Agilent 6890 5975

HS‐SPME and GC‐MS were performed using a 50‐/30‐μm divinylbenzene/carboxen fibers purchased from Supelco Inc. (Bellefonte, PA, USA). Before analysis, the fiber was conditioned for 2 hr by inserting it into an Agilent 6890–5975 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) at 250°C to prevent pollution. Each sample following a 6‐day fermentation (3 g) was, respectively, placed in a 15‐mL phial at room temperature along with a magnetic stirrer and tightly covered with a Teflon‐faced silicone septum. The sample was balanced for 10 min at 60°C in a water bath and extracted at the same temperature with continuous stirring for 50 min. After extraction, the fiber was pulled into the needle sheath, the SPME unit was removed from the phial and inserted into the injection port of the gas chromatograph, and the fiber was desorbed for 5 min at 250°C. All experiments were performed in triplicate.

FFAs and FAMEs were extracted by addition of 6 mL of a 2:1 chloroform/methanol mixture (spiked with 0.15 mg/L of either methyl tridecanoate or methyl heptadecanoate as an internal control) to 5 ml of culture. For consistency in data analysis, 1 mL of dodecane layer was similarly treated with 6 mL of a 2:1 chloroform/methanol mixture before gas chromatography (GC) analysis. Quantification of FAs/FAMEs was conducted by GC-FID using an HP 5890 Series II gas chromatograph equipped with an HP-Innowax Column (0.32 mm x 30 m x 0.25 μm, Agilent). All samples were analyzed using the following parameters: inject: 1 μl; inlet temperature 250 °C with split ratio 1:1; carrier gas: helium; flow: 5 ml/min; oven temperature: initial temperature of 160 °C, hold 3 min; gradient to 255 °C at 5 °C/min; hold 3 min; inlet temp: 270 °C, detector temp: 330 °C. The amount of FAs/FAMEs was determined by comparison to a standard curve of various FAs and FAMEs and methyl tridecanoate or methyl heptadecanoate concentrations. To identify all FA/FAME products, GC/mass spectrometry analysis was additionally performed using an Agilent 6890-5975 equipped with HP-Innowax Column (0.32 mm x 30 m x 0.25 μm, Agilent). Peak identification was performed through comparison with GC retention time, known standards and mass spectra with the National Institute of Standards and Technology (NIST) database.

DHPs-1 was methylated according to Wang et al. (7 (link)). The sample was methylated, hydrolysed, reduced and acetylated. The partially methylated alditol acetates (PMAAs) were determined by a gas chromatography/mass (GC/MS) system (Agilent 6890/5975, USA). Briefly, 5 mg of polysaccharide was dissolved in DMSO/NaOH under nitrogen and then methylated with CH₃I. The fully methylated sample was hydrolyzed with 2.5 mol/L TFA at 121°C for 1.5 h. After reduction and acetylation of the hydrolysates, the partially methylated alditol acetate (PMAA) derivatives were analyzed with a GCMS system (Agilent 7890A/5975C, USA) fitted with a HP-5 ms quartz capillary colum (30 m × 0.25 μm × 0.25 mm).