Total bile acid assay kit

Serum TC level was determined using reagent kits purchased from Abcam (Cat No: ab65390; Cambridge, UK). TBA level in human serum was measured using commercial Total Bile Acid Assay Kit (Cell Biolabs, San Diego, CA, USA). Serum ALP level was examined using Alkaline Phosphatase Assay Kit (Abcam, Cambridge, UK).

Plasma glucose, TG, NEFA, cholesterol, total bile acid, and FGF21 levels were measured using Autokit Glucose CII (Fujifilm Wako Pure Chemical, Osaka, Japan), LabAssay Triglyceride (Fujifilm Wako Pure Chemical, Osaka, Japan), LabAssay NEFA (Fujifilm Wako Pure Chemical, Osaka, Japan), LabAssay Cholesterol (Fujifilm Wako Pure Chemical, Osaka, Japan), Total Bile Acid Assay Kit (Cell Biolabs, San Diego, CA, USA), and Mouse/Rat FGF21 Quantikine ELISA kit (R & D Systems, Minneapolis, MN, USA), respectively. All assays were performed in accordance with the manufacturers’ protocols.

Bile acids were measured using the total bile acid assay kit from Cell Biolabs, Inc. following the manufacturer’s protocol.

Bile acid levels in fecal samples were measured with Total Bile Acid Assay Kit (Cell-Biolabs Inc., San Diego, CA, USA, STA-631) according to the manufacturer’s instructions.

To determine the bile acid retention capacities of the BG, the total bile acid assay kit (No. STA-631, Cell Biolabs, Inc., San Diego, CA, USA) was used on the chyme samples after in vitro digestion. The preparation of all solutions, standards, as well as the assay were carried out according to the instructions given in the manual. In brief, 2 × 20 µL of appropriately diluted sample or bile acid standard were combined with 150 µL of thio-NAD⁺ solution, using a 96-well microtiter plate, and homogenized. After incubation (5 min, 37 °C), 50 µL of a solution containing 3α-HSD and NADH were added to the standards and one half of the sample wells, while 50 µL of NADH solution were added to the other half of the sample wells. The plate was incubated at 37 °C for 40 min, before the absorbance was read at 405 nm and 630 nm, using a microtiter plate reader (Biotec Synergy, Thermo Fisher Scientific, Schwerte, Germany). All calibrations were found to be linear in the respective calibration range (0–25 µM, R² > 0.99), when plotting the absorbance difference (at 405 nm and 630 nm) against the standard concentration. The available bile acids in the chyme were calculated based on the calibration, according to the manual. The bile acid content of digested swb was used as baseline, to determine the reduction due to the addition of cBG or hBG to the breads.

A portion of the powdered, dried feces was used to measure total fecal bile acid content using a commercial assay kit (Total Bile Acid Assay Kit, Cell Biolabs Inc. Sand Diego, CA, USA). The assay measured bile acid content through a colorimetric enzyme driven reaction with 3ɑ-hydroxysteroid dehydrogenase, NADH and thio-NAD⁺. A second portion of feces was used to measure TCA content using a commercial assay kit (Taurocholic Acid (TCA) ELISA Kit, Abbexa LTD, Cambridge, UK). This ELISA used a competitive inhibition assay with a colorimetric detection.