Calcium phosphates in Ca(2+)-fortified milk: phase identification and quantification by Raman spectroscopy

Calcium phosphate nanoclusters (CPNs) are important for the structure, function, and nutrient density of many dairy products. Phosphorylated amino acids in caseins stabilize calcium phosphate as nanoclusters which are amorphous to X-ray diffraction and exist within casein micelles, and these CPNs play a key role in micelle stability. Addition of calcium to milk results in further calcium phosphate removal from the serum, and there is uncertainty about the nature of the material formed and its stability. In this work we investigate both the solution and colloidal phases in CaCl2 enriched bovine milk to identify, quantify, and determine the solubility of the calcium phosphate material formed in response to calcium addition to milk. The P-O stretching bands are quite distinct in the Raman spectra of the main synthetic calcium phosphate mineral phases, including the amorphous calcium phosphate phase. In response to adding between 5 and 40 mM CaCl2 to milk, the serum phosphate concentration decreased asymptotically from 7.5 ± 0.2 to 0.54 ± 0.05 mM. Using Raman spectroscopy with a combination of internal and external standards, it was possible to show that the calcium phosphate material formed after Ca(2+) addition to milk was the same as amorphous calcium phosphate nanoclusters present in the absence of added calcium. The use of an internal standard allowed a quantitative analysis of the spectra which demonstrated that the amorphous calcium phosphate formed accounted for all of the calcium and phosphate that was removed from solution in response to calcium addition.