Sulfuric acid

Tartary buckwheat seeds (Hefeng No. 1) were cultivated in Datong, Shanxi Province, China, and were acquired from Beijing Green Valley Sprout Co., Ltd. (Beijing, China). Rutin, gallic acid, and γ-Amino butyric acid standard were purchased from Shanghai solarbio Bioscience & Technology Co., Ltd. (Shanghai, China). The analytical grade chemicals, such as Folin-Ciocalteu reagent, gallic acid (GA), sodium carbonate, sodium hypochlorite, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, phosphoric acid, glucose anhydrous, aluminum chloride, potassium acetate, o-phthalaldehyde, copper sulfate, potassium sulfate, sulfuric acid, boric acid, ethanol, ethyl acetate, hydrochloric acid, trichloroacetic acid, sodium acetate, phenol, potassium persulfate, sodium hydroxide, and aluminum chloride were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). In addition, 2,2-diphenyl picryl hydrazyl (DPPH), acetone, and methanol (HPLC grade) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Total antioxidant capacity (T-AOC) kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Sulfuric acid, hydrochloric acid, ethanol, silver nitrate, potassium chloride, ammonium hydroxide solution, ammonium tetrathromolybdate and sodium sulfide nonahydrate were purchased from Macklin. Copper foam (200 × 100 × 1.5 mm, 99.8% purity) was obtained from ZhongNuo Advanced Material (Beijing) Technology Co., Ltd. Deionized water (DI water) (resistivity > 18.2 MΩ cm⁻¹) was prepared by a pure water equipment (TTL-6B). All chemical reagents were used as received.

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).

Furfural residue (FR) was obtained from Jinan Shengquan Company (Jinan, China), Shandong Province. Urea, KOH, Hydrochloric acid (36% HCl), Sulfuric acid (98%), and ethanol (99.8% CH₃CH₂OH) were all purchased from Shanghai Macklin Biochemical Technology Co., Ltd., (Shanghai, China) and were used without being purified. Deionized water was adopted for all experiments.

The seeds of G. uralensis utilized for the experiment were provided by the Institute of Licorice at Shihezi University (Shihezi, China). The standards of glycyrrhizic acid (Batch No.: C11654946), glycyrrhizin (Batch No.: C11602211), glycyrrhizin (Batch No.: C10828731), methanol, formic acid, anhydrous ethanol, 2,3,5-triphenyl tetrazolium chloride (Batch No.: C298-96-4), ethyl acetate, KH₂PO₄, 2,4-dinitrophenol indicator, NaOH, ammonium vanadate, aluminum trichloride, and sulfuric acid were purchased from Macklin Biochemical (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China); acetonitrile was purchased from Thermo Fisher Scientific (LCMS-level, Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA); nitric acid and hydrogen peroxide were purchased from Adamus Reagent (Adamas Reagent Co., Ltd., Shanghai, China).

Fecal samples for SCFA analysis were frozen at −80°C within 3 h of voiding. Six analytes were targeted for SCFA analysis, namely, acetic acid (Dr. Ehrenstorfer, Germany), propionic acid (Dr. Ehrenstorfer, Germany), butyric acid (Dr. Ehrenstorfer, Germany), isobutyric acid (Supelco, United States), valeric acid (Nu-Chek, United States), and isovaleric acid (Sigma-Aldrich, United States). Feces were homogenized in 1.0 mL of ultrapure water containing an internal standard, 2,2-dimethylbutyric acid (Dr. Ehrenstorfer, Germany). After centrifugation, the supernatant was transferred into a new tube. Then, 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 solution was vortexed for 1 min and then centrifuged for 10 min at room temperature. The ether layer was collected for gas chromatography with mass selective detection (5977B GC/MSD, Agilent Technologies, Santa Clara, CA, United States) measurement (Supplementary Table S2). The GC/MS data were acquired and analyzed using MassHunter Workstation software (Agilent Technologies) running on Windows 7 (Microsoft, Redmond, WA, United States). The concentrations of fecal SCFAs were calculated with the use of external standards and are expressed as micromoles per gram of wet feces.