Effect Of Ultrasonic Assisted Enzymatic Hydrolysis On Antioxidant Activity Of Walnut Protease Hydrolysate

In this study,Xinjiang thin-skinned walnut was used as raw material,walnut protein isolate was pretreated by ultrasonic wave to obtain walnut protease hydrolysate with good antioxidant activity,and response surface was used to optimize walnut protease hydrolysate process,and the kinetic model of walnut protease hydrolysate was established,and alkaline protease was used for enzymolysis.In addition,the structural characteristics,amino acid composition,molecular weight distribution and antioxidant changes of walnut protease hydrolysates during enzymolysis were explored.The separation,purification and structural identification of walnut protease hydrolysates were carried out,and ten antioxidant peptides were screened out.Four of them were found to be novel peptides after database search.Molecular docking technique was used to dock the novel peptide with ABTS radical to determine the specific antioxidant active sites and interaction mode of walnut antioxidant peptide,so as to explore the antioxidant mechanism of peptide.The main research contents and results are as follows:（1）Optimization of ultrasonic assisted enzymatic hydrolysis of walnut protein and its kinetics by response surfaceFive proteases（alkaline protease,flavor protease,trypsin,pepsin and neutral protease）were selected to hydrolyze walnut protein in order to obtain the enzymatic hydrolysis products with good antioxidant activity.With the free radical scavenging rates of DH and DPPH hydrolysis degree as the evaluation index,the alkaline protease was selected as the best protease,and the enzymatic hydrolysis process was optimized by response surface.The optimal enzymatic hydrolysis process and parameters were determined as follows:ultrasonic time 20 min,ultrasonic power 200 W,substrate concentration 2%（w/w）,enzyme dosage 3%,enzymatic hydrolysis temperature 55℃,enzymatic p H 9.0,enzymatic hydrolysis time 120 min.In addition,the kinetic study of enzymatic hydrolysis of walnut protein by alkaline protease showed that the degree of hydrolysis increased with the increase of initial enzyme concentration and decreased with the increase of initial substrate concentration.The hydrolysis kinetic model was as follows:DH=3.72 ln[1+（6.68 E₀/S₀+0.08）t],can more accurately control the process of walnut separation protease hydrolysis,and promote the promotion and application of walnut peptidylysis preparation technology.（2）The structural properties,amino acid composition,molecular weight distribution and antioxidant activity of enzymatic hydrolysis products were studied researchThe structural changes of walnut protein isolate during enzymolysis（0 min,30 min,60 min,90 min,120 min,150 min）were studied by ultraviolet absorption spectrum,endogenous fluorescence spectrum and Fourier infrared spectrum.The results showed that enzymolysis significantly changed the structure of walnut protein isolate.The hydrophobic groups of walnut protein isolate were exposed by enzymolysis,which led to the increase of ultraviolet absorption and endogenous fluorescence intensity.After enzymolysis,the secondary structure of walnut protein isolate gradually changed from the orderedα-helix form to the disorderedβ-angle and random curling form,which showed more loose and flexible,indicating that the structure of walnut protein was destroyed and the active site was exposed,which was conducive to improving the antioxidant potential of walnut protein.The amino acid composition analysis showed that hydrophobic amino acid content increased with the hydrolysis time.With the increase of hydrolysis time,the high molecular weight polypeptides of walnut protein isolate were gradually hydrolyzed to low molecular weight small peptides and even free amino acids.The antioxidant activity of walnut protein isolate and its enzymolysis products was evaluated by measuring the antioxidant indexes of DPPH radical scavenging,ABTS radical scavenging,hydroxyl radical scavenging and reducing power.The results showed that moderate enzymolysis（enzymolysis time 0-120 min,DH<23.09%）could effectively improve the antioxidant activity of walnut protein isolate.While excessive enzymatic hydrolysis（enzymatic hydrolysis time>120min,DH>23.09%）can reduce the antioxidant activity.（3）The walnut antioxidant peptide purification,Structural identification and molecular dockingAfter separation and purification by DEAE-52 cellulose ion exchange chromatography,two components（F1 and F2）were obtained.The antioxidant activity of F1 was found to be the best through determination of DPPH free radical scavenging,ABTS free radical scavenging,hydroxyl free radical scavenging and reducing power.LC-MS/MS mass spectrometry was used to identify this component.Ten peptide sequences were screened,among them,HADMVFY,PSYQPTP,NHCQYYL and NLFHKRP were identified as novel peptides by BIOPEP database search.Molecular docking was used to study the interaction between the novel peptides and ABTS free radicals.The results showed that the specific amino acid residues of walnut antioxidant peptides could bind to ABTS free radicals through hydrogen bonding and hydrophobic interaction,so as to exert antioxidant effects.The results provided theoretical basis for further study of the structural-activity relationship of walnut antioxidant peptides.