Peanut Protein Hydrolysates from Oil Cake: Enhanced Antioxidant Activity, Techno-Functional Properties, and Safety Evaluation.

The valorization of agro-industrial by-products into bio- and techno-functional ingredients is a key strategy for sustainable food systems, yet peanut oil cake (POC), a protein-rich residue of peanut oil extraction, remains underexploited. Its protein can be recovered as an isolate (PPI) and converted through enzymatic hydrolysis into antioxidant peanut protein hydrolysates (PPH). This study therefore aimed to optimize the enzymatic preparation of PPH from PPI using Alcalase® 2.4 L to maximize antioxidant activity (AA), and to carry out a comprehensive screening of their polypeptide profile, techno-functional properties, immunoreactivity, and cytotoxic/cytoprotective effects - an integrated assessment not previously reported for POC-derived hydrolysates. Hydrolysis was optimized by response surface methodology; the optimal conditions (49.2 °C, 197 min, enzyme-to-substrate ratio 0.024) yielded a degree of hydrolysis of 19.95 and 44.72% ABTS•+ scavenging activity. Relative to PPI, PPH exhibited significantly higher AA (ABTS•+ scavenging, 2.3-fold; •OH scavenging, 1.6-fold; and metal-chelating activity, 3.6-fold lower EC₅₀), together with increased solubility (98.8%) and oil-binding capacity, but lower foaming and emulsifying capacities. Immunoreactivity, measured with a peanut-specific polyclonal serum, was reduced ~ 42-fold (to 1.17% of detected protein), and PPH showed no cytotoxicity up to 1000 µg/mL while protecting intestinal cell models, normal rat ileum epithelial cells (IEC-18) and human colorectal adenocarcinoma cells (Caco-2), against menadione-induced oxidative stress. Overall, POC-derived PPH are safe, multifunctional antioxidant ingredients with potential for functional foods and beverages, adding value to an agro-industrial by-product within a sustainable framework.