2.4. In Vitro Investigation of Glucose Metabolism by B. fragilis Strain SNBF-1

In the α-amylase assay [23], 500 μL of CFS and CFE from isolates were mixed with an equal volume of 0.1 M PBS containing α-amylase (0.5 mg/mL, pH 6.4) and incubated at 25 °C for 10 min. Next, 500 μL of 1% starch in 0.1 M PBS (pH 7.4) was added, followed by a further 10 min incubation at the same temperature. The reaction was stopped with 1 mL of DNS reagent and heated in a boiling water bath for 5 min. After cooling to room temperature and diluting with 10 mL of water, the absorbance was measured at 540 nm to calculate α-amylase inhibition:

where A540 (Control) is the absorbance at 540 nm of the control sample without protein extract, and A540 (Sample) is the absorbance at 540 nm of the test sample.

In the α-glucosidase assay [24], yeast α-glucosidase (100 U/mg) was used. Test samples (CFS and CFE100 μL) were mixed with 50 mM PBS (pH 6.8) and incubated for 10 min. α-glucosidase (100 μL, 0.25 U/mL) was then added and pre-incubated at 37 °C for 15 min. This was followed by the addition of 100 μL of 5 mM pNPG and a further 30 min incubation at 37 °C. The reaction was stopped with 1000 μL of 0.1 M Na₂CO₃, and the absorbance of 4-nitrophenol was measured at 405 nm to calculate percent inhibition.

where A405 (Control) is the absorbance at 540 nm of the control sample without protein extract, and A405 (Sample) is the absorbance at 540 nm of the test sample.

To establish an insulin resistance (IR) model, 2 × 10⁵ HepG2 cells/well were cultured until adherence and induced with insulin-containing DMEM for 48 h [25,26]. Cultured HepG2 cells were divided into groups to assess the influence of CFS and CFE on glycogen synthesis, which was determined using a glycogen assay kit following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institution). The activities of hexokinase (HK) and pyruvate kinase (PK) in IR-HepG2 cells were measured with specific assay kits. The inhibitory effects of various strain supernatants on α-amylase and α-glucosidase were assessed. Changes in glucose levels in IR-HepG2 cells were monitored post-supernatant treatment. Protein extracts and Acarbose were prepared in water. Porcine α-amylase inhibition was measured with dinitrosalicylic acid. After pre-incubation and starch addition, reactions were stopped and diluted, and their absorbance at 540 nm was measured to calculate percentage inhibition.