Screening And Mechanism Of Probiotics With Anti-obesity Function For Children

Obesity is a chronic metabolic disease caused by many factors.Children obesity will seriously affect children’s physical and mental health.At present,the global incidence of childhood obesity is increasing year by year.It is urgent to explore healthy and safe anti obesity methods.The existing research results show that some probiotics have anti-obesity effect.However,the anti-obesity mechanisms were different from probiotics to probiotics,and many have not been clearly reveal-ed by researchers.Therefore,this study aimed to screen probiotics with excellent ability of anti-obesity for children and revealed its mechanism in vivo and in vitro.The specific contents include:(1)revealing the features of intestinal flora of children with obesity,(2)secreening the strains with potential anti-obesity function in vitro,(3)evaluating the anti-obesity effect of strains in vivo,(4)revealing the mechanism of anti-obesity strains.Firstly,we compared and analyzed the intestinal flora structure of obese and normal weight children in Harbin by high-throughput sequencing.The results showed that there were significant differences in the structure of intestinal microflora between obese childr en and normal weight children.Specifically,the species richness and diversity of intestinal microflora in obese children decreased,the contents of Firmicutes,Lactobacillus and Bifidobacterium decreased,and the contents of Bacteroidetes,Bacteroides and Prevotella increased.Combined with these results and the analysis of the existing literature,Lactobacillus and Bifidobacterium was used as candidate strains of anti-obesity functional probiotics.In order to screen probiotics with anti-obesity function,Lactobacillus and Bifidobacterium from children’s fecal samples were screened by the selective medium.Next,the surface hydrophobicity,the ability to promote PYY secretion of L cells,the ability to produce bile salt hydrolase(BSH),the tolerance to artificial intestinal juice and gastric juice and its cell surface adhesion were evaluated.The results showed that among 808 strains of Lactobacillus and Bifidobacterium,656 strains were eliminated due to less than 50% hydrophobicity and the remaining 152 strains were preserved.Among the 152 strains,12 strains were finally retained,all of them had good tolerance to artificial intestinal juice,gastric juice and adhesion cells.In addition,five of them could promote L cells to secrete PYY(PYY p athway strain),five of them had good ability to produce BSH(BSH pathway strain),and two of them had both(BSH-PYY pathway strain).After 16 S r RNA identification,12 strains were identified as Lactobacillus,including 5 Lactobacillus plantarum,2Lactobacillus acidophilus,1 Lactobacillus rhamnosus,2 Lactobacillus gardneri and1 Lactobacillus salivarius.In order to evaluate its effect of 12 strains with potential anti-obesity function in vivo,15 groups of animal experiments were set up in male C57BL/6J mice,including low-fat feeding blank control group,high-fat feeding model control group,orlistat and high-fat feeding positive drug control group,probiotics(one group for each strain)and high-fat feeding experimental group.At the end of the 8-week experiment,the body weight,body fat,white adipose tissue weight and histological morphology,liver histological morphology,serum glucose and lipid metabolism related indicators,liver lipid deposition and inflammatory indicators of mice in each group were evaluated.The results showed that the anti-obesity effect of L.plantarum H-87 and L.salivarius LCK11 were better than that of orlistat.They were identified as excellent strains with anti-obesity effects in BSH pathway and PYY pathway,respectively.Specifically,both H-87 and LCK11 can significantly relieve the increase of body weight,body fat,white adipose tissue cells,liver weight,liver cholesterol and triglyceride content induced by high-fat diet,alleviate insulin resistance and serum lipid metabolism disorder(serum triglyceride,cholesterol,low density lipoprotein and low density lipoprotein levels).In order to reveal the anti-obesity mechanism of LCK11 and H-87,fluorescence imaging,high performance liquid chromatography-mass spectrometry,spectrophotometer,ELISA,western blot,measurement,q RT-PCR,high-throughput sequencing and cytological methods were used.The results showed that H-87 could be customized in the ileum of mice,significantly promoting the hydrolysis of glycine chenodeoxycholic acid(GCDCA)and tauroursodeoxycholic acid(TUDCA)and the production of chenodeoxycholic acid(CDCA)and ursodeoxycholic acid(UDCA).In addition,due to the hepatointestinal circulation of bile acids,a significant decrease in GCDCA and a significant increase in CDCA were observed in the liver.Furthermore,the decrease of the concentrations of TUDCA and GCDCA in ileum alleviated insulin resistance by reducing the secretion of glucagon like peptide-1 in L cells of ileum.The increase of CDCA in liver inhibited hepatic lipid synthesis by activating farnesoid X receptor related lipid metabolism related pathways.In addition,H-87 could prevent obesity by alleviating the disorder of intestinal flora induced by high-fat diet.LCK11 could also colonize in the intestinal tract of mice,and its cell wall component PGN could inhibit toll like receptor2/nuclear factor κB pathway of intestinal L cells to promote the secretion of PYY to inhibit appetite.In addition,similar to H-87,LCK11 could also effectively alleviate the disorder of intestinal flora induced by high-fat diet,and its effect was better than that of H-87.In conclusion,this project established an efficient and reliable screening method for probiotics with anti-obesity function,which provides a new theoretical basis for research in related fields.In addition,we obtained two probiotics with anti-obesity function and revealed their anti-obesity mechanism,which enriched the research results of anti-obesity probiotics.