# Statistical Approach to Potentially Enhance the Postbiotication of Gluten-Free Sourdough
## 3 Results and Discussion
### Sourdough Fermentation Process Optimization
#### 3.1.1 Selection of the Most Important Fermentation Parameters by Plackett-Burman Design (PBD)
Additionally, malic, tartaric and fumaric acids were identified in a wheat-soybean sourdough fermented with different mixed starters of _Lactobacillus_ spp. [95]. It is well-known that the amount and type of organic acids found after the fermentation of different matrices depend largely on the inoculum strain(s) and the composition of the fermentation substrate [72; 96]. The relatively high level of organic acids eluted as peaks 1 and 2 in the unfermented sourdough (Figure 5b) proves that it originated from the floors, as it was reported in some works [97; 98]. Indeed, the accumulation of organic acids in the pseudocreal and legume seeds is a stress adaptation to inadequate environmental conditions [99]. \begin{table}
\begin{tabular}{l c c} \hline \hline
**Response** & **Predicted Value** & **Experimental Value** \\ \hline TTA, mL NaOH 0.1N & 33.12 & 40.21 \(\pm\) 2.16 \\ Antifungal activity against _Aspergillus niger_ I\% & 70.20 & 71.42 \(\pm\) 1.00 \\ Antifungal activity against _Aspergillus flavus_ I\% & 94.30 & 95.00 \(\pm\) 0.50 \\ Antifungal activity against _Penicillium spp._ I\% & 94.70 & 95.71 \(\pm\) 0.70 \\ Antibacterial activity against _Bacillus_ spp. I\% & 100.00 & 100.00 \(\pm\) 0.00 \\ Composite desirability & 0.94 & \\ \hline \hline \end{tabular}
\end{table} 
Table 5: Predicted versus experimental results for the optimized sourdough. Inhibition ratios obtained according to Equation (2). The composition of the optimized gluten-free sourdough was also investigated by RP-HPLC-DAD and compared to the unrefmented sourdough, to observe the differences in the composition of the most important bioactive compounds. The lactic acid fermentation process clearly modified the concentration of the flavonoids and polyphenols determined at wavelengths of 280 nm (Figure 6) and 320 nm (Figure 7), respectively. In the uninoculated sourdough sample, 27 bioactive compounds were separated (Figures 6b and 7b), showing a higher diversity of these compounds when compared to the fermented sourdough (Figures 6a and 7a). The gallic acid content was higher than quercetin in the unrefmented sample (Figures 6b and 7b). In contrast, more than 10 flavonoids and polyphenols can be observed in the chromatograms presented in Figures 6a and 7a. In the fermented optimized sourdough, only some bioactive compounds with the highest peaks' intensity could be separated from the baseline. The peaks' intensities corresponding to caffeic and vanillic acids were higher in the control sample, their maximum absorbances being identified at 280 nm. On the other hand, ferulic acid was eluted at its maximum intensity in the fermented sourdough when the wavelength of 320 nm was used. The high number of  
Figure 5: RP-HPLC-DAD (\(\lambda\) = 210 nm) chromatograms for the organic acids in the optimized sourdough (**a**) and unrefmented optimized sample (**b**), according to the elution pattern and chromatographic conditions described in Section 2.4. The injection volume (IV) was 20 \(\mu\)L and the concentration of the sample was 400 mg/mL. Peak’s identification: 3—lactic acid; 5—acetic acid; 6—propionic acid. Figure 6: RP-HPLC-DAD (\(\lambda\) = 280 nm) chromatograms for the flavonoids and polyphenols in the optimized sourdough (**a**), and the unrefmented optimized dough (**b**), according to the elution pattern and the chromatographic conditions described in Section 2.4. The injection volume (IV) was 10 \(\mu\)L and the concentration of the sample was 400 mg/mL. Peak’s identification: 9—caffeic acid; 10—vanillic acid; 12—ferulic acid; 8′—chlorogenic acid; 9′—caffeic acid; 10′—vanillic acid; 23′—quercetin; 24′—gallic acid; 26′—epictechin. peaks eluted between 4-19 min (Figure 6a), corresponding to ferulic, gallic, cinnamic acid or quercetin derivatives, that were the effect of the metabolic activities of the selected LAB used as starter cultures, were preliminarily identified by comparing the retention times to those reported in the literature [100,101].