Advance spectrometer

Faecal water was prepared to quantify short chain fatty acids in stool. Briefly, samples (13 mL) taken from colonic batch fermentations were centrifuged at 3220×g for 15 min at 4 °C. 100 μl NMR buffer (0.26 g NaH₂PO₄ and 1.41 g K₂HPO₄ made up in 100 ml D₂O, containing 0.1% NaN₃ (100 mg), and 1 mM sodium 3-(Trimethylsilyl)-propionate-d4, (TSP) (17 mg) as a chemical shift reference) was added to 900 μl supernatant and analysed using ¹H NMR spectroscopy (this mixture is defined as ‘faecal water’). The ¹H NMR spectra were recorded at 600 MHz on a Bruker Advance spectrometer (Bruker BioSpin GmbH, Germany) running Topspin 2.0 software and fitted with a cryoprobe and a 60-slot autosampler. Each ¹H NMR spectrum was acquired with 256 scans, a spectral width of 12,300 Hz, and an acquisition time of 2.67 s. The “noesypr1d” pre-saturation sequence was used to suppress the residual water signal with a low-power selective irradiation at the water frequency during the recycle delay and a mixing time of 10 ms. Spectra were transformed with a 0.3 Hz line broadening, and were manually phased, baseline corrected, and referenced by setting the TSP methyl signal to 0 ppm. The metabolites were quantified using the software Chenomx® NMR Suite 7.0™.

All 1D NMR experiments were performed at 500.12 MHz for ¹H (11.7 T) on a Bruker Advance spectrometer with a Bruker narrow bore H/C/N MAS probe (fig. S9A). The ¹³C/¹⁵N MAS NMR spectra were acquired using CP from ¹H to ¹³C/¹⁵N nuclei with heteronuclear decoupling at a sample spinning rate of 10 kHz (fig. S9B). All CP experiments were conducted at a recycle delay of 2 s with a radio frequency field strength of ∼25 to 70 kHz, with a contact time of 2 ms. The quantitative ¹³C direct excitation experiments were carried out with heteronuclear decoupling during detection and sufficiently long recycle delays, 200 s, allowing the nuclei to fully relax between scans. The 2D ¹H-¹³C HETCOR experiments were conducted at 16.4 T (700 MHz for ¹H) using a Bruker 3.2-mm MAS probe at a spinning rate of 15 kHz with a radio frequency field strength of ∼80 kHz. The CP (¹H to ¹³C) pulse sequence was 90° (¹H) − t₁ − CP − t₂, with a contact time of 100 μs. A ¹H 90° one-pulse sequence was used for ¹H NMR experiments. ¹H, ¹³C, and ¹⁵N chemical shifts were calibrated using ¹³C-labeled adamantane and ¹⁵N-labeled glycine, referenced to 1.85 ppm (adamantane; ¹H), 38.5 ppm (adamantane, tertiary carbon; ¹³C), and 33.4 ppm (glycine; ¹⁵N), respectively.