## 3 Results
### Binary Protein Mixture Ultrafiltration at pH 3.4  
In Figure 5, the fluxes obtained during the ultrafiltration of binary protein mixture (0.5, 1.0, 2.0 g\(\cdot\)L\({}^{-1}\)) at pH 3.4 are reported. When the initial protein concentration was 0.5 g\(\cdot\)L\({}^{-1}\), a constant flux was observed for about three hours (65 L\(\cdot\)h\(\cdot\)m\({}^{-2}\)); after that, the flux starts to decrease, reaching a value of 50 L\(\cdot\)h\(\cdot\)m\({}^{-2}\) after 5 h of continuous UF process in concentration mode. However, after washing the membrane with the buffer, the initial pure water permeance (6.70 \(\times\) 10\({}^{-8}\) (\(\pm\)1.68 \(\times\) 10\({}^{-9}\)) m\(\cdot\)Pa\({}^{-1}\)\(\cdot\)s\({}^{-1}\)) was restored (6.68 \(\times\) 10\({}^{-8}\) (\(\pm\)1.60 \(\times\) 10\({}^{-9}\)) m\(\cdot\)Pa\({}^{-1}\)\(\cdot\)s\({}^{-1}\)), demonstrating that in this case also, no irreversible fouling did occur. If the initial protein concentration was doubled to 1 g\(\cdot\)L\({}^{-1}\), a constant flux was observed (Figure 5) (64 L\(\cdot\)h\({}^{-1}\)\(\cdot\)m\({}^{-2}\)) for about two hours; after that it started to decrease, reaching a value of about 10 L\(\cdot\)h\({}^{-1}\)\(\cdot\)m\({}^{-2}\) after 4.3 h of UF process. If the initial protein mixture concentration was further increased to 2 g\(\cdot\)L\({}^{-1}\), a constant flux was observed for about 1.8 h; after that, a severe flux decrease was observed (Figure 5). Although a flux decrease was observed for both the highest concentrations used, the initial pure water permeance, also in this case, was completely recovered. The absence of irreversible fouling can be attributed to proteins and membrane electrostatic repulsion which avoids a possible chemical interaction between membrane and protein. The flux decrease was mainly due to reversible fouling, caused by the accumulation of the most retained protein on the retentate side of the membrane. In Figure 6, the electrophoretic profile related to permeates collected as a function of time for all the three initial protein concentrations tested is reported. When 0.5 g\(\cdot\)L\({}^{-1}\) was used (Figure 6a), just ALA can pass through the membrane, increasing its concentration as a function of time. Until a VRF of about 1.5, 95% of pure ALA was obtained with a recovery factor of 33% (Table 3). Figure 4: SDS-PAGE carried out on (**a**) standard solutions used to carry out molecular weight (MW) determination and quantification of proteins in binary protein mixture. 1: solution containing both ALA and BLG (1 g\(\cdot\)L\({}^{-1}\)); 2: wide range molecular weight marker (1:20 dilution); 3: internal molecular weight standard (formed by ALA, BLG, and BSA); (**b**,**c**) on permeates collected as a function of time, when UF process was carried out at pH 3. IS: initial solution concentration. The dilution of permeates samples and IS of (**c**) is: 1.2. Figure 5: Time course of the UF flux of binary protein mixture (ALA + BLG), carried out in concentration mode through charged membrane by using different initial mixture concentration; pH: 3.4, T: 25 \({}^{\circ}\)C, ionic strength: 0.1 M.  
Figure 6: SDS-PAGE carried on permeates collected as a function of time when the UF process was carried out at pH 3.4. IS: initial solution concentration. (**a**) IS: 0.5 g:L\({}^{-1}\); (**b**) IS: 1 g:L\({}^{-1}\); (**c**) IS: 2 g:L\({}^{-1}\) (IS and samples dilution: 1:2). Using 1 g\(\cdot\)L\({}^{-1}\) as initial protein concentration, in this case just ALA was also present in all the collected permeates (Figure 6b), and its concentration increased as a function of time. Despite the abovementioned flux decrease after two hours of UF, no BLG was present in the permeate, but it was retained on the retentate side of the membrane, causing reversible fouling and flux decrease.