# 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)
Taking into consideration that the fermentation temperature had a significant impact on the antibacterial activity of the sourdoughs against the _Bacillus_ spp. strain, and simultaneously proved to be an important factor regarding other generated models (viz., TTA and growth inhibition of _Aspergillus flavus_), the antibacterial activity of the sourdoughs was, therefore, included in the further studies by RSM. Overall, by analyzing all the models with the responses of interest using the PBD, factor \(E\) (_Leu. mesenteroides_ ssp. _mesenteroides_ MIUG BL 40 inoculum) previously identified as an important exopolysaccharideacharide producing strain ([PERSON] and [PERSON], 2018), it has not been statistically significant for the responses studied in this work. Thus, six factors were proved to be significant on the sourdough properties, chiefly dough yield, \(L\). _paracasei_ ssp. _paracasei_ MIUG BL 21 and _L. parabuchneri_ MIUG BL 24 strains' inoculum concentration, okara amount, fermentation temperature and time--thus being selected for the further optimization studies by RSM. 1.2 Optimization of Gluten-Free Sourdough's Solid-State Fermentation (SSF) by Response Surface Methodology (RSM)  
The factors \(A\), dough yield; \(B\), _L. paracasei_ ssp. _paracasei_ MIUG BL 21 strain inoculum (% _v/w_); \(C\), _L. parabuchneri_ MIUG BL 24 strain inoculum (% _v/w_); \(D\), okara (% _w/w_); \(E\), fermentation temperature (\({}^{\circ}\)C); and \(F\), fermentation time (h) (Table 2) previously selected (see Section 3.1.1) were used for the RSM analysis, the results for each response being listed in Table 4.  
(1) Acidification Capacity (TTA)  
Regarding the acidification capacity of the studied LAB strains in the sourdoughs, it can be observed from Table 4 that the highest values for the TTA were achieved at 35 \({}^{\circ}\)C after 60 h of fermentation with different inoculum types and concentrations. Analyzing the results (Table 4) in the sourdough samples with the lowest consistency (i.e., highest DY of 700), an increase in TTA was observed with increasing temperature and okara concentration, when the sourdough was inoculated uniquely with the _L. parabuchneri_ MIUG BL 24 strain. Moreover, the _L. paracasei_ ssp. _paracasei_ MIUG BL 21 strain produced higher amounts of organic acids when the fermentation time ranged between 60 h and 96 h. A synergistic effect on the TTA values was detected when the studied lactobacilli strains were used together and, simultaneously, when the fermentation medium was supplemented with 10% (_w/w_) okara powder and 96 h of fermentation was applied. The used indicator molds and the bacterial strains (i.e., _Aspergillus niger_, _Aspergillus flavus_, _Penicillium_ spp. and _Bacillus_ spp., respectively), were inhibited in different manners by the obtained samples of the sourdough formulated based on the optimization design and principles. The negative inhibition ratios were calculated. Thus, these values helped for a better discrimination of the results towards an optimized sourdough formulation. The most acidified sourdough sample--obtained with the co-culture of both selected lactobacilli strains determined a total inhibition of _Aspergillus niger_, _Penicillium_ spp. and _Bacillus_ spp., whereas only a growth inhibition ratio of 75.9% was attained for _Aspergillus flavus_. Another promising result was found when the sourdough was fermented with a co-culture of 2% (_v/w_) of each _L. paracasei_ ssp. _paracasei_ and _L. parabuchneri_ strain's inoculum at 35 \({}^{\circ}\)C for 60 h. The combination of these parameters along with the use of 16.9% (_w/w_) of okara powder provided a sourdough with the capacity to fully inhibit the growth of _Aspergillus flavus_, _Penicillium_ spp. and _Bacillus_ spp. Each mold and spoilage bacteria strains used as the indicators in this study were fully inhibited by specific sourdoughs prepared under certain combinations of factors obtained by the RSM model.