## 2 Materials and Methods
### Experimental Plan and Sample Collection for Sugarcane Fermentation  
Juices extracted from nine sugarcane varieties were then mixed in equal proportions to obtain a single batch of 24 L of juice, with the aim of ensuring chemical and physical homogeneity between fermentation treatments. The juice was divided into 16 experimental treatments (SC1-SC16), with each treatment being conducted in three independent 500 mL fermentation units.  
Trials from SC1 to SC4 did not include any chemical treatment, trials from SC5 to SC8 included the addition of 5 g per hectoliter (g/hL) potassium metabisulfite, trials from SC9 to SC12 were acidified with sulfuric acid to pH 3.5 [15], while trials from SC13 to SC16 were acidified with lactic acid to pH 3.5. The different treatments were applied to three different strains of inocula and a spontaneous fermentation. Specifically, the strains used were _S. cerevisiae_ SPF21, _S. cerevisiae_ MN113, and the commercial control _S. cerevisiae_ EC1118. Finally, sixteen experimental trials were set up as shown in Figure 1.  
Prior to yeast inoculation, all trials were supplemented with 80 g/hL diammonium phosphate (Chimica Noto s.r.l., Partinco, Italy) and the respective treatment as described above. Each strain was cultured in YPD broth (yeast extract 10 g/L, peptone 20 g/L, dextrose 20 g/L) in 250 mL Erlenmeyer flasks containing 100 mL of medium. Cultures were incubated at 28 \({}^{\circ}\)C for 48 h under orbital shaking at 120 rpm. After incubation, cells were harvested by centrifugation at 4000 rpm for 10 min, washed twice with sterile saline solution (0.9% NaCl), and resuspended in the same solution. The concentration of the resulting suspension was estimated by OD\({}_{600}\) and adjusted to approximately 2 \(\times\) 10\({}^{8}\) CFU/mL. A volume of 5 mL of this suspension was inoculated into each 500 mL fermentation unit, resulting in a final concentration of approximately 2 \(\times\) 10\({}^{6}\) CFU/mL in the sugarcane juice. The cell count was confirmed by plate counting on WL nutrient agar [26]. Fermentation was performed at 20 \({}^{\circ}\)C in hermetically sealed glass fermenters (total capacity: 750 mL), each containing 500 mL of sugarcane juice and equipped with a sluice valve. At each sampling point (day 0, 6, 12, and at the end of fermentation),  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Chemical Parameters** & **Wort** \\ \hline D-fructose (g/L) & 13.19 \(\pm\) 0.12 \\ D-glucose (g/L) & 23.01 \(\pm\) 0.22 \\ D-sucrose (g/L) & 168.50 \(\pm\) 1.35 \\ Glycerol (g/L) & 0.32 \(\pm\) 0.05 \\ Acetic acid (g/L) & 0.09 \(\pm\) 0.06 \\ Lactic acid (g/L) & 0.06 \(\pm\) 0.05 \\ Malic acid (g/L) & 0.02 \(\pm\) 0.02 \\ Tartaric acid (g/L) & 0.03 \(\pm\) 0.01 \\ Ammoniacal nitrogen (mg/L) & 0.00 \(\pm\) 0.00 \\ Alpha-amino nitrogen (mg/L) & 74.08 \(\pm\) 0.26 \\ pH & 5.11 \(\pm\) 0.12 \\ Total acidity (TA; g/L tartaric acid) & 2.85 \(\pm\) 0.10 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Chemical parameters identified in the sugarcane word before treatments.  
Figure 1: Experimental plan to produce fermented sugarcane juice.  
10 mL of fermented juice was aseptically collected from each fermenter for microbiological and physicochemical analyses. Sugar concentration was monitored daily throughout the process to precisely assess sugar consumption and fermentation dynamics. The process of fermentation was considered to have reached its conclusion when no variation in residual sugar levels was observed over the course of two consecutive days. After this, all samples were subjected to immediate analysis.