Study On Preparation And Structure Analysis Of DPP-4 Inhibitory Peptide From α-Lactalbumin And It’s Functional Evaluation

Diabetes is one of the three major epidemics in the world,with type 2 diabetes(T2DM)accounting for more than 90% occurrence.In recent years,hypoglycemic substances from food sources such as cereal proteins,fish protein and milk proteins,having more advantages than chemical synthetic drugs have been developed and applied.According to literature reports,α lactalbumin has a high potential as a source of DPP-4 inhibitory peptide.Therefore in this paper,the commercial α-lactalbumin was used as the raw material,and the sample was hydrolyzed by the central combination-response surface experiment(CCD-RSM).After separation and purification,the pre-synthesized sample was identified to obtain the DPP-4 active peptide sequence,and the invitro inhibitory activity was subsequently analyzed.Functional chemical verification and model cell validation was also determined.The main conclusions are as follows:1.α-Glucosidase and Dipeptidyl Peptidase Ⅳ(DPP-4)are two important targeting enzymes in the treatment of type Ⅱ diabetes.most commercially available enzymes are mainly of microbial sources,and are somewhat different from the properties of those obtained from mammalian sources It is difficult to objectively evaluate the screening of inhibitors of these two enzymes.In this study,fresh porcine intestinal mucosal secretions and epithelial cells were collected.Commercial enzymes were used as controls to analyze the α-glucosidase and DPP-4 enzyme activities as well as the specific activities of different small intestinal mucosal extracts from different parts.The dilution factor and the effective fraction were established.The enzyme activity assay system for porcine intestinal-inducing α-glucosidase and DPP-4 was established,which provided a reliable method for the invitro screening of their inhibitors.2.In order to most effectively obtain DPP-4 inhibitory peptide derived from α-lactalbumin source,the single factor experiment was designed and a center point of four factors was obtained.On this basis,30 groups analyzed by central combination experiment(CCD)were obtained.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(P < 0.0001).This indicates that the model can be used to screen for hydrolysis conditions.According to the predictions by this model,optimal enzymatic hydrolysis conditions of temperature,time,amount of trypsin and α-lactalbumin concentration were obtained at 41.5 o C,129 min,4537 U/g pro and 7% respectively.3.In order to further discriminate peptides with DPP-4 inhibitory activity in α-lactase hydrolysate,this study used membrane tandem protein separation and purification system to separate and purify the hydrolysates of the enzyme.The protein peptide sequence was identified using LC-m S/mS.Sequence scoring,six sets of peptide sequences derived from α lactalbumin were obtained.Simultaneously,the six groups of peptides were synthesized by chemical solid phase,and their invitro inhibitory activities were verified.The results showed that the DPP-4 inhibitory activity of the active peptide LDQWLCEKL was the highest,with a half-inhibitory concentration(IC50)of 131.07μm.In addition,the mode of inhibition of LDQWLCEKL as analyzed by Lineweaver-Burke double reciprocal proved to be a non-competitive type.4.NCI-H716 cells were used as model cells,and the relative activities of DPP-4 and GLP-1 were measured at different times using different concentrations of synthetic peptides and cells.The positive drug LGT n effectively inhibits the expression and relative activities of DPP-4 and GLP-1,and is concentration and time dependent.The effects of active peptides on DPP-4 and GLP-1 were different.LDQ and AEP reduced the relative activity of DPP-4 in cells.However,no inhibition was observed with the other four groups of active peptides.In terms of GLP-1,active peptides LDQ,RAP,YPK promoted the secretion and expressions of GLP-1.On the other hand,the active peptides;ILD,AEP and TPK were unable to promote the expression and secretion of GLP-1.