# 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)
Different culture medium constituents, fermentation temperatures and incubation times or inoculum sizes were screened via Plackett-Burman Design (PBD), Response Surface Methodology (RSM) or Box-Behnken Design (BBD) to identify the most suitable conditions for the specific enzyme biosynthesis [63; 74]. The statis  
Figure 4: Surface (**a**) and contour (**b**) plots for the two-way interactions between the significant fermentation variables \(B\) and \(C\), on the antibacterial activity (inhibition ratio, %) against _Bacillus_ spp. The mean of the factors \(A\) to \(F\) is given in Table 2.  
Figure 3: Surface (**a**) and contour (**b**) plots for the two-way interactions between the significant fermentation variables \(B\) and \(C\), on the antifungal activity (inhibition ratio, %) against _Penicillium_ spp. The mean of the factors \(A\) to \(F\) is given in Table 2.  
process optimization are very common in food science and other biotechnological fields, to decrease the number of time-consuming experimental runs, costs and technical resources. In particular, various factors were screened by PBD and RSM in order to establish those with significant effects on the TTA levels of a sourdough fermented with _L. paracasei_ ssp. _paracasei_[75]. A stable matrix able to ensure the _L. curvatus_ viability during storage was optimized by RSM, being successfully used in the sourdough technology [76]. Moreover, the variation of the dough yield between 175-225 was statistically studied to conclude that this factor positively affects the textural characteristics of a sourdough cake [77]. Sourdough bread's physicochemical and textural properties (viz. TTA, porosity, chewiness and hardness) were optimized after the RSM analysis by [PERSON] et al. (2019) [78]. The regression coefficients reported in the above-mentioned study varied between 43.4-97.9% and 29.5-95.2% for R\({}^{2}\) and adjusted R\({}^{2}\) (R\({}^{2}\)adj), respectively. In our RSM analyses, values in the same range were attained (viz. R\({}^{2}\) 71.4-87.7% and R\({}^{2}\)adj 40.4-74.4%). The antifungal effect of LAB metabolites has been intensively studied by several researchers to conclude that the fungal growth is related to multiple biotechnological factors. The most important factors have been claimed to be the type of exogenous LAB starter strains used for fermentation and their initial concentration. _Aspergillus flavus_, _Aspergillus niger_ and _Aspergillus tubingensis_ indicator strains were successfully inhibited by _Lactobacillus_ spp. and _Leuc. mesenteroides_ strains studied by [PERSON] et al. [67] and [PERSON] et al. [79]. In addition, the fungal contamination of bread by _Penicillium_ spp. strains was delayed by 3 days when a whole wheat sourdough inoculated with selected strains of _L. plantarum_ was used [56]. The same effect was observed when 30% (\(w/w\)) of a commercial sourdough was used [80] or essential oils from aromatic plants [81], respectively, when a mixture of eugenol-cital was added to the wheat bread doughs [82]. A delayed bacterial spoilage determined by _Bacillus subtilis_, _B. cereus_ and _B. licheniformis_ was observed after the in vitro assays using _Lactobacillus_ spp. strains for the sourdough bread making [79; 83]. Finally, the study conducted by [PERSON] et al. [84] led to the conclusion that bread ropy spoilage can be effectively delayed by up to 3 days when the moisture content and pH value are controlled. The scientific works mentioned above reinforce the LAB strains' effects that contribute to sustainability and innovation in the production of food and feed that positively affect quality of life [85]. Moreover, sustainable approaches were reported by some authors regarding the utilization of the microorganisms from the sourdough microbiota as starters for other fermentation processes, results with positive impact on the economic efficiency and diversification of the products [86; 87].