Trichloroacetic 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).

BSH activity was determined by measuring the amount of amino acids released from conjugated bile salts, as previously described with minor modifications (Kumar et al., 2012 (link)). BSH activity of BDX-01 was measured in vitro, and Lactobacillus plantarum strain lp91 was used as a positive control. Fecal bacterial BSH activity was assayed according to CA production from fecal TCA. Briefly, protein extracts were prepared by sonicating 0.5 g of stool samples in 1 mL of PBS. The samples were centrifuged at 12,000 × g for 10 min and 1 mL of supernatant was collected and incubated with 1.8 mL of PBS and 0.1 mL of 0.1 M trichloroacetic acid (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) at 37°C for 30 min. Reactions were stopped by adding with 0.1 mL of trichloroacetic acid for 1 min. The mixtures were centrifuged and 1 mL of supernatant collected was mixed with 1 mL of 2 M trichloroacetic acid buffer and 1 mL of ninhydrin reagent [0.5 mL of 1% (w/v) ninhydrin in 0.5 M citrate buffer (pH 5.5), 1.2 mL of 30% (v/v) glycerol, and 0.2 mL of 0.5 M citrate buffer (pH 5.5)]. The mixture was vortexed to mix well, boiled for 15 min, and then cooled on ice. The taurine standard was then read for absorbance at 570 nm. Unit activity of BSH was expressed as the amount of enzyme that releases 1 mmol amino acid (AA)/min from the substrate.

NaOH, AlCl₃, NaNO₂, 2,2′-Diphenyl-1-picrylhydrazyl (DPPH), gallic acid, Na₂CO₃, 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), potassium persulfate, potassium ferricyanide, ferric chloride, and trichloroacetic acid were obtained from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China) Rutin, methyl thiazolyl tetrazolium (MTT) and Vitamin C (Vc) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China), while foline-phenol, β-sitosterol, and vanillin were purchased from Sigma-Aldrich Corporation (St. Louis, MI, USA). Assay kits for LDH, MDA, SOD, and GSH-PX were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). RPMI-1640 culture medium, phosphate buffer solution (0.01 M, pH 7.2), dimethyl sulfoxide (DMSO), and 0.05%Trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA), while fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (Huzhou, China). ROS assay kit and 30% H₂O₂ solution were gained from Beijing Solarbio Technology Co., Ltd. (Beijing, China). All organic reagents used in the experiments, including petroleum ether (bp. 60–90 °C), ethyl acetate, n-butanol, and 95% ethanol, were of analytical grade.

Aptamers (5′-TTAGTTGGGGTTCAGTTGG-3′) were synthesized and HPLC-purified by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). Chloroauric acid (HAuCl₄·4H₂O), trisodium citrate, and amoxicillin (AMO) were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. Ampicillin (AMP), cefixime (CFM), cefotaxime (CEF), chloramphenicol (CHL), tetracycline (TET) and ionic salts were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Trichloroacetic acid (10% w/v) was purchased from Shanghai Macklin Biochemical Co., Ltd. All chemicals used were analytical reagent grade, and the solutions were prepared with ultrapure water (18.2 MΩ cm⁻¹, 25 °C), which was purified with Milli-Q Integral (Millipore, Eschborn, Germany).

Hibiscus manihot L. was provided and identified by Yulin Xinbang Pharmaceutical Co., Ltd. (Yulin, China). It was dried in a tray at 65 °C, destemmed and crushed (0.42 mm), and stored at 4 °C for further use.
The fermentation strain was Rhizopus arrhizus JHK31. We obtained it through pressure screening. It is stored in the China Center for Type Culture Collection under the deposit number CCTCC M 2023722.
Reagents including rutin (P98%), vitamin C (P99%), aluminum nitrate nonahydrate (P99%), DPPH (P96%), iron trichloride hexahydrate (P99%), trichloroacetic acid (P99%), dibasic sodium phosphate (P99%), and sodium dihydrogen phosphate (P99%) were from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China) provided other chemicals such as ferrous sulfate (P98%), green vitriol (P99%), sodium hydroxide (P96%), Tris (P99.8%), and salicylic acid (P99%). Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China) provided hyperoside (P98%), isoquercitrin (P98%), myricetin (P98%), and quercetin (P98%). Other chemicals such as sodium nitrite (P99%), potassium ferricyanide (P99.5%), and pyrogallol (P99%) were analytically pure. Acetonitrile and methanol were of chromatographic grades. The resin used was LX-83 macroporous resin from Sunresin New Materials Co., Ltd. (Xi’an, China). We used ultrapure water in all experiments.