Extraction of walnut protein using deep eutectic solvent aqueous two-phase system: Investigation of structural and functional properties

Walnut protein (WP) exhibits high nutritional value and excellent techno-functional properties, making it a promising candidate for broad applications in the food industry. It is commonly extracted from the meal after oil processing of walnut through alkali extraction with isoelectric precipitation (AE-IP). In this study, an aqueous two-phase system (DES-ATPS) consisting of betaine-urea/K 2 HPO 4 was used for the extraction of WP from walnut kernels and cold-pressed walnut meal. Comparison with AE-IP, WP extracted through DES-ATPS exhibited enhanced color characteristics and there was no significant difference in protein subunit composition. The WP extracted by DES-ATPS displayed lower Surface hydrophobicity (H 0 ) and intrinsic fluorescence intensities. Conversely, the exposure of internal groups in WP extracted by AE-IP resulted in a higher Zeta potential and a decrease in protein particle size. The comparative analysis revealed fundamentally distinct structural impacts between the two extraction methodologies. Notably, DES-ATPS-isolated WP demonstrated superior solubility under acidic conditions (pH 2).