Emulsion-enhanced synergy of walnut-derived hexapeptide (EPEVLR) and DHA in mitigating D-galactose-induced cognitive decline in mice

• The peptide EPEVLR and DHA (DHA@EP) can synergistically alleviate D-gal-induced cognitive dysfunction in mice through emulsification. • The interaction between EPEVLR and DHA has been confirmed through FTIR and DFT. • DHA@EP forms stable emulsion via hydrogen bonding between EPEVLR and DHA. • W/O emulsion enhances intestinal stability of EPEVLR during digestion. This study explored the synergistic effects of walnut-derived peptide (Glu-Pro-Glu-Val-Leu-Arg, EPEVLR) and docosahexaenoic acid (DHA) on ameliorating D-galactose (D-gal) induced cognitive dysfunction in mice. EPEVLR and DHA were integrated using ultrasonic mixing at a 2:1 molar ratio, resulting in a stable water-in-oil (W/O) emulsion labeled DHA@EP. DHA@EP was more effective than DHA or EPEVLR alone in alleviating D-gal-induced mice cognitive deficits. The underlying mechanisms included reducing oxidative stress, mitigating neuroinflammation, and stabilizing the cholinergic system. Structural characterization and theoretical calculations confirmed that EPEVLR and DHA formed a stable emulsion with droplet diameters below 10 μm, driven by hydrogen bonding between the nitrogen (N) and oxygen (O) atoms of EPEVLR and the hydrogen (H) atoms of DHA. In vitro digestion simulation experiments indicated that the W/O emulsion structure of DHA@EP significantly enhanced the intestinal stability of EPEVLR, likely due to the protective effect of the oil phase against enzymatic degradation in the gastrointestinal tract. These findings highlight the potential of DHA@EP as a functional food ingredient for cognitive improvement and provide insights into the synergistic use of bioactive peptides and fatty acids.