Preparation And Effect Of DPP-Ⅳ Inhibitory Peptides From Acetes Chinensis

In recent years,dipeptidyl peptidase-Ⅳ(DPP-Ⅳ)inhibitors have received more and more attention as novel anti-diabetic drugs.Food-derived DPP-Ⅳ inhibitory peptides have attracted much attention as potential functional food raw materials because they were derived from natural proteins and have many advantages,such as high safety,favorable absorption by the human body and selective hypoglycemia.Acetes Chinensis is a high-quality aquatic raw material with high protein,low fat and rich mineral elements.The Acetes Chinensis protein has a complete range of amino acids and is rich in hydrophobic amino acids,which is a good source for the preparation of biological peptides.In this paper,using Acetes Chinensis as raw material,the process parameters of enzymatic hydrolysis of Acetes Chinensis were optimized first,and DPP-Ⅳ inhibitory peptides were separated and purified from the enzymatic hydrolysis products of Acetes Chinensis,and the hypoglycemic mechanism of DPP-IV inhibitory peptide was explored.Finally,the stability of Acetes Chinensis polypeptides in simulated digestion was analyzed.(1)Taking the degree of hydrolysis,nitrogen recovery rate and molecular weight distribution as evaluation indicators,the parameters of enzymatic hydrolysis of Acetes Chinensis were finally determined through the selection of optimal protease,single factor test and response surface design:enzymatic hydrolysis time of 5.1 h,material-to-liquid ratio(W/V)of 1:10.5 g/mL,and the amount of enzyme added of 5100 U/g.At that time,the degree of hydrolysis in the enzymatic hydrolysis solution was the highest,and the actual value was 45.71±1.04%,which was close to the predicted value.The DPP-Ⅳinhibition rate of the enzymatic hydrolysate of Acetes Chinensis hydrolysates under the optimal conditions was 44.32±2.78%at a concentration of 20 mg/mL,which proved that the response surface optimization of this experiment could improve the degree of hydrolysis of the enzymatic hydrolysate of Acetes Chinensis.The enzymatic hydrolysates with DPP-IV inhibitory activity were obtained.(2)Tthe enzymatic hydrolysate of Acetes Chinensis was purified by membrane separation,Sephadex G-15 and RP-HPLC in 3 steps to obtain the highly active F2-3 fraction,which was structurally identified by LC-MS/MS,and finally screened for five DPP-Ⅳ inhibitory peptides,YGGQFPTRPD,PALPSIPH,IFTAPQVP,PAFPHPLP and LGDAPGEVP,by database search and comparison of structural features.(3)The DPP-Ⅳ inhibitory peptides were synthesized by solid-phase synthesis method.By verifying the DPP-Ⅳ inhibitory activity of the synthetic peptides,the DPPⅣ inhibitory activities of PALPSIPH and PAFPHPLP were higher among the five peptides,with IC50 values of 2.68 ± 0.06 mmol/mL and 1.61 ± 0.06 mmol/mL respectively.Subsequently,the inhibitory mechanism of the highly active DPP-Ⅳ inhibitory peptide was explored.The results of enzyme inhibition kinetics showed that the inhibition types of DPP-Ⅳ inhibitory peptides PALPSIPH and PAFPHPLP with higher activity and DPPⅣ were competitive inhibition and competitive/non-competitive mixed inhibition,respectively.Molecular docking results showed that PAFPHPLP combined with the Gln553 residue in DPP-Ⅳ to form a hydrogen bond,and the binding energy was smaller,indicating that the docking effect of the peptide and DPP-Ⅳ was better,and the inhibitory ability of DPP-Ⅳ was greater;docking of PALSIPH The second effect is that the polypeptide is combined with Arg358,Try547 and Ser209 of the active center of DPP-Ⅳthrough hydrogen bonding.(4)To explore the stability of the structure and activity of Acetes Chinensis polypeptide(ACHP)using an in vitro simulated gastrointestinal digestion model.Overall,the effects of simulated intestinal digestion on ACHP structure and activity were greater than those of gastric digestion.The digestibility of Acetes Chinensis peptides in the gastric digestion stage is lower than that of the intestines,indicating that the stomach has better resistance to digestion.The secondary structure,molecular weight distribution and UV scanning results also showed that the structure of the peptides changed greatly during the intestinal digestion stage.The DPP-Ⅳ inhibitory activity and antioxidant activity of Acetes Chinensis peptides were improved to varying degrees after simulated gastric digestion,while the two activities were greatly weakened in the intestinal digestion stage,which also confirmed the effect of simulated intestinal digestion in vitro on the effect of shrimp peptides was greater.In order to protect the Acetes Chinensis peptides from being changed in the gastrointestinal environment,β-cyclodextrin microcapsules and entericcoated capsules were used to protect the polypeptides respectively.The protective effect of inhibitory activity and antioxidant activity is better than that of microcapsules.