Studies On The Response Of Bifidobacterium Pseudolongum To Fructooligosaccharides In Mice

Fructooligosaccharides（FOS）have been identified as an important functional oligosaccharide.They cannot be digested and absorbed in the stomach and small intestine,so FOS reach the colon and are utilized by intestinal bacteria.As an internationally recognised prebiotic,FOS significantly promote the proliferation of beneficial bacteria in the intestine,enhance the mucosal barrier function and modulate immunity,etc.In the previous studies,FOS were reported to significantly promote the proliferation of bifidobacteria in C57BL/6J mice.The analysis further revealed that FOS significantly promoted the proliferation of Bifidobacterium pseudolongum in the genus Bifidobacterium.However,a variety of FOS-utilizing bacteria colonize the intestine.The systematic pattern and mechanisms of why FOS selectively promote B.pseudolongum,and how B.pseudolongum responds to FOS are not available.Considering the differences in the composition of the intestinal microbiota of mice with different genotypes,the effect of FOS on the composition of intestinal microbiota and the genus Bifidobacterium of mice with different genotypes was investigated.Subsequently,C57BL/6J mice were used to investigate the correlation between the abundance of B.pseudolongum and the dose of FOS,as well as the temporal dynamics of B.pseudolongum to FOS,so as to elucidate the response pattern of B.pseudolongum to FOS in mice.The mechanisms of the significant proliferation of B.pseudolongum was analyzed from the aspects of its capacity to utilize FOS,initial abundance and immunogenicity.The main results are shown as follows:16S r DNA sequencing was used to analyze the effect of FOS on the composition of intestinal microbiota and Bifidobacterium spp of C57BL/6J,Balb/c,Institute Cancer Research（ICR）and Kunming（KM）mice.The intestinal microbiota of the four genotypes of mice exhibited differences in the composition.After FOS intervention,the relative abundance of Bifidobacterium in the four genotypes of mice increased significantly.B.pseudolongum dominated in Bifidobacterium and FOS significantly promoted the proliferation of B.pseudolongum in the four genotypes of mice,with the most significant proliferation in C57BL/6J mice.C57BL/6J mice were used to investigate the correlation between B.pseudolongum and FOS doses,as well as the temporal dynamics of B.pseudolongum to FOS.The composition of intestinal microbiota were examined after FOS intervention at doses of 0,2.5,5.0,7.5,and 15g/kg.A strong positive correlation and linearity between the relative abundance of Bifidobacterium and the FOS doses were observed.Especially,the relative abundance of B.pseudolongum was positively correlated with FOS doses（R²=0.95,y=0.044x+0.0076,y is the abundance of B.pseudolongum and x is the dose of FOS）.To investigate the temporal dynamics of B.pseudolongum to FOS,the mice were gavaged with 2.5 and 7.5g/kg FOS for 14 days and eluted for a fortnight.The abundance of B.pseudolongum increased daily in a gradual accumulative pattern within 0-7 days of intervention,reaching a stable abundances on day 7（3.85×10⁹ CFU/g in the high dose group）,and remained after 14 days of elution.Within 24 h after FOS intervention,the abundance of B.pseudolongum significantly increased at 4 h,remained to 12 h,and reverted at 24 h.The response mechanism of B.pseudolongum to FOS was analyzed by comparing the patterns and ability of B.pseudolongum and other intestinal bacteria to utilize FOS in vitro.FOS-utilizing species were isolated from the mouse feces,including B.pseudolongum,Bifidobacterium breve,Bifidobacterium longum,Ligilactobacillus murinus,Lactiplantibacillus plantarum,Lacticaseibacillus rhamnosus,Lacticaseibacillus paracasei,Lacticaseibacillus casei,Enterococcus faecalis,Enterococcus hirae,Enterococcus gallinarum,and Streptococcus alactolyticus.The FOS-utilizing bacteria exhibited the intracellular hydrolysis of FOS.Bifidobacterium used major facilitator superfamily（MFS）transporter,Lactobacillus,Enterococcus and S.alactolyticus used the phosphotransferase system（PTS）system to transport FOS to be hydrolyzed byβ-fructofuranosidase intracellularly.The capacity of the 12species to utilize FOS varied considerably.B.pseudolongum,B.longum,B.breve,S.alactolyticus and E.gallinarum showed shorter generation times,faster rates of FOS hydrolysis and higherβ-fructofuranosidase activity to utilize FOS.However,B.pseudolongum had no clear advantage in utilizing FOS compared to other FOS-utilizing species.The response of intestinal microbiota and B.pseudolongum to FOS was investigated after increasing the initial abundance of FOS-utilizing species by gavage with the species.Before FOS intervention,the initial number of B.pseudolongum was the highest compared to other FOS-utilizing species,and the abundance of B.pseudolongum significant increased after FOS intervention.The response of FOS-utilizing species to FOS was enhanced by an initial increase in the abundance of the species.After the initial increase in the abundance of B.pseudolongum,B.pseudolongum showed a stronger response to FOS,with an increase in abundance from3.57×10⁹ CFU/g to 4.99×10¹¹ CFU/g.Moreover,the initial abundance of FOS-utilizing species and B.pseudolongum were not significantly different,resulting in a significant proliferation of both species to the abundance that was not significantly different.The initial abundance of other FOS-utilizing species was significantly higher than that of B.pseudolongum,resulting in the significantly reduced response of B.pseudolongum to FOS.Furthermore,B.pseudolongum was co-cultured with other FOS-utilizing species at different initial abundance in the medium.The high abundance of other FOS-utilizing species enabled the species to proliferate significanltly but inhibited the growth of B.pseudolongum.Therefore,the initial abundance of FOS-utilizing species showed a strong influence on the response of B.pseudolongum to FOS.The higher initial abundance of B.pseudolongum contributed to its significant proliferation,and high initial abundance of B.pseudolongum was a potential mechanism by which FOS significantly promoted the proliferation of B.pseudolongum.The immunogenicity and intestinal immune stimulation of B.pseudolongum and other FOS-utilizing bacteria were evaluated to elucidate the mechanism of response of B.pseudolongum to FOS.After 10¹⁰ CFU of the FOS-utilizing bacteria intervention,the content of serous immunoglobulin G（Ig G）was significantly increased by E.gallinarum.The FOS-utilizing bacteria except B.pseudolongum significantly increased colonic secretory immunoglobulin A（s Ig A）,and the contents of the recombinant regenerating islet derived protein 3γ（Reg3γ）significant increased after the intervention of L.murinus,S.alactolyticus and B.longum.According to the contents of proinflammatory and anti-inflammatory cytokines in colon,the contents of interleukin（IL）-4,transforming growth factor（TGF）-β,IL-6,tumour necrosis factor-α（TNF-α）,IL-17 and IL-22 in colon after the intervention of B.pseudolongum were not significantly different from those in the control group,wheras L.plantarum,S.alactolyticus and E.gallinarum showed stronger colonic immunostimulatory.Therefore,compared to other FOS-utilizing bacteria,B.pseudolongum showed lower immunogenicity and less immune stimulation on the colon.The significant increase in the abundance of B.pseudolongum did not induce immune response in the intestine,which might be one of the mechanisms by which FOS significantly promoted the proliferation of B.pseudolongum.