Study On The Improvement Effects And Mechanism Of Probiotic Fermented Milk On Type 2 Diabetes In Mice

The biological function of probiotics is related to their probiotic effects and biogenic effects.Probiotics have been proven to be effective in preventing and improving type 2 diabetes（T2DM）.Milk is the best matrix for the growth of probiotics,probiotic fermented milk is a daily consumption food,whether its long-term intake has the positive effect of preventing and improving T2DM is a scientific issue worthy of further study.In this research,conventional fermented milk FM1（Streptococcus thermophilus ST447 and Lactobacillus bulgaricus LB4）was used as a control,the improvement effects and mechanism of probiotic fermented milk FM2（S.thermophilus ST447,L.bulgaricus LB4 and Lactobacillus paracasei IMC 502）and FM3（S.thermophilus ST447,Lactobacillus acidophilus NCFM,Lactobacillus rhamnosus LGG and Bifidobacterium lactis HN019）and their probiotics（FM2-C,FM3-C）and milk-based fermentation metabolites（FM2-S,FM3-S）on T2DM mice were investigated.Firstly,the enumeration of viable bacteria and the characteristics of milk protein,free amino acids,free fatty acids,peptides,volatile and non-volatile metabolites in probiotic fermented milk during storage（4℃for 27 d）were studied.The counts of probiotics in FM2 and FM3 were decreased by 54.09%and 59.00%over the entire storage period,respectively.The continue d degradation of caseins and whey proteins during storage led to the increase in the content of non-protein nitrogen and total free amino acids.A total of 760peptides were identified,the type and concentration of peptides were FM3>FM2>FM1.The abundance ratio of bioactive peptides to total peptides in FM2and FM3 was 10.34%and 9.33%,respectively.The sum of DPP-IV inhibitory peptides,α-glucosidase inhibitory peptides andβ-glucuronidase inhibitory peptides in FM2 and FM3 accounted for 3.11%and 2.62%of the total peptide abundance,respectively.A total of 17821 non-volatile metabolites were detected,and compounds that regulate lipid and sugar metabolism accounted for more than 83.51%of the total functional metabolites.The abundance of phosphorylcholine（1.36%,1.40%）,L-carnitine（0.37%,0.61%）,betaine（0.36%,0.40%）and D-tagose（0.29%,0.30%）was higher in FM2 and FM3.During storage,the concentration of peptides in FM2 and FM3 increased by 14.04%and14.01%,respectively.The abundance ratio of bioactive peptides to all peptides in FM2 and FM3 decreased by 9.96%and 11.68%,respectively.The abundance ratio of functional metabolites to all metabolites in FM2 and FM3 decreased by27.38%and 16.01%,respectively.T2DM mice were successfully established by a long-term high-fat diet and one-time low-dose streptozotocin injection and the effects of probiotic fermented milk（FM1,FM2 and FM3）,probiotics（FM1-C,FM2-C and FM3-C）and probiotic milk-based metabolites（FM1-S,FM2-S and FM3-S）on T2DM mice were evaluated.No obvious amelioration of FM1,FM1-S and FM1-C on typical pathological indicators of T2DM mice was observed.FM2,FM2-C,FM3,FM3-S and FM3-C significantly relieved the typical pathological symptoms of T2DM mice,reduced the fasting blood glucose concentration from 10.35mmol/L to 7.50～9.30 mmol/L,the glucose area under the curve(AUC _glucose)by14.64%～33.03%.the insulin concentrati on by 13.36%～26.32%and the insulin resistance index（HOMA-IR）by 25.66%～41.59%.The overall improvement of these indexes was FM3>FM2>FM3-C≈FM2-C>FM3-S.The concentrations of serum total cholesterol in FM2,FM2-S,FM3 and FM3-S treated mice were decreased to 5.13～5.55 mmol/L.Additionally,FM3significantly downregulated the m RNA expression of liver TNF-αand upregulated the IL-10 m RNA expression（p<0.05）and the m RNA expression of IL-6 was increased to normal.FM2 and FM3 activated the PI3K/Akt/GSK-3βinsulin signaling pathway and downregulated the m RNA expression of the key rate-limiting enzymes in gluconeogenesis（G6Pase and PEPCK）in T2DM mice to slow down the gluconeogenesis process.FM3 also downregulated the m RNA expression of ACC,FAS and SREBP-1 to inhibit the process of de novo lipogenesis.FM2-S,FM2-C,FM3-S and FM3-C downregulated the expression of key rate-limiting enzymes in gluconeogenesis（PEPCK,G6Pase）and/or key rate-limiting enzymes in de novo lipogenesis（ACC,FAS and SREBP-1）to inhibit gluconeogenesis and/or de novo lipogenesis to different degrees.The fecal gut microbiota of mice was analyzed using 16S r RNA gene sequencing.FM2,FM2-C,FM2-S,FM3,FM3-C and FM3-S optimized the structure of gut microbiota in T2DM mice to varying degrees.FM2 and FM3intervention induced the increase in the richness of the gut microbiota community,manifested by the downregulation of the relative abundance of Deferribacteres（75.44%,77.19%）,Peptococcaceae（51.31%,67.67%）,Parabacteroides（43.07%,46.44%）and Rc4-4（53.34%,80.45%）and the upregulation of S24-7（1.02-fold,1.57-fold）and Lactobacillaceae（2.46-fold,6.02-fold）,which also increased the total amount of fecal short-chain fatty acids（37.53μmol/g and 46.87μmol/g）in T2DM mice.FM2 enhanced the secretion of serum PYY and raised the level of serum GLP-1 to normal.FM3 decreased the contents of lipopolysaccharide and D-lactic acid in the blood.FM2-C,FM3-C and FM3-S enhanced the production of fecal short-chain fatty acids（33.98,28.19 and 33.17μmol/g）,FM3-C can reduce the content of D-lactic acid（13.01μmol/L）in the blood.Probiotics and probiotic milk-based fermentation metabolites have positive effects on relieving the pathological symptoms of T2DM mice.The probiotic effects of probiotics and the biogenic effects of functional metabolites together promote the beneficial effects of probiotic fermented milk,and these effects are strain specific.FM2 improved the pathological symptoms of T2DM mice by regulating the structure of gut microbiota,promoting the production of intestinal short-chain fatty acids,stimulating the secretion of downstream intestinal hormones（GLP-1 and PYY）and inhibiting the gluconeogenesis process.FM3reduced the blood glucose and lipid levels in T2DM mice by optimizing the structure of gut microbiota,promoting the production of intestinal short-chain fatty acids,reducing intestinal permeability,activating PI3K/Akt pathway and inhibiting gluconeogenesis and de novo lipogenesis.