2.4. Angiotensin-I Converting Enzyme (ACE)-Inhibitory Activity and Inhibition Kinetics

The ACE-inhibitory activity was evaluated using HHL as a substrate by monitoring the released hippuric acid using a spectrophotometric method [22]. Briefly, the reaction system comprised 50 µL of ACE (25 mU), 150 µL of 8.3 mM HHL, and 50 µL of sample solutions. After incubation in a shaking water bath (37 °C) for 1 h, 1 M HCl (250 µL) was added to stop the reaction. The hippuric acid formed was subsequently extracted by 1.4 mL ethyl acetate. After centrifugation, 1 mL of the supernatant was collected and evaporated at 80 °C for 60 min in a vacuum. The residue was then dissolved in 2 mL of distilled water and the absorbance at 228 nm was read. The inhibitory activity was calculated as follows:

where A_C is the absorbance of the ACE solution without an inhibitor and A_S is the absorbance of the mixture-contained samples. The IC₅₀ value, the inhibitor concentration required to inhibit half the ACE activity, was acquired by nonlinear regression from a plot of percentage ACE inhibition versus different inhibitor concentrations.

Lineweaver–Burk plots of 1/V versus 1/HHL in the presence of the inhibitor were used to analyze the ACE inhibition kinetics of the peptides identified in QBAH. The ACE enzyme activity was measured with various HHL concentrations (0.76, 1.52, 3.04, 3.80 and 7.60 mM) and peptide concentrations (0, 0.1, 0.2 and 0.5 mg/mL).