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).