WPI solution

WPI (Bipro, Davisco, lot # JE 198 ‐1‐420, MN, USA) powder (containing 97.5 wt% proteins) was first dissolved in an HCl solution (Merck, Germany) of pH 2 with stirring at 4 °C overnight. The concentration of the WPI in the solution is about 15 wt%. The concentration of WPI used in this study is the concentration of the WPI powder used for sample preparation. After dissolution, the pH of the WPI solution was adjusted to pH 2 with a 6 M HCl solution. We added the HCl drop by drop and made sure each drop of HCl is sufficiently mixed by stirring with a magnetic stirrer and the pH did not change further before we added the next drop. Subsequently, the solution was filtered using a syringe filter (Hydrophilic PES 0.45 µm, Millipore Millex‐HP, Germany) to remove undissolved protein. UV spectrophotometer (Cary 50 Bio, Varian, USA) and a calibration curve of known WPI concentration at a wavelength of 278 nm was used to determine the protein concentration in the solution (Kroes‐Nijboer et al., 2012). We prepared a concentration series of WPI solutions from a stock WPI solution with known concentration and measured the absorbance of each concentration, the calibration curve was constructed from the results. The WPI concentration of the final stock solution was determined by diluting to concentrations low enough to allow for UV absorbance measurement. The WPI stock solution was left at 4 °C before further sample preparation.