Db 1 column

To investigate if the carbon flux was really redistributed to the newly constructed EP-bifido pathway, 13C-MFA was performed using 100% 1-¹³C₁ glucose as the feeding substrate was added to a concentration of 10 g/L. Cells at the exponential growth phase were harvested by centrifugation at 7000 g for 5 min at 4 °C. The cell pellet was then washed twice with chemical defined medium and hydrolyzed in 6 M HCl for 24 h at 120 °C (Schwender et al. 2006). The resulting proteinogenic acids were derivatized with N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide containing tert-butyldimethylchlorosilane in acetonitrile at 105 °C for 1 h, and then analyzed by a GC–MS [Agilent 7890 A GC and 5975 C Mass Selective Detector (Agilent Technologies, Santa Clara, USA)] equipped with a DB-1column (Agilent Technologies). The data obtained from GC–MS were corrected by reduction of the natural abundance ratio of C, H, O, N, and Si isotopes [30 (link)]. Metabolic fluxes were estimated by minimizing the residual sum of squares between experimentally measured and model predicted ¹³C-enrichment using ¹³C-Flux software obtained from Dr. Wiechert [33 (link)].

After centrifuging the fecal and slurry samples at 3134× g for 20 min at 20 °C, 4 mL of supernatant was collected. The supernatant was placed in a 20-mL glass vial, and then 4 mL of chloroform (Merck, Darmstadt, Germany) and 60 μL of 4 M sodium hydroxide solution (Sigma-Aldrich, St. Louis, MO, USA) were also added to the glass vial and mixed with supernatant. The mixture in the glass vial was centrifuged at 3134× g for 20 min at 20 °C, and the chloroform layer was collected and placed into a 2.0-mL gas chromatography vial (Agilent, Santa Clara, CA, USA). Phenols and indoles in the chloroform layer were determined using a gas chromatograph (6890N, Agilent, Santa Clara, CA, USA) that was equipped with a DB-1 column (30 m × 0.25 mm × 0.25 μm, Agilent, Santa Clara, CA, USA) and a flame ionization detector. A sample of 2.0 μL was injected at a 5-to-1 split ratio. The gas chromatography oven was initially set at 40 °C for 5 min, increased to 230 °C at a rate of 10 °C per min, and finally held at 230 °C for 2 min. The injection and detection ports of gas chromatography were maintained at 250 °C.

The coupled GC–electrophysiology system, in which the effluent from the GC column is simultaneously directed to the antennal preparation and the GC detector, has been described previously.41 Separation of the collected palm wine alcohol extract was achieved by high‐resolution GC (6890N, Agilent Technologies, Stockport, UK) equipped with a cool on‐column injector and flame ionization detector (FID). A capillary GC column was used, 50 m × 0.32 mm i.d. DB‐1 column (Agilent J & W, Agilent Technologies, Stockport, UK). The oven temperature was maintained at 30 °C for 2 min and then programmed to increase at 10° min⁻¹ to 250 °C. The carrier gas was helium. The outputs from the EAG amplifier and the FID were monitored simultaneously and analysed using the software package (Syntech). A peak was deemed to be electrophysiologically active if it elicited responses on three or more antennal preparations.

The dried extracts from above were derivatized using previously published methods [22 (link), 23 (link)]. Briefly, samples were reacted at 60 °C for 1 h with 100 μL of methoxyamine hydrochloride in pyridine (20 μL/mL). The sample was then silylated with 100 μL of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) (70 °C/1 h). GC/MS conditions were as follows: the gas chromatograph, model 6890GC (Agilent Technologies, Santa Clara, CA, USA) was equipped with a mass spectrometer detector 5975 and an apolar stationary-phase DB-1 column (100% dimethylpolysiloxane); (30 m × 0.25 mm; i.d. 0.25 μm film thickness). The starting oven temperature of the column at the time of injection, 70 °C, was maintained for 4 min and then ramped up at a rate of 10 °C/min until it reached a final temperature of 300 °C. The carrier gas (helium) had a flow rate of 1 mL/min. The injector and the detector were maintained at 250 °C. Injection volume was 1 μL, after being split 25 to 1. The mass spectrometer was set to electron ionization mode (70 eV). Identification of components was by both retention time, and comparison of the mass spectra to a commercial library [24 ].