## 4 Materials and Methods
### Wine Volatile Analysis  
Wine volatile compounds were analyzed with headspace gas chromatography-flame ionization detector (HS-GC-FID) and headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) [43]. Each sample was analyzed in triplicate. Highly volatile compounds, including acetaldehyde, ethyl acetate, propanol, isobutanol, and isoamyl alcohol were analyzed through HS-GC-FID. In brief, 0.5 mL wine was diluted two-fold with Milli-Q water and 20 \(\upmu\)L methyl propionate (2.5 mg/L) was added as an internal standard. Extraction and injection were conducted in a Gerstel MPS (multi-purpose sampler, Linthicum, MD, USA) autosampler equipped with a 2.5 mL headspace syringe. Before injection, samples were incubated in a thermostatic bath at 50 \({}^{\circ}\)C for 15 min. The injection volume was 0.5 mL with a 1:10 split ratio. Analysis was performed on Agilent 6890 N GC coupled with a flame ionization detector (Agilent Technologies Inc., Santa Clara, CA, USA). Compounds were separated on a ZB-WAX column (30 m \(\times\) 0.25 mm i.d., 0.5 \(\upmu\)m film thickness, Phenomenex Inc., Torrance, CA, USA). The column flow rate was 1.5 mL/min with helium as the carrier gas. The initial oven temperature was 35 \({}^{\circ}\)C held for 4 min, then increased by 10 \({}^{\circ}\)C/min to 150 \({}^{\circ}\)C and held at 150 \({}^{\circ}\)C for 5 min. Injection port temperatures was 250 \({}^{\circ}\)C.  
Major volatile compounds were analyzed with HS-SPME-GC-MS. Two-milliliter wine sample was diluted 5-fold with 0.2 M citrate buffer (pH 3.5) saturated with sodium chloride. Isotope-labeled internal standard solution and a magnetic stir bar were added to the vial. Sample vials were equilibrated at 50 \({}^{\circ}\)C in a thermostatic bath for 10 min and then the headspace was extracted with a 2 cm divinylbenzene/carbouxan/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 \(\upmu\)m film thickness, Supelco, Bellefonte, PA, USA) SPME fiber for 50 min at the same temperature under stirring (500 rpm). Desorption was performed by introducing the SPME fiber into the GC injection port for 5 min with splitless mode. Analysis was performed on Agilent 6890 N GC coupled with a 5973 N mass selective detector (Agilent Technologies Inc., Santa Clara, CA, USA). Compounds were separated on a ZB-WAX column (30 m \(\times\) 0.25 mm i.d., 0.5 \(\upmu\)m film thickness, Phenomenex Inc., Torrance, CA, USA). The column flow rate was 1.5 mL/min with helium as the carrier gas. Initial oven temperature was 40 \({}^{\circ}\)C and held for a 4 min, then increased to 230 \({}^{\circ}\)C at a rate of 4 \({}^{\circ}\)C/min, with a 10 min hold at the final temperature. Methoxypyrazines analysis was conducted on a HP-5 column (30 m \(\times\) 0.25 mm i.d., 0.5 \(\upmu\)m film thickness) under SIM. The following oven program was used for methoxyprazines: initial oven temperature was 40 \({}^{\circ}\)C and held for a 2 min, increased to 120 \({}^{\circ}\)C at a rate of 3 \({}^{\circ}\)C/min, then increased to 230 \({}^{\circ}\)C at a rate of 6 \({}^{\circ}\)C/min with a 10 min hold at the final temperature. Monitored selected ions: 2-isopropyl-3-methoxypyrazine (IPMP), _m/z_ 137; 2-sec-butyl-3-methoxypyrazine (SBMP), _m/z_ 138; 3-isobutyl-2-methoxypyrazine (IBMP), _m/z_ 124; IBMP-d\({}_{3}\), _m/z_ 127. Injection port, MS transfer line, and ion source temperatures were 250, 280, and 230 \({}^{\circ}\)C, respectively. Electron ionization mass spectrometric data from _m/z_ 40 to 250 were collected, with an ionization voltage of 70 eV. Compound identification was performed by comparison to retention times and matching mass spectra of analytical standards under identical conditions. Compounds were quantified with internal standard calibration curves which were built with gradient dilution of known amounts of the standards.