Ultrasonic pretreatment remodels alkaline protease-mediated peptide release from pecan protein for enhanced xanthine oxidase inhibition and antioxidant protection

Defatted pecan [Carya illinoinensis (Wangenh.) K. Koch] meal is a protein-rich by-product of pecan oil processing and a potential source of multifunctional food-derived peptides. This study evaluated whether ultrasonic pretreatment could alter the physicochemical organization of pecan protein and enhance its susceptibility to alkaline protease hydrolysis. Among the four proteases tested, alkaline protease produced the highest degree of hydrolysis (DH; 23.26 ± 1.08% at 240 min) and the strongest combined xanthine oxidase (XOD)-inhibitory and antioxidant activities. Ultrasonic pretreatment further increased DH from 23.55 ± 0.74% to 30.11 ± 0.25%, enhanced XOD inhibition, radical-scavenging activity, and ferric reducing antioxidant power, reduced adenosine/XOD-driven uric acid accumulation in HK-2 culture supernatants, and attenuated H₂O₂-induced oxidative stress in Caco-2 cells. Structural analyses showed that ultrasound altered aggregation morphology, particle-size distribution, surface hydrophobicity, amide I components, and the microenvironment of aromatic residues, consistent with enhanced susceptibility to proteolysis. LC-MS/MS peptidomics revealed an increase in the number of detectable peptide entries from 5768 to 7029 and in the proportion of 2–5-residue peptides from 50.45% to 53.96%. An integrated workflow combining comparative peptidomics, relative-abundance analysis, in silico prediction, target-specific molecular docking, and experimental validation prioritized YRAF, FLADAF, and EWVAF as UP-CiPAH-associated peptides. FLADAF showed the strongest XOD inhibition (IC₅₀ = 1.24 ± 0.06 mM), whereas YRAF exhibited the strongest DPPH scavenging activity (IC₅₀ = 0.448 ± 0.030 mM). These findings support an association among ultrasonic pretreatment, physicochemical remodeling of pecan protein, altered peptide profiles, and enhanced multifunctional activity, highlighting ultrasonic pretreatment as a promising strategy for valorizing pecan oil-processing by-products.