Experimental and molecular dynamics simulation study on the mechanism of β-lactoglobulin self-aggregation induced by ultra-high temperature

Ultra-high temperature (UHT) treatment effectively prevents milk spoilage, yet it can cause protein aggregation, thereby impacting milk stability. β-Lactoglobulin (β-Lg) is a thermally unstable whey protein, but the mechanism of its self-aggregation induced by UHT treatment remains unclear. Herein, experimental approaches and molecular dynamics simulations were performed to elucidate β-Lg aggregation under UHT. The physicochemical properties of the aggregates were initially characterized after different UHT treatments. Size exclusion chromatography and multi-angle static light scattering showed β-Lg aggregates with molecular weights from 20 to 900 kDa and particle sizes from 100 to 350 nm, with bead string and irregular morphologies. Multispectroscopy showed that UHT treatment caused the unfolding of the β-Lg. Molecular dynamics simulations further observed hydrogen bond disruption and β-barrel opening, enhancing the electrostatic interactions and hydrophobicity of β-Lg surfaces, leading to non-covalent aggregation. Furthermore, SDS-PAGE confirmed the involvement of disulfide bonds in aggregate formation, and mass spectrometry identified the disruption of original disulfide bonds under UHT, promoting intermolecular disulfide bonding involving residues Cys119, Cys121, and Cys160. These findings provide a comprehensive understanding of β-Lg aggregation under UHT conditions, which is essential for improving the stability and quality of UHT-treated milk products. • Hydrophobic and electrostatic interactions promote non-covalent aggregation of β-Lg. • Hydrogen bond disruptions and β-barrel opening in β-Lg led to self-aggregation. • Cys119, Cys121, and Cys160 formed new covalent disulfide bonds in β-Lg aggregates.