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To extract fecal metabolites, 100 mg of fecal samples were transferred into 2 mL screw-cap vials, and 1.5 mL of 50% methanol was added. Samples were homogenized with beads for 1 min followed by a 30 s rest, and this cycle was repeated five times. After homogenization, samples were centrifuged at 5,000 × g for 15 min, and the supernatant was transferred to 8 mL vials. Samples were concentrated overnight using a SpeedVac concentrator. For LC–MS analysis, dried samples were reconstituted in 400 μL of 70% methanol and filtered through a 0.22 µm PVDF filter prior to transfer into LC–MS vials. , To extract fecal metabolites, 150 mg of fecal samples were transferred into 2 mL screw-cap vials, and 1.5 mL of 70% methanol was added. Samples were homogenized with beads for 1 min followed by a 30 s rest, and this cycle was repeated five times. After homogenization, samples were centrifuged at 5,000 × g for 15 min, and the supernatant was transferred to 14 mL glass tubes. Acetone was added to the supernatant at a 1:4 (v/v) ratio for protein precipitation, and samples were stored at −20 °C for 1 h. The protein-precipitated samples were then centrifuged at 5,000 × g for 15 min, and the resulting supernatant was transferred to 8 mL vials. Samples were concentrated overnight using a SpeedVac concentrator. For LC–MS analysis, dried samples were reconstituted in 400 μL of 70% methanol and filtered through a 0.22 µm PVDF filter prior to transfer into LC–MS vials. , Fecal short-chain fatty acids (SCFAs) were extracted following a modified isobutanol-based protocol. Briefly, 100–150 mg of fecal samples were placed into a 2 mL screw-cap tube containing beads. Subsequently, 1 mL of 10% isobutanol was added, and the samples were homogenized twice for 20 seconds at 6,000 rpm with a 30-second interval between homogenizations. The homogenates were centrifuged at 21,000 × g for 5 minutes, after which 675 μL of the supernatant was transferred to a new 1.5 mL microcentrifuge tube. To the supernatant, 125 μL of 20 mM NaOH and 400 μL of chloroform were added. The mixture was vortexed thoroughly and centrifuged at 21,000 × g for 2 minutes. Subsequently, 400 μL of the upper aqueous phase was transferred into a new 2 mL screw-cap tube. Then, 80 μL of isobutanol and 100 μL of pyridine were added, and the total volume was adjusted to 650 μL with 70 μL of distilled water (DIW). To minimize bubble formation during the derivatization reaction, a boiling chip was added to each tube.

Chromatographic separation was performed using a Phenomenex HPLC C18 column (250 × 4.6 mm, 5 µm particle size; Phenomenex, USA) equipped with a SecurityGuard C18 cartridge (4 × 2.0 mm; Phenomenex, USA). The column temperature and flow rate were set to 35 °C and 0.7 mL/min, respectively. The mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient program was as follows: 90:10 (A:B) at 0 min, linearly ramped to 0:100 (A:B) over 30 min, held at 0:100 (A:B) until 33 min, and then returned to the initial conditions of 90:10 (A:B). The injection volume was 5 µL, and samples were injected using an autosampler. , Chromatography separation was achieved using an HP-5ms capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness). Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. Samples were injected in split mode with a split ratio of 50:1, and the injector temperature was maintained at 260 °C. The oven temperature program was set to an initial temperature of 40 °C held for 5 minutes, followed by a ramp of 10 °C/min to a final temperature of 310 °C. The transfer line temperature was set to 280 °C. The ion source temperature and quadrupole temperature were maintained at 250 °C and 150 °C, respectively.

Metabolite samples were analyzed using an Agilent 6546 Q-TOF mass spectrometer coupled with a UPLC system (Agilent Technologies, USA), and LC-MS/MS spectral data were obtained. , The analysis of derivatized SCFAs was performed using a gas chromatography–mass spectrometry (GC/MS) system consisting of an Agilent 7890N gas chromatograph coupled to a 5975C mass selective detector (MSD).