Aqueous dispersions of demineralized beta-lactoglobulin (beta-lg) were held at 85 degrees C for 15 min at a constant protein concentration of 1 wt % in the pH range of 3.0-7.0. This led to denatured protein content ranging from 20% (pH 3.0) to 90% (pH 5.0). The protein aggregates formed were characterized as to their stability to sedimentation (turbidity), morphology, size, surface charge, ANS surface hydrophobicity, and content in accessible thiol groups. Additionally, the changes in secondary structures of the protein upon heating were followed by Fourier transform infrared spectroscopy (FTIR). Stable dispersions (no sedimentation for 10 min) of individualized beta-lg microgels were obtained at specific pH 4.6 and 5.8, corresponding to an aggregation yield of about 80%. The width of the pH region leading to these microgels was 0.3 pH unit below or above the two specific pH values. Microgels were characterized by a spherical shape and remarkably low polydispersity in size (<0.2). Their z-average hydrodynamic diameter determined by dynamic light scattering (DLS) was between 160 and 220 nm, and their zeta-potential was +30 or -40 mV, depending on the initial pH before heating. Microgels obtained at pH 4.6 displayed a lower binding capacity for ANS and a lower content of accessible thiol groups as compared to those obtained at pH 5.8. Both types of microgels might therefore differ in their internal and interfacial structures. Between pH 4.6 and 5.8, large sedimenting protein particulates were obtained, whereas soluble aggregates were formed at pH <4.6 or >5.8. Interestingly, DLS experiments showed that before heating, beta-lg was mainly present in an oligomeric state at pH 4.6 and 5.8. This result was confirmed by FTIR measurements indicating the stronger contribution of the 1616-1624 cm(-1) spectral band corresponding to intermolecular beta-sheets in the pH range of 4.0-6.0. Upon heating, FTIR spectroscopy revealed that individualized microgels were obtained under pH conditions where a balance between attractive forces arising from protein unfolding leading to the formation of intermolecular beta-sheets (1616-1624 cm(-1 )band) and the repulsive electrostatic forces due to the initial protein net charge was achieved.

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