Nelson 1020

SCFA (acetate, propionate, and butyrate) content was determined by gas chromatography (Nelson 1020, Perkin-Elmer, St Quentin en Yvelines, France). The samples were extracted with water (wt g/vol), centrifuged at 17,000 × g for 10 min, and the supernatant collected. The proteins were precipitated using a phosphotungstic acid saturated solution. A volume of 0.1 mL of the supernatant was analyzed using a gas–liquid chromatograph (Autosystem XL; Perkin Elmer, Saint-Quentin-en-Yvelines, France). All samples were analyzed in duplicate. The data was collected and peaks integrated using Turbochromv6 software (Perkin Elmer, Courtaboeuf, France).

The SCFA (acetate, propionate and butyrate) levels in the culture supernatants were determined by gas chromatography (Nelson 1020, Perkin Elmer, St Quentin-en-Yvelines, France). The overnight culture was centrifuged, and the supernatant was collected. The proteins were precipitated using a saturated phosphotungstic acid solution. A volume of 0.1 mL of the supernatant was analysed using a gas–liquid chromatograph (Autosystem XL; Perkin Elmer, Saint-Quentin-en-Yvelines, France). All the samples were analysed in duplicate. The data were collected, and the peaks were integrated using Turbochromv6 software (Perkin Elmer, Courtaboeuf, France).

SCFA (acetic, propionic and butyric acid) were analysed and concentrations were determined with gas chromatography (Nelson 1020, Perkin‐Elmer, St Quentin en Yvelines, France) as described previously^{76 (link)}. Results are expressed in relative percentage of each SCFA.

The concentrations of short-chain fatty acids (acetate, propionate, and butyrate) in the fecal content were analyzed after water extraction of acidified samples using gas-liquid chromatography (Nelson 1020; Perkin-Elmer, St. Quentin en Yvelines, France), as described previously (Lan et al., 2008 (link)). Fecal SCFA concentrations are expressed as millimolar (mM). The concentration of D and L-Lactates was measured in fecal samples using the Biosentec D/L lactic acid enzymatic kit according to the manufacturer's instructions (Biosentec, Toulouse, France) and as we described previously (Rul et al., 2011 (link)). Fecal D and L-Lactate concentrations are expressed as millimolar (mM).

Samples were water-extracted, and proteins were precipitated with phosphotungstic acid. A volume of 0.1 μl of the supernatant was analyzed for SCFAs on a gas–liquid chromatograph (Nelson 1020; Perkin-Elmer, St. Quentin en Yvelines, France) equipped with a split-splitless injector, a flame-ionization detector and a capillary column (15 m × 0.53 mm, 0.5 μm) impregnated with SP 1000 (FSCAP Nukol; Supelco, Saint-Quentin-Fallavier, France). The carrier gas (He) flow rate was 10 ml/min, and the inlet, column, and detector temperatures were 175, 100, and 280 °C, respectively. 2-Ethylbutyrate was used as the internal standard. Data were collected and peaks integrated using Turbochrom v6 software (Perkin Elmer, Courtaboeuf, France). Cecal SCFA concentrations are expressed as μmol/g of stool.

Weighted fecal samples were reconstituted in 200 µL MilliQ^® water, homogenized, incubated at 4 °C for 2 h, and then centrifuged at 12,000× g at 4 °C for 15 min. Supernatants were weighed, saturated phosphotungstic acid (100 µL for 1 g of fecal content) was added, and samples were incubated at 4 °C overnight. After centrifugation, short chain fatty acid (SCFA) concentrations (including butyrate, propionate, and acetate) were determined by gas chromatography (Nelson 1020, Perkin-Elmer, St. Quentin en Yvelines, France). Briefly, chromatographic separation was achieved on DB-FFAP columns (30 m × 250 μm, 0.25 μm). The injector temperature was 250 °C and the injection volume was 1 μL. The initial oven temperature was 100 °C, and then was increased to 250 °C (10 °C/min) and held for 5 min. The carrier gas was helium at a constant flow of 7 mL/min. Samples were injected using the split mode at a ratio of 10:1. Detection was performed with a flame ionization detector (FID).

The concentrations of short-chain fatty acids (SCFA), namely acetate, propionate, butyrate, valerate, caproate, isobutyrate, isovalerate, and isocaproate, were determined in the cecal contents by gas–liquid chromatography (Nelson 1020, Perkin-Elmer, St Quentin en Yvelines, France) as previously described [35 (link)]. SCFA concentrations are expressed in mM.