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

ABSTRACT

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.