The Regulatory Mechanism Of Hypoglycemic And Lipid-lowering Efficacy In Probiotics Based On The Multi-omics

Studies have shown that probiotic can improve the regulation of glucose and lipid metabolism,but the mechanism of different effects of probiotic on health is still unclear.Therefore,four strains of Lactobacillus plantarum 84-3,Lactobacillus fermentum 87-1,Lactobacillus plantarum 61-1 and Lac.tobacillus delbrueckii 42-1 were isolated from traditional fermented dairy products.The microbial diversity and function prediction of different kinds of fermented dairy products were analyzed,and probiotics were isolated and identified;The effects of different probiotics and resistant starch on simulated intestinal fermentation in vitro were studied;The difference in hypoglycemic,lipid-lowering and inflammatory factors in type 2 diabetes mellitus rats was compared with different probiotics.Multi omics(16S rRNA gene high-throughput sequencing+non targeted metabolomics+transcriptomics+whole genome sequencing)were used to analyze the regulation mechanism of glucose and lipid metabolism of probiotic,so as to lay a theoretical foundation for probiotic as a new microbial agent.The main research contents,results and conclusions are as follows:(1)16S rRNA gene high throughput sequencing technology was used to study 51 traditional fermented dairy products from China(Inner Mongolia,Xinjiang),South Africa and Sri Lanka.The results showed that the composition and function of bacterial community of traditional fermented dairy products were significantly affected by geographical sources and sample types.The main bacterial genera in dairy products in Inner Mongolia were Lactobacillus,Streptococcus and Lactococcus,and the microbial composition of butter in is complex,including Rhodococcus and Pseudomonas,with relative abundances of 42.1%and 15.9%,respectively;The main bacterial genera in dairy products in Xinjiang was Lactobacillus,its relative abundance in milk pimple,yogurt and kumiss reached 57.4%,67.6%and 97.7%respectively;The main bacteria in dairy products in South Africa and Sri Lanka was Streptococcus,its relative abundance reached 27.1%and 98.4%in cheese and yogurt in South African,and 61.4%in cheese in Sri Lanka,respectively.The results of functional prediction analysis show that Rhodococcus erythropolis and Lactobacillus helveticus play an important role in glucose and lipid metabolism.(2)The fermentation characteristics of resistant starch was evaluated by simulating human fecal fennentation in vitro and rat experiment in vivo.The results showed that Lactobacillus plantarum 84-3 could significantly promote the fermentation of resistant starch,and increase the production of acetic acid,propionic acid and butyric acid,and promote the growth of short chain fat acids producing bacteria,such as Lactobacillus and Brucella(P<0.05);Compared with the resistant starch group,Lactobacillus plantarum 84-3 combined with resistant starch also significantly increased short chain fat acids producing bacteria in rat feces(Fecobacteria,Parabacteroides,Alistipes and Anaeroplasma)(P<0.05),significantly increased glycolysis/gluconeogenesis,pyruvate metabolism and citric acid cycle in carbohydrate metabolism,fatty acid biosynthesis and primary bile acid biosynthesis in lipid metabolism,branched chain amino acid metabolism and tryptophan metabolism in amino acid metabolism,and significantly increased tryptophan synthase(EC:4.2.1.20)and ?-glucosidase(EC:3.2.1.21)related genes(P<0.05);Metabonomics showed that Lactobacillus plantarum 84-3 could significantly enrich the degradation of branched chain amino acids and the metabolic pathway of aromatic amino acids.(3)Type 2 diabetes model was induced by intake of high sugar and high fat diet combined with low-dose streptozocin.Lactobacillus plantarum 84-3 and Lactobacillus fermentum 87-1 were used as intervention group.Wistar rats were intervened for 13 weeks,and physiological and biochemical indexes related to blood sugar,blood fat and inflammatory factors were determined.The results showed that after feeding Lactobacillus,the level of fasting blood sugar was significantly reduced,the glucose tolerance of type 2 diabetic rats was improved.The contents of glycosylated hemoglobin and glucagon in serum were reduced,and increased the levels of insulin and glucagon like peptide-1(GLP-1),decreased the activities of dipeptidyl peptidase ?(DPP-IV)and?-glucosidase(P<0.05);reduced the level of total cholesterol,triglyceride and low-density lipoprotein cholesterol,decreased leptin level,increased adiponectin level(P<0.05),and reduced the level of proinflammatory factors in diabetic rats(CRP,endotoxin,TNF-?,interleukin-6)(P?0.05);reduced the contents of serum cysteine,isoleucine,branched chain amino acids and the ratio of Fischer,increased the level of tyrosine and aromatic amino acids(P<0.05);increased the production of acetic acid,propionic acid,butyric acid and valeric acid in feces(P<0.05).After the intervention of Lactobacillus plantarum 84-3,simultaneously increased the content of propionic acid,butyric acid,isobutyric acid and isovaleric acid in feces.