Study On Antioxidant Activity And Dipeptidyl Peptidase-? Inhibitory Activity Of Walnut Protein Hydrolysates

Defatted walnut meal,as the main by-product of walnut oil extraction,is composed of more than 50%proteins.In addition to proteins,defatted walnut meal contains appreciable amounts of phenolic compounds and dietary fiber that provide several nutritional and health benefits.However,defatted walnut meal is underutilized and its higher value-added product is urgently needed to be created in order to improve the economic value.In this paper,defatted walnut meal was used as a raw material to study the walnut protein composition and characteristics of enzymatic hydrolysis;the preparation process for walnut peptides was optimized;the antioxidant activities of walnut protein hydrolysates were evaluated,especially the individual contribution of naturally present phenolic compounds and walnut peptides to the total antioxidant capacity of the hydrolysates was further investigated;and the DPP-? inhibitory activity of walnut peptides was further studied.The research is aimed to supply the method and theoretical basis for the preparation of walnut bioactive peptides.The major results are as follows:Firstly,due to the ambiguity of walnut protein composition,reducing SDS-PAGE and MALDI-TOF/TOF MS were used and the results showed that walnut protein was mainly composed of vicilin?7S?and legumin?11S?;with the combination of diagonal electrophoresis and two-dimensional electrophoresis,the results showed that 11S globulin subunit?56000?58000?was formed by disulfide bonds-linked polypeptides with molecular weights of32000?35000?pI 5.5?7.5?and 19000?22000?pI 9.0?10.0?.Based on the protein composition study,the effects of five proteases on the hydrolysis properties of walnut protein were further investigated.The results showed that Alcalase was the most efficient protease to hydrolyze walnut protein.After 1 h of hydrolysis,7S globulin and 11S-A peptide chain were mostly hydrolyzed,while 11S-B peptide chain had a strong resistance to hydrolysis.The Alcalase-derived hydrolysate attained significantly higher degree of hydrolysis?21.70%?,trichloroacetic acid-nitrogen soluble index?84.23%?and yield?81.58%?,and peptides with molecular weights below 2000 accounted for 72.69%.Hence,Alcalase showed to be the best enzymes for preparing walnut peptides.Then,the walnut peptides preparation process from defatted walnut meal was determined,including acid-precipitation,high-speed shear treatment,enzymatic hydrolysis with Alcalase,desalination with ultrafiltration and spray-drying.The yield and the protein content of the prepared walnut peptides product were 76.74%and85.12%,respectively.And the peptides with molecular weights below 2000 accounted for81.60%.Secondly,the in vitro antioxidant activity of walnut protein hydrolysates was evaluated using chemical assays.The free radical scavenging activity of Alcalase-derived hydrolysate was higher than other hydrolysates,and the IC₅₀0 value of the DPPH,ABTS and hydroxyl radical scavenging activities were 0.109 mg/mL,39.890?g/mL and 1.174 mg/mL,respectively.The Alcalase-derived hydrolysate also exhibited ferric reducing activity,with a reducing power of 1.2 at 1.0 mg/mL concentration,which showed dose-dependent behavior.Furthermore,walnut protein hydrolysates could effectively protect glutathione from Fenton reagent-induced oxidation and regenerate glutathione after oxidation.The contribution of naturally present phenolic compounds and walnut peptides to total antioxidant capacity of hydrolysates was also investigated.Results indicated that in the walnut protein hydrolysates,its antioxidant activity is mainly mediated by peptides which were the main responsible for approximately 80%of the free radical scavenging activity,while the presence of phenolic compounds led to an increase of 10%?25%on radical scavenging activity and nearly 50%on ferric reducing activity.Finally,the DPP-? inhibitory activities of walnut protein hydrolysates were evaluated.The Alcalase-derived hydrolysate exhibited the highest DPP-? inhibitory activity with IC50value of 0.90 mg/mL.The DPP-? inhibitory peptides were isolated from Alcalase-derived hydrolysate by ultrafiltration and SP Sephadex C-25,with the activity increased about 3-fold.The peptide fraction collected was rich in basic amino acids,accounting for 34.35%,which was believed to contribute to the high DPP-? inhibitory activity.Thermal?25?121°C?and pH 1.0?11.0 treatments did not alter DPP-? inhibitory activity of the peptides,and DPP-? inhibitory peptides from walnut protein maintained their activity after simulated gastrointestinal digestion.