Gc2010a

SCFAs were extracted from the fecal samples by using methanol solution. Briefly, 0.5 g of a fecal sample was suspended in 2 ml methanol, and the pH value was adjusted to 2.0 by sulfuric acid solution. Subsequently, the suspensions were placed in ice water for 20 min and instantly homogenized using a vortex mixer. The suspensions then were centrifuged at 12,000 × g for 15 min at 4°C. Finally, the supernatants were filtered and collected for further analysis. The contents of SCFAs were determined using a Shimadzu GC2010A (Kyoto, Japan) gas chromatography instrument coupled to a MS-QP2010 mass spectrometer. The inlet temperature was set at 220°C, and 1.0 μl of the sample was injected into the GC-MS system. The run time of the analysis was set to 17.5 min for each sample. The detection conditions were nitrogen gas set at a flow rate of 1.0 ml/min; ionization voltage, 70 eV; inlet temperature, 220°C; and detector temperature, 250°C. The total ion chromatograms were compared to the standard GS-MS chromatograms to identify the profiles of SCFAs according to peak similarities and m/z.

Samples of fermented and dried cocoa beans (20 g) of each fermentation treatments were subjected to the simultaneous distillation/extraction process for 2 h, using pentane (Sigma-Aldrich, St. Louis, MO, USA) as a solvent [44 ,45 ,46 ]. The volatile concentrate obtained was analysed by GC-MS (model QP-2010 Plus, Shimadzu, Tokyo, Japan), equipped with a DB-5MS column (30 m × 0.25 mm × film thickness = 0.25 µm). The oven temperature was adjusted from 60–250 °C, using a ramp of 3 °C/min, injector temperature of 250 °C. Helium gas was used as a mobile phase, with a flow rate of 1.2 mL/min. An electron ionization mass spectrometer (model GC-2010A, Shimadzu, Tokyo, Japan) at 70 eV with the ion source temperature and other parts at 220 °C was used. Quantitative analysis of the chemical constituents was performed by peak-area normalization using a flame ionization detector (FID—Shimadzu, QP 2010 system) under the same conditions as GC-MS, except that nitrogen was used as a mobile phase. The components were identified based on the retention index (RI), which was calculated using the retention times of a homologous series of n-alkanes (C8–C40, Sigma-Aldrich, St. Louis, MO, USA). The pattern of fragmentation observed in the spectra was compared with existing data in the system library and with data from the literature [44 ,45 ,46 ].