Identification of peptides obtained via hydrolysis of bovine casein by chymosin using HPLC and mass spectrometry.

The pH 4.6-soluble products of hydrolysis of whole bovine casein by chymosin were analysed using reversed-phase high-performance liquid chromatography and electrospray-ionisation mass spectrometry off-line. The molecular masses, together with information about the amino acid sequences of caseins and the specificity of the chymosin towards bovine caseins were used to identify peptides. In some cases, partial fragmentation in the ion source allowed the C-terminal amino acid residue to be identified. Among the proteolysis products we found kappa-caseinomacropeptide fractions containing up to 3 glycosidic residues. Fragments alpha s1-CN 1-23, with an antibacterial and immunomodulating activity, and beta-CN 193-209, with an immunomodulating activity were also found as major products of proteolysis. Identification of the latter fragment was confirmed via second-derivative UV spectroscopy. This fragment contains 2 different aromatic amino acid residues and was a well-separated and homogeneous fraction, thus allowing such an identification. Some typical products of plasmin action on beta-CN were also detected. They could be associated with casein micelles during the precipitation.