Study Of Preparation And Identification Of DPP-4 Inhibitory Peptides From Glycomacropeptide(cGMP20) Protein

In recent years,food derived bioactive peptides have been developed and applied to treat diabetes.This study was designed to investigate the potential of glycomacropeptide as a natural source of DPP-? inhibitor.To investigate the DPP-4 inhibitory peptide derived from glycomacropeptide,glycomacropeptide(CGMP₂₀)was used as a raw material and digested with trypsin and papain.Fresh murine small intestine mucosal secretions and epithelial cells were collected to analyze the activities of DPP-4 enzymes.In order to obtain DPP-4 inhibitory peptides derived from glycomacropeptide source more efficiently,a single factor experiment was conducted to determine the center point of four factors.The response values were determined in turn for DPP-4 inhibitory activity and then response surface analysis was performed for quadratic nonlinear regression fitting.The regression model was highly significant.The model had a good correlation?R2=0.6876?and the accuracy of the test was 13.477.The optimal conditions predicted according to this model were combined temperature:40 ^oC,time?1h?,enzyme amount?4500 U/g?pro,protein concentration 4%.The concentration of glycomacropeptide glycoside was hydrolyzed by papain and by using a semi-preparative chromatographic separation.One of the seven collected fractions showed significant activity of the DPP-? inhibitor.These fractions were analyzed by HPLC and mass spectrometry?HPLC-MS/MS?to determine the peptides responsible for the observed activity.The strongest fragment sequences NQDKTEIPT f?130–140?and NQDKTEIPTIN f?140–150?corresponded to glycomacropeptide which IC50 value was 563.817?M and 1063.60.The result showed that glycomacropeptide derived peptides would be beneficial components to prevent type2 diabetes.Different concentrations of the test material and cell culture were used at different times to determine the relative activity of DPP-4 and GLP-1content in the cells.It was found that the positive drug LGT can effectively inhibit the expression and relative activity of DPP-4 and GLP-1.It also showed good concentration and time dependence.The effects of active peptides were not the same.LDQ and AEP can reduce the relative activity of DPP-4 in cells,but the other four groups of active peptides do not inhibit DPP-4.In terms of GLP-1,active peptides LDQ,RAP,YPK It can promote the secretion and expression of GLP-1 while the active peptides ILD,AEP and TPK cannot promote the expression and secretion of GLP-1.