Db wax capillary column

A GC–MS (7890A-7000, Agilent Technologies Inc., Santa Clara, CA, USA) instrument combined with an olfactory detection port (ODP4, Gerstel, Germany) was used to identify volatile odor compounds. Separation of odor-active substances in samples was performed on a polar DB-WAX capillary column (30 mm × 0.32 mm, 0.25 µm film thickness; J & W Scientific, Folsom, CA, USA). The gas chromatographic instrument condition includes an initial column temperature setting of 40 °C, holding for 3 min, followed by an increase in temperature up to 230 °C at 4 °C/min and holding for 3 min. Ultra-pure helium (99.999%, Beijing AP BAIF Gas Industry Co., Ltd., Beijing, China) was used as the carrier gas. The electron impact mass spectra were generated at an ionization energy of 70 eV with an m/z scan range of 25–370 amu. The temperatures of the mass spectrometer source and quadrupole were programmed at 230 °C and 150 °C, respectively. Moisture gas was delivered to the olfactory detection port through a blank capillary column.

Headspace collections were also analyzed on an Agilent 7890A GC equipped with a DB-WAX capillary column (30 m × 0.25 mm × 0.25 μm; J & W Scientific, Folsom, CA, USA) and interfaced with an Agilent 5975C mass selective detector. Column and oven temperature programs were identical to those described above. Helium was used as a carrier gas (1.5 ml/min) and the purge valve was opened 1 min after injection. The inlet was maintained at 250°C in splitless mode. Compounds that repeatedly elicited antennal responses were identified by comparing the retention times of the respective synthetic standards and mass spectral fragmentation patterns in an MS database (NIST08.L).

A GC-MS (7890A-7000, Agilent Technologies Inc., Santa Clara, CA, USA) instrument combined with an olfactory detection port (ODP4, Gerstel, Germany) was used to identify volatile odor compounds. Separation of odor-active substances in samples was performed on a polar DB-WAX capillary column (30 mm × 0.32 mm, 0.25 μm film thickness; J & W Scientific, Folsom, CA, USA). The gas chromatographic instrument condition includes an initial column temperature setting of 40 °C, holding for 3 min, followed by an increase in temperature up to 230 °C at 4 °C/min and holding for 3 min. Ultra-pure helium (99.999%, Beijing AP BAIF Gas Industry Co., Ltd., Beijing, China) was used as the carrier gas. The electron impact mass spectra were generated at an ionization energy of 70 eV with an m/z scan range of 25–370 amu. The temperatures of the mass spectrometer source and quadrupole were programed at 230 °C and 150 °C, respectively. Moisture gas was delivered to the olfactory detection port through a blank capillary column.

The open reading frame lacking the region encoding the plastid transit peptide of HpDTC1 was ligated into the KpnI and EcoRI sites of the pCold I vector (Takara Bio, Japan) using two sets of primers, HpDTC1-ORF. The recombinant HpDTC1 protein was produced in Escherichia coli strain M-15 as a His₆-tagged protein according to the manufacture’s protocol and our previous work27 (link). Enzyme products were identified using a GC-MS system (Bu-25, Jeol, Tokyo, Japan) equipped with a DB-WAX capillary column (0.25 mm ϕ, length 30 m, and film thickness 0.25 μm; J&W Scientific).
Enzyme reactions and preparation of samples for analysis were performed as reported previously27 (link). To identify an intermediate during the bifunctional cyclization reaction from GGDP to syn-pimara-7,15-diene, an amino acid-substituted mutant of HpDTC1 was prepared by PCR using two sets of primers (double-mutation-A and double-mutation-B in Supplementary Table S1) as described previously27 (link)39 (link). The products obtained from wild type HpDTC1 and the HpDTC1 mutant were determined by using a GC-MS system equipped with a DB-1 capillary column (0.25 mm ϕ, length 15 m, and film thickness 0.25 μm; J&W Scientific).

Strawberry samples (whole fruit) with different impact extents and storage times in three duplicates were analyzed by headspace solid-phase microextraction coupled to gas chromatography–mass spectrometry (HS–SPME/GC–MS). The GC–MS analysis was carried out using a 7890A gas chromatograph coupled to an Agilent 5975C mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Same as the e-nose measurement, before extracting VOCs, strawberry fruits were placed in a 100 mL glass beaker sealed by Parafilm PM996 for 30 min at 15 °C. Subsequently, the SPME fiber coated with 65 μm of polydimethylsiloxane and divinylbenzene (PDMS-DVB) (Supelco, Bellefonte, PA, USA) was inserted into the beaker to collect the VOCs. After 30 min of extraction, we inserted the VOC-adsorbed fiber into the injection pore, and it was desorbed at 240 °C for 5 min for the GC-MS measurement. Then, the VOCs were separated on a DB-WAX capillary column (30 m × 0.25 mm × 0.25 µm, J&W Scientific, Folsom, CA, USA). The main parameters were: high-purity helium as the carrier gas, with a flow rate of 1.0 mL/min; the initial column temperature was 40 °C, then increased to 100 °C at a rate of 3 °C/min, and then increased to 245 °C at a rate of 5 °C/min.

Fatty acids methyl esters (FAME) were prepared according to Bandarra et al. [39 (link)]. Samples were lyophilized (−60 °C and 2.0 hPa) to a constant weight. The FA methyl esters (FAME) preparation was carried out under acid conditions according to Donato et al. [11 (link)] using 0.3 g of freeze dried D. vlkianum-biomass, chow, and tissues except for fat samples where 0.1 g was used instead. FAME were concentrated to a final volume of 25 μL in n-heptane, and 2 μL of the sample was injected on a capillary DBWax capillary column (30 m × 0.25 mm ID × 0.25 μm film thickness; J&W Scientific, Agilent, Santa Clara, CA, USA) in a CP-3800 gas chromatograph (Varian, Palo Alto, CA, USA) equipped with a flame ionization detector. The injector and detector temperatures were set at 250 °C. Adequate separation was obtained over a 40-min period, with 5 min at 180 °C, followed by an increase of 4 °C/min until 220 °C, and kept at this temperature for 25 min. Authentic standards were used for fatty acid identification. Individual fatty acids were expressed as percentage of the total fatty acids.

Strains were grown in YPD media in both aerobic flasks and semi-anaerobic vials until steady state was reached. 2 ml samples were passed through a 0.22μ filter into gas-chromatography (GC) vials and analysed by GC-FID using an Agilent 6850A GC system with an automatic injector, sampler and controller (Agilent 4513A). A DB-WAX capillary column (30 m × 0.25 mm, 0.25 μM, J & W Scientific) was used for separation. Samples were quantified relative to a standard of ethanol, isoamyl alcohol and isobutanol and butanol.