2.2.1 Alkaline extraction-isoelectric precipitation
The fermented (72 h) CP and HE
meals (100 g) were extracted at a 1:10 (w/v, meal:water) ratio at pH 11
(adjusted using 2 N NaOH) for a 1-h period at room temperature (21-23℃)
according to Aider and Barbana (2011). The suspension was then
centrifuged (Sorvall RC Plus Superspeed Centrifuge, Thermo Fisher
Scientific, Asheville NC, USA) (4,500 × g at 4℃ for 15 min), and
the supernatant was vacuum filtered with No. 1 Whatman filter paper
(Whatman International Ltd., Maidstone, UK) to remove any floating
particles. The supernatant was then adjusted to pH 5 using 2 N HCl to
precipitate the proteins. The protein precipitate was collected as a
pellet after centrifugation at 4500 × g at 4℃ for 15 min and
freeze dried. The crude protein content of meals and protein products
was determined using the micro-Kjeldahl method (AOAC 960.52) with a
conversion factor of 6.25 (AOAC, 2005).