Effect of Oxidative Modification by Peroxyl Radical on the Characterization and Identification of Oxidative Aggregates and In Vitro Digestion Products of Walnut (Juglans regia L.) Protein Isolates

Abstract

Walnut protein is a key plant protein resource due to its high nutritional value, but walnuts are prone to oxidation during storage and processing. This article explored the oxidative modification and digestion mechanism of walnut protein isolates by peroxyl radical and obtained new findings. SDS-PAGE and spectral analysis were used to identify structural changes in the protein after oxidative modification, and LC-MS/MS was used to identify the digestion products. The findings demonstrated that as the AAPH concentration increased, protein carbonyl content increased from 2.36 to 5.12 nmol/mg, while free sulfhydryl content, free amino content, and surface hydrophobicity decreased from 4.30 nmol/mg, 1.47 μmol/mg, and 167.92 to 1.72 nmol/mg, 1.13 μmol/mg, and 40.93 nmol/mg, respectively. Furthermore, the result of Tricine-SDS-PAGE in vitro digestion revealed that protein oxidation could cause gastric digestion resistance and a tendency for intestinal digestion promotion. Carbonyl content increased dramatically during the early stages of gastric digestion and again after 90 min of intestine digestion, and LC-MS/MS identified the last digestive products of the stomach and intestine as essential seed storage proteins. Oxidation causes walnut proteins to form aggregates, which are then re-oxidized during digestion, and proper oxidative modification may benefit intestinal digestion.

Protein oxidation‐induced changes in the physicochemical properties and quality of plant food

“…Cys can be oxidised to cystine (protein cross‐linking agent), sulphate acid, sulphite acid and sulphonic acid. It was found that the decay rate of amino acids in oxidised walnut protein from high to low was Cys, Met, Tyr, Lys, His, Val and Pro (Zhao et al ., 2022). Moreover, Cys‐S‐S linkage produced by the reaction of cysteine with free sulphydryl groups may cause soluble protein aggregation.…”

“…Studies have shown that when the time of oxidation of soybean protein by 2,2′‐azobis (2‐amidinopropane) dihydrochloride (AAPH) increased from 0 to 4 h, more internal hydrophobic groups were exposed, but with the further strengthening of oxidation, the oxidative attack changed the interaction and cross‐linking reaction of soybean protein and promoted the conversion of free sulphydryl groups into disulphide bonds, which induced the formation of polymer flexible oxidised aggregates with larger particle size (Guo et al ., 2022). Similarly, the oxidation results of walnut protein showed that tryptophan and free sulphydryl group decreased with increasing concentrations of oxygens, producing a large number of soluble aggregates, which may be due to the Cys‐s‐s linkage formed by the reversible reaction of free sulphydryl groups with cysteine (Zhao et al ., 2022).…”

“…Also, with the increase of oxidation degree, the FC and FS of walnut protein increased first and then decreased, which may be due to the interaction between hydrophobic residues caused by the expansion of peptide chain, resulting in lower surface tension and faster protein diffusion to the air–water interface, thereby promoting the formation of bubbles. However, excessive oxidation made the structures of walnut protein and rice protein more unstable and disrupts the interaction at the interface, thereby reducing FC and FS (Wu et al ., 2020a; Zhao et al ., 2022).…”

Effect of Alkyl Peroxyl Radical Oxidation on the Oxidative Stability of Walnut Protein Emulsions and Their Adsorbed Proteins

“…Finally, the walnut isolate protein were placed in the freeze dryer at 4 • C for freezing. The protein content was measured as 86.53% by Kjeldahl (SKD-3000, Xi'an Yupu Instrument Co., Xi'an, China) [21].…”