3.1.1. Carbonyl Groups

The process of peroxy radical oxidation of protein is complex and accompanied by corresponding products generation, among which carbonyl group is the most widely used indicator for preliminary evaluation of oxidation degree [10]. The effect of peroxy radical on carbonyl content of WPI is shown in Table 1. Carbonyl content of oxidized WPI was significantly (*p* < 0.05) increased with the concentration of AAPH, and a maximum (1.48 nmol/mg) at 10 mmol/L AAPH was observed. As reported by previous studies [26], peroxy radicals were generated linearly with the decomposition of AAPH, in turn attacking the main peptide chain or side chain of WPI, and thus carbonyl derivatives such as aldehydes and ketones were formed accordingly. These results were similar to that of AAPH oxidized casein [27] and ovalbumin [17].

**Table 1.** Carbonyl, free sulfhydryl, total sulfhydryl, and free amino groups of WPI oxidized with different concentration of AAPH.


WPI = walnut protein isolates, AAPH = 2, 2- -azobis (2-amidinopropane) dihydrochloride. Different letters within a column indicate significant differences (*p* < 0.05).

#### 3.1.2. Sulfhydryl Groups

Cysteine residues are sensitive to oxidation, and are susceptible to corresponding modification, therefore, the degree of protein oxidation can be reflected by the content of sulfhydryl groups. As shown in Table 1, with the increase of AAPH concentration, the total and free sulfhydryl groups of WPI decreased significantly (*p* < 0.05), and the minimums were achieved (1.25 and 114.26 nmol/mg) respectively when AAPH concentration was 10 mmol/L. This result is similar to that of peroxyl radical oxidized chickpea protein reported by Zhu et al. [23], the sulfhydryl groups were oxidized to both disulfide bonds and irreversible sulfonic and sulfonic acid groups by radicals [28]; moreover, other covalent cross-linking might have occurred during oxidation [18].
