*2.5. Determination of Protein Carbonyl Content*

The carbonyl content of the OVM samples was determined according to the method of Zhang et al. [18] with slight modifications. The measurements were performed by monitoring the reaction between 2,4-dinitrophenylhydrazine (DNPH) and the carbonyl group of the protein. An amount of 0.2 mL of OVM (4 mg/mL) was mixed with 0.4 mL of DNPH (10 mmol/L, in 2 mol/L HCl) and the mixture was incubated at 37 ◦C in the dark for 1 h. Followed by 0.5 mL of 20% trichloroacetic acid was added and the solution was incubated for 5 min. The sample was then centrifuged at 12,000 rpm and 4 ◦C for 15 min. The supernatant was removed, and the precipitate was retained. The sediment was washed using 1 mL of ethyl acetate/ethanol (1:1) mixture and then centrifuged with the method described above three times. The obtained protein was incubated with 1 mL of guanidine hydrochloride solution (6 mol/L), and the solution was centrifuged to remove precipitation. Absorption at 370 nm was determined using a U-2910 ultraviolet-visible spectrophotometer. The blank was applied with the same method except using HCl (2 mol/L) instead of DNPH. The carbonyl content was calculated with a molar extinction coefficient of 22,000 mol−1·cm<sup>−</sup>1, and the result was expressed as nmol/mg protein.

#### *2.6. Determination of Surface Hydrophobicity*

The surface hydrophobicity of the OVM samples was determined by an 8-aniline-1 naphthalene sulfonic acid (ANS) fluorescence probe method [19]. The samples were diluted to 1, 0.5 and 0.25 mg/mL, and the fluorescence intensities were determined by mixing 4 mL of each sample with 20 μL of ANS solution (8 mmol/L). The measurement conditions were as follows: excitation wavelength of 390 nm; scanning emission wavelength range of 400–600 nm; slit width of 5 nm. A linear regression equation was prepared according to the fluorescence intensities of the samples at each concentration for curve fitting. The obtained slope was the surface hydrophobicity of the samples.
