Enhanced Unfolding of Bovine β-Lactoglobulin Structure Using Microwave Treatment: a Multi-Spectroscopic Study

Abstract

Commercial whey protein hydrolysates containing bovine β-lactoglobulin (β-Lg) may have residual allergenicity due to the inaccessibility of some sequential epitopes to proteases. Microwave may enhance unfolding pathways in protein structure due to its non-thermal effects. This research compared the effects of microwave heating (MW) and conventional heating (CH) on the unfolding in the secondary and tertiary structures of β-Lg over a temperature range of 40-90 °C using circular dichroism (CD), fluorescence spectroscopy, and two dimensional (2D) 1H nuclear magnetic resonance (NMR) spectroscopy. Above 50 °C, β-sheet and α-helical secondary structures decreased during MW and CH, with a higher decrease being observed during MW. The near-UV spectra of MW β-Lg showed lower intensity suggesting higher tertiary structure loss than in CH β-Lg at all temperatures. The fluorescence spectra of MW β-Lg showed increased exposure of tryptophan residues to solvent as compared to CH β-Lg and suggested greater unfolding in tertiary structure in MW β-Lg at 60 °C than in CH β-Lg at 70 °C. 2D 1H NMR spectra confirmed more extensive H-D exchange in MW β-Lg explained by the exposure of β-sheets (C, G, and H) at 50 °C under microwave treatment, which are thermally resistant to H-D exchange up to 75 °C during conventional heating. These results revealed a substantial enhancing effect of microwave treatment on the thermal unfolding and exposure of buried amide groups in β-Lg compared to conventional heating. Microwave processing could be a promising alternative to produce hydrolysates with lower allergenicity and improved bioactivity through structure modification.