Db ffap 125 3237

SCFAs were extracted from cecal content (~2 g) and analyzed by using gas-liquid chromatography (GLC) as described previously [34 (link),35 (link)]. Briefly, the cecal content was homogenized and protonated with hydrochloric acid, by using 2-ethylbutyric acid as internal standard. Samples were centrifuged before injection onto a fused-silica capillary column (DB-FFAP 125-3237; J&W Scientific, Agilent Technologies Inc., Folsom, CA, USA). SCFAs in serum were pre-enriched and extracted by hollow-fiber before being injected and analyzed with GLC using the same column as for SCFAs extracted from cecum. GC ChemStation software (Agilent Technologies Inc.,Wilmington, DE, USA) was used for evaluation of the SCFA analysis. For analysis of lactic and succinic acids ion-chromatography was employed according to Jakobsdottir et al. [10 (link)]. The same extraction as for SCFAs was used.

Short-chain fatty acids of grass carp hindgut samples were extracted and determined using gas chromatography as previously described (Schneider et al., 2006 (link); Zhao et al., 2006 (link)). In brief, 200 mg of the sample was suspended in 1.6 mL sterile distilled water with an internal standard hexanoic acid (5 μL). 50% aqueous sulfuric acid (0.4 mL) and diethyl ether (2 mL) were then added. The sample was mixed for 45 min with an orbital shaker and centrifuged for 5 min at 3000 rpm at room temperature. Concentrations of the SCFAs in the supernatant were determined using an Agilent 7890A gas chromatograph equipped with flame ionization detection (Agilent, United States). A fused-silica capillary column (30 m, 0.52 mm, 0.50 mm) with a free fatty acid phase (DB-FFAP 125-3237, J&W Scientific, Agilent Technologies Inc.) was used.

Mouse fecal pellets were collected at week 1, 2 and 3 and frozen until analyzed. Single pellets were weighed and homogenized in 100 µL of deionized water for 3 min. The pH of the suspension was adjusted to 2–3 by adding 5 M HCl at room temperature for 10 min with intermittent shaking. The suspension was transferred into a polypropylene tube and centrifuged for 20 min at 3,000 g, yielding a clear supernatant. The internal standard, 2-ethylbutyric acid (TEBA), was added into the supernatant at a final concentration of 1 mM. Chromatographic analysis used the Agilent 7890 (Agilent). A fused-silica capillary column (30 m, 0.52 mm, 0.50 mm) with a free fatty acid phase (DB-FFAP 125-3237, J&W Scientific, Agilent Technologies Inc.) was used for analysis. Helium was the carrier at a flow rate of 14.4 mL min⁻¹. The initial oven temperature (100°C) was maintained for 30 s, raised to 180°C at 8°C min⁻¹ and held for 60 s, then increased to 200°C at 20°C min⁻¹ and held for 5 min. The flame ionization detector and injection port were kept at 240 and 200°C, respectively. The flow rates of hydrogen, air, and nitrogen were 30, 300 and 20 mL min⁻¹, respectively. The injected sample volume for GC analysis was 1 µL, and each analysis had a run time of 32 min [17] (link).