Structural Transitions of β-lactoglobulin Aggregates Observed Under Controlled Heat Treatment

β-Lactoglobulin (β-Lg), the major whey protein in bovine milk, readily undergoes structural transitions leading to amyloid fibril formation under acidic conditions and elevated temperatures. This study investigated the influence of different thermal treatment conditions on the aggregation and fibrillation behavior of β-Lg at pH 2.

β-Lg solutions were subjected to three different thermal conditions: stepwise heating from 35o C to 90o C for 12h (Sample A), cyclic heating and cooling from 35o C to 90o C for 24h (Sample B), and constant heating at 90o C for 24h (Sample F). Untreated β-Lg served as the control (Sample M). Structural and morphological changes induced by these treatments were characterized by using intrinsic tryptophan fluorescence spectroscopy, Thioflavin T (ThT) fluorescence assay, circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FE-SEM) and atomic force microscopy (AFM).

Spectroscopic and microscopic analyses revealed that stepwise heating (Sample A) promoted amyloid fibril formation, whereas constant heating (Sample F) resulted in extensive aggregation and the development of highly ordered β-sheet-rich fibrillar structures. In contrast, cyclic heating and cooling (Sample B) produced reduced fibril formation, partial retention of native β-Lg structural changes, and distinct aggregate morphologies. Fluorescence, CD, FTIR, and microscopic analyses consistently indicate that Sample F represents the most advanced fibrillar state, while Sample B remained in a partially unfolded and hydrated state with limited changes in the structure.

The observed differences in aggregation and fibrillation behavior demonstrated that thermal treatment strongly influences the balance between structural rearrangement and irreversible protein aggregation. Controlled thermal cycling appears to modulate the aggregation pathway by stabilizing intermediate conformational states and limiting the formation of highly ordered amyloid assemblies.

The method of heating plays a critical role in determining the structural change of βLg. While prolonged heating promotes extensive fibrillation and aggregation, controlled heatingcooling cycles reduce the extent of amyloid formation and preserve partially native-like structural characteristics. These findings provide valuable insights into the thermal regulation of β-Lg aggregation and may contribute to the development of processing strategies for controlling protein selfassembly in food and biomaterial applications.