Determination of Bioactive Peptides in Milk

Milk contains several bioactive proteins and peptides as original minor constituents. Peptides that are inactive within the sequence of major milk proteins (caseins, whey proteins) are of particular interest. Such peptides are e.g. casomorphins, caseinophosphopeptides and casokinins. These peptides may be released from the appropriate protein precursor during intestinal proteolysis or the manufacture of milk products.Regarding the significance of bioactive peptides from milk as exogenous regulatory substances, different analytical techniques have been developed for the determination of peptide fragments that are liberated as a result of technological treatment or during intestinal proteolysis. In order to detect bioactive sequences that are hidden within milk proteins an enzymatic in vitro-proteolysis (e.g. with trypsin) has to be performed. The analysis of minor peptide concentrations demands an enrichment of the analyte as well as the application of methods with a low detection limit. For example, the solid phase extraction using C18 cartridges is suitable for the concentration of hydrophobic casomorphins; anion exchange chromatography is useful to isolate phosphopeptides. Reversed phase high-performance liquid chromatography (HPLC) has proved itself to be a versatile procedure for the determination of peptides.Immunochemical analyses using enzyme-immunoassay (ELISA) provide an advantage regarding specifity and sample throughput as compared with liquid chromatographic methods. Anti-peptide-(lgY)antibodies can be easily prepared from the egg yolks of immunized laying hens and used in ELISA for the determination of peptides. Suitable standard substances and immunogens, respectively, are obtained by solid phase peptide synthesis (Fmoc strategy).Further studies are needed to examine which bioactive peptides are ingested in physiologically significant amounts from milk and milk products. Nevertheless, the relatively new area of investigation in bioactive peptides have introduced a new criterion for defining the protein and food quality.