## 2 Materials and Methods
### Analysis of Volatile Compounds by GCMS  
Volatile compounds analysis was carried out using TRACETM 1300 Gas Chromatographer coupled to ISO 700 TriPlus quadrupole mass spectrometer (Thermo Fisher Scientific Inc., Bartlesville, OK, USA) equipped with TG-WAXMS A capillary column (60 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m film thickness; Thermo Fisher Scientific, Bartlesville, OK, USA). Solid Phase Microextraction Arrow used for isolation of analyte was carried out using RSH Triplus autosampler (Thermo Fisher Scientific Inc., Brookfield, MO, USA). Prior to extraction, the Arrows were preconditioned in the injector port of the gas chromatograph according to the manufacturer's instructions. The SPME Arrow procedure was performed according to [27] with fibers (Supelco, Bellefonte, PA, USA) coated with stationary phase and film thicknesses as follows: divinylbenzene-carboxen-polydimethylsiloxane 50/30 \(\upmu\)m (DVB/CAR/PDMS) Arrow system. A total of 5 mL of wine and 2.5 g of NaCl salt were added to the glass vials closed with polytetrafluoroethylene (PTFE) coated silicone rubber septum. Equilibration of the wine sample was performed at 55 \({}^{\circ}\)C for 10 min. Adsorption of the analyte was performed at 55 \({}^{\circ}\)C for 60 min. Desorption was performed in a liquid chromatograph injector at 250 \({}^{\circ}\)C for 7 min. The volatile compounds injected into the inlet were delivered to the column at a splitless mode, and helium was used as a carrier gas at a constant flow rate 1 mL/min. Chromatographic analysis was performed at temperature program in the range of 40 to 210 \({}^{\circ}\)C. Mass spectrum recording was performed by monitoring the current of all ions in the range of 20 to 500 \(m/z\) while the ionization energy was 70 eV. The data obtained were processed using ChromleonTM Data System (Thermo Fisher Scientific Inc., Bartlesville, OK, USA). Identification of volatile compounds was performed by comparing the recorded mass spectrum with the data available in Wiley Registry 12 th Edition/NIST (National Institute of Standards and Technology) Mass Spectral Library and the calculation of the retention index of the analytes. The Retention index (RI) was calculated using alkane standards C8-C20 (Sigma Aldrich, St. Louis, MO, USA) as described [27; 28].  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Compound** & **L** & **S** & **N** \\ \hline Alcohol (\% \(v/v\)) & 14.93 \(\pm\) 0.06 b & 15.13 \(\pm\) 0.06 ab & 15.23 \(\pm\) 0.12 a \\ Reducing sugar (g/L) & 7.13 \(\pm\) 0.81 a & 4.73 \(\pm\) 0.38 b & 5.27 \(\pm\) 1.27 ab \\ Extract (g/L) & 25.43 \(\pm\) 0.61 a & 24.77 \(\pm\) 0.95 a & 25.43 \(\pm\) 0.23 a \\ Ash (g/L) & 2.81 \(\pm\) 0.22 a & 3.11 \(\pm\) 0.01 a & 2.91 \(\pm\) 0.05 a \\ pH & 3.56 \(\pm\) 0.03 b & 3.59 \(\pm\) 0.03 b & 3.68 \(\pm\) 0.02 a \\ Total acidity (g/L as tartaric acid) & 5.90 \(\pm\) 0.00 a & 5.60 \(\pm\) 0.00 b & 5.30 \(\pm\) 0.10 c \\ Volatile acidity (g/L as acetic acid) & 0.50 \(\pm\) 0.00 a & 0.40 \(\pm\) 0.00 b & 0.40 \(\pm\) 0.00 b \\ \hline \hline \end{tabular} Values are means with standard deviations of three separate repetitions. Values with different letter (a, b, c) in the same row are significantly different, according to the [PERSON]’s LSD test (\(p<\) 0.05).  
\end{table}
Table 2: Physicochemical composition of the Plavac Mali wines produced by sequential L, conventional S and spontaneous N fermentation.