| In recent years,the probiotic effects of intestinal microbes on gastrointestinal health and some metabolic syndrome are gradually recognized and accepted by human organs.As an important part of human intestinal microorganism,probiotics have the characteristics of safety and high efficiency,and have great application value in food,medicine,and other fields.With the improvement of people’s living standard,the incidence of many metabolic syndrome is increasing day by day.Diabetes is one of the most common and harmful metabolic syndrome,which is characterized by hyperglycemia and relative deficiency of insulin.However,the application prospect and material basis of probiotics in diabetes have not been widely explored.Therefore,it is an important task to find efficient natural hypoglycemic probiotics and their metabolites.Inhibition of α-glucosidase activity is an important strategy to control blood glucose concentration.In this paper,using probiotics from food sources at home and abroad as the screening object,the α-glucosidase inhibitory activity of bacterial suspension,inactivated bacterial suspension,cell-free supernatant(CFS)and cell-free extracts(CFE)were determined by establishing α-glucosidase screening model.Finally,the CFS of L.rhamnosus LB1lac10 was determined to have the highest α-glucosidase inhibition ability.To further determine the material basis for L.rhamnosus LB1lac10 hypoglycemic ability,the extracellular polysaccharide(EPS)of the bacterium was extracted and purified,and its ability to inhibit α-glucosidase was further identified.Its structure and composition were analyzed by congo red test,gel chromatography(GPC),high performance liquid chromatography(HPLC),uv full wavelength scanning(UV),infrared spectroscopy(FT-IR),nuclear magnetic resonance(NMR),etc.The main results are as follows.(1)Isolation,screening and identification of lactic acid bacteria in characteristic functional foods.This experiment utilizes traditional microbial isolation and purification methods,combining modern molecular biological means to separate,screening and identifying potential probiotics in domestic and foreign feature foods.According to the phenotypic characteristics of the strain separated in the experiment and the results of 16 S r DNA-based Blast comparison,22 strains were divided into 12 species: 8 Lactobacillus rhamnosus,2 Lactobacillus casei,2 Lactobacillus helveticus,2 Lactobacillus plantarum,1Streptococcus thermophilus,1 Lactobacillus mobilis,1 Lactobacillus buchneri,1Lactobacillus pentosus,1 Lactobacillus brevis,1 Pediococcus pentosaceus,1 Lactobacillus acidophilus.(2)Establish an external screening system of α-glycosidase to define the potential hypoglycemic ability of probiotic strains.The α-glucosidase inhibition ability of 11 strains of bacteria suspension,inactivated bacteria suspension,CFS and CFE were determined.The CFS of L.rhamnosus LB1lac10 finally had the strongest inhibitory ability(27.74%),indicating that the strain L.rhamnosus LB1lac10 may be a potentially effective natural source for lowering blood glucose.(3)Using the third generation of Nanopore sequencing techniques to take whole genome sequencing,and the genome function comments were performed.In the genome function comment results,Nr database alignment showed that the strain had 97.15% likelihood of Lactobacillus rhamnosus;In COG function comments,the gene of carbohydrate transport and metabolism was relatively numerous;the metabolic process of KEGG shows that the gene of ABC Transporters and carbon metabolism were highly enriched.In addition,the Cazy(carbohydrate active enzyme database)was shown that this strain contained a large amount of glycoside hydrolase and glycosyltransferase,indicating that the carbohydrate metabolism in the strain machine was active,especially the hydrolysis and glycosyl groups of glycosidism.The results showed that the strain had 8 functional genes related to extracellular polysaccharide synthesis.These molecular-based conclusions are reasonable to some extent for the potential hypoglycemic capabilities of L.rhamnosus LB1lac10.(4)Isolation,purification and structural function analysis of extracellular polysaccharide EPS1-1.The exopolysaccharide of LB1lac10 was extracted,and the output of crude extracellular polysaccharide EPS-1 was calculated to be 8.24 g/L.After that,the EPS-1 was purified by molecular sieve chromatography and the pure extracellular polysaccharide was freeze-dried,named EPS1-1.The EPS1-1 also was demonstrated efficient inhibition ability ofα-glucoside.The composition and structural characteristics of EPS1-1 were further analysis.The results of gel chromatography(GPC)and monosaccharide composition determination showed that the EPS1-1 of L.rhamnosus LB1lac10 had a molecular weight of 88650 Da,which was mainly composed of mannose,glucuronic acid,glucose,xylose,galactose and arabinose.The results of infrared spectroscopy(FT-IR)and nuclear magnetic resonance(NMR)showed that the EPS1-1 had typical polysaccharide structural functional groups and contained two glycosidic bonds with α-configuration of pyranose.the main glycosidic bonds corresponded to →4)-α-D-Glcp-(1→.Congo red test and thermodynamic stability study showed that EPS1-1 had three-strand spiral structure and high heat resistance,which could meet the requirements of hot product processing.To sum up,the L.rhamnosus LB1lac10,found in this study as a food source of lactic acid bacteria,has an efficient ability to α-glucosidase inhibition.The extracellular polysaccharide EPS1-1 produced by L.rhamnosus LB1lac10 has also been shown to own potential hypoglycemic function.In the future,they can be used as a natural functional product to regulate blood sugar in daily diet. |
