2.5.4. Surface Hydrophobicity (H0)

The surface hydrophobicity (H0) of WPI was determined by the 1-anilino-8 naphthalenesulfonic acid (ANS) fluorescence probe method [24]. Each protein sample was diluted to 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mg/mL in PB (10 mM), respectively, then a 2 mL portion of the aforementioned solution was mixed well with 20 μL ANS solution (in 10 mM PB) and allowed to stand for 3 min at room temperature. The fluorescence intensity was measured at 390 nm excitation wavelength and 470 nm emission wavelength. Next, a linearity curve was drawn with the protein concentration as the abscissa and fluorescence intensity as the vertical coordinate. The slope of the curve was calculated as surface hydrophobicity of protein sample. This experiment was repeated with three different batches of samples.

#### 2.5.5. Fourier Transformed Infrared Spectroscopy (FT-IR)

Protein powder samples were mixed with potassium bromide and pressed into a thin tablet, followed by full-band scanning from 4000 to 400 cm−<sup>1</sup> using a Fourier transform infrared spectrometer (Thermo Fisher Scientific, Nicolet IS50, Waltham, MA, USA), and scans were averaged three times for each spectrum. The data were analyzed and fitted with Peak fit 4.12 and OMNIC 8.2 software to obtain the proportions of the secondary structure (α-helix, β-sheet, β-turn, and random coil), respectively [15].
