| Diabetes Mellitus(DM)is caused by insufficient insulin secretion or poor insulin action.It is a metabolic disease characterized by high blood sugar,in which type 2 diabetes is caused by insulin insensitivity(ie insulin resistance)as the main pathogenesis(Type 2 Diabetes Mellitus,T2DM)patients are mostly.In recent years,as people's dietary structure has undergone tremendous changes,high-calorie,high-fat,and high-sugar daily diets are accompanied by high-intensity life and work pressures,which has made diabetics younger and the number of diabetic patients has increased sharply.At present,the commonly used treatment methods for patients with T2DM are oral sulfophthalamides and biguanide drugs.These drugs may have side effects on patients.Therefore,there is an urgent need to develop natural,efficient,and safe functional products that relieve the symptoms of diabetic patients to improve the quality of patients'life.Exopolysaccharides(EPS),as an active product of secondary metabolism of lactic acid bacteria,endows it with unique physical and chemical properties and functional activity due to its structural characteristics.It can not only improve the sensory quality of the product,but also has probiotic functions such as blood sugar regulation.Its advantages such as safety,greenness and no side effects have been widely recognized in experimental research and clinical applications,and have broad application development space in the food field.However,the blood glucose regulation mechanism of EPS is not clear at present,so more in-depth research on the mechanism of its blood glucose regulation is needed.In this study,the EPS yield of the strain was used as the evaluation index.From 20strains of Lactobacillus paracasei isolated from traditional fermented dairy products in Tibet,L.paracasei TD 062 with high EPS production was selected as the test strain.Based on the genetic point of view,the functional genes of L.paracasei TD 062 strain were annotated by whole-gene sequencing technology,the way to synthesize EPS was analyzed,the available carbon source was predicted.And the fermentation conditions for EPS production were optimized based on the results,in order to increase the output of EPS under laboratory conditions.Based on the molecular perspective,the molecular structure of the different components of EPS separated by column chromatography was analyzed,the molecular schematic structure was clarified,and its structure-activity effect was analyzed.From the two levels of in vitro and in vivo,clarify the hypoglycemic function of EPS,analyzed the expression of genes and key proteins related to hypoglycemic effect,and clarify its role in regulating blood sugar mediated by affecting the intestinal flora,and the hypoglycemic mechanism of EPS was analyzed in-depth.Provide a theoretical reference for the development and research of new functional products with safety,efficiency and no side effects.The following results are obtained through the above research:(1)L.paracasei TD 062 with high EPS production was screened out,and its genome-wide characteristics were clarified,functional genes were annotated,the biosynthetic pathway of EPS was analyzed,and the carbon source available for EPS production was inferred.By comparing the EPS production capacity of 20 strains of L.paracasei isolated from traditional fermented dairy products in Tibet,it was found that L.paracasei TD 062 had the highest EPS yield,with a yield of 0.609 g/L and a polysaccharide content of 90.57?g/m L.Using whole-genome sequencing technology to analyze the genomic characteristics of the strain,it was found that L.paracasei TD 062 was composed of a circular chromosome of2867519 bp and 6 plasmids.A total of 3064 coding genes were annotated,including 3 related to bacterial secondary metabolism.Through CAz Y database function annotation,276 coding genes were involved in the transport and metabolism of carbohydrates,and 48 coding genes were involved in glycolysis/gluconeogenesis pathways.The related genes in the L.paracasei TD 062 strain were responsible for encoding the phosphoglucose transferase system could transport fructose,mannose,trehalose,glucose,galactose and lactose into the cytoplasm to form the precursor glycosides for the synthesis of EPS.There was an EPS gene cluster composed of 35 genes on plasmid 4,which was responsible for the functions of EPS synthesis regulation,chain length,repeat unit and output.From the perspective of functional genes,L.paracasei TD 062 had a strong ability to produce EPS,and it predicted the available carbon source of the strain by analyzing the synthesis pathway of EPS.(2)The carbon source that could be used in the process of producing EPS by the strain was used as a composite optimized carbon source,and the production conditions(time,temperature,initial p H)of EPS were optimized,which increased the output of EPS by 7.78times.For the purpose of increasing EPS production,through genome analysis,it was determined that fructose,mannose,trehalose,glucose,galactose,and lactose were used as the composite optimized carbon source to optimize the fermentation time,fermentation temperature,and initial p H of the fermentation required to produce EPS,in order to achieve the purpose of high-yield EPS.It was found that using a compound carbon source medium,fermented for 26 h at a fermentation temperature of 30?,the initial p H of the fermentation was 6.0,the experimental output of EPS increased from 0.609 mg/m L to 5.351 mg/m L,and the output rate increased by 7.78 times.