**1. Introduction**

Walnut (*Juglans regia* L.) is one of the common tree nuts widely grown worldwide. The planted area and production of walnuts in China ranks first in the world [1]. It has been reported that walnut kernels are rich in unsaturated oils, protein, vitamins, minerals and some bioactive compounds which confer antioxidant, cholesterol reduction, arterial improving, immunomodulatory and other biological activities [2,3]. Therefore, walnut kernels are widely consumed in China (409,608 metric tons in 2019) and even worldwide (914,383 metric tons in 2019) for their health promotion and delicious taste [4].

In view of the high-cholesterol and saturated fatty acids content in animal-derived foods, plant proteins are gaining increasing interest for their specific advantages. Soy protein, peanut protein and walnut protein, etc. have been studied for their nutritional, processing and physiological properties. Among them, as one of the main by-products

**Citation:** Zhang, X.; Yang, X.; Li, Y.; Wang, Z.; He, X.; Sun, J. Effect of Peroxyl Radical-Induced Oxidation on Functional and Structural Characteristics of Walnut Protein Isolates Revealed by High-Resolution Mass Spectrometry. *Foods* **2022**, *11*, 385. https://doi.org/10.3390/ foods11030385

Academic Editor: David Bongiorno

Received: 11 December 2021 Accepted: 24 January 2022 Published: 28 January 2022

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of the oil industry, walnut protein has great potential of high value utilization. Currently, there have been gradually increasing studies on walnut protein due to its large amount of consumption, such as classification and characterization [5], characteristics analysis and functional properties modification [3,6–9].

Oxidation is one of the three major chemical reactions. In contrast to other forms of modification, oxidation can occur during the harvesting, storage and processing of foods [10]. It has been reported that radical-induced oxidation could change the properties, conformation, digestibility and allergenicity of food proteins [11–14]. As we know, walnut kernels contain high amounts of unsaturated fatty acids, which could generate radical intermediates and secondary products through oxidative stress, and then induce the oxidized walnut protein, in turn, to undergo corresponding changes.

Peroxy radical (ROO·) is an important radical during the lipid oxidation process. However, the effect of peroxy radical on the properties and structure of walnut protein remains unclear. Thus, in this study, walnut protein isolates (WPI) were prepared and oxidized by 2,2- -azobis (2-amidinopropane) dihydrochloride (AAPH), a water-soluble azofree radical initiator, and then, the effect of peroxy radical on the main groups, structure, functional properties, and antigenicity of WPI were investigated. Meanwhile, the oxidation site and modifications of WPI were analyzed by proteomics technology. This study hopes to provide advice on quality control and utilization of walnut protein.
