Fused silica capillary column

Cecal chyme samples were mixed evenly with double steam water at 1:5 and centrifuged at 2000 rpm for 12 min at 4 °C. The supernatants (1 mL) were collected and added to 0.2 mL of deproteinizing-acidifying solution (metaphosphoric acid [25% w/v] and crotonic acid [0.65% w/v]) for SCFAs measurement using a gas chromatography method (Agilent 7890B, Agilent, CA, USA). The samples were separated on a fused silica capillary column (Agilent, Santa Clara, CA, USA) at 110 °C, 3 min; 110–150 (40 °C/min) column temperature program, a 200 °C injection temperature, and 220 °C FID detector temperature with N2 as a carrier gas.

The GC/MS analysis was carried out on an Agilent 6980 GC system equipped with a fused-silica capillary column with a 0.25-μm HP-5MS stationary phase (Agilent, Shanghai, China). We used the same operational methods as our previous studies [20 (link)].

GC-MS analysis of the biological extracts was performed on a Agilent 7890A GC split/split less injector (280°C) linked to a Agilent 5975C MSD (electron voltage 70eV, source temperature 230°C, quad temperature 150°C multiplier voltage 1200V, interface temperature 310°C). The acquisition was controlled by a HP Compaq computer using Chemstation software, initially in full scan mode (50–600 amu/sec) or in selected ion mode (30ions 0.7cps 35ms dwell) for greater sensitivity. The sample (1ul) in diethyl ether was injected by an Agilent7683B auto sampler and the split opened after 1 minute. Separation was performed on an Agilent fused silica capillary column (30m x 0.25mm i.d) coated with 0.25um dimethyl polysiloxane (HP-5) phase. The GC was temperature programmed from 50–310°C at 5°C min and held at final temperature for 10 minutes with Helium as the carrier gas (flow rate of 1ml/min, initial pressure of 50kPa, split at 30 mls/min). Peaks were identified and labelled after comparison of their mass spectra with those of the NIST05 library if > 90% fit or from their elution order from biochemical literature.

Total lipid content of the cells was determined in triplicate by the modified Bligh & Dyer [23 (link)] method using 1 : 2 chloroform : methanol and presented as weight percentage (%) [23 (link)]. Fatty acids were determined as fatty acid methyl esters (FAMEs) in triplicate after acidic transesterification of lipids [24 ]. The resulting FAMEs were analysed by gas chromatograph mass spectrometry (GC-MS; Thermo Scientific ITQ 700, USA) equipped with a flame ionization detector (FID) and a fused silica capillary column (60 m × 0.25 mm × 0.25 µm; Agilent Technologies, USA). The injector and detector temperatures were maintained at 270 and 280°C, respectively, with an oven temperature gradient of 50–170°C at 40°C min⁻¹ after a 1 min hold time at 50°C, then with an oven temperature gradient of 170–210°C at 18°C min⁻¹ after a 1 min hold. All parameters of the FAME were derived from the calibration curves generated from the FAME standard mix (Supelco 37 component FAME mix, Sigma-Aldrich, USA) [25 (link)].

Gas chromatography (GC)- Mass spectrometry (MS) analysis was carried on a 7820A GC/5977E MSD (Agilent, USA) fitted with an HP-5 (30 m × 0.25 mm ID, film thickness 0.25 µm) fused-silica capillary column (Agilent, USA). The carrier gas was helium at flow rate of 1.0 ml/min. The mass spectra were obtained with an ionization voltage 70 eV, trap current 250 μA, and ion source temperature of 200 °C. The conditions of programmed oven temperature were similar to those described for GC. Samples were injected using the splitless mode. The column temperature was maintained at 35 °C for 2 min and programmed as follows: increase rate 5 °C/min to 250 °C and finally hold for 10 min at 250 °C.
Each Mentha plants (2.0 g) were put into a 15 mL thermostated vial and the SPME fiber was introduced for 12 h into the thermostated vial (RT) with a rubber septum containing 2.0 g of the three fresh aerial parts of each Mentha species during the SPME extraction procedure. A 1 cm long 50/30 µm polydimethylsiloxane/divinylbenzene/carboxen-coated fiber was utilized for analysis. The fiber was adjusted in a GC injection port at for 1 min prior to use. The absorbed component was injected into a GC by desorption at 250 °C for 2 min in the injector (splitless mode). SPME procedure was carried out three times and the results were presented as the mean ± standard deviation.