Structure of [beta]-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 [beta]-lactoglobulin ([beta]lg) solutions at pH 5.9 by small-angle X-ray scattering (SAXS) and light scattering. First, unheated [beta]lg solutions were characterized at different pH values between 2.0 and 7.0. At pH 5.9, [beta]lg solutions contain mainly dimers (with a radius of approx. 2 nm), which coexist with a small number of larger oligomers (approx. 4 nm). Afterwards, [beta]lg microgels, which form upon heating, were studied. They exhibit an average hydrodynamic radius around 130 +/- 20 nm and an average molar mass around 7 x 10[super]8 g mol[super]-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 [beta]lg microgels were removed, and the solvent. After an hour of heating at 85 [deg]C the maximum yield of the [beta]lg 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 [beta]lg into [beta]lg microgels that exhibit less buffering capacity than native protein. The remaining soluble fraction consists of native [beta]lg and some small aggregates, whose number increases on the cost of native [beta]lg as heating time proceeds. We propose that the formation of these lower molecular mass aggregates is triggered by the increased pH. AB: