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.