Isolation and characterization of angiotensin I-converting enzyme (ACE) inhibitory peptides from Ulva rigida C. Agardh protein hydrolysate

Ulva rigida protein was hydrolysed with pepsin plus bromelain after a screening of nine enzymes for optimal proteolysis. This hydrolysate, presenting ACE-inhibitory activity with an IC50 value of 0.483 mg/mL, was fractionated by ultrafiltration membranes into three molecular weight ranges (<1 kDa, 1-3 kDa and >3 kDa). The <1 kDa fraction that exhibited the highest activity (IC50: 0.095 mg/mL) was purified using size-exclusion chromatography and reversed-phase high-performance liquid chromatography, yielding two active ACE-inhibitory purified peptides. Edman degradation revealed its amino acid sequences to be IP and AFL with IC50 values of 0.020 and 0.023 mg/mL, respectively. Both peptides were synthesized to confirm the structure and to validate their ACE-inhibitory activities. Lineweaver-Burk plots suggest that IP acts as a non-competitive and AFL as a competitive ACE-inhibitors. Stability assays showed that both peptides are heat-stable and AFL is hydrolysed by intestinal mucosa peptidases to FL with IC50 value of 0.004 mg/mL that acts as a non-competitive ACE-inhibitor. The results suggest that these peptides might have a potential use in the preparation of antihypertensive drugs or functional foods.