Structure and adsorption behavior of high hydrostatic pressure-treated ß-lactoglobulin.

HYPOTHESIS: High hydrostatic pressure treatment causes structural changes in interfacial-active ß-lactoglobulin (ß-lg). We hypothesized that the pressure-induced structural changes affect the intra- and intermolecular interactions which determine the interfacial activity of ß-lg. The conducted experimental and numerical investigations could contribute to the mechanistic understanding of the adsorption behavior of proteins in food-related emulsions. EXPERIMENTS: We treated ß-lg in water at pH 7 with high hydrostatic pressures up to 600 MPa for 10 min at 20 °C. The secondary structure was characterized with Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD), the surface hydrophobicity and charge with fluorescence-spectroscopy and ζ-potential, and the quaternary structure with membrane-osmometry, analytical ultracentrifugation (AUC) and mass spectrometry (MS). Experimental analyses were supported through molecular dynamic (MD) simulations. The adsorption behavior was investigated with pendant drop analysis. FINDINGS: MD simulation revealed a pressure-induced molten globule state of ß-lg, confirmed by an unfolding of ß-sheets with FTIR, a stabilization of α-helices with CD and loss in tertiary structure induced by an increase in surface hydrophobicity. Membrane-osmometry, AUC and MS indicated the formation of non-covalently linked dimers that migrated slower through the water phase, adsorbed more quickly due to hydrophobic interactions with the oil, and lowered the interfacial tension more strongly than reference ß-lg.

Conformational state and charge determine the interfacial stabilization process of beta-lactoglobulin at preoccupied interfaces.

Amphiphilic properties enable proteins like ß-lactoglobulin to stabilize oil/water-interfaces and provide stability in food-related emulsions. During emulsification, the protein undergoes three stages: (I) migration through bulk phase, (II) adsorption, and (III) interfacial rearrangement at the oil/water-interface - the kinetics of which require further research. Therefore, the aim of our study was the analytical and computational investigation of stage (I) and (II) as a function of the interfacial preoccupation, conformational state and charge of ß-lactoglobulin. For this purpose, the adsorption of ß-lactoglobulin (at pH 7, pH 7 containing 0.1 M NaCl, and pH 9) at increasingly preoccupied oil/water-interfaces has been compared through measuring interfacial tension and ζ-potential and through running molecular dynamics simulations. With increasing interfacial preoccupation, (I) the migration via lag time increased and (II) the adsorption rate decreased. The (II) adsorption rate was highest for ß-lactoglobulin containing NaCl, due to dense packing and electrostatic screening. ß-lactoglobulin at pH 7 reached a lower adsorption rate than the more negatively charged ß-lactoglobulin at pH 9, due to exposure of hydrophobic regions that had a greater effect on adsorption rates than electrostatic repulsion. Our research contributes to a profound understanding of the interfacial stabilization mechanism of proteins at oil/water-interfaces, necessary to characterise and control emulsification processes.