Preparation And Structure Identification Of Antioxidant Peptide From Almond Meal Protein And Analysis Of Its Activity In Vivo

As one of the characteristic dried fruits in Xinjiang,almond contains high-quality protein in its processing by-products.At present,almond is only in the initial processing state in the market,and it does not fully reflect its resource advantages and value.The development and utilization of ingredients can bring more benefits to the almond resources.This study optimized the enzymatic hydrolysis process for preparing antioxidant peptides of almond meal protein.Microwave technology was used to treat the protein to enhance the antioxidant properties of the enzymatic hydrolysis product peptides,and the antioxidant peptides were further separated and purified and amino acid sequence identification was performed.Oxidized peptides were analyzed for in vivo activity.The main findings are as follows:(1)Preparation of antioxidant peptides by enzymatic hydrolysis of almond meal protein.The optimal hydrolysis enzyme was selected from alkaline protease,neutral protease,trypsin,papain,and complex protease as alkaline protease based on the hydrolysis degree and DPPH·clearance of the enzymatic hydrolysis product.Based on the single-factor test,the four-factor three-level response surface method was used to determine the enzymatic preparation process as follows:the pH was 9.1,the amount of enzyme added was 10000 U/g,the enzymolysis temperature was 58?,and the enzymolysis time was 4 h.At this time,the DPPH·clearance of the enzymolysate was 74.45%.It was suitable for preparing antioxidant peptides of almond meal protein.(2)The microwave technology was used to pretreat the almond meal protein.The single-factor test determined that the microwave treatment of almond meal protein conditions the power was 210 W and the time was 1.5 min.Compared with the untreated enzyme,the DPPH·clearance of the hydrolyzed product was increased by 18.8%,the·OH clearance was increased by 8.52%,the ABTS+·clearance was increased by 3.25%,and the reducing power was increased.The microstructure,Zeta sites,fluorescence spectra and FTIR spectra were observed.It is concluded that the microwave technology can effectively stretch the protein structure and generate bonded stretching vibration,which is conducive to subsequent enzymolysis and improves its antioxidant activity.(3)Taking DPPH·clearance,·OH clearance,ABTS+·clearance and reducing power as indexes and GSH as positive control,it was determined that MW<1 kDa after ultrafiltration was the best.Three components were separated by cation exchange resin HW-55F,and the antioxidant activity of component F-I was determined to be better.The components of F-I were identified by using ul-tra high pressure nano upgrading liquid chromatography tandem electrospray ionization mass spectr-ometry(LC-MS/MS)system.Three sequences were identified as follows:Gly-Asp-Val-Ile-Arg-Val-Pro-Ala-Gly-Ala(GDVIRVPAGA),Ala-Val-Pro-Val-Leu-Asp-Arg(AVPVLDR)and Phe-Val-Gln-ProPro-Arg(FVQPPR),with molecular weights of 954.53 Da,769.46 Da and 743.41 Da,respectively.(4)The mice in the blank group were gavage with deionized water,and the mice in the positive control group were gavage GSH,low dose,medium dose and high dose(100,250,500 mg/kg·bw)group were gavage with MW<1 kDa almond meal protein antioxidant peptide,and the gavage period was 28 d.The results showed that the activity of T-SOD,GSH-Px and MDA in the high dose group were increased to 153.89 U/ml,901.62 U/ml and 3.82 nmol/ml,respectively,compared with the blank group.The serum non-target metabonomics of mice was studied by UPLC-Q-TOF MS,and significant differential metabolites and their metabolic pathways related to the antioxidant mechanism of antioxidant peptides in vivo were screened out.The results showed that antioxidant peptide from almond meal protein could improve the metabolism of glycerophosphingolipids,sphingolipids,histidine and the levels of phosphatidylethanolamine,lysophosphatidylcholine,sphingomyelin,3-dehydsphingosine and 1-methylhistidine in mice by regulating 12 metabolic pathways corresponding to 9 different metabolic markers.The results showed that antioxidant peptide from almond meal protein could improve oxidative damage and antioxidation in vivo.