Right after evacuation, the stools were assigned a faecal quality score using a 5-points visual scale with 0.5 score interval ranging from 1 (hard and dry faeces) to 5 (liquid diarrhoea) [22 ]. Scores of 2–3 were considered the optimum, consisting in firm but not dry stool, with moderate segmentation visible, holding form when picked up leaving none or minimal residual on the ground.
After thawing, 2 g of faeces were mixed with 1/1 deionized water and pH measured using a Mettler Toledo InLab® Expert Pro pH meter. The analysis of short chain fatty acids (SCFA) (2:0, acetic; 3:0, propionic; 4:0, butyric; iso 4:0, isobutyric; 5:0, valeric; iso 5:0, isovaleric) and lactic acid of faecal samples was performed by HPLC according to the following procedures: 3 g of faeces was diluted with 150 mL of 0.1 N H2SO4 aqueous solution and homogenized for 2 min by UltraTurrax (IKA®-Werke GmbH & Co. KG, Staufen, Germany). The mix was centrifuged (5,000 × g for 15 min at 4 °C) to separate the liquid phase from the solid residuals and the liquid phase subsequently microfiltered (SLMV033RS, 0.45-μm Millex-HV, Merck-Millipore, Billerica, MA). The resulting sample was directly injected in the HPLC apparatus using an Aminex 85 HPX-87 H ion exclusion column (300 mm × 7.8 mm; 9-μm particle size; Bio-Rad, Milan, Italy) kept at 40 °C; the detection wavelength was 220 nm. The analyses were carried out applying an isocratic elution (flux 0.6 mL/min) with a 0.008 N H2SO4 solution as mobile phase; the injection loop was 20 μL. Individual SCFA and lactic acid were identified using a standard solution of 4.50 mg/mL of lactic acid, 5.40 mg/mL of acetic acid, 5.76 mg/mL of propionic acid, 7.02 mg/mL of butyric acid and isobutyric acid, 8.28 mg/mL of valeric acid and isovaleric acid in 0.1 N H2SO4 (69775, 338826, 402907, B103500, 58360, 75054, 129542, respectively; Sigma-Aldrich, Milano Italy). Quantification was done using an external calibration curve based on the standards described above.
After thawing, 2 g of faeces were mixed with 1/1 deionized water and pH measured using a Mettler Toledo InLab® Expert Pro pH meter. The analysis of short chain fatty acids (SCFA) (2:0, acetic; 3:0, propionic; 4:0, butyric; iso 4:0, isobutyric; 5:0, valeric; iso 5:0, isovaleric) and lactic acid of faecal samples was performed by HPLC according to the following procedures: 3 g of faeces was diluted with 150 mL of 0.1 N H2SO4 aqueous solution and homogenized for 2 min by UltraTurrax (IKA®-Werke GmbH & Co. KG, Staufen, Germany). The mix was centrifuged (5,000 × g for 15 min at 4 °C) to separate the liquid phase from the solid residuals and the liquid phase subsequently microfiltered (SLMV033RS, 0.45-μm Millex-HV, Merck-Millipore, Billerica, MA). The resulting sample was directly injected in the HPLC apparatus using an Aminex 85 HPX-87 H ion exclusion column (300 mm × 7.8 mm; 9-μm particle size; Bio-Rad, Milan, Italy) kept at 40 °C; the detection wavelength was 220 nm. The analyses were carried out applying an isocratic elution (flux 0.6 mL/min) with a 0.008 N H2SO4 solution as mobile phase; the injection loop was 20 μL. Individual SCFA and lactic acid were identified using a standard solution of 4.50 mg/mL of lactic acid, 5.40 mg/mL of acetic acid, 5.76 mg/mL of propionic acid, 7.02 mg/mL of butyric acid and isobutyric acid, 8.28 mg/mL of valeric acid and isovaleric acid in 0.1 N H2SO4 (69775, 338826, 402907, B103500, 58360, 75054, 129542, respectively; Sigma-Aldrich, Milano Italy). Quantification was done using an external calibration curve based on the standards described above.
Free full text: Click here
