## 3 Results
### Yeasts' Molecular Characterization and Phylogenetic Analysis  
The analysis of the RFLP-PCR of ITS-5.8S rDNA and the yeasts' amplicons digestion with _Hae_III allowed us to detect two different profiles (Table 3). Specifically, all the isolates belonging to samples A showed the same profile characterized by two bands of 230 and 400 base pairs (bp). As reported in the literature, this profile was tentatively attributed to the species _K. humilis_ (formerly _Candida milleri_) [13; 14]. Regarding the isolates from samples B, 12 showed a profile with two bands, as before, whereas a profile with three bands of 150, 180, 230, 320 bp were detected in the remaining ones. These isolates were preliminarily attributed to _S. cerevisiae_ species according to [PERSON] et al. [38]. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Samples** & **PHA** & **Water Activity** & **Total Titractable Acidity** \\  & & **(a\({}_{\mathrm{w}}\))** & **(mL NaOH 0.1N/10 g)** \\ \hline MA & 3.90 \(\pm\) 0.03 d & 0.9855 \(\pm\) 0.002 d & 10.85 \(\pm\) 0.45 b \\ IMPA & 5.44 \(\pm\) 0.05 b & 0.9636 \(\pm\) 0.001 b & 3.35 \(\pm\) 0.25 a \\ FINA & 5.35 \(\pm\) 0.02 b & 0.9441 \(\pm\) 0.002 a & 3.55 \(\pm\) 0.45 a \\ MB & 3.81 \(\pm\) 0.02 d & 0.9894 \(\pm\) 0.001 d & 10.45 \(\pm\) 0.45 b \\ IMPB & 5.30 \(\pm\) 0.07 ac & 0.9562 \(\pm\) 0.002 c & 3.45 \(\pm\) 0.30 a \\ FINB & 5.21 \(\pm\) 0.03 a & 0.9411 \(\pm\) 0.003 a & 4.05 \(\pm\) 0.05 b \\ \hline \hline \end{tabular} Values are means \(\pm\) standard deviation of three replicates (n = 3). Values within a column with different superscript letters are significantly different (\(p<0.05\)).  
\end{table} 
Table 1: Physicochemical parameters detected on the samples collected during the Panettone production. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Samples** & **Means Counts * on the Isolation Media** \\ \hline  & **YPDA** & **MRS** & **SDB** \\ \hline MA & 6.81 \(\pm\) 0.25 b & 6.15 \(\pm\) 0.14 ab & 6.17 \(\pm\) 0.21 b \\ IMPA & 7.81 \(\pm\) 0.05 a & 5.39 \(\pm\) 0.12 a & 5.65 \(\pm\) 0.21 ab \\ FINA & 7.83 \(\pm\) 0.17 a & 5.46 \(\pm\) 0.07 a & 5.45 \(\pm\) 0.19 ab \\ MB & 7.56 \(\pm\) 0.03 a & 6.87 \(\pm\) 0.42 b & 6.16 \(\pm\) 0.15 b \\ IMPB & 8.02 \(\pm\) 0.01 a & 5.57 \(\pm\) 0.06 a & 5.27 \(\pm\) 0.09 a \\ FINB & 7.89 \(\pm\) 0.17 a & 5.64 \(\pm\) 0.04 a & 5.64 \(\pm\) 0.02 ab \\ \hline \hline \end{tabular}  
* expressed as log of colony forming unit (cfu)/ g of sample \(\pm\) standard deviation. Values are means \(\pm\) standard deviation of three replicates (n = 3). Values within a column with different superscript letters are significantly different (\(p<0.05\)). YPDA medium (yeast extract, peptone, dextrose, agar); MRS medium (de Man Rogosa Sharpe); SDB medium (Sourdough Bacteria agar).  
\end{table} 
Table 2: Colony counts on the growth media used for the isolation of yeasts and lactic acid bacteria from dough samples. For strain clusterization, the (GTG)\({}_{5}\) rep-PCR fingerprinting techniques were performed. Figure 2 reports the dendrogram obtained from UPGMA analysis using Pearson's coefficient. The discrimination of biotypes was performed considering a similarity threshold of 89%, which allowed the grouping of yeast isolates into 25 clades. The resulting discrimination power, calculated using the Simpson's index, was 0.92. The clades with the highest number of strains (from 5 to 13) included those preliminarily attributed to _K. humilis_. Considering a similarity threshold of 40%, all the tentative _K. humilis_ strains (42 in total) were grouped closely in a major clade clearly distinct from a minor clade that included the remaining 15 strains preliminarily attributed to _S. cerevisiae_. A total of 43 high-quality sequences were used to infer the phylogeny of yeast isolates. Reference sequences belong to _Saccharomyces_ and _Kazachstania_ genera, detected after alignment in the YeastIP database. After all-vs.-all sequence alignment, sequences were trimmed all at the same length, resulting in a total length of 599 bp.