Identification by MALDI-ToF of B-lactoglobulins of buffalo mozzarella cheese: as authenticity index

Italian Mozzarella is a typical fresh “pasta filata” cheese made from raw water buffalo’s milk only using natural whey cultures as fermentation starters. It received the European protected denomination of origin (PDO) certification under the designation “Mozzarella di Bufala Campana”. The seasonal increase in market demand occurring every summer and, on the other hand, the limited productions of buffalo milk may induce fraudulent addition of bovine milk during manufacturing of Mozzarella. A number of protein-based methods was developed to spot this fraud and to assure products’ genuinity to both producers and consumers. The current legislation in EU and in Italy, based on official control methods which rely on the isoelectrofocusing (IEF) of gamma-caseins after plasminolysis and on HPLC analysis, respectively, tolerates a maximum addition of 1% bovine milk. In addition, several other techniques to detect bovine milk in water buffalo mozzarella were developed. Identification of bovine β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) was performed by a capillary zone electrophoresis procedure for basic proteins; this method is limited by the presence of interfering peaks in the area of α-LA and the variability of β-LGA/β-LGB ratio. Immunoblotting analysis to determine the presence of bovine milk in water buffalo Mozzarella was optimized by Addeo et al. and consists in detecting the bovine -casein and peptides by the use of polyclonal antibodies against β-casein. This analysis confirmed the results obtained by IEF. Recently, a new method based on the determination of protein molecular masses was developed. Bovine whey protein (α-LA and β-LG) were detected in water buffalo Mozzarella by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. On the other hand, DNA-based methods relying on conventional PCR technology are very reliable and suitable to routine detection of bovine DNA but, since these techniques are not quantitative assays, they’re not appropriate to provide information on the observance of the 1% law threshold, notwithstanding a quantitative PCR method was recently developed to detect fraudulent addition of bovine milk in ovine cheeses. The aim of this work is to detect fraudulent addition of bovine milk in Italian Mozzarella cheese by using MALDI-ToF technique.