The effect of protein aggregate morphology on phase separation in mixtures with polysaccharides.

The morphology of aggregates formed by heating the globular protein β-lactoglobulin (β-lg) changes with the addition of a small amount of CaCl2, from small strands to larger spherical aggregates (microgels). We investigated the effect of this morphological transition on the structure of mixtures of β-lg aggregates with the polysaccharide κ-carrageenan (κ-car), using confocal laser scanning microscopy and dynamic light scattering. The change in the morphology of the β-lg aggregates strongly reduced the κ-car concentration at which the system phase separated. As a consequence a dramatic change in the structure of the mixtures occurred over a narrow range of the CaCl2 concentration. Phase separation leads to the formation of micron-sized protein rich domains that have a tendency to stick together in large flocs. There is a big difference between the protein concentrations in the two phases, but the κ-car concentration is only weakly lower in the protein rich phase. A comparison is made between mixtures prepared at room temperature, after separately heating β-lg, and heated mixtures of native β-lg and κ-car. The micro-phase separated structure of the two systems is similar, but the aggregates disperse upon dilution in the former case, while they are covalently bound within the domains in the latter case. Other, more subtle, differences were also observed. The results explain the very high sensitivity of the structure of β-lg/κ-car mixtures to calcium ions.