The safeguard of river buffalo Mozzarella cheese, a Protected Designation of Origin dairy product, has prompted an analytical study to trace the milk and curd used as raw material in cheesemaking. This is to prevent the illegal use of milk or curd from different geographical areas outside of those indicated in the official production protocol. For this purpose, we studied primary proteolysis occurring in fresh and frozen milk and curd to identify a molecular marker that could indicate the raw material used. Whole casein from frozen river buffalo milk was separated using cation-exchange chromatography and sodium dodecyl sulfate-PAGE, and a protein component with an estimated molecular weight of 15.3 kDa was detected. This protein component was revealed in fresh river buffalo milk as a faint electrophoresis band, which drastically increased in intensity in refrigerated and frozen milk as well as in curd and was found to be associated with beta-CN through hydrophobic interaction. By using matrix-assisted laser desorption/ionization-time of flight peptide mass mapping, this component was identified as the C-terminal fragment f(69-209) of beta-CN (expected molecular weight of 15,748.8 Da). beta-Casein f(69-209), originating from the early hydrolysis of Lys(68)-Ser(69) by plasmin, has no counterpart in bovine milk. The increased rate of hydrolysis by plasmin toward the cleavage site Lys(68)-Ser(69) has to be ascribed to the elevated proline content of the peptide 61-73. The favored production of beta-CN f(69-209) has also drawn attention to the complementary proteose peptone beta-CN f(1-68) that is presumed to play a physiological role in inducing milk secretion similar to that of beta-CN f(1-29). The higher in vivo and in vitro production rate, compared with gamma(1)-CN formation, indicates that beta-CN f(69-209) and its complementary fragment are candidate molecular markers to evaluate milk and curd freshness. We suggested [corrected] indirect ELISA analysis based on the determination of remaining nonhydrolyzed beta-CN to perform a quantitative evaluation of proteolysis.

The guarantee of the origin and quality of raw material is essential for the protection and valorization of Campana buffalo mozzarella cheese. The risk of utilization of semifinished products and stored milk in substitution for fresh milk is increasing, due to the continuous desire to reduce production costs. A proteomics approach and electrophoresis survey of retail mozzarella cheeses indicated different rates of proteolysis in the production of dairy industries. The use of fresh milk and correct cheesemaking protocol yielded only γ-caseins, which are derived from β-casein by plasmin, and para-κ-casein, which is derived from κ-casein by chymosin. The detection of abnormal hydrolysis resulting in β- and αS1-casein fragments, identified by mass spectrometry, indicates the use of stored milk or stored and pressed curd, or the reuse of unsold mozzarella cheese, to produce mozzarella. The formation of γ-caseins and other fragments during a long storage of raw materials at room or refrigeration temperature was ascribed to plasmin (endogenous milk enzyme), whereas formation of αS1-casein fragments, mainly αS1-I(6P)- and αS1-I(7P)-casein during the storage of curd was ascribed to the action of chymosin (exogenous enzyme) from rennet. Sodium dodecyl sulfate-PAGE and alkaline urea-PAGE permitted us to evaluate the freshness of the raw materials used in the manufacturing of buffalo mozzarella cheese and to reveal possible inappropriate preservation.