8890 gc system

Volatile compounds were analysed by GC‒MS (Agilent Technologies 8890 GC system) as described previously [7 (link), 18 (link)]. Prior to split-less injection, the loaded SPME fibre was transferred at 2 cm s⁻¹ into the injector port (250 °C) and maintained there for a 3 min desorption interval. After every injection, the fibre was cleaned (20 min, 270 °C). The volatiles were separated on an HP-5MS column (30 m, 250 µm, 0.25 µm, Agilent Technologies) using helium 5.0 as the carrier gas (0.5 mL min⁻¹). Chromatograms were recorded by monitoring the total ion current (TIC) over a mass range from 30 to 300 m/z, followed by deconvolution of the obtained signals (MassHunter Workstation Software, Agilent Technologies). For each analyte i, measurement and data processing yielded a peak area (A_i) that corresponded to the cleaned mass spectrum of the eluting compound, reflecting its absolute abundance. Its relative abundance a_i was then obtained by normalization of A_i to the total peak area (A_total) of all volatiles detected in the sample according to Eq. 1. $${\mathrm{a}}_i=A_i/A_{\mathit{total}}\times 100$$

Extraction of aroma compounds in cider was performed using a headspace solid-phase micro-extraction (HS-SPME) according to an existing method with minor modifications (Niu et al., 2019 (link)). In brief, 5 ml cider, 2 g sodium chloride, and 10 μl 3-octanol (10 mg/L, internal standard) were placed in a 20 ml micro-reaction vial. The vial was put into a thermostatic water bath at 70°C for 30 min. The 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS; Sigma-Aldrich, United States; 57348-U) fiber was exposed to the sample headspace (about 1 cm above the liquid surface) at 70°C for 30 min, injected in the GC injector and kept for 3 min, and analyzed using an Agilent 8890 GC system equipped with a 5977B mass spectrometer (MS) according to a modified method (Niu et al., 2019 (link)). Chromatographic separations were performed using a DB-wax column (20 m × 0.18 mm, Agilent, Santa Clara, CA, United States). The column flow rate of helium was 1.5 ml/min. Electron energy of 40 Ev and mass range of m/z 30–450 were applied during MS analysis. The initial temperature was maintained at 40°C for 6 min, increased to 100°C at 4°C /min rate, then increased to 230°C at 10°C/min rate, and held for 20 min. Each sample was measured in triplicate.

Farnesene determination was performed by gas chromatography (GC), using an Agilent 8890 GC System with a flame ionization detector (FID) (Agilent, Santa Clara, CA, USA. Two different standard farnesene solutions (low and high concentrations) and the samples were injected after performing a farnesene extraction protocol. Briefly, farnesene extraction was applied by mixing fermentation broth with a solution containing 10% 2-butoxyethanol, 0.25% tetradecane, and 90% methanol at a proportion of 1:20 or 1:40 (shake-flask or reactor samples, respectively). Samples were injected in a separation column (Phenomenex ZB-5HT Inferno; Phenomenex, Torrance, CA, USA, with 30 + 5 m guardian, 0.25 mm and 0.25 µm film) with the column temperature ranging from 60 to 325 °C. Hydrogen was used as sample carrier gas. Data acquisition was performed using Agilent OpenLab CDS–Build 2.205.0.1344 (Agilent, Santa Clara, CA, USA.