(4)16S rRNA gene high throughput sequencing technology was used to study the effects of different lactic acid bacteria on the composition of colonic faecal flora in type 2 diabetic rats.The mechanism of lowering blood sugar and lipid-lowering was elucidated.The results showed that the type 2 diabetes reduced the relative abundance of Firmicutes,and increased the relative abundance of Bacteroides,Actinobacteriota,Desulfobacterota,Proteobacteria(P<0.05);Increased the relative abundance of Lactobacillaceae,Peptostreptococcaceae,Bacillus,and Christensenellaceae;decreased the relative abundance of Muribaculaceae,Bifidobacteriaceae,Desulfovibrionaceae,Prevotellaceae,Bacteroidaceae and Erysipelotrichaceae(P<0.05);increased relative abundances of Lactobacillus,Romboutsia,Bacillus,Ruminococcaceae NK4A214 group(P<0.05).After feeding lactic acid bacteria,it significantly enriched genes related to carbohydrate metabolism,amino acid metabolism and lipid metabolism,especially Lactobacillus plantarum 84-3 increased the metabolism of starch,sucrose,galactose and propionic acid,glycerol lipid metabolism,fatty acid degradation,biosynthesis of secondary and primary bile acids,down regulated the biosynthesis of phenylalanine,tyrosine and tryptophan,branched chain amino acid and histidine metabolism.(5)Non-targeted metabonomics with the technology of LC-MS was used to study the changes of metabolites in feces of type 2 diabetic rats treated by Lactobacillus plantarum 84-3.Results showed that compared with model group(DC),there were 57 different metabolites in feces of rats after Lactobacillus plantarum 84-3 intervention,lithocholic acid,purine and pyrimidine compounds(guanine,uracil and thymine),p-hydroxybenzaldehyde were increased significantly in rat feces(P<0.05),while the content of cholic acid and choline(oleoyl carnitine,myristoyl carnitine,stearoylcarnitine)were reduced(P<0.05).In addition,the intervention of Lactobacillus plantarum 84-3 significantly increased the content of L-glutamine(P<0.05).13 metabolic pathways were significantly enriched in feces of rats after Lactobacillus plantarum 84-3 intervention,including fatty acid degradation,unsaturated fatty acid biosynthesis,alanine,aspartate,glutamate metabolism,arginine biosynthesis,alanine metabolism and tyrosine metabolism were included.(6)RNA-seq high throughput transcriptome technology was used to screen the differentially expressed genes and metabolic pathways of Lactobacillus plantarum 84-3 in the liver tissue of type 2 diabetic rats.The results showed that type 2 diabetes caused abnormal expression of liver genes.After the intervention of Lactobacillus plantarum 84-3,there were 51 differential genes in 84-3 groups compared with diabetic model group,among which 37 genes were up-regulated and 14 genes were down-regulated;Lactobacillus plantarum 84-3 affects the endocrine system and lipid metabolism by regulating the expression of genes related to signal transduction pathway.The important pathways related to glucose and lipid metabolism mainly include JAK STAT signal pathway,AMPK signal pathway,FOXO signal pathway,insulin signal pathway and PPAR signal pathway;it is the key to the above pathways of functional genes related to glucose and lipid metabolism may include IRS1,Kir6.2,AKT2,BCL6,PEPCK,Fas,SCD1,cyp4al,idax and frizled.(7)The whole genome and metabolome were used to analyze the hypoglycemic and lipid-lowering mechanism of Lactobacillus plantarum 84-3.The results showed that the genome of Lactobacillus plantarum 84-3 contained genes related to carbohydrate metabolism,lipid metabolism and amino acid metabolism,among them,genes involved in L-glutamine metabolism contained Abc.gln1.A and glnA,and contained four gene clusters of secondary metabolites(terpene,T3PKS,cyclic lactone autoinducer and RiPP-like),without virulence or drug resistance genes;its fermentation supernatant contained genes related to aromatic amino acid metabolism(tryptophan,phenylalanine and tyrosine),branched chain amino acid degradation,L-glutamine metabolism;short chain fatty acid metabolism(propionic acid and butyric acid)and lipid metabolism(glycerol phospholipid metabolism),L-glutamine may be an important biomarkers for Lactobacillus plantarum 84-3 to exert its effects of hypoglycemic and lipid-lowering.In summary,probiotic,especially Lactobacillus plantarum 84-3,can reshape the intestinal flora of type 2 diabetic rats,at the same time,it can change the abnormal expression of colon fecal metabolites and liver genes caused by type 2 diabetes,so that the metabolism of glucose and lipid can be recovered,and then the function of lowering blood sugar and lipid can be achieved.