4.3. Preparation of Enzymatic Hydrolysates from Velvet Antler

Enzymatic hydrolysis was performed using five commercial proteases (trypsin, pepsin, α-chymotrypsin, Neutrase, and Alcalase) at their optimal conditions (pH and temperature) as described previously [24]. Briefly, one gram of dried velvet antler powder was added into 100 mL of distilled water. Each enzyme was then added to have a substrate to enzyme ratio of 100:1. Enzymatic hydrolysis was conducted under optimal conditions for 24 h, after which the hydrolysate was boiled at 100 °C for 10 min to inactivate the enzyme. These hydrolysates were clarified by centrifugation at 3000× g for 20 min to remove any unhydrolyzed residue. The supernatant of each hydrolysate was filtered, adjusted to pH 7.0, and stored for subsequent use in experiments.