Preparation of GAS protein hydrolysate
GAS protein hydrolysate was prepared using the method described by Adler-Nissen (1986) with slight modification. About 22 g of minced GAS flesh was mixed in 121 g of distilled water and minced in a blender. The mixture was then placed in a water bath. Temperatures were set to 45, 55 and 65 ̊C while the pH was adjusted 8, 9 and 10 (Table 1). Once the temperatures and the pH stabilized, alcalase at enzyme-to-substrate ratios (E/S) of 2, 3 and 4% were added. Hydrolysis processing times were set for 60, 120 and 180 min. pH were kept constant throughout the hydrolysis period by adding 4N NaOH. After each mixture completed its specified hydrolysis period they were inactivated at 90 ̊C for 15 minutes. The mixture was then centrifuged at 5000 rpm at 4 ̊C for 20 minutes. Respective supernatants were collected then freeze dried (Labconco, USA) and stored in freezer at -80 ̊C for further analysis.