Experimental evidence for previously unclassified calcium phosphate structures in the casein micelle

1H-31P Cross-polarization magic angle spinning (CP-MAS) measurements of 40-d-old Mozzarella cheese and 20 mM EDTA-treated casein micelles revealed that each sample had immobile phosphorus with the same spectral pattern, which did not match that of native casein micelles. To identify the immobile phosphorus bodies, 1H-31P CP-MAS spectra and cross-polarization kinetics measurements were undertaken on native casein micelles, EDTA-chelated casein micelles, and reference samples of β-casein and hydroxyapatite. The results showed that the immobile phosphorus bodies in the mature Mozzarella cheese had the following characteristics: they are immobile phosphoserine residues (not colloidal calcium phosphate); they are not the product of phosphoserine to colloidal calcium phosphate bridging; the phosphate is complexed to calcium; their rigidity is localized to a phosphorus site; their rigidity and bond coupling is unaffected by protein hydration; and the immobile bodies share a narrow range of bond orientations. Combining these observations, the best explanation of the immobile phosphorus bodies is that bonding structures of phosphorus-containing groups and calcium exist within the casein micelle that are not yet clearly classified in the literature. The best candidate is a calcium-bridged phosphoserine-to-phosphoserine linkage, either intra- or inter-protein.