Hydration-mediated stabilization and anti-aggregation of soy and whey protein isolates, insulin, and collagen in hydrophilic deep eutectic solvents

Hypothesis In deep eutectic solvent (DES)–H 2 O systems, protein stability is not determined solely by chemical composition but by a general colloid–interface mechanism: there exists a moisture-dependent hydrogen-bonding network reorganization point at which interfacial hydration forces and bulk DES structure are co-optimized. At this “critical point”, protein–solvent interfacial properties—wettability, hydration layer structure, and interfacial tension—are tuned to form stable colloid systems. Experiments We investigated three hydrophilic DESs (choline chloride:glycerol, betaine:glycerol, l -carnitine:glycerol) across a moisture gradient. Bulk and interfacial properties were probed via viscosity, density, refractive index, conductivity, contact angle, interfacial tension, and AFM force spectroscopy. Aggregation and secondary structure of protein colloid systems were evaluated for four proteins using TEM, PSD, UV–Vis, FTIR, and CD. MD simulation was used to demonstrate the supramolecular interaction between proteins and DESs. Findings Hydrophilic DES–H₂O systems undergo hydrogen-bonding network reorganization at ~16 wt% moisture content, where bulk and interfacial properties of all DESs display marked changes. These interfacial transitions correlate directly with maximal anti-aggregation effects and preservation of native protein structures. Our results suggest a mechanism whereby water actively reorganizes the internal hydrogen-bonding network of hydrophilic DESs, leading to protein colloids that are both structurally and colloidally stable. This study established a general colloid–interface framework for protein stabilization in DES–H₂O systems, with potential applications in biomedical, pharmaceutical, cosmetic, and food products.