakened when capsular polysaccharide were added to a certain amount.(5)Ropy EPS producing B.longum YS108 R were used to produce fermented milk,and the alleviative effect of YS108 R fermented milk on colitis were studied.YS108 R could decreased whey syneresis and increased water-holding capacity of fermented milk,and it could also improve the texture of ferment milk.Combined utilization of YS108 R and commercial starter could eliminate the undesirable flavor appeared with YS108 R singly fermented milk and improve the mouthfeel of fermented milk.YS108 R fermented milk could alleviate the symptom of colitis by anti-inflammation,maintenance of the normal mucosal barrier and reverting the change of microbiota.