Effects Of Different Substrates On Gut Microbiota Of Autism In In Vitro Batch Fermentation

Objective:In vitro batch fermentation,combined with high-throughput sequencing technology and gas chromatography,was used to investigate the effects of different substrates on fecal microbiota and short chain fatty acids(SCFAs)in children with autism spectrum disorder(ASD),and find the substrate which has the potential to improve gut microecological niche and clinical symptoms.Methods:?From December 2016 to October 2017,fifteen children with ASD were enrolled voluntarily from pediatric department of Chinese PLA General Hospital,and seven of them were treated with Fecal Microbiota Transplantation(FMT).Fifteen matched neurotypical children were selected as the control group.?Feces of 15 children with ASD(group A),7 autistic children after FMT(group B),and 15 neurotypical children(group N)were collected and diluted into 10%fecal slurries,then inoculated into in vitro batch fermentation systems,which contained YCFA as basic medium and added mucin(MUC),xylitol(XYI),mannitol(MAI),xylo-oligosaccharides(XOS),fructo-oligosaccharides(FOS),inulin(INU)and starch(STA)as the sole substrate.Each sample was fermented for 48H.?Barometer was used to measure the gas pressure after 24H fermentation.SCFAs after 24H and 48H fermentation were detected by gas chromatography.16S rDNA of fecal microbiota after 48H fermentation were measured using high-throughput sequencing technology.Results:?Fecal microbiota:Compared with group N,diversity of fecal microbiota in group A decreased significantly.Relative abundance of Streptococcus,[Ruminococcus]and Enterococcus increased significantly,while relative abundance of Roseburia,Faecalibacterium and Megamonas decreased significantly in group A.After FMT treatment,the diversity of microbiota in group B increased as compared with group A,and there was no significant difference between group B and group N.? Microbiota after fermentation:In group A,microbiota after fermentation in XYI,which had the highest alpha diversity,was the closest to the structure of fecal microbiota in group N.After fermentation in YCFA and MUC,alpha diversities of microbiota in group A were significantly lower than those in group N,while those fermented in MAI,XOS,FOS,INU and STA were not significantly different among groups but lower than XYI.XYI increased the relative abundance of Blautia,Anaerofustis,Odoribacter,Butyricimonas and reduce the relative abundance of Ruminococcus,Lactobacillus,Streptococcus,Bifidobacterium and Lactococcus in group A.After FMT treatment,varying degrees of increases of alpha diversities were found in mediums of group B,and the microflora structure was improved to be no significant difference with group N.?SCFAs of feces:There was no significant difference in short-chain fatty acids of feces between groups A,B and N.?Gas pressure after 24H fermentation:Gas pressures in YCFA and XYI in group A were significantly lower than those in group N.MUC and MAI had significantly higher gas pressures than YCFA in group A.Growth rates of gas pressure in MUC,MAI,XOS,FOS and INU to YCFA were significantly higher in group A than those in group N.After FMT treatment,gas pressures in YCFA and XYI had no significant difference between group B and N.Growth rates of gas pressure in MUC,MAI and XOS did not differ between group B and N and differences of FOS and INU to group N reduced.?SCFAs after fermentation:After 24H fermentation,total acid,acetic acid,propionic acid and butyric acid of YCFA in group A were all lower than those in group N,while the differences disappeared after 48H.No significant difference in SCFAs of YCFA was found between group B and group N.In comparison of MUC,XOS,FOS,INU and STA,the production and the growth rate of SCFAs in group A were higher than those in group N.In group A,MAI engendered a higher production of propionic acid than YCFA and a higher growth rate than that in group N.When comparing the ratio of propionic acid to butyric acid and the difference between propionic acid and butyric acid,MAI was higher than other mediums.Total acid,acetic acid,propionic acid and butyric acid of XYI in group A after 24H fermentation were lower than those in group N,while differences disappeared after 48H.The ratio of propionic acid to butyric acid and the difference between propionic acid and butyric acid were lower than other mediums.Conclusion:?Fecal microbiota of children with ASD has a deficient ability of protein fermentation and an exorbitant ability of carbohydrate fermentation.?XYI is a beneficial substrate which has the potential to improve the structure of gut microbiota and SCFAs ratio of children with ASD.?MAI was potentially harmful to children with ASD as the promotion of propionic acid.?Gut microbiota of children with ASD can produce more propionic acid,and maintain the difference to healthy children with the existence of mucin.?FMT can effectively improve the structure of gut microbiota and its ability to ferment substrates in children with ASD,and improve the clinical symptoms of children with different degrees.? Simply testing metabolites in feces can not explain the metabolic activity of gut microbiota.In vitro fermentation can eliminate differences of intestinal mucosal absorption,food residues and other interference factors to display the metabolic activity of gut microbiota more accurately.Hence,clinical diet guidance can be given properly.