# Statistical Approach to Potentially Enhance the Postbiotication of Gluten-Free Sourdough
## 2 Materials and Methods  
### Materials, Microorganisms and Culture Conditions  
The LAB strains used in this study were previously selected based on specific functional properties, respectively, the acidification capacity, the production of exopolysaccharidecharides and the antimicrobial spectra at different pH values and temperatures [45]. Specifically, four LAB strains as part of the Microorganisms Collection of Dunarea de Jos University of Galati (acronym MIUG), corresponding to three _Lactobacillus_ spp. strains (coded MIUG BL 21, MIUG BL 24 and MIUG BL 38) and one _Leuconostoc_ spp. strain (coded MIUG BL 40) were tested. The presumptive identification at species level was carried out by Biolog Microbial Identification System (Biolog Inc., Hayward, CA, USA) using 94-well Gen III MicroPlates for aerobes. The manufacturer's instructions were carefully followed, as previously described [46; 47; 48]. The LAB strains were presumptively identified as follows: MIUG BL 21 being _Lactobacillus_ spp. _paracasei_; MIUG BL 24 being _Lactobacillus parabuchneri_; MIUG BL 38 being _Lactobacillus brevis_ and MIUG BL 40 being _Leuconostoc mesenteroides_ ssp. _mesenteroides_. The stock cultures of LAB were reactivated in de Man, Rogosa and Sharpe (MRS) broth. After 48 h of incubation at 37 \({}^{\circ}\)C (Binder BF4000, Tuttlingen, Germany) under aerobiosis, each inoculum was diluted in MRS broth to reach an optical density at the wavelength of 600 nm (OD\({}_{600\text{nm}}\)) of 2.0, using a spectrophotometer (Libra S22 UV-VIS, Biochrom, Cambridge, UK) [49]. The cell viable counts of LAB in each inoculum was also determined using saline (0.9%, \(w/v\)) serial dilutions inoculated in MRS agar plates [50; 51]. For the established optical density (2.0 at \(\lambda\) = 600 nm) and incubation conditions (48 h, 37 \({}^{\circ}\)C, aerobiosis), an inoculum size of ca. 1 \(\times\) 10\({}^{7}\) colony-forming units per milliliter (CFU/mL) was obtained for all LAB strains. The mold strains used in this study as the indicator microorganisms for the antifungal effect were isolated and purified as reported in our previous work [45]. Prior to their use, the stock cultures of _Aspergillus niger_, _Aspergillus flavus_ and _Penicillium_ spp. strains were reactivated on Potato Glucose agar (PGA) slants at 25 \({}^{\circ}\)C (Pol-Eko Aparatura, Poland) for 96 h under aerobiosis. The indicator microorganism used for the antibacterial activity was a _Bacillus_ spp. strain previously isolated from quinoa seeds, as reported elsewhere [45]. This bacterial strain was routinely reactivated from stock cultures by cultivation overnight on Nutrient broth at 37 \({}^{\circ}\)C under aerobiosis. Whole seeds of chickpea, white quinoa, buckwheat, and non-genetically modified soybeans were purchased from Solaris Plant S.R.L, Bucharest, Romania. The seeds were finely ground with a coffee mill for approximately 5-7 min (DeLonghi, Bucharest, Romania), and the respective flours were further used for the experiments in this study. The soybeans were previously soaked in tap water for 12 h at room temperature. Afterwards, soymilk was obtained from the soaked beans and water 1:4 (\(w/v\)) using a blender (Bosch, Cluj, Romania) for 5 min [33]. Finally, the solid residue from the previous soymilk's preparation, called okara, was collected, freeze-dried ([PERSON], Osterode am Harz, Germany) and stored at 4 \({}^{\circ}\)C in dark airtight jars. For the formulation of the fermentation medium, the chickpea, quinoa, and buckwheat flours were mixed in equal proportions (1:1:1, \(w/w\)) together with the amount of okara powder ranging between 0-16.9 g% (\(w/w\)) according to the PBD or RSM runs and sterilized at 121 \({}^{\circ}\)C for 15 min (Panasonic MLS-3871L, Bucharest, Romania). The distilled water was sterilized separately from the dry ingredients. The fermentation substrates were made by mixing the sterilized ingredients under aseptic conditions in a laminar-flow chamber (SafeFast, Faster, Cornaredo, Italy). The dough yield (DY) was calculated using Equation (1).