*2.5. Determination of Structural Characteristics of APPH-WPI*

#### 2.5.1. Particle Size Distributions

The particle size distribution of protein samples was determined by laser scattering particle size distribution analyzer (Partica, LA-960, Kyoto, Japan) with the relative refractive index and absorption set as 1.414 and 0.001, respectively. Briefly, the protein samples were suspended in distilled water to obtain a 5 mg/mL solution, and then the particle size distribution and the mean diameter of sample solutions were assessed and repeated at least three times, and then made into graphs [20].

#### 2.5.2. Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis

SDS-PAGE was performed under non-reducing and reducing conditions according to the previous reports [21,22]. Briefly, an amount of 15 μL of sample solution (1 mg/mL) was mixed with 5 μL of 4 × protein loading buffer, followed by heating in a 100 ◦C water bath for 5 min, and then immediately placed in an ice bath. Next, the sample solutions were centrifuged (10,000× *g*, 5 min) and a 10 μL portion of supernatant was taken for electrophoresis (Bio-rad, Powerpac Basic, Hercules, CA, USA) using 12% separating gel and 5% stacking gel, with standards of known molecular weight ranging from 10 to 200 kDa. The gels were decolored and photographed for further analysis. This experiment was repeated with three different batches of samples.

#### 2.5.3. Intrinsic Fluorescence

The protein sample was dispersed in PB (0.01 mol/L, pH 7.9) to prepare a solution at 2.5 μg/mL, followed by centrifugation for 10 min at 10,000× *g*, 4 ◦C. The fluorescence intensity of supernate was scanned three times (Thermo Fisher Scientific, Lumina, Waltham, MA, USA) from 290 to 500 nm with excitation at 283 nm [23].
