Functional characterization and structural analysis of walnut protein-whey protein complexes prepared via pH cycling

Walnut protein isolate (WP) exhibits limited solubility and poor emulsifying ability, restricting its application in the food industry. To improve its functional characteristics, this study prepared a dual-protein complex by combining WP with whey protein isolate (WPI) using a pH-cycling method, and investigated the impact of different WP-to-WPI mass ratios on the complex's functional properties. The results showed that at a mass ratio of 1:1.5 (WP:WPI), the complex achieved the highest solubility (87.6%) and emulsifying activity index (EAI: 68.4 m 2 /g). Structural analysis revealed that the complex underwent rearrangement of its secondary structure, suggesting the involvement of hydrophobic interactions and disulfide bond formation. This led to an increase in hydrophilic groups, free sulfhydryl content (from 2.1 to 6.8 μmol/g) and disulfide bonds (from 1.5 to 4.2 μmol/g), as well as a higher absolute zeta potential (from −18.5 mV to −35.2 mV). Moreover, the complex formed smaller flaky structures (average diameter: 156 nm) and demonstrated enhanced thermal stability, with the denaturation temperature increasing from 72.3 °C to 81.6 °C. This study provides both theoretical insights and a technical foundation for the development of high-performance dual-protein functional ingredients.