Anhydrous ether

Oilseeds such as rapeseed, soybean, peanut, and sesame were collected. After removing broken, mildew seeds and foreign matters, these samples were pulverized using a grinder and refrigerated them at 4 °C until further processing. Corn flour, infant formulas, and edible oils including soybean oils, rapeseed oils, olive oils, safflower seed oils, Camellia oils, linseed oils, grapeseed oils, sesame oils, peanut oils, and corn oils, were purchased from local supermarkets.
Vitamin K₁ and vitamin K₁-D₇ were purchased from the Toronto Research Chemicals (Ontario, Canada). Methanol and n-hexane (HPLC grade) were purchased from Thermo Fisher Scientific Technology Co., Ltd. (Shanghai, China). Formic acid (98% purity) and ammonium formate (98% purity) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Anhydrous ether (99.8% purity) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Nitrogen (99.99% purity) was purchased from Minghui Gas Technology Co., Ltd. (Wuhan, China). Ultra-pure water (18 mΩ) was obtained from a Milli-Q water purification system (Millipore Co., Ltd., Milford, MA, USA) and was used to prepare all aqueous solutions.

Auricularia polytricha No.10 strain was purchased from Jiangsu Tianda Institute of Edible Fungi (Jiangsu, China).
Bovine serum albumin (BSA), Coomassie brilliant blue G–250, and ascorbic acid were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Concentrated sulfuric acid, phosphoric acid, salicylic acid, trichloroacetic acid (TFA), anhydrous alcohol, acetone, anhydrous ether, glucose, sucrose, fructose, maltose and lactose, urea, peptone, ammonium sulfate, yeast and corn starch, neutral protease, 30% hydrogen peroxide (H₂O₂), phenol, Vitamin C (Vc) sodium hydroxide, ferrous sulfate (FeSO₄), ferric chloride (FeCl₃), sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium ferricyanide [K₃Fe(CN)₆] were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The monosaccharide Standards including fucose (Fuc), rhamnose (Rha), arabinose (Ara), mannose (Man), galactose (Gal), glucose (Glc), xylose (Xyl), fructose (Fru), galacturonic acid (GalA), glucuronic acid (GlcA), 2,2–diphenyl–1–picrylhydrazyl (DPPH), and potassium bromide (KBr, Spectrum pure grade) were purchased from Sigma Chemical Co. (St. Louis, MO, United States). All chemicals and solvents used in the current work were of analytical grade.

SCFAs in fecal samples were determined via gas chromatography–mass spectrometry (Agilent Technologies, Palo Alto, CA, USA) using the method of Han et al. [19 (link)] with some modifications. About 0.1 g of fecal samples were homogenized in 600 μL ultrapure water and acidified using 50% concentrated sulfuric acid (Sinopharm, Beijing, China) prior to centrifugation (5000× g, 10 min, 4 °C). The supernatants were taken and vortexed with anhydrous ether (Sinopharm, Beijing, China) at 1: 1 (v/v), after which the samples were centrifuged (5000× g, 10 min, 4 °C), and the ether layer was collected for analysis. SCFAs were separated using an HP-FFAP column (30 m × 250 μm × 0.25 μm; Agilent Technologies, Palo Alto, CA, USA). The initial temperature was 90 °C, which was elevated to 150 °C at 12 °C/min. Then, the temperature was elevated to 220 °C at 20 °C/min and held for 4.5 min. Acetic acid, propionic acid and butyric acid (Macklin Biochemical, Shanghai, China) were the standard solutions.