Agilent 6890a

GC-MS analyses were carried out with an Agilent 6890A gas chromatograph equipped with a Mass Selective Detector MSD 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) and fitted with a DB-WAX capillary column (polyethylene glycol: 30 m × 0.25 mm i.d., 0.33 µm film thickness) (Agilent Technologies, Palo Alto, CA, USA). The transfer line temperature was set at 240 °C. The column carrier gas was helium at a constant flow rate of 2 mL/min. The mass spectrometer was operated in the electron impact mode (EI) at 70 eV, scanning the range 35–350 m/z at a scan rate of 2.36 scans/s and the ion source temperature was set at 230 °C. Samples (2 μL) were injected manually onto the GC in the split mode at a 25:1 ratio. Solvent delay time was set at 8 min. Gas chromatographic conditions were as reported in the previous paragraph. The volatile constituents were tentatively identified by comparing their elution order and mass spectra with data from the NIST library (Version 2.0f, National Institute of Standards and Technology, Gaithersburg, MD, USA, 2008) and the published literature [35 ].

The phytosterol contents were determined as follows: 10 ml of 2 mol/L KOH in ethanol was used to saponify 0.2 g (accuracy 0.0001 g) of the tested oil samples and 0.5 ml of 0.5 mg/ml 5α‐cholestane (internal standard) at 60°C for 60 min; the unsaponifiable compositions were obtained with hexane. The hexane layer was dried over anhydrous sodium sulfate and silylated using 100 µl N, O–bis (trimethylsilyl) trifluoroacetamide + 1% trimethylchlorosilane (BSTFA + TMCS) at 105°C for 15 min. The mixture was then dissolved in 1 ml hexane for further analysis on an Agilent 6890A gas chromatography system (Agilent) equipped with a DB–5HT column (30 m × 0.32 mm, 0.1 μm; Agilent). The nitrogen (carrier gas) flow rate was 2.0 ml/min, while the detector and injection temperatures were maintained at 320°C. The oven temperature was programmed as follows: an original temperature of 60°C for 1 min, increased to 310°C at 4°C /min and maintained at this temperature for 10 min. The split ratio was 25:1, and the injection volume was 1 μl.

The cell concentration was measured using the optical density at 620 nm. Concentrations of the reducing sugar in inulin were determined by the dinitrosalicylic acid method [61 (link)], and the total sugar was firstly hydrolyzed by 0.2 M H₂SO₄ at 100°C for 1 h, then measured by the methods of Miller [61 (link)] after adding an equivalent amount of 0.4 M NaOH.
The ethanol was analyzed by gas chromatography (Agilent 6890A, Agilent Technologies) as previously described [19 (link)]. Briefly, a hydrogen flame ionization detector and isothermal capillary column (solid phase: cross-linked polyethylene glycol, carrier gas: nitrogen, and injector temperature: 250°C) were operated at 250 and 120°C, respectively.