Covalent and Non-Covalent Interactions of Walnut Protein and Ferulic Acid: Impact on Physicochemical and Structural...

Walnut protein (WP) is a high-quality plant protein source, yet its limited solubility and functional properties severely restrict its application in the food industry. This study aimed to overcome these challenges by systematically comparing the impacts of covalent and non-covalent interactions with ferulic acid (FA) on the physicochemical and structural characteristics of WP. The objective was to elucidate how different interaction mechanisms and FA concentrations (0.5%-4.0%, w/v) dictate the functional performance of WP-FA complexes. Results revealed that WP-FA covalent complexes had a higher binding capacity of 157.1 µg/mL compared with their non-covalent counterparts. Solubility data showed that WP-FA covalent complexes achieved a maximum solubility increase of 93.8%, significantly higher than the 56.0% maximum enhancement for WP-FA non-covalent complexes. In addition, particle size and zeta potential analyses indicated that covalent complexes presented larger average particles (2.44 µm) and exhibited higher absolute zeta potential (-38.4 mV). Furthermore, the covalent WP-FA complex (at 2.0% FA) exhibited the highest radical scavenging rates (2,2-diphenyl-1-picrylhydrazyl [DPPH]: 73.3%; 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) [ABTS]: 89.8%), whereas the non-covalent complex required a higher concentration (4.0% FA) to achieve comparable levels (DPPH: 70.2%; ABTS: 84.5%). Multi-spectroscopic analyses confirmed that covalent binding (driven by hydrogen bonding and electrostatics, binding sites (n) = 1.62) had higher affinity than non-covalent binding (hydrophobic interactions, n = 1.21). In conclusion, covalent modification with FA represents an effective approach for enhancing WP properties. This study facilitates the targeted development of WP-based functional ingredients, such as high-performance emulsifiers, antioxidant carriers, and edible coatings designed to enhance preservation.