The effect of pH on the structure and phosphate mobility of casein micelles in aqueous solution

Abstract

The mobility of phosphate groups in aqueous solutions of casein micelles and sodium caseinate from bovine milk was determined with magic angle spinning (MAS) 31P NMR as a function of the pH between pH 4 and pH 8. The chemical shifts and the relative amplitudes of the signals from mobile inorganic phosphate (orthophosphate) and mobile organic phosphate (phosphorylated serines) as well as that of immobile phosphate (colloidal calcium phosphate, and immobile phosphorylated serines) were determined. Sodium caseinate contained very little orthophosphate and all phosphates were mobile over the whole pH range. In micellar casein solutions most of the phosphate was immobile at pH > 6.0, but the fraction of mobile organic and inorganic phosphate increased sharply between pH 5.5 and pH 4.5, showing the disintegration of the CCP nanoclusters. Protonation of the phosphates with decreasing pH was determined from the chemical shift and was related to their mobility. The signal of mobile organic phosphate was different for micellar casein solutions and sodium caseinate demonstrating the influence of calcium phosphate in the former. The microscopic structure of protein solutions was investigated with confocal laser scanning microscopy. Large protein clusters were observed below pH 5.2 with a density that increased with decreasing pH down to pH 3.9. The mobility of either organic or inorganic phosphate at pH 6.8 was not significantly different after the pH had been reduced to 4.8 and subsequently increased to 6.8, but the microstructure was strongly influenced by the pH-cycling.