Effect of calcium on the aggregation behaviour of caseinates

Abstract

Insights into the structure of caseinate aggregates have been obtained from small angle X-ray scattering (SAXS) with complementary measurements obtained by turbidity and dynamic light scattering (DLS). The caseinate precipitation method produced a cold insoluble and cold soluble caseinate (CN) fraction. The caseinates were then recombined in ratios of these fractions from 0% to 100% cold soluble CN (3.3% w/w total protein), and calcium was added up to 22mM. This allowed individual structural features due to calcium and/or protein to be identified as the relative concentrations of each component were varied in a controlled fashion. SAXS analysis indicated the presence of scattering objects on three different length scales in the system: a feature from the largest objects which was ascribed to the overall aggregate size; a small feature at q>0.1Å−1 (where q is the magnitude of the scattering vector) corresponding to protein inhomogeneities with a radius of gyration of 1.9–5.9nm, and a mid-range feature at q=0.02Å−1 which corresponds to a protein cluster interaction distance of 22–28nm. DLS and turbidity measurements showed that the overall aggregate size and turbidity increased with increasing calcium and cold insoluble CN concentration. The hydrodynamic diameter for the cold insoluble CN increased from 190nm to 295nm with 0mM and 22mM calcium, respectively. In contrast, a cold soluble CN with 22mM calcium had a diameter of 140nm. Collectively this is consistent with calcium binding to the phosphoserine clusters via ionic bonding, leading to particle growth; the higher ratio of cold insoluble CN allowed the aggregates to grow to larger sizes, which is likely due to different structuring of κ-CN around the aggregates.