Enhancement of the In Vitro Bioactivities of Limpet (Patella vulgata) Proteins: Effect of Pre-Enzymatic Hydrolysis Followed by Simulated Gastrointestinal Digestion

Bioactive hydrolysates were generated from limpet (Patella vulgata) protein concentrate (LPC) utilising Alcalase™ and Flavourzyme™ followed by simulated gastrointestinal digestion (SGID). Samples were analysed using gel electrophoresis (SDS-PAGE) and gel permeation chromatography (GP-HPLC), while in vitro antioxidant (FRAP, ORAC and ABTS^•+) and angiotensin-converting enzyme (ACE) inhibitory activities were employed to evaluate bioactivity. Alcalase was more effective than Flavourzyme in hydrolysing LPC, achieving a significantly higher (p < 0.05) amino nitrogen content after digestion, i.e., 29.50 ± 0.69 vs. 21.06 ± 1.04 mg g⁻¹ protein, respectively. The hydrolysates exhibited enhancements in some biological activities following SGID, with significant (p < 0.05) increases in antioxidant (except for the ORAC and FRAP activities in the Alcalase hydrolysates) and ACE inhibitory activities. The antioxidant activity of Alcalase-generated hydrolysates declined significantly during gastric digestion but was subsequently enhanced in the intestinal digestion phase. In contrast, the Flavourzyme-generated hydrolysates exhibited a progressive increase in antioxidant activity throughout SGID, with the highest levels observed following pepsin and Pancreatin™ incubation. In comparison with digestion where only SGID was conducted, the inclusion of a pre-enzymatic hydrolysis step with Alcalase prior to SGID led to significant improvements in both antioxidant (as measured by the ABTS assay, 360.92 ± 18.32 μmol Trolox Equivalents g⁻¹ protein) and ACE inhibitory activity (81.5% inhibition, at a final sample concentration of 0.2 mg mL⁻¹). These findings highlight the potential health benefits of protein hydrolysates from limpet and their applicability in the food and health industries, underscoring the value of marine-derived proteins in developing bioactive compounds.