# Proteomics Analysis in Dairy Products: Cheese, a Review
## 9 Adulteration and Proteomics
[10] to develop a new laboratory approach through an analytical detection method using the MALDI-TOF-MS data of signature peptides from wild and variant WB and bovine CN, thus allowing the identification of milk adulterations with non-Italian WB or bovine milk in commercial Mozzarella di Bufala Campana cheese samples [10]. A great increase in Mozzarella di Bufala Campana production demands has been observed in the past few years, which has not been followed by an increase in Mediterranean WB milk availability [21]. Furthermore, the price of buffalo milk varies within each year, creating an unstable economic environment for Mozzarella's producers. In order to deal with these challenges, dairy owners turn to non-compatible with PDO rules and techniques. In more detail, they use frozen curd as a substitute for fresh milk, something highly prohibited by European Union laws, resulting in different flavor and other traits compared to the original product. To restrict this fraudulent procedure new laboratory methods were applied [194,195], which identified the marker \(\beta\)-casein fragment (69-209) [194, 195, 196], resulting from protein proteolysis as result of the activity of endogenous plasmin [197, 198, 199], in curd. Dedicated controls in Italy from the Anti-Sophistication Nucleus for foods revealed recently this technological approach to become progressively adopted for Mozzarella DOP production [21]. In fact, this adulteration practice allows for several stable proteolytic and lipolytic enzymes from psychrotrophic bacteria to induce non-desired coagulation of milk proteins and production of unwanted organic compounds [200, 201, 202, 203], affecting final Mozzarella organoleptic characteristics. Nineteen discriminant marker signals in frozen buffalo milk with respect to the fresh counterpart were linked to specific polypeptides/proteins based on literature data [194, 196, 204, 205], mass value calculations, and additional mass spectrometric investigations. Among protein/polypeptide marker signals identified by MALDI-TOF-MS profiling and worth mentioning are those related to \(\alpha\)-lactalbumin, \(\beta\)-lactoglobulin and \(\beta\)-casein coupled with the ones associated with GLYC-AM1- and \(\beta\)-casein-derived fragments which possibly originated as a result of the activity of proteolytic enzymes in buffalo milk. In view of this fact and in order to find a parameter to assess the presence of cow or ewe milk in samples of water buffalo Mozzarella cheese, attention was focused on the mass region of the whey proteins. As the molecular masses of cow, ewe and water buffalo whey proteins are very different and due to the MALDI-TOF-MS resolution in this mass region (14-19 kDa) being high enough to resolve them, lactalbumins and lactoglobulins have been used as biological markers for the evaluation of possible fraudulence in water buffalo Mozzarella cheese production. The mass difference between buffalo and ewe whey proteins is 3/478 Da for lactalbumins and 3/4116 Da for lactoglobulins, therefore making it possible to use both proteins for the evaluation of ovine milk sophistication of fresh water buffalo Mozzarella cheese [206]. The situation is very similar for the MALDI-TOF-MS analysis of Mozzarella prepared with a 90:10 mixture of water buffalo and ewe milk. Even in the case of two mass ranges presented (14-18 kDa), the mass signals of water buffalo and ovine milk proteins are well separated, and the existence of ewe milk can be readily evaluated. The study of other Mozzarella cheese samples obtained from different percentages of ewe milk added to water buffalo milk demonstrate that the response of the instrument is linear up to a limit of 2%. The EU reference methodology to detect bovine proteins in dairy products is based on gel isoelectric focusing of g-caseins after plasminolysis (EC Regulation No. EC 273/2008) with a detection limit for bovine milk in buffalo cheese products of about 1%.