2.3.1 Solubility
Protein solubility was studied as a function of pH according to Can Karaca et al. (2011) and Chang et al. (2015) with minor modifications. In brief, 20 mL of 0.25% (w/w) protein (corrected by protein content) solution at the required pH (3, 5, and 7) was prepared and allowed to stir for 1 h at room temperature (21-23℃). The protein solution was then transferred to a 15-mL centrifuge tube and centrifuged (VWR clinical centrifuge 200, VWR International, Mississauga, ON, Canada) at 4,180 × g for 10 min at room temperature. The supernatant was carefully collected, followed by the Bradford method to determine the protein content. In detail, a 50 µL of supernatant was added to a 1.5-mL centrifuge tube and 1.5 mL of Coomassie dye (Thermo Fisher Scientific, Madison, WI, USA) was added. The mixture was vortexed for 10 s vigorously and then left to stand for 5 min. The absorbance of the sample was measured at 595 nm using a UV-visible spectrophotometer (Genesys 20, Thermo Scientific, Madison, WI, USA), and Milli-Q water was used as a blank while a mixture of 50 µL of 0.5 N NaOH and 1.5 mL of Coomassie dye was used as the sample blank. Different concentrations of bovine serum albumin (BSA) were used to create a standard curve. The total protein content was determined by dissolving 20 mg of dry protein extract in 20 mL of 0.5 N NaOH solution and allowing the solution to stir for 1 h at room temperature, followed by centrifugation at 12,100 ×g for 30 min, and the total protein content of the supernatant was determined using the Bradford method. The protein solubility (%) was determined by dividing the protein content in the solutions at different pH levels by the total protein in the sample (N% × 6.25), multiplied by 100%. All measurements were reported as the mean ± one standard deviation (n=3).