Collective weak interactions of formic acid with hemp protein: Mechanism and application in Pickering emulsion stabilization.

Anti-solvent precipitation using formic acid (FA, 0-98%, v/v) was developed to fabricate hemp protein nanoparticles (HPNs) for Pickering emulsion stabilization. FA ≥10% (v/v) increased hemp protein solubility from 38.0% to 86.9% by solubilizing storage globulins, yielding HPNs with tunable sizes (100-780 nm), high ζ-potentials (>30 mV), and adjustable contact angles (57.7°-126.6°). HPNs prepared at 40% FA exhibited optimal interfacial properties, including the lowest interfacial tension and highest emulsifying activity index (∼1800 m2/g), forming Pickering emulsions with 3-week stability and solid-like rheology. Spectroscopic analysis revealed concentration-dependent conformational changes: invariant Raman spectra confirmed intact primary bonds, while fluorescence and UV-vis indicated initial unfolding (10-40%) followed by refolding (50-98%), and FT-IR showed that β-sheet content peaked at 61% (40% FA), which correlated with optimal interfacial performance. Molecular dynamics simulations elucidated a dynamic "besieging effect" where numerous FA molecules collectively disrupted edestin's hydrogen-bond network via weak interactions (binding energy -3.1 kcal/mol), inducing transient conformational expansion. This work demonstrates the feasibility of FA as a solvent system for hemp protein nanoparticle fabrication and provides mechanistic insights into unconventional protein-solvent interactions, offering a foundation for future food-grade applications.