# 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)
Thus, the stabilized sourdoughs could be used as artisanal starters for the fermentation processes associated with the agirofood byproducts bio-valorization through multiple bioprocessing techniques (fermentations, bioconversions) that support the circular economy and the environmental protection [88; 89]. Finally, the postbiotics produced by LAB have multiple implications in the food and feed preservation and safety assurance. The in vivo effects of the fermented products as a valuable source of biotics (prebiotics, postbiotics) are intensively studied regarding the health effects on both humans and animals, considering also the microbiome complexity and functionality [90]. In the current study, the RSM model was validated by the multi-response optimization tool, considering all the analyzed response variables (see Table 2). Based on the mathematical modelling studies, the following optimal combination of independent variables was established: 475.0 dough yield; co-inoculation of 2.9% (\(v/w\)) of _L. paracasei_ ssp. _paracasei_ MIUG BL 21 strain and 5.0% (\(v/w\)) of _L. parabuchneri_ MIUG BL 24 strain; 16.9% (\(w/w\)) of okara; 31.4 \({}^{\circ}\)C fermentation temperature; and 66.1 h of fermentation time. The results for the considered responses under the optimized conditions are listed in Table 5. This optimized sourdough variant was experimentally produced, and the parameters analyzed. These experimental results excellently fit with the predicted ones (Table 5), as proved by the composite desirability (94%)--which allowed the validation of the statistically optimized model [91]. Such value for the composite desirability is comparable to that of 98% obtained in a study for the formulation of gluten-free noodles [42]. Our results for the antimicrobial properties and TTA (Table 5) are in agreement to those reported in the literature. Particularly, inhibition ratios between 20 and 60% against _Aspergillus_ spp. and _Penicillium_ spp. indicator strains were determined for a chickpea sourdough [28], whereas a TTA value of 43.4 was obtained for a hemp sourdough fermented for 24 h at 30 \({}^{\circ}\)C [92]. The satisfactory results, determined by the optimized gluten-free formulation from our work, suggest that this fermented product, obtained in controlled SSF fermentation conditions involving selected strains of LAB, has valuable properties with impact on enhancing the antimicrobial and technological properties of the gluten-free sourdough bread that will be assessed in the future. The Organic Acids, Flavonoids and Polyphenolic Compounds Bioactive Composition of the Optimized Sourdough  
Under the optimal fermentation conditions, the resulting gluten-free sourdough was subjected to chromatographic analysis, by RP-HPLC-DAD, to investigate its profile of organic acids, flavonoids and polyphenic compounds, as depicted in Figures 5-7. Following the established optimized variant, the LAB strains involved in the fermentation of the complex substrate made of chickpea, quinoa, buckwheat and okara increased the amount of lactic (peak 3) and propionic acids (peak 6), after 66 h of fermentation at 31.4 \({}^{\circ}\)C (Figure 5a), in comparison to the unfermented sample (Figure 5b). On the other hand, based on the peak's intensity, the optimized fermented sample showed a profile where a slight increase of the amount could be observed for the non-identified peaks 7 and 8 in Figure 5a, when compared to the same peaks in Figure 5b. Taking into consideration the organic acids that eluted from the fermented sourdough (Figure 5a), it can be stated that the mixed inoculum of the selected LAB starter used for fermentation displayed a heteroformative metabolism that could increase the antimicrobial and technological features of baked goods [93]. A dairy product fermented by _L. rhamns_ showed antimicrobial properties due to the acetic, lactic, citric, propionic and succinic acids synthesized in the fermented medium [94].