Acoustic Cavitation-Induced Unfolding and Solubilization of Velvet Antler Protein for Antioxidant Peptide Release: Substrate Modification Kinetics, Quantum Chemistry, and Keap1/Nrf2-Associated Cellular Responses.

The targeted extraction of bioactive peptides from structurally robust biological matrices, such as velvet antler protein (VAP), is severely hindered by high steric hindrance and profound conformational stability. This study established a multiscale analytical framework to investigate ultrasound-assisted VAP modification and its association with antioxidant peptide release and Keap1/Nrf2-related cellular responses. High-intensity ultrasound pretreatment (optimized at 450 W, 30 min, 30 mL/g) effectively deconstructed the dense interfacial architecture of VAP, sub-micronizing the particle size to ∼175 nm and promoting an ultrasound-associated conformational transition from α -helices to random coils. Thermodynamic deconstruction revealed that acoustic shear forces significantly attenuated the denaturation enthalpy (ΔH) and elevated surface hydrophobicity (H0), alleviating steric constraints and driving an increase in targeted peptide yield from 44.82% to 68.32%. By integrating peptidomics, machine learning, and density functional theory (DFT), five representative antioxidant lead candidates were prioritized and subsequently synthesized for validation. DFT analyses suggested that these selected sequences may possess favorable electron-donating properties, potentially associated with aromatic residues, adjacent hydrophobic residues, and Pro-related conformational restriction. Furthermore, molecular docking suggested that these peptides may interact with the Keap1 Kelch domain, while in vitro cellular assays showed that the selected peptides restored endogenous antioxidant enzymes (SOD, CAT, GSH-Px) in H2O2-challenged RAW264.7 macrophages. These findings provide physicochemical and cellular evidence supporting ultrasound-assisted VAP modification and antioxidant peptide discovery, offering a laboratory-scale, mechanistically guided strategy for discovering functional peptides from structurally dense biological resources. Nevertheless, further pilot-scale validation is required before industrial translation.