# 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)  
Following the combinations of factors at different levels (Table 1) obtained by PBD, the variations between the experimental results for the studied responses were observed, as it is shown in Table 3. The acidification capacity expressed as TTA varied between 5.90 to 38.90 mL NaOH 0.1N. As expected, the lowest TTA value was determined for the uninoculated dough incubated for 24 h at 25 \({}^{\circ}\)C. The most acidified sample was obtained with a mixed inoculum containing the LAB strains of \(L\). _paracasei_ ssp. _paracasei_ MIUG BL 21, \(L\). _brevis_ MIUG BL 38 and _Leu_. _mesenteroides_ ssp. _mesenteroides_ MIUG BL 40 after the fermentation of a medium containing 10% (_w/w_) okara powder. For some samples, a mixed inoculum did not determine a high level of TTA, most likely due to the competitive and/or antagonistic effects between the studied LAB strains. Similar results were reported by [PERSON] et al. (2018), who reported differences between the TTA values when various strains of _Lactobacillus_ spp. and _Pediococcus pentosaceus_ were co-inoculated as starter cultures for the wheat-based sourdoughs. For some LAB strains, a fermentation temperature of 35 \({}^{\circ}\)C caused TTA values below 4.0 mL NaOH 0.1N, whereas a mixed inoculum composed by the four studied LAB strains caused a significant increase in TTA values to 13.0 mL after the sourdough's fermentation at 28 \({}^{\circ}\)C. The reported results underline the importance of the fermentation temperatures on the microbial and enzymatic activities of pseudocoreal and legume flours. \begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c} \hline \hline \multirow{3}{*}{**Run**} & \multicolumn{10}{c}{**Factors**} & \multicolumn{10}{c}{**Respenses**} \\ \cline{2-13}  & \multirow{2}{*}{\(A\)} & \multirow{2}{*}{\(B\)} & \multirow{2}{*}{\(C\)} & \multirow{2}{*}{\(D\)} & \multirow{2}{*}{\(E\)} & & \multirow{2}{*}{\(F\)} & & & & & **TTA**, & **L \%** & **I, \%** & **I, \%** & **I, \%** \\  & & & & & & & & & **0.1N** & _A. niger_ & _A. flavus_ & _Penicillium_ & _Bacillus_ \\  & & & & & & & & & & **0.1**N & _A. niger_ & _21_ & _A. flavus_ & _Penicillium_ & **spsp.**1 \\ \hline
1 & 250.00 & 4.00 & 4.00 & 0.00 & 4.00 & 0.00 & 25.00 & 24.00 & 11.23 & 21.27 & 56.36 & 6.90 & 2.23 \\
2 & 700.00 & 0.00 & 0.00 & 0.00 & 4.00 & 10.00 & 45.00 & 24.00 & 11.00 & \(-\)0.81 & 43.18 & \(-\)1.55 & \(-\)9.48 \\
3 & 700.00 & 0.00 & 4.00 & 0.00 & 0.00 & 0.00 & 45.00 & 96.00 & 10.67 & 41.64 & 45.45 & 8.90 & \(-\)34.50 \\
4 & 250.00 & 4.00 & 0.00 & 0.00 & 0.00 & 10.00 & 45.00 & 96.00 & 13.73 & 31.26 & 57.73 & 29.83 & \(-\)36.07 \\
5 & 700.00 & 4.00 & 0.00 & 4.00 & 4.00 & 0.00 & 45.00 & 24.00 & 14.77 & \(-\)8.88 & 51.82 & 16.21 & 2.69 \\
6 & 250.00 & 4.00 & 4.00 & 4.00 & 0.00 & 10.00 & 45.00 & 24.00 & 10.23 & \(-\)6.43 & 51.36 & 55.00 & \(-\)16.42 \\
7 & 700.00 & 4.00 & 0.00 & 4.00 & 0.00 & 0.00 & 25.00 & 96.00 & 27.10 & \(-\)7.97 & 51.82 & 21.55 & 16.60 \\
8 & 700.00 & 0.00 & 4.00 & 4.00 & 0.00 & 10.00 & 25.00 & 24.00 & 22.87 & \(-\)32.96 & 29.09 & \(-\)2.41 & \(-\)11.59 \\
9 & 250.00 & 0.00 & 4.00 & 4.00 & 4.00 & 0.00 & 45.00 & 96.00 & 11.60 & 10.03 & 50.00 & 23.45 & \(-\)5.46 \\
10 & 250.00 & 0.00 & 0.00 & 4.00 & 4.00 & 10.00 & 25.00 & 96.00 & 28.77 & 10.76 & 43.18 & 24.83 & 25.55 \\
11 & 250.00 & 0.00 & 0.00 & 0.00 & 0.00 & 0.00 & 25.00 & 24.00 & 5.90 & 13.55 & 15.00 & \(-\)21.03 & 25.27 \\
12 & 700.00 & 4.00 & 4.00 & 0.00 & 4.00 & 10.00 & 25.00 & 96.00 & 38.90 & \(-\)6.72 & 50.91 & 26.90 & 65.22 \\ \hline \hline \end{tabular}  
* Negative values resulting from the calculation of the inhibition ratio. These values have statistical relevance for the mathematical modelling.  
\end{table} 
Table 3: Experimental results of the total titratable acidity (TTA) and inhibition ratio (_I_, %, defined by the Equation (2)) obtained by Plackett–Burman Design (PBD) analysis.