Thermo trace 1300

The volatile components in GWK were analyzed on a column (30 m × 0.32 mm × 0.25 μm) of Agilent J&W GC DB-1 (Agilent, USA) by the Thermo Trace 1300 gas chromatography system coupled with Thermo ISQ LT single quadrupole mass spectrometer (Thermo Fisher, USA). The program was as follows: the column temperature was increased with a rate of 10°C/min from 50°C to 80°C (held for 2 min), 5°C/min to 140°C (held for 1 min), and 20°C/min to 280°C (held for 1 min) and then reduced with a rate of 50°C/min to 50°C (held for 2 min). The injector temperature was maintained at 280°C with splitless injection (25 : 1) and helium (purity 99.99%) as the carrier gas at a flow rate of 1.0 mL/min. The mass spectrometer was performed at a scan range of 50-500 m/z with 70 eV ionization energy and applied 280°C and 200°C of ion source and quadruple temperature, respectively. Identification of the detected peaks was conducted by comparing the mass spectrum with the individual peak to those in the NIST MS Search 2.2 standard spectral library.

Participants were asked to collect a fecal sample in the morning using insulated fecal collection containers with surrounding ice. After arriving at the laboratory, it was stored at −80°C prior to progressing. The analysis of short‐chain fatty acids was performed following routine operations by Tinygene Bio‐Tech (Shanghai) Co., Ltd. Take 50 mg of feces in the 1.5 mL centrifugal tube, add 500 μL of water, add 100 mg of glass beads, 1 min for homogenate, centrifuge for 10 min at 4°C with 13,200 g, and then 200 μL supernatant was collected. Next, add 100 μL 15% phosphate, 20 μL 375 μg/mL internal standard (4‐methylic acid) solution and 280 μL ether, 1 min for homogenate, centrifuge for 10 min at 4°C with 12,000 rpm, and the supernatant was collected and used to detect SCFAs. Concentrations of SCFAs were determined using gas chromatography–mass spectrometry (GC–MS) with Thermo Trace 1300 and Thermo ISQ 7000 under the full scan and SIM mode. For targeted fecal metabolomic detection, internal standard references of amino acids and related metabolites were purchased from Cambridge Isotope Laboratories (US), and the methods of metabolite extraction, instrument, and data analyzing of target metabolomic detection were conducted as previously described¹⁶ with modification.

Identification and relative quantitation of main constituents of EOs under study was carried out using a Thermo Trace 1300 GC coupled to a Thermo ISQ 7000 MS (Thermo Fisher Scientific, Waltham, MA, US) (Pitsch 2020). Prior analysis, EOs were diluted 1:100 with methyl-tert-butyl ether. Chromatographic separation of EOs was achieved using a TRACE TR-5MS column (0.25 mm, 0.25 µm, 30 m; Thermo Fisher Scientific, Waltham, MA, US). Injector port temperature was kept at 200 °C. GC column temperature was kept at 45 °C for 1 min and was increased from 45 °C to 210 °C at a rate of 5 °C∙min⁻¹ and held for 5 min. During measurements, transfer line was kept at 220 °C and ion source at 200 °C, respectively. GC was operated with helium (99.999%) at a constant flow rate of 1.0 mL/min. Each sample was determined in triplicate via 1:20 split injection of 1.0 µL. Total ion current (TIC) mode from m/z 50–500 was used for measurement. Data processing was carried out with Chromeleon 7.2.10 software (Thermo Fisher Scientific, MA, US). Identification of single constituents was carried out using standard substances and by comparing mass spectra obtained from the total ion chromatogram with NIST and MoNa mass spectrometry data library.