Study On Dipeptidyl Peptidase-Ⅳ(DPP-Ⅳ)inhibitory Peptides Derived From Antarctic Krill Protein

Diabetes is becoming the third metabolic disease after malignant tumor and cardiovascular,threatened to people’s health and life.It could cause cerebrovascular disease,coronary heart disease,retinopathy and other complications,even death.Nowadays,the control and treatment of diabetes are mainly dependent on food control and drugs.Diabetic drugs used are mainly synthetic drugs,which all have side effects to some extent.DPP-Ⅳ inhibitors,a kind of incretin,have been arose people’s attention because of its good and safe effects in blood control.DPP-Ⅳ inhibitory peptides derived from food proteins have advantages of wide sources and safety,which has become a hotspot for attention and research.The Antarctic krill biomass was tremendous and the potential resource reserve was about 600 million to 1 billion tons.Antarctic krill had high protein content and protein content of the single shrimp was about 70.58%(dry basis).It was rich in 20 kinds of amino acids for human health,which was good resource for DPP-Ⅳ inhibitory peptides.This studied was aimed to preparation,separation and purification,evaluation of activity and mechanism of action of DPP-Ⅳ inhibitory peptides from Antarctic krill protein,and to provide technique bases for development of hypoglycemic function food ingredient.The main research contents and results were as follows:1.With DPP-Ⅳ inhibition rate as main index,Antarctic krill was hydrolyzed by using animal proteolytic enzyme.Firstly,the effect of four single factors such as enzymolysis time,temperature,pH and amount of Animal protein hydrolase on the DPP-Ⅳ inhibition rate and soluble protein of Antartic krill enzymatic hydrolysate were investigated,and then response surface method was used to optimize these four factors based on single factor experiment results.Results showed that optimal enzymolysis parameters of DPP-Ⅳ inhibitory peptide from Antarctic krill were as follows: enzymolysis time 3.85 h,temperature 45 ℃,pH 7.6,enzyme added 237.6 U/g.The DPP-Ⅳ inhibition rate was 64.82% under the above conditions.2.Using DPP-Ⅳ inhibitory rate determined by the method of fluorescence substrate(reagent kit method)as a evaluation specification,Fluorine ion of Antarctic krill enzymatic hydrolysates was removed by calcium acetate,and separated into four fractions(>5000Da,3000Da-5000 Da,100Da-3000 Da,<100Da)by ultrafiltration membranes with molecular weight(MW)cut-offs 5,000 Da,3,000 Da and 100 Da.The fraction with molecular mass weight of 3000-100 Da,which exhibited strong DPP-Ⅳ inhibitory activity,was separate into 3 sub-fractions(F1,F2,F3)on Sephacryl S-100 column.F1 sub-fraction with high DPP-Ⅳ inhibitory activity was further purified into 3 peptides(F1-1,F1-2,F1-3)by reversed-phase high performance liquid chromatography.The amino sequences of 3 purified peptides were identified by amino acid analyzer and QTOF-MS.They were Ala-Pro(AP),Ile-Pro-Ala(IPA)and Ile-Pro-Ala-Val-Phe(IPAVF),respectively.The IC50 values of DPP-Ⅳ inhibitory activity were 0.0530 mg/m L,0.0370 mg/mL and 0.0730 mg/m L,respectively.3.AP,IPA,IPAVF were synthesized by peptide solid-phase synthesis method.The identity of 3 synthetic peptides was verified by MS and HPLC.Results showed that 3 synthetic peptides and 3 DPP-Ⅳ inhibitory peptides purified from Antartic had the same amino acid sequence and high purity.Diabetic zebrafish model was established quickly by combining 20 g/L glucose solution immersing and 10% cholesterol feeding for 20 days.The vivo hypoglycemic activities of AP,IPA and IPAVF were evaluated by this model.The evaluation physicochemical indexes include DPP-Ⅳ inhibitory activity,glucose,triglyceride,cholesterol.The evalution gene indexes include Insa,Glucagon and Pck1.Results indicated that AP,IPA and IPAVF all had the hypoglycemic action on diabetes zebrafish.The concentrations of the peptides used were positively correlated with lowering blood sugar effect.The IPA exhibited the best hypoglycemic action among 3 peptides.4.Inhibition kinetics of 3 kinds of DPP-Ⅳ inhibitory peptides(AP、IPA、IPAVF)on DPP-Ⅳ were studied.Results displayed that inhibitory types of AP、IPA、and IPAVF on DPP-Ⅳ were all competitive reversible.The interations of 3 kinds of DPP-Ⅳ inhibitory peptides(AP、IPA、IPAVF)and DPP-Ⅳ were investigated by computer simulation software and molecular docking method.3 models of IPA-DPP-Ⅳ,IPA-DPP-Ⅳ and IPAVF-DPP-Ⅳ have been simulated.IPA-DPP-Ⅳ molecular docking model was best,which indicated that IPA inhibitory activity on DPP-Ⅳ was strongest.Main docking locations of IPA on DPP-Ⅳ molecular were situated in 91-96 and 101-105 amino acids residuals.5.With physicochemical indexes(DPP-Ⅳ inhibitory activity,glucose,triglyceride,cholesterol)and relative gens(Insa,Glucagon and Pck1)expression levels as evaluation indexes,hypoglycemic effect of Antarctic krill hydrolysate(AKH)with molecular mass weight of 3000-100 Da was evaluated by using diabetic zebrafish model established.Combined with physicochemical indexes and gene indexes,conclusions were drawn as follows.The hypoglycemic effect of AKH(3000-100 Da)at the concentration of 1.35 g/L and 2.7 g/L was not obvious.AKH at the concentration of 5.4 g/L exhibited certain effect on hypoglycemic action,but not as good as purified AP,IPA and IPAVF.