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.

Two angiotensin converting enzyme (ACE)-inhibitory peptides were isolated from enzyme modified cheese (EMC) and their amino acid sequences were identified as Leu-Gln-Pro and Met-Ala-Pro. The EMC was prepared by a combination of Protease N, Umamizyme, and Flavourzyme 500L. Both peptides were derived from β-casein, f 88-90 and f 102-104, respectively. Met-Ala-Pro showed strong ACE inhibitory activity (IC50=0·8 μm) and antihypertensive activity in spontaneously hypertensive rats (SHR) after single oral administration. The IC50 value of Met-Ala-Pro was not affected by pre-incubation with ACE, suggesting that this peptide was a true ACE-inhibitory peptide. We report here, for the first time antihypertensive peptides from EMC.

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.