## 3 Results
### Low-Molecular-Weight Peptides Fractionation  
WSEs obtained from sourdoughs and breads prepared with the different combination of microorganisms and flours were freeze-dried, re-suspended in water with 0.1% TFA added, and finally, 500-mL aliquots were applied on reverse-phase chromatography (Figure 1). A separation between low and high molecular species is clearly visible in the chromatographic layouts, thus enabling us to collect fractions by hand-picking: the LMW peptides were recovered either for doughs (Figure 1a) and breads (Figure 1b) based on the retention times. For each combination, the 5-20-min fractions were collected and analyzed by SDS-PAGE (Figure 1c). On the basis of their retention times and the protein content, the fractions were called LMW peptides, assayed to determine the protein content, lyophilized, and finally re-suspended in bi-distilled water at the same protein concentration (0.05 mg/mL) useful to treat cell cultures according to Table 3. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Dough** & **Final pH** & **\(\Delta\)pH** & **Final TTA** & **\(\Delta\)V/V0 \(\times\) 100** & **LAB (CFU/g)** & **Yeasts (CFU/g)** \\ \hline Aubusson & 4.37 \(\pm\) 0.20 & 0.64 \(\pm\) 0.11 & 5.10 \(\pm\) 0.6 & 67 \(\pm\) 5 b  & (8.75 \(\pm\) 0.83) \(\times\) 10\({}^{8}\) & (5.83 \(\pm\) 0.73) \(\times\) 10\({}^{6}\) \\ \hline Skorpion & 4.39 \(\pm\) 0.25 & 0.70 \(\pm\) 0.11 & 5.00 \(\pm\) 0.1 & 94 \(\pm\) 8 c  & (8.15 \(\pm\) 0.92) \(\times\) 10\({}^{8}\) & (7.55 \(\pm\) 0.82) \(\times\) 10\({}^{6}\) \\ \hline Rondo & 4.42 \(\pm\) 0.16 & 0.67 \(\pm\) 0.04 & 4.35 \(\pm\) 0.5 & 11 \(\pm\) 0 a  & (8.65 \(\pm\) 1.34) \(\times\) 10\({}^{8}\) & (6.45 \(\pm\) 0.64) \(\times\) 10\({}^{6}\) \\ \hline \end{tabular} Values in the same column with different letters (a–c) are significantly different (\(p\) \(<\) 0.05).  
\end{table} 
Table 2: Acidification (pH, total tittatable acidity), volume increase (%, \(\Delta\)V/V0), and microorganism concentrations (LAB and yeast) of the doughs at the end of the 4-h fermentation. \(\Delta\): difference between the final and the initial value. Aubusson: dough with a sourdough made with wheat flour cv Aubusson; Rondo: dough with a sourdough made with barley flour cv Rondo. Results are expressed as average \(\pm\) standard deviation. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Flour** & **Bacteria and Yeasts** & **mg/mL** \\ \hline \multirow{3}{*}{Doughs} & \multirow{3}{*}{Aubusson} & _L. plantarum_ Fi13 & \multirow{3}{*}{4.44} \\  & & _L. brevis_ LD66 & & \\  & & _S. cerevisiae_ D20Y & \\ \hline \multirow{3}{*}{Skorpion} & _L. plantarum_ Fi58 & \multirow{3}{*}{5.17} \\  & & _L. santhrancisensis_ Fi33 & \\  & & _S. cerevisiae_ L6Y & \\ \hline \multirow{3}{*}{Rondo} & _L. plantarum_ Fi13 & \multirow{3}{*}{3.76} \\  & & _L. santhrancisensis_ Fi33 & \\ \cline{1-1}  & & _S. cerevisiae_ L6Y & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: LMW peptides from doughs and breads: flours, microorganism used for fermentation, and peptide concentration. Protein was determined by the BCA method and expressed in mg/mL. Figure 1: HPLC fractionation of WSEs from doughs **(a)** and breads **(b)**. First, 500- \(\mu\)L aliquots of each sample (Aubusson, Skorpion Rondo) were applied on a reverse phase column (Vydac, 4.6 \(\times\) 250 mm; 5 \(\mu\)m; 300 A) and eluted with a non-linear gradient (Solvent A: H\({}_{2}\)O + 10 mM TFA; Solvent B: CH\({}_{3}\)CN + 10 mM TFA. Gradient: 0–10 min 30%B, 10–50 min 50%B, 50–60 min 100%B). **(c)** SDS-PAGE analysis of extracts and peptides from breads. 1 Bio-rad All Blue Standard; 2 Aubusson; 3 Aubusson 5–20 min; 4 Rondo; 5 Rondo 5–20 min; 6 Skorpion; 7 Skorpion 5–20 min.