Determination of the minimum inhibitory concentration (MIC)

The MICs were determined by using the micro-dilution method as previously described [33]. Briefly, each GRA derivative was adjusted to 4,096 mg/L in TSB, and 2-fold serial dilutions were prepared in a 96-well microplate (Thermo Fisher Scientific, Roskilde, Denmark). Overnight bacterial cultures were adjusted to an OD₆₆₀ of 1.0 (1x10⁹ cells/ml) and diluted to 1:100 with TSB (1x10⁷ cells/ml). Ten microliters of the bacterial culture (1x10⁵ cells/well) was applied to each well (100 μl total volume). The MICs of glycyrrhetinic acid and its derivatives were determined after the plate was incubated for 24 h at 37°C.