Preparation,Purification And Characterization Of DPP-Ⅳ Inhibitory Peptide Derived From Eel Protein

Eel is a resource-based fish with high economic value,and the processing of eel produces a large number of scraps with great nutritional benefits,which are less utilized by eel processing enterprises at this stage.Therefore,how to efficiently develop these processing scraps has become a key point to improve the utilization of eel resources.Influenced by the improvement of living conditions,dietary habits and genetic factors,the number of patients with type II diabetes is increasing year by year and has become one of the chronic diseases that pose a serious threat to human health.In the development of glucose-lowering drugs,researchers have found that dipeptidyl peptidase Ⅳ（DPP-Ⅳ）inhibitors that aim at intestinal insulin can exert better glucose-lowering effects.However,in monitoring the clinical use of these drugs,researchers have found that some chemically synthesized DPP-Ⅳ inhibitors are associated with adverse effects during administration,and the finding of food protein derived DPP-Ⅳ inhibitory peptides has been an important breakthrough in addressing the side effects of these drugs.Therefore,this thesis demonstrated the potential of eel protein as a precursor protein for the preparation of DPP-Ⅳ inhibitory peptide using eel scraps as raw material,optimized the enzymatic preparation process of eel-derived DPP-Ⅳ inhibitory peptide,and then purified and isolated the enzymatic product and identified the sequence of DPP-Ⅳ inhibitory peptide using DPP-Ⅳ inhibition rate as approach to assessment,based on which,molecular docking and enzymatic kinetic analysis were applied to explore the Finally,we investigated the gastrointestinal digestive stability,thermal stability,metal ion effect and cytotoxicity of the eel-derived DPP-Ⅳ inhibitory peptide.The main studies and results are as follows:（1）Firstly,the nutrient composition of eel and amino acid composition of eel protein were analyzed,and crude eel protein was extracted from eel processing offal.Then,four food-grade proteases were used to hydrolyze the crude eel protein with the DPP-Ⅳ inhibition rate and hydrolysis degree as indicators,and the best protease was finally screened as the composite protease.Then the optimum temperature and p H of the complex protease were adjusted,and the three influencing factors of enzyme addition,hydrolysis time and substrate concentration were tested separately in a single-factor test,and the data from the single-factor test were used as the basis for optimising the conditions of the enzymatic digestion process of eel protein using the response surface methodology.The optimum enzymatic process was determined as follows:temperature 50℃,p H 7.0,substrate concentration 2%（w/v）,enzyme addition 1%（w/w）and reaction time 2 h.The DPP-Ⅳ inhibition rate of the enzymatic products prepared under these conditions was verified to be 49.41±1.07%（2.5 mg/m L）.（2）Firstly,the enzyme digestion products were separated by ultrafiltration,and the enzyme digestion products were passed through 10 k Da and 3 k Da ultrafiltration membranes successively,and the three ultrafiltration fractions obtained:MU1（<3 k Da）,MU2（3 k Da～10k Da）and MU3（>10 k Da）all had certain DPP-Ⅳ inhibition activity,among which MUI had the highest activity,and its DPP-Ⅳ inhibition rate was 32.17±0.21%（1 mg/m L）,the half inhibition concentration(IC₅₀)was 1.19±0.17 mg/m L,and the ultrafiltration product yield was 46.17±1.52%.Then the MU1 fractions were separated by a Superdex^TM G-15 gel chromatography column.Six fractions（P1,P2,P3,P4,P5 and P6）were obtained,and the highest inhibition rate of 54.75±1.02%（0.5 mg/m L）and IC₅₀ of 0.4151±0.09 mg/m L were detected for the P4 fraction,and 12 fractions were collected by RP-HPLC and named as F1～F12 in order.The highest inhibition rate was 74.51±1.57%（0.25 mg/m L）for the F4fraction,with an IC₅₀of 0.0923±0.06 mg/m L.Forty-two peptide sequences were identified by mass spectrometry analysis of the F4 fraction,and three DPP-Ⅳ inhibitory peptide sequences were screened:Phe-Pro-Arg（FPR）,Tyr-Pro-Pro-Ser-Phe-Ser（YPPSFS）,and Tyr-Pro-Tyr-Pro-Ala-Ser（YPYPAS）,with IC₅₀s of 62.14±1.47μmol/L,102.65±4.57μmol/L and 68.30±3.85μmol/L,respectively.（3）The binding mechanism of FPR,YPPSFS and YPYPAS to DPP-Ⅳ enzyme was investigated using molecular docking,and the free binding energies of the inhibitory peptides were determined to be-8.3 0 kcal/mol,-9.20 kcal/mol and-8.60 kcal/mol,respectively,mainly through hydrogen bonding to DPP-Ⅳ enzyme.Lineweaver-Burk double inverse plotting was used to analyze the inhibition patterns of FPR,YPPSFS and YPYPAS and it was found that FPR and YPPSFS were competitive DPP-Ⅳ enzyme inhibitors,while YPYPAS was a combination of competitive and non-competitive inhibitor.（4）The stability of DPP-Ⅳ inhibitory peptides under the digestion of pepsin and trypsin was investigated using in vitro simulated digestion experiments.FPR and YPYPAS were able to maintain their structural stability under the action of both pepsin and trypsin,and their inhibition rates hardly decreased,which proved their better anti-digestion ability.In contrast,YPPSFS showed degradation in the presence of pepsin,and the inhibition rate of DPP-Ⅳ was significantly different from that of the blank group,but the retention rate and the inhibition rate of DPP-Ⅳ did not decrease in the presence of subsequent trypsin.Then,the thermal stability of the active peptides was investigated.YPYPAS was more thermally stable,with a small decrease in retention and DPP-Ⅳ inhibition after heating at 95℃for 4 h,while FPR and YPPSFS were less thermally stable and began to show structural degradation above 70℃.The effect of p H on the stability of the inhibitory peptide was investigated.FPR and YPYPAS maintained good stability in acidic and alkaline environments,while YPPSFS was less stable in acidic environment and more stable in alkaline environment.The effects of metal ions on the stability of FPR,YPYPAS and YPPSFS were investigated.Under the action of different metal ions,YPYPAS all had good stability,while FPR and YPPSFS had poor stability.The cytotoxicity of FPR,YPYPAS and YPPSFS was investigated using Caco-2 cells.Compared with the blank group,FPR and YPYPAS at varying concentrations（0.05 mg/m L,0.1 mg/m L,0.25 mg/m L,0.5 mg/m L and 1 mg/m L）showed cell growth promoting effects with cell survival rates exceeding 100%,while YPPSFS showed cell growth inhibiting effects although the cell survival rate of FPR and YPYPAS was more than 85%.