A Study on the Formation and Mechanisms of Worm-Like β-Lactoglobulin Self-Assemblies and Their Role in Regulating Fibrillar Self-Assemblies

This study investigated the formation, structural characterization, cross-nucleation behavior, and functional properties of worm-like β-lactoglobulin (β-Lg) self-assemblies induced by heat treatment at 85°C under neutral pH conditions. The assembly process was monitored using SDS–PAGE, free thiol content determination, transmission electron microscopy (TEM), thioflavin T (ThT) fluorescence, ζ-potential, surface hydrophobicity, Fourier transform infrared spectroscopy (FTIR), and molecular dynamics (MD) simulations. The results indicate that heating promotes disulfide bond rearrangement and conformational changes, leading to the formation of a stable worm-like self-assembled structure. This is specifically reflected in the ζ-potential shifting from −16.8 ± 0.2 to −36.5 ± 0.3, an increase in surface hydrophobicity (from 810 ± 8.5 to 2643 ± 10.2), and a decrease followed by an increase in β-sheet content (from 36.12% to 28.35% to 29.25%), revealing structural changes during the formation of the worm-like self-assembled structures. Molecular dynamics simulations revealed that heating increased structural openness and enhanced residue mobility (Leu87, Ala111, Val128 RMSF > 2 Å). Cross-seeding experiments indicate that worm-like fibers influence the fiberization kinetics by prolonging the hysteresis phase and modulating the fluorescence intensity (depending on the seeding concentration); the fluorescence intensity during the plateau phase increased by 26.30%. Functionally, the appropriate incorporation of worm-like fiber bundles improves emulsification (emulsification capacity increased to 43.53% ± 0.3%) and foaming properties (foaming capacity increased to 142.56% ± 0.1%), whereas excessive aggregation weakens these properties. These findings provide new insights into the controlled formation of β-Lg worm-like assemblies and their potential as functional ingredients in food systems.