## 2 Materials and Methods
### Identification of the Isolates
#### 2.3.2 Molecular Identification Methods  
Isolation of total DNA. The DNA of the resultant cultures was isolated using the E.Z.N.A. Bacterial DNA Kit (OMEGA Bio-tek, Norcross, GA, USA) in accordance with the manufacturer's instructions. A 24 h broth culture obtained in MRS was used.  
RAPD. Strain differentiation was performed by a random amplified polymorphic DNA (RAPD) analysis [45] through PCR amplification with the primers rapd-4 (5\({}^{\prime}\)-AAGAGCCC GT-3\({}^{\prime}\)) or opp-7 (5\({}^{\prime}\)-GTCCATGCCA-3\({}^{\prime}\)) used separately. The PCR reactions (25 \(\upmu\)L) contained diluted VWR Taq 2\(\times\) Masterum, 2.0 mM MgCl\({}_{2}\) (VWR), 20 pmol of primer, and 50 ng of genomic DNA. The amplification program consisted of 95 \({}^{\circ}\)C for 3 min, 40 cycles of 95 \({}^{\circ}\)C for 30 s, 34 \({}^{\circ}\)C for 1 min, and 72 \({}^{\circ}\)C for 2.5 min, and a final elongation at 72 \({}^{\circ}\)C for 7 min. The resulting products were visualized on 1.5% agarose gel at 100 V for 90 min and visualized with ethidium bromide.  
ARDRA. Isolates with different RAPD profiles were then grouped using Amplified rDNA Restriction Analysis (ARDRA) [46] by the PCR amplification of a 168 rDNA gene region with the universal primers 27F (5\({}^{\prime}\)-AGAGTTTGATCMTGGCCTAG-3\({}^{\prime}\)) and 1492R (5\({}^{\prime}\)-TACGYTACCTTGTACGACT-3\({}^{\prime}\)). The PCR reactions (25 \(\upmu\)L) contained diluted VWR Taq 2\(\times\) Masterumix, 2.0 mM MgCl\({}_{2}\) (VWR), 10 pmol of each primer, and 50 ng of genomic DNA. The amplification program consisted of 95 \({}^{\circ}\)C for 3 min; 30 cycles of 95 \({}^{\circ}\)C for 30 s, 55 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 1 min, and a final elongation at 72 \({}^{\circ}\)C for 7 min. The restriction polymorphism of the amplicons was determined by digesting 10 microliters of the PCR product with 0.5 U of _Hae_III (Invitrogen at 37 \({}^{\circ}\)C) for 1 h. The obtained fragments were separated on 2% agarose and visualized with ethidium bromide.  
Preparation of the amplified 16S rDNA genes for sequencing. For partial sequencing of the 16S rDNA gene, PCR amplifications were performed as described above for ARDRA,and the PCR product was purified with Clean-Easy PCR and a Gel Purification kit (Canvas Biotech, Valladolid, Spain) according to the manufacturer's instructions. The sequencing was performed by Macrogen Inc. (Seoul, Republic of Korea) on an automatic sequencer (Applied Biosystems Inc., Foster City, CA, USA) with the di-deoxy termination procedure in both directions using the universal primers 27F and 1492R. The resulting sequences were processed with the _CLC Sequence Viewer_ program v.6.6.1 (www.clcbio.com). The resulting sequences were run entries in the GenBank database of the National Center for Biotechnology Information (NCBI) with the BLASTN 2.2.25+ search engine [47]. A phylogenetic tree of the most common _Lactobacillus_ species found in sourdough and the isolates obtained in this study was built based on a ca. 1356 bp partial sequence of the 168 rRNA gene. The tree was generated by the CLC Sequence Viewer software ver. 6.6.1 (www.clcbio.com, CLC bio A/S) by the UPGMA algorithm. Bootstrap values were obtained based on 100 replicates. Sequences of type strains were derived from GenBank (NCBI).  
Species-specific PCR. Additionally, in order to differentiate between _Lactiplantiobacillus plantarum_, _Lp. Pentosus_, and _Lp. paraplantarum_ isolates, species-specific PCR targeting the _recA_ gene was performed as described by [PERSON] et al. (2001) [33].