# 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)
Similarly, in the unfermented sample (Figure 6b), the \(p\)-coumaric acid (peak 11\({}^{\prime}\)), vanillic (peak 12\({}^{\prime}\)) and caffe acid (peak 16\({}^{\prime}\)) derivatives were identified [102,103]. The acidic conditions of the fermented medium and other microbial enzymes may significantly affect the composition of flavonoids and polyphenols in the final sourdough, thus resulting in structural changes. Another reason for this difference between the uninoculated and fermented sourdoughs can be related to the ratio between the bounded and free phenolic compounds and flavonoids, the first ones being more stable. The concentration of the phenolic acids in some gluten-free cereals was studied by [PERSON] et al. [104] using HPLC methods, to conclude that the content of bounded phenolic acids was higher than the free phenolics. Flavonoids (quercetin, catechchin and kaempfer), tannins (proantocyanidins), phenolic acids (caffeic, chlorogenic, ferulic, gallic, vanillic) and their derivatives were identified in buckwheat [105,106], quinoa, and chickpea seeds [107,108,109]. The intensity of each compound is different based on the wavelength used for the HPLC data acquisition [110], our results being supported by these above-mentioned works. The antioxidant properties of pseudocoreals and legumes are related to the applied processing methods [111,112]. Consequently, it was highlighted by [PERSON] et al. [113] that the levels of saponins and antinutritional factors depend on the milling process of quinoa. Furthermore, the antioxidant properties of buckwheat were modified after ultrasound treatment [114] whereas the importance of the HPLC elution solvent (acetonitrile) for the extraction of isoflav from okara was stated by [PERSON] et al. [115]. [PERSON] et al. [116] and [PERSON] et al. [117] affirmed that the molecular complexes between bioactive compounds and starch or proteins from flours can be formed in food systems, and their stability and properties are affected mainly by the pH, temperature and nutritional composition of the matrix [118,119]. The above-mentioned conclusion would probably be the reason why the bioactive composition of our samples was different, along with the fact that the fermentation substrate was obtained by combining several flours, with different biologically active compound profiles. Figure 7: RP-HPLC-DAD (\(\lambda\) = 320 nm) chromatograms for the flavonoids and polyphenols in the optimized sourdough (**a**), and the unfermented 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: 4—vanillic acid; 9—ferulic acid; 5\({}^{\prime}\)—chlorogenic acid; 7\({}^{\prime}\)—vanillic acid; 13\({}^{\prime}\)—ferulic acid; 23\({}^{\prime}\)—quercetin, 24\({}^{\prime}\)—gallic acid. Finally, it is obvious that different chromatographic profiles of the fermented optimized sourdough and control sample (unfermented sourdough) were obtained in this work, such changes being justified by the specific metabolic activity of the selected LAB strains co-culture (_L. paracasei_ ssp. _paracasei_ MIUG BL 21 strain and _L. parabuchneri_ MIUG BL 24 strain), in correlation with intrinsic and extrinsic factors' variation, under the optimized fermentation conditions established by RSM. Some researchers found that _Lactobacillus_ spp. strains can synthesize enzymes (oxidases, esterases, decarboxylases and dehydrogenases) able to destabilize specific complexes and affect the individual structures of the polyphenic compounds [120,121]. These results confirm that, by RSM, the interactions between the previously selected independent variables were established in order to determine the optimal conditions to achieve a gluten-free sourdough with enhanced antimicrobial properties against spoilage molds and bacteria, correlated to the organic acids content, phenolic content and functional properties.