Tracetm1300

The 15 mL of in vitro fermentation fluid of all syringes at 24 h of incubation was taken to determine the concentration of straight‐short‐chain fatty acids (SCFA), branched short‐chain fatty acids (BSCFA), and ammonia‐nitrogen. The ammonia‐nitrogen concentration was determined using a commercial kit (Megazyme, Ammonia Assay Kit – Catalogue no: Product code: K‐AMIAR, Wicklow, Ireland) in a UV‐VIS spectrophotometer (SI Analytics – Xylem Analytics Germany Sales GmbH & Co. KG, Mainz, Germany). The SCFA compositions [acetic acid – AA (C2:0), propionic acid – PA (C3:0), butyric acid – BA (C4:0), valeric acid – VA (C5:0), hexanoic acid – HEXA (C6:0), heptanoic acid – HEPA (C7:0)] and BSCFA [iso‐butyric acid – IBA (C4:0i), iso‐valeric acid – IVA (C5:0i) (do not include 2‐methylbutyric acid) and iso‐caproic acid – ICA (C6:0i)] of fermentation fluid were determined in a GC‐FID (TRACE^TM1300, Thermo Fisher Scientific, Orlando, USA). Helium was used as a carrier gas, and hydrogen was used for combustion. A polyethylene glycol column (length: 60 m, i.d.: 0.25 mm, film thickness: 0.25 μm; TG‐WAXMS, Thermo Scientific, Orlando, FL, USA) was used for this analysis in the device, and the analysis procedure was performed according to Ersahince and Kara (2017 (link)).

All chemicals were purchased from Alfa Aesar or Sigma-Aldrich.
Gas chromatography-mass spectrometry (GC-MS) analyses were carried out by using a TRACE^TM 1300 apparatus equipped with an ISQ^TM QD mass spectrometer detector (Thermo Fisher Scientific). The following chromatographic system was used for analytical experiments: GC-MS (Thermo TG-5SilMS, 20 m, 0.18 µm) with hydrogen as carrier gas [80 °C (0.5 min), followed by linear gradient from 80 to 280 °C (5 min)] at a flow rate of 0.8 mL/min.