Urea Polyacrilamyde Gel Electrophoresis (Urea-PAGE)
This technique separates proteins according to their mass-to-charge ratio, using the naturally occurring negative charges that each protein shows when solubilized in alkaline buffers. All the casein fractions of milk are well resolved using this technique. On the other hand, whey proteins are not resolved at all; they interfere with the resolution of caseins and therefore, they are generally removed before the analysis. Looking from the top to the bottom of the gel, we find y- CN, p-CN, aS2’ and aS1-CN (Figure 220.127.116.11). aS2-CN appears as a series of bands just above aS1. This latter is generally split into two bands as a result of eight or nine phosphoserine residues in the sequence (Fox & McSweeney, 1998).
During the seasoning of cheese and with the progress of proteolysis, y-CN become more evident on the upper part of the urea gel and at the same time the proteolitc
Figure 18.104.22.168 Urea-PAGE of casein fractions of mozzarella cheeses identified as reported by Faccia et al. (2014). The first three lanes are samples of mozzarella cheese made with not fresh curd and the last three are mozzarella cheeses made with fresh milk.
products of aSi appear in the lower part of the gel, just below the asi. Since all caseins and their degradation products are well resolved, this technique is a very useful tool to evaluate the proteolytic process in cheese.
The proteolysis in cheese is affected by many factors such as technology, seasoning conditions, and duration. The analysis of polypeptides formed by this biochemical process could be used in the characterization of protected designation of origin (PDO), protected geographical indication (PGI), and traditional specialities guaranteed (TSG) cheeses. In fact, the Urea-PAGE together with the analysis of water-soluble fraction by reversed phase high-performance liquid chromatography, followed by principal component analysis, is proposed as a method to ascertain cheese authenticity (Guerreiro et al., 2013). The use of Urea-PAGE as a tool for process control was proposed by Faccia et al. (2014). These authors suggested the use of the aS1-I casein (f24-199), produced by the action of the residual rennet enzyme, as a possible marker to reveal the use of stored curd in Fiordilatte cheese.
The aS1-I (f24-199) naturally occurs in curd, but the quantity found in mozzarella cheese is proportional to the elapsed time between the rennet addition to the milk and the stretching phase in the production process. The amounts of aS1-I does not increase during shelf life in the mozzarella; in fact, the high temperature at which the curd is submitted during stretching inactivates chymosin. In other cheeses, chymosin remains active during the storage, and therefore aS1-I increases progressively. This fragment is easily detectable by Urea-PAGE (Figure 22.214.171.124) and 2-DGE. Faccia et al. (2014) identified the spots of 2-DGE by trypsin digestion and Matrix Assisted Laser Desorption Ionization Time of Fly (MALDI-ToF). In this work the quantitative analysis of aS1-I casein was performed by densytometric image analysis, and calculated as relative quantity. Due to the peculiarity of this test, it was only suitable on fresh-made “pasta filata cheeses" This method is suggested for the protection of high-quality mozzarella cheese.
The primary structural alignment of cow, sheep, and goat as1 casein is shown in Figure 126.96.36.199. Positively and negatively charged residues, together with phosphoserine, are marked with colors. The differences in the amino acid residues among species were previously discussed. The aS1-CN has a different number of negative charges among the species considered, leading to a different Urea-PAGE profile (Figure 188.8.131.52). This could be a useful tool to identify mixtures of milks belonging to different species, as suggested by Mayer (2005), and Veloso, Teixeira, and Ferreira (2002).