Alcalase–Flavourzyme Red Seaweed Hydrolysates as Antioxidants to Enhance Oxidative Stability of DHA Nanoemulsions

This study evaluated Palmaria palmata hydrolysates produced using Alcalase® and Flavourzyme® at 1, 2, 3, 4, 5, and 10% (w/w biomass protein, corresponding to AF1-10), and their performance in oil-in-water nanoemulsions under iron-induced oxidation. AF4 showed significantly higher total amino acid and phenolic contents and the strongest Fe2+-chelating activity (IC50 = 1.64 ± 0.22 mg/mL, p < 0.05) and was therefore selected for nanoemulsion stabilization. Nanoemulsions exhibited high physical stability with no significant changes in droplet sizes (D3,2 ~77–79 nm), ζ-potential (~−18 to −19 mV), and viscosity (~1.2–1.5 cP) (p > 0.05). Dynamic interfacial tension measurements and confocal laser scanning microscopy (CLSM) indicated limited interfacial activity of AF4, with most components remaining in aqueous phase. Compared to the control, AF4 significantly reduced peroxide formation (~100–164 vs. 289–357 meq/kg at Day 4–8, p < 0.05) and partially preserved tocopherols. It also delayed the formation of some volatiles during intermediate stages of storage. However, it was less effective than ethylenediaminetetraacetic acid (EDTA). Increasing the AF4 concentration did not further improve oxidative stability. These findings suggest that antioxidant efficacy depends on composition, interfacial behavior, and spatial distribution. Antioxidants with limited interfacial activity may therefore exhibit different modes of action within emulsified systems.