Non-monotonic concentration-dependent hydrogel formation of α-Lactalbumin induced by glycyrrhizin: Mechanistic insights from multi-spectroscopic and molecular dynamics approaches

First phase diagram of α-lactalbumin/glycyrrhizin hydrogels defines concentration-dependent gelation windows. • Gy induces controllable hydrophobic exposure and a reduction in antiparallel β-sheet content. • Hydrophobic interactions dominate network assembly. • Gelable systems exhibit shear-thinning rheology, a critical viscosity threshold, and enhanced bound water content. • MD simulations reveal Gy increases α-La–Gy H-bonds and lowers protein/water diffusion coefficients. The non-monotonic concentration-dependent formation mechanism of α-lactalbumin (α-La) and glycyrrhizin (Gy) composite hydrogels was systematically investigated using multi-technique approaches combined with molecular dynamics simulations. Phase diagrams showed that gelation occurred only within specific windows (α-La 0.1–1.0% w / v , Gy 3.0–15.0 mM), with Gy thresholds increasing from 3.0 to 15.0 mM as α-La rose, whereas no gel formed at α-La ≥2.0%. Hydrophobic interactions dominated network assembly (56–79% contribution), accompanied by conformational rearrangements. All gels exhibited shear-thinning rheology and enhanced bound water (LF-NMR). Simulations revealed Gy-dependent increases in H-bonds, and the solvent-accessible surface area of α-La peaked at an optimal Gy number then slightly decreased. Gy also reduced diffusion coefficients of both α-La and water. Overall, gelation relies on moderate hydrophobic exposure within a strict non-monotonic window, excess protein paradoxically inhibits gelation. This finding could provide a molecular foundation for designing green hydrogels with precisely tunable behavior Download: Download high-res image (477KB) Download: Download full-size image