| Walnut protein has high nutritional value and is an important vegetable protein resource.Walnut protein has high nutritional value and is an important vegetable protein resource.In the process of processing walnuts into walnut oil,the residual lipids are easy to cause the oxidation of protein in walnut skim meal,which leads to a change in protein structure and digestive characteristics,thus resulting in a decline in protein nutritional quality.In this thesis,2,2’-amidinopropane(2,2’-azobis(2-amidinopropane),AAPH)thermal decomposition was used to produce peroxide radicals,and 1,1,3,3-tetraethoxypropane(1,1,3,3-tetrathoxypropane,TMP)malondialdehyde(MDA)prepared by acid hydrolysis represents lipid free radicals and active aldehydes produced by lipid peroxidation,respectively.The structural changes of proteins modified by oxidation and the identification of digestive products in vitro were studied.The results provide a theoretical basis for the quality control of walnut protein and walnut related products.(1)Walnut protein isolate under different oxidation concentrations of AAPH(0,0.04,0.2,1,5,and 25mmol/L)was used as the research object,and the oxidation degree of walnut protein was characterized by the content of the carbonyl group and sulfhydryl group.The solubility,secondary structure,surface hydrophobicity,endogenous fluorescence,NFk(N’-formyl-L-canurine)and Schiff bases,SDS-PAGE,molecular weight,in vitro digestibility,carbonyl value during digestion,molecular weight,antioxidant value of digestive products,and other indexes were analyzed,characterized,and determined.The results showed that with the increase in AAPH concentration,protein carbonyl content increased from 2.36 nmol/mg to 5.12nmol/mg,free sulfhydryl group decreased,α-helix and β-folding content decreased,random curling increased,surface hydrophobicity increased first and then decreased,and tryptophan fluorescence intensity decreased.The contents of Schiff base and NFk increased,and SDS-PAGE showed that the structure of walnut protein expanded to form soluble aggregates and insoluble aggregates.The results of in vitro digestion showed that AAPH oxidation could significantly reduce the digestibility of walnut protein,and Tricine-SDS-PAGE indicated that AAPH oxidation could cause gastric digestion resistance.The carbonyl content was dynamically changed during digestion and tended to increase during intestinal digestion.The results of molecular exclusion chromatography showed that oxidation decreased the dominant peptide(500–1000 Da)of walnut protein digestive products and decreased the antioxidant activity of digestive products.(2)Walnut protein isolates at different concentrations of MDA(0,0.01,0.1,1,and 10 mmol/L)were used as research objects to determine the corresponding indexes in the oxidized protein system with AAPH.The results showed that compared with the AAPH oxidizing protein system,with the increase of malondialdehyde concentration,protein carbonyl content increased from 2.22 nmol/mg to 9.42 nmol/mg,free sulfhydryl group decreased more obviously,secondary structure destruction was significant,α-helix tended to 0,oxidation strength,and corresponding characterization results were more obvious.The results showed that the variation in digestive properties of walnut protein oxidized by MDA was similar to that of walnut protein oxidized by AAPH.(3)Walnut protein with an AAPH concentration of 5 mmol/L was used as the research object,and amino acid analysis and LC-MS/MS identification were performed for walnut protein and digestive products.The results showed that the oxidation of amino acids was selective.Cysteine is the most vulnerable amino acid and is usually the first to be oxidized.The decay rate of amino acids in walnut protein before and after oxidation was as follows: Cys(50.87%),Met(5.35%),Tyr(4.02%),Lys(3.25%),His(2.58%),Val(2.40%),and Pro(2.10%).The amino acid decay rates of walnut protein before and after digestion were as follows:Lys(14.07%),Met(13.50%),Pro(6.46%),Glu(6.18%),Ile(5.90%),Ala(5.41%),Val(3.33%).LC-MS/MS showed that compared with oxidized walnut protein,unoxidized walnut protein has better digestibility.11 S globulin seed storage protein,legumin,vicilin,and 2S sulfur-rich seed storage were the main proteins that were detected to resist gastric and intestinal digestion in the digestive products of oxidized walnut samples and other important seed storage proteins. |
