Barbel muscle (500 g), in 500 ml distilled water, was first
minced using a grinder (Moulinex Charlotte HV3, France) then
cooked at 90 C for 5 min to inactivate endogenous enzymes The
cooked muscle sample was then homogenised in a Moulinex
blender for about 2 min. The sample was adjusted to optimal pH
and temperature for Alcalase (pH 8.0; 50 C). The hydrolysis reaction
was started by the addition of the enzyme at a 1:1 (U/mg)
enzyme/protein ratio. The reaction was conducted at 50 C and pH
8.0 for 2 h. During the reaction, the pH of the mixture was maintained at the desired value by continuous addition of 4 N
NaOH. After the required digestion time, the enzymatic hydrolysis
was stopped by addition of disodium tetraborate (0.32 M, pH 12.7)
up to a final pH of 12. Protein hydrolysates were then centrifuged at
5000 g for 20 min to separate soluble and insoluble fractions.
Finally, the soluble fractions, referred to as protein hydrolysates,
were freeze-dried at 50 C and 121 mbar (Modulyod-230,
Thermo-Fisher Scientific, Waltham, MA) and then stored at 20 C
for further use.