Introduction
 Olive Seeds is a major by-product of the olive oil industry, produced in millions of tons annually. It contains significant amounts of protein (approximately 13.5%) along with fiber, lipids, and phenolic compounds. However, native proteins have low solubility and complex structures that limit their functionality. Enzymatic hydrolysis is an eco-friendly method to release bioactive peptides with antioxidant, ACE-inhibitory, and cholesterol-lowering activities. The choice of enzyme is crucial; alcalase (a broad-specificity serine protease) and trypsin (a specific protease cleaving at lysine and arginine residues) produce different peptide profiles. Process variables such as hydrolysis time and enzyme-to-substrate (E/S) ratio significantly affect the degree of hydrolysis and antioxidant activity. Response Surface Methodology (RSM) is a powerful statistical tool for optimizing such multi-variable processes. Moreover, the stability of hydrolysates during storage, thermal processing, freeze-thaw cycles, and pH changes is essential for industrial applications. This study aimed to optimize the hydrolysis conditions (time and E/S ratio) for olive seeds protein using alcalase and trypsin via RSM, evaluate the antioxidant activities, and assess the stability of the optimal hydrolysates under different pH, heat, refrigeration, freeze-thaw, and ambient storage conditions.
Materials and Methods
Olive seeds were obtained from local market at Gorgan then dried, ground, defatted with hexane (AOAC method), and protein was isolated by alkaline extraction (pH 11) followed by isoelectric precipitation (pH 4). Protein isolate (5% w/v) was hydrolyzed in Tris-HCl buffer (pH 8 for alcalase, pH 7 for trypsin) at 50°C (alcalase) or 37°C (trypsin) with E/S ratios of 1–3% and 30–210 min according to a Central Composite Design (CCD) approach. The reaction was stopped by heating at 85°C for 30 min, then centrifuged and freeze-dried. Antioxidant activities were measured by different methods namely: DPPH radical scavenging, reducing power, and total antioxidant capacity (phosphomolybdenum method). Stability tests included: pH stability (pH 3–8, 30 min), thermal stability (95°C for 15–75 min), refrigeration storage stability (4°C for 15 days), freeze-thaw cycles stability (15 days), and ambient storage (25°C for 7 days). Data were analyzed by Design Expert, ANOVA followed by Duncan's multirange test (SPSS), and graphs prepared by Excel software.
Results and Discussion
RSM analysis showed that both time and E/S ratio had significant quadratic effects on DPPH, FRAP, and TAC for both enzymes. For alcalase, response surfaces exhibited a dome-shaped central peak with optimal conditions around 150 min and E/S 2%. For trypsin, a rising quadratic plateau was observed, with optimal conditions around 139 min and E/S 2%. These patterns confirm that excessive hydrolysis reduces antioxidant activity due to over-degradation of active peptides into free amino acids. Alcalase hydrolysates consistently showed higher antioxidant activities than trypsin hydrolysates, attributed to alcalase's preference for hydrolyzing at hydrophobic/aromatic amino acids (Phe, Tyr, Trp, Leu, Val) which are strong electron donors. Stability studies revealed, pH: Maximum TAC at pH 7–8, minimum at pH 3–4 (near isoelectric point). Alcalase hydrolysate was more stable. Heat (95°C): Gradual time-dependent decrease in both FRAP and TAC; alcalase hydrolysate retained higher activity after 75 min. Refrigeration (4°C, 15 days): Progressive decline in TAC and FRAP; alcalase hydrolysate showed slower decay. Freeze-thaw (15 days): Significant reduction after each cycle, mainly due to ice crystal damage and cryoconcentration; alcalase hydrolysate more resistant. Ambient (25°C, 7 days): Faster decline than refrigeration; alcalase hydrolysate.
Conclusion
The optimal hydrolysis conditions for olive seeds protein were determined using RSM. Alcalase-produced hydrolysate exhibited superior antioxidant activity and stability compared to trypsin-hydrolysate due to the release of hydrophobic/aromatic-rich peptides. Stability decreased in all tested conditions (heat, freeze-thaw, storage), but alcalase hydrolysateconsistently showed higher retention. These findings support the valorization of olive seeds protein as a source of stable antioxidant peptides for food and pharmaceutical applications.