Agilent 5977b

The samples were equilibrated at 45 °C for 20 min in a water bath before headspace-SPME analysis. The SPME fiber was a DVD (Divinylbenzene) /Car/PDMS (Polydimethylsiloxa) fiber (50/30 µm thickness, Supelco, Bornem). Then, the volatile compounds were extracted at 50 °C for 30 min. GC–MS analyses were performed by using an Agilent 8890 GC coupled with an Agilent 5977B quadrupole mass selective detector (MSD, Agilent Technologies, Diegem, Belgium) with a Varian DB-1701 capillary column (30 m length × 0.25 mm i.d.; 0.25 µm film thickness). The working conditions of GC–MS were as follows: the transfer line to MSD (Mass Selective Detector) was maintained at 250 °C; the carrier gas (He) flow rate was 1.0 mL/min; the electron ionization (EI) was 70 eV; the scanned acquisition parameter ranged from 30 to 550 m/z; the initial oven temperature was 40 °C for 2 min; the temperature program was increased from 40 to 100 °C at 10 °C/min and held for 5 min and then raised to 220 °C at a rate of 10 °C/min and held for 15 min, and the equilibrium time was 0.5 min. The injection port was in split mode, and the split ratio was 30:1.

Concentrations of SCFAs were determined by GC-MS. GC-MS was performed with an Agilent 8890B GC and an Agilent 5977B mass analyzer (Agilent Technologies Inc. CA, UAS) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. Quantification of SCFAs was calculated with external standard curve method and were normalized to sample weight.

The volatile constituents of each essential oil were analyzed by GC-MS as previously reported [68 (link)]. They were processed using a gas chromatograph (Agilent 7890B; Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a capillary column (Agilent HP-5MS; 30 m × 0.25 mm; coating thickness, 0.25 μm; Agilent Technologies Inc., Santa Clara, CA, USA) and a single quadrupole mass detector (Agilent 5977B; Agilent Technologies Inc., Santa Clara, CA, USA). The analytical conditions were as follows: injector temperature, 220 °C; transfer line temperature, 240 °C; oven temperature programmed, from 60 °C to 240 °C at 3 °C/min; carrier gas, helium at 1 mL/min; injection volume, 1 μL (in 0.5% HPLC grade n-hexane solution); split ratio, 1:25. The full scan acquisition parameters were as follows: scan range, 30 m/z to 300 m/z; scan time, 1.0 s (See supplementary materials).
Identification of the constituents was based on comparisons of retention times with those of the authentic standards, with comparisons of their linear retention indices relative to the series of n-hydrocarbons. Computer matching was also used against commercial (NIST 14, Adams) and laboratory developed mass spectra libraries built for pure substances and components of known oils, and against the mass spectrometry literature data [69 ,70 (link),71 ,72 ,73 ,74 ].

The volatile apocaroteniods produced by A10-δ-NtCCD1-3 and the control A10 were analyzed by SPME-GC–MS. The SPME fibre, equipped with 65 μm polydimethylsiloxane/divinylbenzene (Supelco, United States), was put into a headspace vial with 0.15 g cell pellet. Then the vial was stirred with 250 rpm at 80°C for 20 min. The apocaroteniods in the fibre were detected by GC–MS (Agilent 5977B, United States) equipped with DB-5MS column (Agilent, United States, 30 m × 0.25 mm ID, 0.25 μm film thickness) using helium as carrier gas at a flow rate of 2 ml/min. The temperature program was as follows: 40°C held for 1.0 min, up to 250°C at a rate of 2°C/min, and held for 10.0 min. The electron ionization energy was 70 eV. The mass range was recorded from m/z 30–500, and the ion source temperature was 200°C. NIST 2.0 spectral library was used to retrieve the detected apocaroteniods, and β-ionone was identified by comparing mass spectrum and chromatographic mobility with that of the authentic standard.

Fresh fecal samples were obtained from both groups only after 4 weeks of intervention. The fecal samples were weighed and suspended in 1 mL of water with 0.5% phosphoric acid per 0.1 g of sample, then homogenized for 2 min. The samples are then centrifuged (14.8 RPM for 10 min) and the supernatant collected. Next, an aliquot of ethyl acetate (300 μL) was added into the supernatant and mixed well before centrifuging (14.8 RPM for 10 min). The organic phase was collected for GC-MS analysis with an Agilent 5977B coupled with a 7693A autoinjector (Agilent Technologies, Palo Alto, CA, United States). The Nukol Capillary GC Column (30 m × 0.25 mm id, 0.25 μm df) was applied to the GC, and the gas (helium) carrier injected at 1 mL/min. The column temperature stared at 90 °C, then increased to 150 °C at 15 °C/min, to 170 °C at 5 °C/min, and finally to 250 °C at 20 °C/min for 2 min (total time, 14 min). The detector is operated in electron impact ionization mode (electron energy 70 eV), scanning the 30–250 m/z range. The indicated SCFA identification was based on the retention time of standard compounds and the assistance of the NIST 08 and Wiley7N libraries.

Methylation analysis was performed by gas chromatography-mass spectrometry, using a previously published method with slight modifications, to determine the bonding structure of GFPA (21 (link)). A gas chromatography system (Agilent 7890A; Agilent Technologies Inc., Santa Clara, CA, USA) was used with the following conditions: injection volume, 1 μL; split ratio, 10:1; carrier gas, high purity helium and temperature program set at 140°C for 2 min and increasing to 230°C for 3 min at 3°C/min. A quadrupole mass spectrometry detection system (Agilent 5977B, Agilent Technologies Inc.) equipped with a MassHunter workstation, and an electron impact ion source was used to detect the analyte in full-scan mode with a mass scan range of 30-600 m/z. The characteristic fragments of the methylated polysaccharides were compared to those in an existing database to confirm the glycosidic linkages.