## 3 Results
### Differences in 2-DE Spots Depending on Mastication Conditions (CS, CG, P)  
An example of a gel with the protein spots quantified for analysis is shown in Figure 3.  
The changes in the salivary protein spots among the three mastication conditions, as well as the identification of spots by MS, are shown in Table 1.  
Figure 3: Example of a representative 2-DE gel and the selected spots for their quantification and comparison among conditions. Figure 2: Saliva flow rate variation between the periods before (T0) and after (T1) each chewing condition (mean \(\pm\) standard deviation; different upper letters (a \(\
eq\) b) represent differences between periods for \(p<0.05\)). \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \multirow{2}{*}{**Spot No. **} & \multicolumn{3}{c}{**CG**} & \multicolumn{3}{c}{**CS**} & \multicolumn{3}{c}{**P**} \\ \cline{2-10}  & \(p\)**-Value** & **Change** & **Ratio** & **(T1/T0)** & \(p\)**-Value** & **Change** & **Ratio** & **Change** & **Ratio** (T1/T0)** & **Identification** \\ \hline
**25** & 0.5 & & 0.96 & 0.028 & \(\downarrow\) & 0.55 & 0.753 & & 0.98 & Serum albumin \\ \hline
**28** & 0.043 & \(\downarrow\) & 0.54 & 0.028 & \(\downarrow\) & 0.42 & 0.463 & & 0.97 & Serum albumin \\ \hline
**31** & 0.138 & & 0.83 & 0.028 & \(\downarrow\) & 0.42 &  
As can be observed, the effects of chewing on the salivary protein profile were much more pronounced for carrots (CS and CG) than for the non-flavoured control parafilm (P). In fact, only one protein spot (spot 45, previously identified as the Ig \(\alpha\)-1 chain C region) showed significant decreases due to the mastication of parafilm, while the mastication of carrot slices (CS) and grated carrot (CG) resulted in significant differences in the levels of 37 and 9 protein spots, respectively. From these nine protein spots significantly affected by grated carrot mastication, eight of them also changed after the mastication of sliced carrots, while they were not affected by parafilm mastication. Of those, the levels of seven spots increased in saliva after carrot mastication, whereas the levels of one spot (spot 28, previously identified as serum albumin) decreased after carrot mastication (CG, CS). Of the seven spots that increased due to mastication of both shapes of carrots, two of them (spots 143 and 202) were identified as amylase (from Western blot analysis, as further detailed) and two of them (spots 227 and 228) as the Ig kappa chain C region, while three of them (spots 90, 93, and 198) were not identified. \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Spot No.**} & \multicolumn{2}{c}{**CG**} & \multicolumn{2}{c}{**CS**} & \multicolumn{2}{c}{**P**} & \multirow{2}{*}{**Identification**} \\  & \(p\)**-Value** & & **Range** & **Ratio** & & **(T1/T0)** & & \(p\)**-Value** & **Change** & **Ratio** \\ \hline
**301** & 0.893 & & 1.13 & 0.028 & \(\uparrow\) & **2.38** & 0.463 & 1.29 &
\begin{tabular}{c} Prolactin-inducible \\ protein \\ \end{tabular} \\ \hline
**311** & 0.225 & & 1.39 & 0.028 & \(\uparrow\) & **2.49** & 0.116 & 1.54 & n.i. \\ \hline
**312** & 0.345 & & 1.87 & 0.028 & \(\uparrow\) & **2.84** & 0.917 & 1.11 &
\begin{tabular}{c} Prolactin-inducible \\ protein \\ \end{tabular} \\ \hline
**313** & 0.345 & & 0.92 & 0.028 & \(\uparrow\) & **2.02** & 0.6 & 1.09 & n.i. \\ \hline
**334** & 0.043 & \(\uparrow\) & 2.73 & 0.917 & & 0.99 & 0.917 & 1.05 & Cystatin SN \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._  
Figure 4: Examples of images of membranes incubated with anti-alpha-amylase primary antibody, highlighting the presence of several different spots of this protein. Among the 37 spot proteins that showed significant differences after the consumption of CS, 9 of them presented significant decreases after the consumption of slices of carrot, whereas 28 spots presented significant increases.