*2.3. Oxidation of WPI*

The oxidation of WPI was performed according to Duan et al. [15] with some modifications. Briefly, dried WPI was dispersed in phosphate buffer (PB, 0.01 mol/L, pH 7.4) to prepare 25 mg/mL protein suspension, then mixed with various concentrations of AAPH (0, 0.04, 0.20, 1.00, 3.00, 5.00 and 10.00 mmol/L), followed by incubation for 24 h at 37 ◦C under light protection. The reaction solutions were centrifuged for 30 min at 1000× *g* and 4 ◦C. The supernatants were dialyzed (molecular weight cut-off 7 kDa) against distilled water for 72 h at 4 ◦C. Next, the dialysates were freeze-dried at −80 ◦C for 24 h to obtain AAPH oxidized WPI (APPH-WPI) and were stored frozen at −80 ◦C for spare use.

#### *2.4. Determination of Oxidation Degree of AAPH-Oxidized WPI (APPH-WPI)*

#### 2.4.1. Determination of Carbonyl Content

The carbonyl contents of WPI and APPH-WPI were measured by 2,4 dinitrophenylhydrazine (DNPH) assay. Briefly, 1.5 mL protein dispersion (10 mg/mL) was mixed with 1 mL DNPH solution (10 mmol/L, containing 2 mol/L HCl), after incubation in the dark for 1 h at room temperature, then 1 mL of trichloroacetic acid (TCA, 20%) was added and it was mixed well, followed by centrifugation at 8000× *g* for 15 min. The precipitate was washed with 1 mL of ethyl acetate-ethanol (1:1, *v/v*) three times, and then dissolved in 4 mL urea (6 mol/L) for 20 min at 37 ◦C. After centrifugation at 8000× *g* for 15 min, absorbance of the supernate was read at 370 nm by a microplate reader (Biotek, Winooski, VT, USA), and the carbonyl content of protein was expressed as nmol/mg protein using the molar extinction coefficient of 22,000 L·mol−1·cm−<sup>1</sup> [16]. Measurement was repeated at least three times to calculate the carbonyl content of the samples.

#### 2.4.2. Determination of Sulfhydryl Group Content

The sulfhydryl group content of WPI and APPH-WPI was measured in triplicate and repeated three times by using the DTNB assay. A 4 mg/mL DTNB solution was first prepared in 0.086 M Tris-Gly buffer (containing 5 mM EDTA, pH 8.0), followed by a suspension of protein samples in Tris-Gly buffer (containing 2% (*w/w*) sodium dodecyl sulfate (SDS)) to obtain a concentration of 10 mg/mL. Then, 1 mL of the sample solution was reacted with 10 μL of DTNB solution for 1 h at room temperature, followed by centrifugation at 10,000× *g* for 10 min, the absorbance of supernate was then measured at 412 nm against the blank (without DTNB and sample). The free sulfhydryl group contents of protein samples were calculated using the extinction coefficient of 13,600 L·mol−1·cm−1, while the total sulfhydryl content was measured as a similar procedure, except that 8 M urea was added to the sample buffer [17,18].

#### 2.4.3. Determination of Free Amino Groups Content

The o-phthaldialdehyde (OPA) method was used to measure the free amino groups content of proteins samples. OPA was first dissolved in methanol solution (0.4 g/mL), followed by addition of 2.5 mL of SDS solution (200 g/L), 25 mL of boric acid solution (0.1 mol/L), and 100 μL of β-mercaptoethanol. The mixture was diluted to a certain volume with distilled water to obtain the OPA working solution for the subsequent analysis. Next, an amount of 200 μL of protein solution was mixed with 4 mL of OPA working solution, and then water bathed at 35 ◦C for 2 min. The absorbance at 340 nm was measured against distilled water as control. This trial was repeated three times to calculate the free amino group content [19].
