The crayfish Cherax quadricarinatus stores calcium ions, easily mobilizable after molting, for calcifying parts of the new exoskeleton. They are chiefly stored as amorphous calcium carbonate (ACC) during each premolt in a pair of gastroliths synthesized in the stomach wall. How calcium carbonate is stabilized in the amorphous state in such a biocomposite remains speculative. The knowledge of the microstructure at the nanometer level obtained by field emission scanning electron microscopy and atomic force microscopy combined with scanning electron microscopy energy-dispersive X-ray spectroscopy, micro-Raman and X-ray absorption near edge structure spectroscopy gave relevant information on the elaboration of such an ACC-stabilized biomineral. We observed nanogranules distributed along chitin-protein fibers and the aggregation of granules in thin layers. AFM confirmed the nanolevel structure, showing granules probably surrounded by an organic layer and also revealing a second level of aggregation as described for other crystalline biominerals. Raman analyses showed the presence of ACC, amorphous calcium phosphate, and calcite. Elemental analyses confirmed the presence of elements like Fe, Na, Mg, P, and S. P and S are heterogeneously distributed. P is present in both the mineral and organic phases of gastroliths. S seems present as sulfate (probably as sulfated sugars), sulfonate, sulfite, and sulfoxide groups and, in a lesser extent, as sulfur-containing amino acids.

A specific and simple competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine bovine β-casein phosphopeptides (β-CPP) in casein phosphopeptides (CPP) or CPP complexes such as casein phosphopeptide amorphous calcium phosphate complexes added into dairy products. The method combines sample pretreatment designed for CPP enrichment and anti-β-CPP(f(1–25)) monoclonal antibody 1A5 (mAb 1A5). The mAb 1A5 bound specifically to the tryptic phosphopeptides from β-casein but not from αs1- or αs2-casein. Reactivity was also influenced by the extent of the phosphorylated form of serine residues. Based on the sequence-specific recognition and contribution of phosphorylated serine residues, the epitope of mAb 1A5 was found to reside within the cluster motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu and the surrounding residues in β-CPP. The competitive ELISA developed here can be used as an alternative to specialised and expensive techniques such as mass spectrometry. In particular, it is suitable for the measurement of CPP or CPP complexes in dairy products, which contain closely related endogenous molecular species.

Casein phosphopeptide amorphous calcium phosphate nanocomplexes (CPP-ACP) in chewing gum, lozenges and mouthrinses have been shown to remineralize enamel subsurface lesions in human in situ experiments. The aim of this double-blind, randomized clinical study was to investigate the capacity of CPP-ACP added to bovine milk to remineralize enamel subsurface lesions in situ. Ten subjects drank milk containing either 2·0 or 5·0 g CPP-ACP/l or a control milk whilst wearing removable appliances with enamel slabs containing subsurface demineralized lesions. Each 200 ml milk sample was consumed once a day for each weekday over three consecutive weeks. After each treatment and one weeks rest the subjects crossed over to the other treatments. At the completion of the treatments the enamel slabs were removed and remineralization determined using microradiography and microdensitometry. The results demonstrated that all three milk samples remineralized enamel subsurface lesions. However, the milk samples containing CPP-ACP produced significantly greater remineralization than the control milk. The remineralising effect of CPP-ACP in milk was dose-dependent with 2·0 and 5·0 g CPP-ACP/l producing an increase in mineral content of 70 and 148%, respectively, relative to the control milk. The differences in remineralization following exposure to the three milk samples were all statistically significant (P<0·001). In conclusion, this study shows that the addition of 2·0–5·0 g CPP-ACP/l to milk substantially increases its ability to remineralize enamel subsurface lesions.