## 2 Materials and Methods
### Chemical Analysis of Must and Wines  
Classic parameters of wines (free and total SO\({}_{2}\) contents, % vol., pH, titratable and volatile acidity, A\({}_{420\text{nm}}\)) were determined according to the international methods of the International Organization of Vine and Wine (OIV) [19]. Nitrogen of \(\alpha\)-amino acid was determined as described by [PERSON] and [PERSON], (1998) [17], while ammonium nitrogen was determined according to [PERSON] (1996) [20]. Glycerol and acetic acid were quantified through high-performance liquid chromatography/HPLC (Waters Association 600E apparatus) with an RI detector (Waters 410, Midland, ON, Canada) and an ion exclusion column (Aminex HPX-87H, Bio-Rad, CA, USA). The column temperature was set at 65 \({}^{\circ}\)C with a flow rate of 0.8 mL/min. The mobile phase was H\({}_{2}\)SO\({}_{4}\). For quantitative analysis, standard solutions of glycerol and acetic acid (Sigma-Aldrich Ltd., Taufkirchen, Germany) were prepared in pure water (Milli-Q, Merk, Taufkirchen, Germany) and wine samples were injected directly into the column. Determinations were performed by means of standard curves [16]. The amino-acid analysis was performed using a liquid chromatographic-triple-quadrupole mass spectrometric method. Analyses were performed on a Thermo Ultimate 3000 ultra-high pressure liquid chromatograph consisting of a 1000 pressure pump, degasser, PAL open-autosampler, and valve interface module. The detection system consisted of a Thermo Quantum Access Max triple-quadrupole mass spectrometer, and for the evaluation, a multiple reaction monitoring option was used. Chromatographic separation was performed in an Intrada Aminoacid (mixed mode, normal phase and ion exchange) column, particle size 3 \(\upmu\)m (150 \(\times\) 4.6 mm), thermostated at 45 \({}^{\circ}\)C through the binary gradient shown in Table 1 (phase A, 74:16:10:0.2 mixture of isopropanol, 25 mM ammonium formate, acetonitrile, and formic acid; phase B, 20:80 mixture of acetonitrile and 100 mM ammonium formate) and a flow rate 0.35 mL/min. For detection, triple-quadrupole mass spectrometry with spray voltage 3000 V, vaporizer temperature 350 \({}^{\circ}\)C, sheath gas pressure 35 psi, ion sweep gas pressure 0.0 psi, aux gas pressure 10 psi, capillary temperature 380 \({}^{\circ}\)C, and capillary offset 35 v was used. The target compounds were identified according to the retention times and the characteristic ions from molecular ion fragmentation. \begin{table}
\begin{tabular}{c c c c c c c} \hline Time (min) & 0–12 & 12–17 & 17–19 & 19–31 & 31–31.5 & 31.5–35 \\ Fluent A (\%) & 100 & 97 & 0 & 0 & 100 & 100 \\ Fluent B (\%) & 0 & 3 & 100 & 100 & 0 & 0 \\ \hline \end{tabular}
\end{table} 
Table 1: Fluent gradient for UPLC determination of amino acids. The identification of the headspace volatiles of produced wines was conducted by means of gas chromatography/mass spectrometry (GC/MS) using the solid phase microextraction method (SPME). GC/MS analysis was performed on a GCMS-QP2010 Ultra (Shimadzu Inc., Japan) system set at 240 \({}^{\circ}\)C in split mode (split ratio 1/20) [21]. The conditions of headspace SPME sampling were as follows: 2 mL of wine sample, 7.5 mL of deionized water, 1 g ammonium sulfite, and 500 \(\upmu\)L internal standard (1-octanol) were transferred into a 20 mL headspace vial fitted with a polytetrafluoroethylene (PTFE)-lined silicone septum. The sealed wine samples were initially stirred (250 rpm) in a tap bath (T = 40 \({}^{\circ}\)C) for 5 min (Thermodyne-Nuova II; stir speed 5) to achieve the appropriate temperature, and then the SPME fiber (DVB/CAR/PDMS, 2 cm; Sigma Aldrich, Germany) was exposed to the headspace for 30 min. The separation of wine volatile compounds took place in a DB-Wax capillary column (30 m \(\times\) 0.25 mm i.d., 0.25 \(\upmu\)m film thickness, Agilent, USA). Helium was used as the carrier gas at a constant linear velocity of 36 cm/s.