2.2.2 Salt extraction-dialysis (SE)
SE described by Klassen et
al. (2011) and Chang et al. (2015) was employed with minor
modification to obtain protein products from fermented CP and HE canola
meals. In brief, a 0.05 M Tris–HCl buffer solution (pH 7.0) containing
0.1 M NaCl was mixed with fermented canola meal at a 1:10 (w/v) ratio
and stirred at 500 rpm for 2 h at room temperature (21-23℃). The
supernatant was then collected by centrifuging (Sorvall RC Plus
Superspeed Centrifuge, Thermo Fisher Scientific, Asheville NC, USA) at
6,000 × g for 30 min, followed by vacuum filtration with No.1
Whatman filter paper (Whatman International Ltd., Maidstone, UK). The
filtrate was dialyzed to remove NaCl and other small molecules using a
Spectra/Por molecular porous membrane tubing (6–8 kDa cut off, Spectrum
Medical Industries, Inc., USA) at 4℃ for 72 h against distilled water
(distilled water was refreshed twice a day). The dialyzed protein
extract was then centrifuged at 3,000 × g for 30 min at 4℃. The
supernatant was discarded, and the protein pellet was collected and
freeze dried. The protein content was determined using the same protocol
for AE-IP samples.