## 3 Results
### Bacteria Molecular Characterization and Phylogenetic Analysis  
The analysis of the 16S-23S rDNA ISR allowed to detect _Fl. sanfranicscensis_ species among the bacterial isolates (Table 4). In particular, PCR products from all the MA and MB isolates yielded similar profiles containing three bands of 600, 700, and 800 bp; these isolates were preliminarily attributed to _Fl. sanfranicscensis_[27]. All the remaining isolates showed a profile with only two bands of 600 and 800 bp. This result was consistent with previously reported data for _Lactobacillus_ 16S-23S ISR organization [27; 42; 43]. The digitized patterns obtained from (GTG)\({}_{5}\) rep-PCR, analyzed with the UPGMA method and using Pearson's coefficient, allowed the construction of the dendrogram shown in Figure 4. The biotypes discrimination was performed considering a similarity threshold of 95%, which allowed the grouping of the strains into 19 different clades; only the strains BIA5 and BIB8 were included in the same clade. The calculated Simpson's index was 0.99, indicating a high diversity among the isolated strains including those preliminarily attributed to the species _Fl. sanfranicscensis_. This is consistent with the evidence reported by several authors that highlighted the great variability among _F. sanfranicscensis_ strains [7; 27]. High-quality sequences obtained from the bacteria dataset were analyzed in order to determine the phylotypes of isolates. After all-vs.-all alignment, sequences were trimmed at the same length, resulting in a total length of 904 bp. Reference sequences were downloaded from NCBI 16S rRNA database, resulting in two genera including _Leuconostoc_ and _Lactobacillus_. The sequence of _Bacillus albidus_ MCCC 1A02146T was used as an outgroup. The average phylogenetic distance among all sequences included in the dataset was 13.17%. The resulting phylogenetic tree was represented in Figure 5. A total of two major clades are depicted, represented by the clade of _Leuconostoc_ genus and the clade including the genera _Furf urilatelobacillus_, _Lactiplantibacillus_, _Lenitalobacillus_, and _Fruticilatobacillus_. In particular, _Leuconostoc_ clade includes five isolates and is branched in two subclades, _Leuc. mesenteroides_ NBRC 100469T and _Leuc. citreum_ ATCC 49370T. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Sample**} & \multirow{2}{*}{**Strain *} & **16S-23S rDNA ISR Size** \\  & & **Fragments (bp)** \\ \hline \multirow{2}{*}{MA} & **BMA2 (UMCC 2989)**, **BMA8 (UMCC 2990)**, & 600–700–800 \\  & **BMA10 (UMCC 2991)** & & \\ \hline \multirow{2}{*}{IMPA} & **BIA2 (UMCC 2992)**, **BIA3**, **BIA5**, **BIA6 (UMCC 2993)**, & 600–800 \\  & **BIA7 (UMCC 2994)**, **BIA8**, **BIA9**, **BIA10 (UMCC 2995)** & \\ \hline \multirow{2}{*}{FINA} & **BFA1 (UMCC 2996)**, **BFA2 (UMCC 2997)** & 600–800 \\ \hline \multirow{2}{*}{MB} & **BMB5 (UMCC 2998)**, **BMB7 (UMCC 2999)** & 600–700–800 \\ \hline \multirow{2}{*}{IMPB} & **BIB7 (UMCC 3002)**, **BIB8** & 600–800 \\ \hline \multirow{2}{*}{FINB} & **BFB3**, **BFB6 (UMCC 3004)**, **BFB8 (UMCC 3006)** & 600–800 \\ \hline \hline \end{tabular}  
* The strains sequenced are shown in bold and in brackets are indicated the correspondent UMCC code.  
\end{table} 
Table 4: PCR amplification of the 16S-23S rRNA intergenic spacer region (ISR). Two strains, BIA7 and BFB6, were grouped with _Leuc. mesenteroides_ NBRC 100469\({}^{\text{T}}\), sharing 100% similarity. Three strains, BFA2, BFB8, and BIA6, were clustered with _Leuc. citreum_ ATCC 49370\({}^{\text{T}}\), sharing 100% of similarity among them and 99.9% of similarity with the reference strain. The phylotype classification was supported by high bootstrap values (ranging between 77% and 100%). The remaining isolated strains were included in the other major clade, represented by four species: _Furfurilactobacillus rossiae_ (formerly _Lactobacillus rossiae_) DSM 15814\({}^{\text{T}}\), _Lacp.