3.1.3. Free Amino Groups

As depicted in Table 1, the free amino groups of oxidized WPI decreased with the AAPH concentration, and the minimum (0.31 nmol/mg) was discerned at a concentration of 10 mmol/L, which could be due to the oxidation of amino-containing side chain (threonine, proline, arginine and lysine residues) in WPI [29], and the formation of Schiff base with carbonyl groups by a covalent reaction [19]. This finding echoed that of the carbonyl result mentioned above and a similar result was obtained with oxidized *Coregonus peled* myofibrillar proteins [29].

#### *3.2. Structural Characteristics Analysis*

#### 3.2.1. Endogenous Fluorescence

The endogenous fluorescence of proteins originates from the emission of tryptophan residues, and the polarity of the micro-environment in which can be reflected by the changes of fluorescence peak. Therefore, endogenous fluorescence was often used to indicate the transformation of protein conformation. The effects of peroxy radical on the endogenous fluorescence intensity and maximum fluorescence emission wavelength of WPI are shown in Figure 1a. With increasing AAPH concentration, the fluorescence intensity of WPI showed a downward trend. This might be attributed to the unfolding of the WPI tertiary structure, and the tryptophan residue is oxidized to kynurenine, thus inducing a reduction in the endogenous fluorescence intensity. In addition, a blue shift of maximum fluorescence peak due to oxidation was also observed in Figure 1a, which is similar to the report by Zhang et al. [18]; hence, it was concluded that the tryptophan residue of WPI was exposed to hydrophobic environment which in turn made hydrophobic interactions by oxidation.

**Figure 1.** Endogenous fluorescence spectra (**a**) and surface hydrophobicity (**b**) of WPI oxidized with different concentration of AAPH. WPI = walnut protein isolates, AAPH = 2, 2- -azobis (2-amidinopropane) dihydrochloride. Different letters indicate significant differences between columns (*p* < 0.05).

#### 3.2.2. Surface Hydrophobicity

Surface hydrophobicity reflects the distribution of hydrophobic amino acid residues on the protein's surface, and characterizes the changes of tertiary structure of protein [30]. The surface hydrophobicity of oxidized WPI increased significantly (*p* < 0.05) with the AAPH concentration, the maximum achieved at 10 mmol/L AAPH, which is 84% greater than the control (Figure 1b), indicating that the peptide chains were moderately unfolded and rearranged [16], and the surface hydrophobic groups were exposed to the polar environment. This result corresponded to those of endogenous fluorescence; meanwhile, oxidized myofibrillar proteins from *Culter alburnus* also showed similar trends [18].

#### 3.2.3. Particle Size Distributions

Particle size is the common indicator to evaluate protein aggregation [31], and can be monitored by dynamic light scattering. Particle size distributions and average particle size of WPI oxidized with different concentrations of AAPH are shown in Figure 2. The particle size of control WPI was mainly distributed between 1–10 μm, while a large peak at 100 μm was observed after oxidation, illustrating formation of large aggregates through the interaction of protein peptides by oxidation [32]. Moreover, there is a rising trend on average particle size with higher AAPH concentration, with maximum values (119.2 μm) at 10 mmol/L AAPH, which is similar to the result of Fu et al. [16]. Overall, the above results suggested that the hydrophobic groups exposure (Figure 1b), as well as the disulfide and secondary bonds cross-linking (Table 1) might be responsible for the formation of larger protein aggregates.

**Figure 2.** Particle size distributions (**a**) and average diameter (**b**) of WPI oxidized with different concentrations of AAPH. WPI = walnut protein isolates, AAPH = 2, 2- -azobis (2-amidinopropane) dihydrochloride. Different letters indicate significant differences between columns (*p* < 0.05).

#### 3.2.4. SDS-PAGE

SDS-PAGE has the advantages of being fast, of high resolution and high sensitivity, and is especially suitable for the analysis and identification of oligomers and their subunits, as well as molecular weight determination of protein. As shown in the non-reducing SDS-PAGE pattern (Figure 3b), the intensity of 7 S conglycinin bands decreased with increasing AAPH concentration; meanwhile, new large molecule weight bands were observed at the top of the gel, indicating that large polymers may be formed by stronger oxidation [19]. Moreover, most of the degraded bands caused by oxidation were recovered in the reducing SDS-PAGE pattern (Figure 3a), and thus we reasoned that disulfide bond polymerization is the predominant manner for aggregates formation, which is similar to the study of Fu et al. [16].

#### 3.2.5. Fourier Transform Infrared (FT-IR) Spectroscopy

The secondary structure of WPI could be reflected by the FT-IR spectrum, specifically, the amide 1 region was processed in de-convolution and second derivative, and the featured wavenumber of α-helix is 1648–1655 cm−1, β-sheet is 1610–1629 cm−1, β-turn is 1660–1681 cm<sup>−</sup>1, and random coil is 1638 cm−<sup>1</sup> [24]. As shown in Figure 4, the percentage of α-helix decreased whereas that of β-sheet and random coil increased with the AAPH concentration. Studies have shown that α-helix is associated with the weak hydrogenbond interaction between amino groups and amidogen, whereas β-sheet is related to the hydrogen-bond interaction between peptide chains [33]. Thus we deemed that the hydrogen-bond interaction between amino bond of WPI was broken by oxidation, and the unfolding peptide chains further re-associated to form a new construction (β-sheet and random coil), which in turn decreased the flexibility of the protein structure [28]. This finding is in accordance with the oxidized chickpea protein isolates reported by Zhu et al. [23].

**Figure 4.** Secondary structure of WPI oxidized with different concentrations of AAPH. WPI = walnut protein isolates, AAPH = 2, 2- -azobis (2-amidinopropane) dihydrochloride.
