PHOSPHOPEPTIDES AS POSSIBLE NUTRACEUTICALS FOR FUNCTIONAL FOODS

A new functional drink has been formulated using as nutraceutical ingredient a preparation of casein phosphopeptides (CPP). Nutraceutical is a term combining the words “nutrition” and “pharmaceutical”. Enzymes at predetermined specificity can digest in vivo or in vitro proteins delivering a number of bioactive casein peptides, among which are comprised the CPP. κ-, αs1-, αs2- and β-casein (CN)-derived CPP are characterized by the consensus sequence SerP-X-SerP/ThrP/Glu/Asp (P means a phosphate group) where X is any amino acid residue, but Pro. CPP as well as other bioactive peptides are encrypted within the sequence of casein and they can be in vivo or in vitro released through enzymatic hydrolysis. CPP for their ability to form soluble organophosphate salts prevent their precipitation as insoluble phosphates. Since CPP function as mineral carriers, an increased level of cations can be specifically absorbed in the small intestine. The literature data suggest the possibility of using CPP as vectors of Ca2+ (or other microelements) in functional foods to increase mineral bioavailability during organism development, bone calcification and osteoporosis prevention. The CPP have the remarkable ability to stabilize calcium phosphate in solution, preventing the growth of amorphous calcium phosphate (ACP) to the critical crystal size for nucleation and precipitation. Other studies have highlighted the importance of CPP in delivering ACP to the tooth surface, preventing tooth enamel demineralization and/or increasing remineralization of enamel subsurface lesions. The CPP-ACP complex is already available as oral care product (RecaldentTM), nutraceutical for chewing gum (Trident White Gum), toothpaste (GC Tooth Mousse) and other products. To demonstrate the CPP feasibility in food technology as well in other fields, fractionation of casein tryptic digests into phosphorylated and non-phosphorylated peptides is presented with the objective to obtain a nutraceutical enriched preparation of CPP immobilized on a solid matrix. Various chromatographic adsorbents for immobilizing metal ion (IMAC) and metal oxide (MO) have been evaluated for obtaining phosphorylated peptides from complex peptide mixtures. None of the analyzed analytical system has resulted suitable for milk fortification with CPP. A new selective technique based on hydroxyapatite (HA) has been developed for CPP enrichment. CPP immobilized on HA microgranules formed a complex that was included in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI) matrix before desorbing directly from the well plate. The HA-bound casein enzymolysis was performed in situ with trypsin to remove non-phosphorylated peptides and isolate the immobilized CPP. The latter were recovered by centrifugation, dried and co-crystallized with a 1% phosphoric acid (PA) solution in the matrix that was appropriate for detecting CPP in MALDI-MS spectra. This approach resulted in the identification of 32 CPP by MALDI-time of flight (TOF). The CPP sequences were confirmed by LC-ESI-MS/MS analysis. Once confirmed the purity of CPP, the preparation in semi-industrial scale was realized for fortifying milk with CPP. In order to reduce production costs, HA was synthesized and the binding capacity of HA evaluated using three different protein mixtures as substrate. The protein mixture was chosen on the basis of the specific industrial needs. The HA-CPP complex was finely ground for fortifying milk. The sensory properties of HA-CPP complex were compatible with the use of the novel formulation as nutraceuticals. In order to quantify CPP bound to HA beads to be added to milk, two synthetic peptides, a natural and the modified counterpart that differed by single amino acid substitution, were used as internal standards (IS). A parallel study on the quality of ovine milks and Grana Padano cheeses was carried out to exploit the casein phosphorylation as index of the protein quality. The phosphoproteomic approach has allowed to detect some proteotypic CPP predictors of abnomalous milk for the high content of somatic cells and of milk quality used for Grana Padano production. Finally, CPP provided significant information on milk and cheese genuineness.