Challenges in Using the Official Italian Method to Detect Bovine Whey Proteins in Protected Designation of Origin Buffalo Mozzarella: A Proteomic Approach to Face Observed Limits

This study critically examines the limitations of the official Italian methodology used for detecting bovine adulteration milk in Protected Designation of Origin (PDO) Mozzarella di Bufala Campana (MdBC). This method focuses on the whey fraction of cheese samples, which comprises about 1% of total MdBC proteins, and is based on a high-performance liquid chromatography (HPLC) quantification of the bovine beta-lactoglobulin A (beta-Lg A) as a marker. Here, we have demonstrated that this official methodology suffers from measurement inconsistencies due to its reliance on raw bovine whey standards, which fail to account for beta-Lg genetic polymorphisms in real MdBC samples and protein thermal modifications during cheesemaking. To overcome these limitations, we propose a dual proteomics-based approach using matrix-assisted laser desorption ionization (MALDI-TOF) mass spectrometry (MS) and nano-HPLC-electrospray (ESI)-tandem mass spectrometry (MS/MS) analysis of MdBC extracted whey. MALDI-TOF-MS focused on identifying proteotypic peptides specific to bovine and buffalo beta-Lg and alpha-lactalbumin (alpha-La), enabling high specificity for distinguishing the two animal species at adulteration levels as low as 1%. Complementing this, nano-HPLC-ESI-MS/MS provided a comprehensive profile by identifying over 100 bovine-specific peptide markers from beta-Lg, alpha-La, albumin, lactoferrin, and osteopontin. Both methods ensured precise detection and quantification of bovine milk adulteration in complex matrices like pasta filata cheeses, achieving high sensitivity even at minimal adulteration levels. Accordingly, the proposed dual proteomics-based approach overcomes challenges associated with whey protein polymorphism, heat treatment, and processing variability, and complements casein-based methodologies already validated under European standards. This integrated framework of analyses focused on whey and casein fraction enhances the reliability of adulteration detection and safeguards the authenticity of PDO buffalo mozzarella, upholding its unique quality and integrity.