High hydrostatic pressure-induced structural remodeling of walnut meal protein and its functional implications:...

Walnut meal protein (WMP) represents an underutilized plant protein resource with considerable application potential. However, the molecular basis underlying its structural responsiveness to high hydrostatic pressure (HHP) remains insufficiently elucidated. This study systematically investigated the effects of HHP (100-600 MPa, 5-20 min) on the structural, physicochemical, and functional properties of WMP by integrating spectroscopic analyses with molecular dynamics simulations. HHP markedly reduced solubility (48.4% to 4.6% at 600 MPa, 20 min) while increasing turbidity and particle size, indicating pressure-induced aggregation. Surface hydrophobicity and absolute zeta potential increased, whereas free sulfhydryl content decreased by up to 64.81%, reflecting unfolding followed by intermolecular association. Circular dichroism revealed reduced α-helix content with increased β-sheet and random coil structures, and fluorescence spectroscopy confirmed tertiary structural perturbation and enhanced hydrophobic exposure. These structural transitions significantly improved functional performance, with emulsifying activity index reaching 33.9 m²/g, foaming ability 47.52%, and marked increases in water- and oil-holding capacities. Molecular dynamics simulations corroborated experimental observations, demonstrating pressure-dependent conformational destabilization, increased solvent-accessible surface area, reduced radius of gyration, and a roughened free-energy landscape. Collectively, these findings elucidate the molecular mechanism underlying HHP-induced structural reorganization of WMP and provide a rational basis for tailoring plant protein functionality through non-thermal processing.