## 2 Materials and Methods
### Analysis of Flavor Compounds of Fermented Chambourcin Juice
#### 2.6.2 Analysis of Volatile Compounds by Gas Chromatography  
Aromatic compounds were analyzed by gas chromatography-mass spectrometry (GC-MS) (7890B System, 5977B MSD, Agilent Technologies, Santa Clara, CA, USA) using the method described here with minor modifications [50]. In total, 2 mL of samples was mixed with 3 g sodium chloride (VWR International, Radnor, PA, USA), 50 \(\upmu\)L internal standard (including 13.7 mg/L 2-octanol and 9.9 mg/L naphthalene-d8 in methanol), and 0.5g D-gluconic acid lactone (Sigma-Aldrich, St. Louis, MO, USA), which inhibits grape \(\beta\)-glucosidase activity during sample preparation and analysis [51]. Samples were then vortexed and analyzed immediately. Each injection was performed in technical triplicates from three biological flasks.  
Solid-phase microextraction (SPME) mixture comprised heptanal (0.452 g/L), octanal (0.484 g/L), nonanal (0.477 g/L), 1-decanol (0.394 g/L), 1-undecanol (0.392 g/L), 1-dodecanol (0.521 g/L), n-decane (0.451 g/L), n-dodecane (0.481 g/L), n-tetradecane (0.497 g/L), methanol (0.511 g/L), (-)trans- and (-)cis-carved (0.778 g/L), (+)-carvone (0.584 g/L), a-pinene (0.553 g/L), b-pinene (0.472 g/L), and p-cymene (0.424 g/L) in acetone. A total of 50 \(\upmu\)L of internal standard as a blank and 10 \(\upmu\)L of SPME mixture as a quality control were used and placed in the first and last order of each batch of samples, respectively. Performance test of GC-system was carried by the mixture of 5 \(\upmu\)L alkane standard solution (\(C_{8}-C_{20}\), -40 mg/L each in hexane; Sigma-Aldrich, St. Louis, MO, USA) and 50 \(\upmu\)L internal standard.  
Samples were incubated at 30 \({}^{\circ}\)C for 5 min under agitation at 250 rpm, and then extracted using a 2 cm Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) SPME fiber assembly (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Separation was carried out with a Rtx-Wax capillary column (25 m \(\times\) 0.25 mm I.D. \(\times\) 0.25 \(\upmu\)m film thickness; Restek Corporation, Bellefonte, PA, USA) in splitless mode. The transfer line and ion source (70 eV) were maintained at 250 and 230 \({}^{\circ}\)C, respectively. The oven temperature was programmed as follows: hold at 30 \({}^{\circ}\)C for 1 min; increase to 250 \({}^{\circ}\)C at rate of 10 \({}^{\circ}\)C/min and hold for 5 min. Helium was used as the carrier gas under constant flow at 1 mL/min. Mass spectra were acquired at a rate of 33-350 amu scan.  
To identify fermentation associated volatile compounds, chromatograms were stripped of common contaminating ions (147, 148, 149, 207, 221, 267, and 281 \(m/z\)) using the Denoising function in OpenChrom followed by the Savitzky-Golay smoothing filter with default settings (width = 15; order = 2) to enhance chromatographic data by reducing noise while maintaining the shape and height of waveform peaks [52; 53; 54]. The PARAFAC2 based Deconvolution and Identification System (PARADISe) computer platform (version 3.9) was used to deconvolute mass spectra with 7000 iterations and non-negativity constraint settings [55]. Retention time intervals were manually selected to increase the resolution of peak identification. Identification based on deconvoluted mass spectra were conducted using the National Institute of Standards and Technology (NIST14) mass spectral library with criteria of match factor over 700, and validated with Kovats retention indices referred to NIST14 library, PubChem ([[https://pubchem.ncbi.nlm.nih.gov/](https://pubchem.ncbi.nlm.nih.gov/)]([https://pubchem.ncbi.nlm.nih.gov/](https://pubchem.ncbi.nlm.nih.gov/))) or literature. Relative abundance of volatile compounds was calculated by dividing the peak area of volatile compounds by the peak area of internal standards (IS) and then subtracted by the relative abundance of such in the blank, 50 \(\upmu\)L internal standard.