Model rid 10a

Glucose, fructose, and sucrose were extracted from the ground samples using a modified method of Zhao et al. (2010) . Briefly, a mixture of 30 mg dried tissue was extracted three times with 80% (v/v) ethanol in an 80 °C water bath for 15 min. Extracts were centrifuged and supernatants were combined, filtered through 0.45 μm pore size nylon membranes, and 20 μl were used for glucose, fructose, and sucrose analysis by HPLC (Shimadzu Corp., Kyoto, Japan) with a refractive index detector (Model RID-10A). The mobile phase consisted of 80% (v/v) acetonitrile in water with a flow rate of 1 ml min⁻¹. A Luna 5 µm NH₂ 100 Å, LC Column 250 × 4.6 mm from Phenomenex (Torrance, CA, USA) was used for the analysis which, was performed with the column maintained at 40 °C. Data acquisition was controlled by LabSolutions software (Shimadzu Corp.).
The pellets remaining from the above extractions were used for starch solubilization according to Zhao et al. (2010) . α-Amylase (Sigma-Aldrich, A3403) and amyloglucosidase (Sigma-Aldrich, A3042) were used to hydrolyse the pellets. The glucose content was assayed as described above by HPLC and used to calculate starch concentration.

GPC analysis was performed with a high performance liquid chromatography system equipped with a Shimadzu refractive index detector (Model RID-10A), and a Shimadzu LC-20AT pump. Polysaccharides were analyzed using two columns connected in series: a Supelco Progel-TKS G4000 and a Waters Ultrahydrogel 250 (300 × 7.8 mm). A degassed solution of 0.05 M NaNO₃ prepared with ultra-pure water containing 0.02% NaN₃ was used as solvent and eluent. One hundred μl of polysaccharide solutions (5 mg/ml) were filtered through a 0.22 μm PVDF membrane (GV, Millipore) and injected into the column using a manual valve. The eluent flow rate was of 0.6 ml/min and the temperature was held at 25 °C. The column was calibrated using dextran standards of different molecular weights (Sigma Aldrich, USA).

In the last week of experiment, the animals were fasted for 12 h and then received, by gavage, 2 mL of the solution (200 mg of lactulose and 100 mg of mannitol); samples of their urine were collected for 24 h. At the end, the collected urine volume was measured, recorded and stored at −80 °C [26 (link)]. Then, for the analysis, the urine was diluted 1:2 with distillated water, filtered (0.45 µm) and analyzed by the HPLC method.
Furthermore, the colonic feces of animals were quantified for their levels of SCFA, acetate, propionate, and butyrate. The extraction of the SCFA was performed by mixing 100 mg of luminal contents with 2 mL of perchloric acid (10%) and centrifuged (9000× g, 10 min, 25 °C). The supernatant was filtered (0.45 µm) and analyzed by HPLC [27 (link)].
The HPLC conditions for the analyses of lactulose, mannitol and SCFA were a Shimadzu HPLC system (Kyoto, Japan) with a degasses (Model DGU-14A), pump (Model LC-10AT), auto-sampler (Model SIL-20A), column oven (Model CTO-10AS), UV–vis detector (Model SPD-10AV), and a refractive index detector (Model RID-10A). The column used was Aminex HPX-87H (300 cm × 8.7 mm; BIO-RAD) in 55 °C with a pressure of 1920 psi, using H2SO4 0.005 mM as a mobile phase under isocratic conditions [28 (link)]. Lactulose, mannitol and SCFA levels (mg/g of feces) were determined by standard curves using commercial standards (Sigma-Aldrich).