Structure of β-lactoglobulin microgels formed during heating as revealed by small-angle X-ray scattering and light scattering

We have investigated the structure of microgels formed during heating of demineralized β-lactoglobulin (βlg) solutions at pH 5.9 by small-angle X-ray scattering (SAXS) and light scattering. First, unheated βlg solutions were characterized at different pH values between 2.0 and 7.0. At pH 5.9, βlg solutions contain mainly dimers (with a radius of approx. 2 nm), which coexist with a small number of larger oligomers (approx. 4 nm). Afterwards, βlg microgels, which form upon heating, were studied. They exhibit an average hydrodynamic radius around 130 ± 20 nm and an average molar mass around 7 × 108 g mol−1. We followed the temporal evolution of the various structures that form after different heating times using subsequent SAXS measurements of the entire sample, the soluble fraction where the βlg microgels were removed, and the solvent. After an hour of heating at 85 °C the maximum yield of the βlg microgels (ca. 70%) is almost reached. Interestingly, the SAXS data show a correlation peak corresponding to a characteristic distance of about 9 nm, indicating an internal organization of the microgels. During the heating procedure the pH increases from pH 5.9 to approximately 6.6, which is induced by the partial conversion of βlg into βlg microgels that exhibit less buffering capacity than native protein. The remaining soluble fraction consists of native βlg and some small aggregates, whose number increases on the cost of native βlg as heating time proceeds. We propose that the formation of these lower molecular mass aggregates is triggered by the increased pH.