| Bifidobacterium longum is the most abundant Bifidobacterium in human intestines and has been used as probiotics for many years,which plays an important role in human health,especially digestive tract health.B.longum is the most abundant species of Bifidobacterium,which has been approved as a “Generally Recognized as Safe(GRAS)” microorganism by United States Food and Drug Administration(FDA),and has been given“Qualified Presumption of Safety(QPS)” status by European Food Safety Authority(EFSA).It was listed in “bacteria that can be used for food” in 2010 by China's Ministry of Health.Therefore,the objective of this study was to screen B.longum strains from people stool samples in different areas of China.The physiological and genomic characteristics of B.longum strains was analyzed.The protective effects of B.longum alleviating colitis DSS-induced and constipation were subsequently investigated in animal models.In addition,the effects of B.longum on functional constipation in adults were studied through random clinical trails.Firstly,people's fecal samples from 14 provinces(17 regions)of different regions,ages and nationalities in China were processed.A total of 158 strains of B.longum have been isolated from feces samples in 17 regions of China.The draft genome was sequenced and assembled for genetic evolution analyzing.A phylogenetic analysis of the strains based on core genes indicated that all B.longum strains are divided into three distinct branches and the evolution of B.longum lineages were not associated with geographical location,age and gender of host.The pan-genome of B.longum contained a total of 11117 genes.The core genome contained 854 genes,most of which were involved in housekeeping functions,particularly carbohydrate transport and metabolism(12.83%)and amino acid transport and metabolism(11.57%).The genomes encoded a total of 32 glycoside hydrolase families,which were higher than other strains in Bifidobacterium,and mainly were involved in glycoside hydrolases that degrade plant-derived polysaccharides,animal-derived polysaccharides and starch.The GH13 family was the most abundant in the genome of B.longum strains,with an average of 10.5 genes.A total of five resistance genes were detected,including ileS,rpoB,tetW,tet(W/N/W)and AAC(6'),among which ile S and rpo B were conserved in all strains.Serpin and bsh gene were conserved in B.longum,while the Pili genes were significantly different among strains.Twenty B.longum strains that were clearly categorized into three sub-phylotypes were randomly selected for further the evaluation of physiological characteristics.The physiological characteristics associated with probiotic function in vitro(e.g.gastrointestinal transit tolerance,oligosaccharide fermentation ability and cell adhesion ability)were investigated.Safety analysis of the strains showed that all the strains were sensitive to erythromycin,gentamicin,linezolid,streptomycin,tetracycline,vancomycin and rifampicin,and some strains had kanamycin and neomycin resistance phenotypes.To evaluate the effect of B.longum on colitis in mice induced by DSS.Oral administration of B.longum FGDLZ8M1 showed protective effects against DSS-induced intestinal inflammation in mice,as they recovered the gut barrier function,alleviated abnormal colonic histopathology,and prevented DSS-induced alterations in the levels of colonic immunomodulatory indicators,while strains FGSZY16M3 and FBJ20M1 had no significant effect.FGDLZ8M1 could significantly increase the content of short chain fatty acid(SCFAs)in colon and the abundance of SCFAs-producing bacteria in intestinal tract,and improve the structure of bacteria destroyed by DSS.The correlation analysis between the physiological and genomic characteristics of the strains and the inflammatory indexes in mice showed that the alleviating effect of B.longum colitis was significantly correlated with the tolerance of the strains to the simulated gastrointestinal environment.The strains that had an alleviating effect on colitis encoded high abundance of genes related to carbohydrate metabolism.Further analysis of the genomes of FGDLZ8M1 showed that FGDLZ8M1 had 12 specific genes,which were mainly related to polysaccharide metabolism,cell stress resistance,and cell wall/membrane synthesis.Comparative genomic analysis indicated that these protective effects may be related to the specific genes of FGDLZ8M1 that were involved in the tolerance to the gastrointestinal environment,short-chain fatty acid production,and host-bacterium interaction.A meta-analysis of clinical trials was conducted to evaluate the efficacy of probiotics in alleviating functional constipation.A meta-analysis of 1189 patients from 15 clinical studies in10 countries showed that probiotics significantly increased defecation frequency by 0.98 bowel movements per week,reduced intestinal transport time by 13.75 h,and improved stool consistency by 1.31 in constipation patients.Based on the subgroup analysis of the number and type of bacteria,the results showed that the probiotics exerted species/strains specific effects on functional constipation and multispecies probiotics were better than single-species in alleviating constipation.Multiple regression analysis showed that the effect of probiotics on increasing defecation frequency was not related to the dose of probiotics or the intervention time,and the effect of probiotics on reducing intestinal transport time decreased with the patient's age.Five strains that were clearly categorized in different sub-phylotypes of the phylogenetic tree were randomly selected for the evaluation of their effects against constipation induced by loperamide in mice.FJSNT70M6,FHNBA4M1 and FGDLZ8M1 significantly increased intestinal transport time,fecal water content and defecation frequency and the contents of short chain fatty acids in colon,while strains FGSZY6M4 and FBJBA20M1 did not.Compared with the less effective strains,the more effective strains shared the specific arabinosanase gene cluster.The metagenomic data of human intestinal microorganism from HMP and Meathit databases showed that the abundance of the arabinosanase gene cluster in the intestinal tract of patients with constipation was significantly lower than that of healthy people,and the gene cluster was mainly distributed in Bacteroides,such as Bacteroides fragilis and Bacteroides ovatus.Comepare to feeding arabinan or B.longum alone,feeding the strain with arabanase gene cluster and arabinan at the same time had more effect on constipation,confirming that this gene cluster plays an important role in relieving constipation.Randomized placebo-controlled clinical trials were conducted to evaluate the effect of B.longum with the arabanase gene cluster on functional constipation in adults.The strains L6 and FGDLZ8M1 with the gene cluster could significantly improve the defecation frequency,fecal traits and life quality of patients with constipation,while the other strain FGSZY6M4 without this gene cluster had no significant effect.Compared with placebo,B.longum L6 and FGDLZ8M1 increased defecation by 1.05 ± 0.95 and 1.15 ± 1.27 bowel movements per week,respectively,while strain FGSZY6M4 only increased by 0.43 ± 0.93 bowel movements per week.Moreover,B.longum L6 and FGDLZ8M1 significantly improved the stool consistency of patients with constipation,while FGSZY6M4 did not. |
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