Isovaleric acid

The concentrations of SFCAs in cecal content were detected using a gas chromatography-mass spectrometry (GC-MS) system (Trace 1300 and ISQ 7000, Thermo Scientific) (BioNovoGene, Suzhou, China). SCFA standards including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid were purchased from Sigma-Aldrich (St. Louis, USA).

The collected mouse feces were added to distilled water, shaken, mixed overnight at 4 °C, and centrifuged at 15,000 rpm for 20 min. The supernatant was mixed with 25% metaphosphoric acid at a volume ratio of 9:1 and left to react at room temperature for 4 h. The supernatant was filtered and added to a gas phase injection vial for gas chromatography–mass spectrometry (GC-MS, Agilent, USA) analysis. The analysis was performed using a free fatty acid phase (FFAP) column with high purity nitrogen as the carrier gas at a flow rate of 0.8 mL/min. The inlet temperature was 250 °C and the injection volume was 1 µL. The initial temperature was 60 °C, which was increased to 220 °C at 20 °C/min and maintained for 1 min. Acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid standards (Sigma-Aldrich) were mixed at different concentrations and measured under the same conditions.

Sweet potato (Zhejiang sweet potato No. 13 variety) are washed, sliced, dried at 50°C until moisture content is below 5%, crushed, sifted through 100 mesh and moisture-proof packaged). Peptone, beef extract and yeast extract were purchased from Beijing Shuangxuan Microbial Culture Medium Products Factory (Beijing, China). Acetic acid (100%), propionic acid (99%), butyric acid (99%), isobutyric acid (99%), valeric acid (90%), and isovaleric acid (99%) were purchased from Sigma Corporation (USA). The enzyme-linked glucose assay kit was purchased from Suzhou Comin Biotechnology Co., Ltd. (Suzhou, China). All other chemical components are of analytical grade.

SCFA was analyzed in the training cohort. 0.2 g of each fecal sample was separated. Six analytes were targeted for SCFA analysis including acetic acid (Dr. Ehrenstorfer, Germany), propionic acid (Dr. Ehrenstorfer, Germany), isobutyric acid (Supelco, United States), butyric acid (Dr. Ehrenstorfer, Germany), isovaleric acid (Sigma-Aldrich, United States), and valeric acid (Nu-Chek, United States). fecal samples were homogenized in 1.0 mL of ultrapure water that contained an internal standard, 2,2-dimethylbutyric acid (Dr. Ehrenstorfer, Germany). The supernatant was transferred into a new tube after centrifugation, followed 10 µL of 50% sulfuric acid and 0.5 g of sodium sulfate (Macklin, China) were added to the tube along with analytically pure diethyl ether (2 mL). The mixture was vortexed for 1 minute and then centrifuged at 5000 rpm (room temperature, 10 minutes). The ether layer was finally collected for gas chromatography with mass selective detection (5977B GC/MSD, Agilent Technologies, Santa Clara, CA, United States) measurement. The Gas chromatography mass spectrometry (GC/MS) data were acquired and analyzed using MassHunter Workstation software (Agilent Technologies) running on Windows 7 (Microsoft, Redmond, WA, United States). Final concentrations were calculated based on internal standards and are expressed as micromoles per gram of wet feces (μmol/g).

Sodium acetate (catalog no. S2889), sodium butyrate (catalog no. 303410), sodium propionate (catalog no. P5436), sodium salt of isobutyric acid (catalog no. I1754), and isovaleric acid (catalog no. 129542) were purchased from Sigma-Aldrich. The compounds were used in the form of sodium salt. Antibodies to acetyl-H3-K9 (Abclonal; catalog no. A7255), acetyl-H3-K18 (Abclonal; catalog no. A7257), trimethyl-H3-K9 (Beyotime; catalog no. AF5707), trimethyl-H3-K27 (Beyotime; catalog no. AF5710), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Abcam; catalog no. AB8245) were obtained commercially. PrimeScript RT master mix (TaKaRa; catalog no. RR047A) and SYBR green PCR master mix (catalog no. Q141-02/03; Vazyme, Nanjing, China) were obtained commercially. Lymphocyte separation reagent was purchased from Tianjin Haoyang TBD (catalog no. LTS1077). EasySep human CD3⁺ selection cocktail II (Stem Cell; catalog no. 17851), human CD19⁺ selection cocktail II (Stem Cell; catalog no. 17854), CD2-APC (BioLegend; catalog no. 300311), and CD19-PE (BioLegend; catalog no. 302207) were purchased from commercial sources. Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Sigma-Aldrich. Immunoblots were detected using an ECL reagent kit (Beyotime) and the ChemiScope 6000 Touch imaging system (Clinx, China).

Chromatographic analysis was carried out using an Agilent 7890a GC system with a DB23 column (Agilent Technologies, Santa Clara, CA, United States) coupled to a flame detector (FID). The temperature at the injection port and at the FID was 250°C. Helium was used as carrier gas at a flow rate of 1.6 mL/min. For the run, 1 μL of the sample was injected with a 1:25 split and a temperature program that consisted of a ramp from 100 to 200°C at a speed of 8°C/min, keeping constant at 200°C for 3 min was used. Calibration curves with standards of glacial acetic acid (1–50 mM), propionic acid (0.5–20 mM), butyric acid (0.5–20 mM), iso-butyric acid (0.5–20 mM), and iso-valeric acid (0.5–20 mM) were prepared (Sigma-Aldrich, San Luis, MO, United States). Organic acids were identified by comparison to standard retention times and quantified with the corresponding peak area using the calibration curve. Differences were statistically tested using One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (p < 0.05) conducted by the GraphPad Prism^® software.

Acetic acid was purchased from Thermo Fisher Scientific (Fair Lawn, NJ). Propionic acid, isobutyric acid, butyric acid, 2-methylbutyric acid, isovaleric acid, valeric acid, 2-methylpentanoic acid, 3-methylpentanoic acid, isocaproic acid, caproic acid, 2-methylhexanoic acid, 4-methylhexanoic acid, heptanoic acid, hexanoic acid-6,6,6-d₃ internal standard, N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), and methoxyamine hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO).