Daidzein Degradation Assay of Bacterial Strains

Bacterial strains were cultured overnight at 28°C in 2 ml of peptone-beef extract medium. Bacterial cells adjusted to OD₆₀₀ = 1.0 were collected by centrifugation at 13000 x g for 1 min and washed twice with MS medium. The resulting bacterial pellet was resuspended in 2 ml of MS medium supplemented with daidzein a concentration of 20 μM and cultured at 28°C. For the time course daidzein degradation assay of the V35 wild-type strain, 100 μl of culture supernatant was collected at 0, 1, 2, 4, and 8 h, immediately mixed with equivalent volume of methanol, and centrifuged at 13000 x g for 1 min. Daidzein degradation of ifc mutants was conducted for 48 h of cultivation. After filtration through a 0.45-μm Minisart RC4 filter (Sartorius, Germany), collected samples were stored at −30°C until UPLC and LC–MS analysis. Detailed methods for UPLC and LC–MS analysis can be found in Supplemental Methods.