A method to detect fraudulent addition of bovine milk in water buffalo Mozzarella cheese by gradient high-performance liquid chromatography (RP-HPLC), relying on the measurement of quantity ratios within beta-lactoglobulin protein family, is described. Analyses were performed on raw milk, cheese matrix and cheese governing liquid using a C4 column and UV detection. This work demonstrated that bovine milk addition during cheesemaking can be detected in governing liquid of Mozzarella down to the EU law limit of 1% as well as in raw milk and cheese matrix. A significant lowering of peaks' areas and heights was observed in cheese matrix and governing liquid samples in comparison with the corresponding milk ones, possibly due to proteins' degradation during the cheesemaking process. The results show that, unlike previous works reported, the use of a matrix-specific calibration curve is essential in order to achieve a proper quantitation of beta-lactoglobulin proteins, thus allowing a reliable estimation of bovine milk addition.
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