(3)Analyzed the fine molecular structure of different components of EPS,clarified its molecular structure,and analyzed its structure-activity effect.The molecular weights of the two EPS components LPP-1 and LPP-2 which separated by column chromatography were determined to be 1.069×10~5 Da and 4.178×10~4 Da,respectively.The chemical functional groups of the two components were determined by infrared spectroscopy,and it is cleared that both LPP-1 and LPP-2 had?and?configurations.The monosaccharide composition results show that LPP-1 was mainly composed of mannose,galactose and glucose;LPP-2 was mainly composed of mannose,galactose,glucose,xylose,trehalose and galacturonic acid.Methylation analysis determined that there were three sugar residues in the LPP-1 component,namely 1,4-linked-Glcp,1,4,6-linked-Manp and T-Galp;there were five sugar residues in the LPP-2 component The sugar residues are T-Glcp,T-Fucp,1,4-linked-Xylp,1,2,6-linked-Galp and 1,4,6-linked-Manp.The one-dimensional and two-dimensional NMR results combined with the results of structural analysis infer the molecular structure of EPS:the molecular structure of LPP-1 is 1-linked-Galp and 1,4-linked-Glcp through?-(1,4)The glycosidic bond and?-(1,6)glycosidic bond are connected with 1,4,6-linked-Manp.The molecular structure of LPP-2 component is 1-linked-Glcp and 1,2,6-linked-Galp through?-(1,6)glycosidic bond and?-(1,4)glycosidic bond with 1,4,6-linked-Manp is connected,1-linked-Fucp and 1,4-linked-Xylp are connected to 1,2,6-linked-Galp through?-(1,6)glycosidic bond and?-(1,2)glycosidic bond.SEM scanning electron microscope images show that the microstructures of the two components of LPP-1 and LPP-2 are quite different,and the highly branched structure and porosity of LPP-2 make it have greater application prospects.(4)LPP-2 had the activity of lowering blood sugar in vitro,can inhibit hepatocyte gluconeogenesis,and improve the state of insulin resistance of hepatocytes.The in vitro screening method was used to analyze the inhibitory effects of the two EPS components of LPP-1 and LPP-2 on the activity of?-glucosidase,and the LPP-2,which had a strong ability to lower blood sugar in vitro,and the?-glucosidase inhibited rate was 57.36%,which was analyzed as a non-competitive type of inhibition,so the LPP-2 component was selected for follow-up research.Hep G2 cells were induced with 0.6 mg/m L palmitic acid solution for 24 h to establish an insulin resistant cell(IR-Hep G2)model.After co-incubation,it was found that LPP-2 could increase the glucose intake of IR-Hep G2 and the synthesis of liver glycogen,and increase the enzyme activity of two key sugar metabolizing enzymes,pyruvate kinase and hexokinase.And LPP-2 could up-regulate the relative expression of AMPK,Akt,PI3K,and IRS1 genes,and down-regulate the relative expression of FOXO1,PEPCK,G-6-Pase,and GSK-3?genes in a dose-dependent manner.It was preliminarily determined that LPP-2could regulate blood sugar by activating AMPK/Akt/PI3K signaling pathway and improve the insulin resistance of liver cells.(5)LPP-2 could regulate blood sugar levels by activating AMPK/Akt/PI3K signaling pathways,thereby alleviating the symptoms of diabetic mice.A diabetic mouse model was constructed through a high-fat and high-sugar diet combined with streptozotocin,and the hypoglycemic effect of LPP-2 was analyzed to clarify the mechanism of hypoglycemic effect in vivo.It was found that LPP-2 could not only reduce the blood sugar level of diabetic mice,inhibited the loss of their weight,and improved their glucose tolerance level,but also down-regulate the levels of basic indicators such as glycosylated hemoglobin and insulin in diabetic mice.LPP-2 could reduce the organ index of diabetic mice,relieved the pathological damage of liver and pancreas,and restored the pancreatic?cells.By analyzing the changes in the expression of related genes in the insulin signaling pathway that regulates blood sugar,it was found that after the intervention of LPP-2,the relative expression of AMPK,Akt,PI3K,IRS1genes in the liver of diabetic mice could be up-regulated,the relative expression of genes FOXO1,PEPCK,G-6-Pase and GSK-3?could be down-regulated.By analyzing the expression of key proteins,it was found that LPP-2 could up-regulate the expression of p-AMPK,Akt and PI3K proteins,and it was dose-dependent.It was confirmed that LPP-2could regulate the level of glucose metabolism in the body by activating the AMPK/PI3K/Akt signaling pathway,and thereby increased the body's sensitivity to insulin,thereby reducing the blood glucose level of diabetic mice and alleviating the symptoms of diabetes.At the same time,it was found that LPP-2 and L.paracasei TD 062 strain compounded to exert a synergistic effect.(6)LPP-2 could affect the structure of the intestinal flora of diabetic mice,increased the relative abundance of Lactobacillus,Bacteroides,Ekmania,etc.,and reduce the relative abundance of Enterococcus,Bacillus,etc.,.LPP-2 could regulate the activity of carbohydrate synthesis,glycan synthesis and metabolism related to alleviating diabetes in the mouse intestine.Analyzing the effect of LPP-2 with hypoglycemic effect on the intestinal flora of diabetic mice,the results showed that the composition of LPP-2 significantly affected the composition of the intestinal flora of mice and increases the?diversity index of intestinal flora.The?diversity analysis showed that LPP-2 component improved the composition of the intestinal flora of diabetic mice,promoted the growth of beneficial bacteria such as Lactobacillus,and inhibited harmful bacteria such as Clostridium.The results of functional prediction analysis showed that the LPP-2 component could regulate the pathway activities of carbohydrate biosynthesis,precursor metabolites,energy production,and polysaccharide synthesis related to the alleviation of diabetes.It showed that LPP-2 components could alleviate the symptoms of diabetes by regulating the structure of intestinal flora and activating related metabolic pathways. |
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