The formation, stability and microstructure of calcium phosphate nanoclusters sequestered by casein phosphopeptides

Abstract

Casein phosphopeptides have been hypothesized to be responsible for stabilizing supersaturated calcium and phosphate ions through the formation of complexes, making them biologically available for intestinal absorption. Considering the importance of phosphopeptides on calcium phosphate salts, this study focused on the formation and stability of calcium phosphate nanocluster (CPN) complexes sequestered by casein phosphopeptides with different concentrations. The complexes formed with phosphopeptides of 2.5 mg/mL were large particles with an uneven size distribution. With addition of 5–7.5 mg/mL phosphopeptides, the complexes formed reached a uniform nano-scaled size after equilibrium, but these nanoclusters gradually aggregated with each other resulting in large particles formed during storage. In contrast, the change of CPN complexes formed with 10 mg/mL phosphopeptides in average particle size was not significant (p < 0.05) with time, showing a good stability. Based on salt analyses and ion activity product (IAP) calculation as a function of pH, the calcium phosphate within nanoclusters was found to be a neutral salt with a chemical formula of Ca(HPO4)0.4(PO4)0.4 and an invariant ion activity product of 8.00 ± 0.03. Small angel x-ray scattering (SAXS) and cryo-TEM were used for microstructural characterization, revealing that most of CPN complexes were of core-shell sphere structure with a core of amorphous calcium phosphate. Besides, the surface of CPN complexes was expected to be rough rather than smooth, and these nanocluster complexes are likely to be of both kinetic stability and relatively thermodynamic stability.