For the alkaline extraction, one part of meal was mixed with 10
parts of distilled H2O with pH adjusted to 8 using 1.0 M NaOH and
stirred for 1 h. pH was checked and adjusted if necessary every
15 min. The slurry was centrifuged (4500g, 4 C, 20 min) and filtered
through 4 layers of cheesecloth, followed by adjusting the
pH of the supernatant to 4 using 0.1 M HCl. pH 4 was selected
rather than 5 or 6 based on immediate protein precipitation and
optimum yield of protein precipitate. After a second centrifugation
(4500g, 4 C, 20 min), protein precipitate was collected and kept at
–30 C before being freeze dried.