## Results and Discussion
### Effect of the Addition of Freeze-Dried GPP on Pasta Gluten Network
#### 3.2.3 Unextractable Polymeric Proteins (UPP) and S-S Bonds Formation Assessment  
The strength of the gluten network was studied by evaluating both the proportion of \"Unextractable Polymeric Proteins\" (% UPP) and the amount of S-S groups formed during pasta making. The percentage of unextractable polymeric proteins (% UPP) in pasta samples was calculated from the peak areas of the SDS-extractable and SDS-unextractable proteins, obtained by proteins fractionation through SE-HPLC and are shown in Figure 3.  
The formation of high-molecular-weight protein aggregates of cooked pasta samples was significantly higher (\(p<0.05\)) than the raw samples, because of the formation of greater polymers between different classes of gluten and non-gluten proteins through covalent bonds during the cooking process [30; 31]. In total, the 100% semolina pasta sample and 5% GP-fresh pasta sample showed the highest and similar values of UPP percentage for both raw and cooked samples (24.37% vs. 23.22% for raw samples and 50.92% and 54.22% for cooked samples, respectively). On the contrary, it was possible to note that the 10% GP-fresh pasta sample showed a significantly lower (\(p<0.05\)) percentage of UPP, confirming a different protein arrangement of this sample, where the SDS-extractable fraction peak area was the highest, as shown in Table 3. This result was confirmed by the amount of S-S bonds (Table 4), which decreased significantly (\(p<0.05\)) from 100% semolina pasta sample to that containing 10% of GPP in the cooked samples (36.19 to 3.44 nmol/mg of proteins, respectively). \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & & **Grape** & **100\% Raw** & **100\%** & **5\% Raw** & **5\% Cooked** & **10\% Raw** & **10\% Cooked** \\  & & **Pomace** & **Semolina** & **Semolina** & **GP-Fresh** & **GP-Fresh** & **GP-Fresh** & **GP-Fresh** & **GP-Fresh** \\  & & **Powder** & **Fresh Pasta** & **Fresh Pasta** & **Pasta** & **Pasta** & **Pasta** & **Pasta** \\ \hline \multirow{2}{*}{**Peak 1**} & mAu+s & 42662.50 \(\pm\) 533.87 & / & 35610.90 \(\pm\) 53.74 & 161.55 \(\pm\) & 25712.60 \(\pm\) & 170 \(\pm\) 5.9 & 23641.60 \(\pm\) & 942.90 \(\pm\) \\  & \% & 21.43 & & 23.90 & 48.44 & 1800.01 & 268.28 & 190.78 \\  & & 21.43 & & 23.90 & 2.18 & 23.07 & 1.06 & 25.44 & 2.13 \\  & & & & & & & & & \\  & & & & & & & & & \\  & & & & & & & & & \\  & & & & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Peaks area of SDS-extractable proteins for semolina flour, grape pomace powder, and raw and cooked pasta samples. However, the decrease in S-S groups also corresponded to a significant decrease (\(p<0.05\)) of SH groups, from 98.98 to 66.03 nmol/mg of proteins, comparing 100% cooked semolina pasta with 10% cooked GP-fresh pasta sample. These results suggested a possible interaction between the gluten proteins and polyphenols occurring in the GPP, also caused by non-disulfide covalent bonds. Moreover, high temperature increases polyphenols oxidation, which in turn, increases the reactivity and interaction with proteins. It is well established that reversible or irreversible covalent bonds can occur in protein-polyphenol conjugates between quinones or semiquinones, enhanced by oxidative and/or thermal conditions, and nucleophilic groups of proteins, such as the \(\varepsilon\)-amino group of -Lys and sulfhydryl (thiol) group of -Cys [36]. This interaction could explain the increase in the antioxidant activity in the cooked pasta samples, as shown in Table 1, since it is well known that protein-polyphenol interaction enhances the antioxidant activity of phenolic compounds. Furthermore, a protein