Preparation And Hypoglycemic Activity Of Enzymatic Peptides From Highland Barley Protein

According to plant classification,highland barley（Hordeumvulgare L.var.nudumhook.f.）is one of the varieties of barley in Gramineae.The nutritional characteristics of high fiber,high protein,high vitamins,low fat and low sugar make it have good hypoglycemic activity.With the increasing incidence of diabetes,the development of natural ingredients from existing natural resources to improve the symptoms of diabetes has attracted much attention in recent years.Therefore,using highland barley（Zangqing 2000）as raw material,the preparation and enzymatic hydrolysis of highland barley protein,the separation and purification of highland barley peptides and their hypoglycemic and antioxidant activities were studied.Type 2 diabetic mouse model was established by intraperitoneal injection of streptozotocin（STZ）combined with high fat diet to study the hypoglycemic effect of highland barley peptides.The main results are as follows:1.The highland barley protein was extracted by alkali dissolution and acid precipitation method.The single factor experiment determined that the isoelectric point of highland barley protein was 4.5.the orthogonal experiment determined the best technological conditions as follows:alkali extraction solution p H 11.5,material-liquid ratio 1:15g/m L,extraction temperature 50℃,extraction time 50min.Under these conditions,the extraction rate of highland barley protein was 64.02%,and the purity of protein was 88.72%.The electrophoretic results of highland barley protein showed 10 bands with molecular weight less than 100 k Da.2.Taking the inhibitory activity ofα-glucosidase as the main index and the degree of hydrolysis as the auxiliary reference index,the optimum protease for enzymatic hydrolysis of highland barley protein is trypsin.According to the inhibitory activity ofα-glucosidase,the optimal technological parameters of enzymatic hydrolysis were determined as follows:the optimum temperature of enzymatic hydrolysis was 35℃,the concentration of substrate was 3%,the amount of enzyme was 12000U/g,and the time of enzymatic hydrolysis was 5 h.Under these conditions,theα-glucosidase inhibitory activity of the hydrolysate was 70.96%.The effects of the three factors on theα-glucosidase inhibitory activity were as follows:substrate concentration>enzyme amount>enzyme hydrolysis time,and the interaction between substrate concentration and enzyme amount was very significant.the interaction between the amount of enzyme and the time of enzymolysis was significant.3.The antioxidant and hypoglycemic activities were determined,and four components were obtained by ultrafiltration,among which the best component was E-4（<3 k Da）.Four components were obtained by Sephadex G-15 filtration chromatography,and the best component was E-43.The IC₅₀values ofα-glucosidase inhibitory activity,α-amylase inhibitory activity,ABTS and DPPH radical scavenging activity were0.26,6.74,2.62 and 4.23 mg/m L,respectively.According to LC-MS/MS analysis,the two octapeptides with high abundance were Gly-Phe-Ser-Gly-Ser-Gly-Gly-Lysand Gly-Val-Gly-Ala-Gly-Ala-Ala-Arg,respectively,the charge-mass ratio was 348.67 and 329.68,and the molecular weight was 695.32 Da and657.36 Da,respectively.4.To establish a type 2 diabetic model to investigate the hypoglycemic effect of highland barley polypeptide（E-4）in vivo.The results showed that highland barley polypeptide alleviated the trend of weight loss of model mice,reduced the value of fasting blood glucose of diabetic mice,protected the organs of mice,and reduced the changes of kidney,liver and spleen caused by diabetes.It is helpful to control the increase of blood glucose after eating,improve the glucose tolerance of diabetic mice and maintain the stability of blood glucose.It decreased the levels of INS,IRI,TG,TC and LDL-C in serum and liver of diabetic mice,and increased the levels of ISI and HDL-C.From the oxidative stress index of liver,highland barley polypeptide decreased the content of MDA and increased the activity of SOD and GSH-Px.5.High-throughput sequencing and bioinformatics analysis of mouse intestinal flora.The results showed that the diversity and richness of intestinal flora in 1 domain,1 kingdom,10 phylum,17 class,43 order,68 family,138 genus,207 species and 400 OTU;model groups decreased.At the gate level,the ratio of thick-walled bacteria to Bacteroides was significantly decreased in the highland barley polypeptide group,and the dominant intestinal flora in the highland barley polypeptide group was similar to that in the normal group;at the generic level,the content of lactic acid bacteria in the highland barley polypeptide group decreased significantly,while the contents of norank＿f＿Muribaculaceae,Lachnospiraceae＿NK4A136＿group and norank＿f＿Lachnospiraceae increased significantly.