Thawed fecal samples (∼100 mg) were weighed and mixed with saline phosphate buffer as described by Le Gall et al. (2011) (link). Samples were homogenized for 60 s, centrifuged (14,000 × g, 15 mins at 4°C), the supernatants filtered through 0.2 μm membrane, and 500 μL of each filtrate transferred to a 5 mm o.d. NMR tube for metabolomic analysis.
1H NMR spectra were acquired on a Bruker Avance 600 MHz NMR spectrometer (Bruker, Rheinstetten, Germany). Sample temperature was controlled at 300 K. The noesypr1d pre-saturation sequence was used to suppress the residual water signal with low power selective irradiation at the water frequency during the recycle delay (D1 = 2 s) and mixing time (D8 = 0.15 s). Spectra were transformed with 1 Hz line broadening and zero filling, and the baseline corrected using the TOPSPIN 2.0 software. The “underground removal tool” of Bruker AMIX software v3.9 was applied to all spectra to remove the broad irregular envelope which extends from 0.7 to 4.5 ppm. The resulting spectra were divided according to the horizontal axis into variable width “buckets” using the AMIX graphical editor, and the intensities within each bucket were summed.
1H NMR spectra were acquired on a Bruker Avance 600 MHz NMR spectrometer (Bruker, Rheinstetten, Germany). Sample temperature was controlled at 300 K. The noesypr1d pre-saturation sequence was used to suppress the residual water signal with low power selective irradiation at the water frequency during the recycle delay (D1 = 2 s) and mixing time (D8 = 0.15 s). Spectra were transformed with 1 Hz line broadening and zero filling, and the baseline corrected using the TOPSPIN 2.0 software. The “underground removal tool” of Bruker AMIX software v3.9 was applied to all spectra to remove the broad irregular envelope which extends from 0.7 to 4.5 ppm. The resulting spectra were divided according to the horizontal axis into variable width “buckets” using the AMIX graphical editor, and the intensities within each bucket were summed.