7890b system

VOAV was extracted from the powdered crude drug by steam distillation according to the procedure recorded in the Chinese Pharmacopoeia (2020 edition) [4 ]. The powdered (15 g, 20 mesh) seed mass of A. villosum was weighed, and then steam distillation was performed with 500 mL of water for 5 h, left for 1 hour to calculate the content of volatile oil.
Powdered (1 g, 50 mesh) seed mass of A. villosum was weighed, added 25 mL of absolute ethanol, sonicated (300 W, 40 kHz) for 30 min, cooled at room temperature and absolute ethanol was used to make up the lost weight, filtered and the filtrate was taken after filtration, which was the test substance solution; and then an appropriate amount of bornyl acetate control sample was taken to prepare a solution of 0.3 mg/mL, which was the control sample solution, and 1 μL each was taken for determination. All experiments were conducted using Agilent Technologies 7890B system(Agilent Technologies, Santa Clara, California, USA). These compounds were separated by a HP-5MS Capillary column(30 m × 0.25 mm × 0.25 μm, Agilent Technologies, USA), the split ratio was 10:1, the inlet temperature, detector (FID) temperature and column temperature were set at 230°C, 250°C, 100°C, respectively.

The profile of volatile organic compounds (VOCs) produced by L. paracasei 85 and L. buchneri 93 were determined using an Agilent 7890bsystem (Santa Clara, CA, USA) coupled with Agilent 5977 Inert XL MSD apparatus. The system was equipped with a fused-silica capillary column coated with a polar stationary phase (OMEGAWAX™250, 30 m × 0.25 mm × 0.25 μm, Supelco, Bellefonte, PA, USA). The bacterial culture with or without ZEN treatment was inoculated with Tryptic Soy Agar (TSA) medium (Sigma-Aldrich, Steinheim, Germany). The vials were placed at an angle into the incubator. The VOCs were extracted with 50 μm polydimethylsiloxane (PDMS)/divinylbenzene (DVB) fiber (Supelco, Bellefonte, PA, USA) at 37 °C for 50 min. The absorbed molecules were desorbed into the injection port for 5 min at 250 °C. VOCs were separated with the following program: held 40 °C for 5 min, heated at 5 °C/min to 185 °C, held for 1 min, heated at 5 °C/min to 200 °C, held for 10 min, and then heated at 10 °C/min to 240 °C. Helium was used as the carrier gas. The MS interface and the ion source were maintained at 250 and 230 °C, respectively. Acquisition was performed in electron impact mode (70 eV) with the mass range of 30–300 m/z. Compounds were identified by matching mass spectra with the NIST14.1 library [2 (link)]. The analyses for each sample were carried out in triplicate.

The SCFA of concentration was measured using the method of gas chromatography, as reported earlier with minor modifications (Zhou et al., 2019 (link)). Stool and cecum content samples were homogenized and diluted with distilled water. After mechanical vibration and centrifugation at 15,000 rpm for 15 min at 4°C, a mixture of supernatant fluid and 25% metaphosphoric acid solution (9:1) was allowed to stand for 3 h. Then, the mixed solution was centrifuged at 15,000 rpm for 10 min and filtered through a membrane filter (pore size 0.45 μm). SCFAs were measured with a gas chromatography (Agilent Technologies 7890B System) equipped with a DB-FFAP column (30 m × 250 μm × 0.25 μm). The carrier gas was nitrogen (flow 0.8 ml/min, split ratio 50:1, volume of sampling 1 μl). The oven, detector, and injector temperatures were 220, 280, and 250°C, respectively. The SCFA content was quantified using an external standard curve method with standard solutions of acetate, propionate, butyrate, isobutyrate, isovalerate, and valerate (Wang et al., 2017 (link)).
The pH of the cecum contents was measured by centrifugation at 8,000 rpm for 1 min at 4°C, then testing 0.8 μl of supernatant with pH paper (pH 5–9) and observing the color change.