Cm 10

All bacterial isolates were cultured individually in TSB (1 L) at 28 °C with shaking at 180 rpm. After 48 h, bacterial pellets were separated from supernatant after centrifuging cultured broth at 10,000×g for 20 min at 4 °C. The resulting supernatant was subjected to fractionation of secondary metabolites by successively adding four different organic solvents (hexane, ethyl acetate, chloroform, and butanol) as described previously by Mollah et al. [35 (link)]. Resulting extracts were resuspended in methanol (0.4 mg/mL) and further diluted with DMSO or methanol to desired concentrations based on experimental purposes. During each fractionation step, the metabolite extraction was subjected to thin layer chromatography (TLC) by spotting it on a silica gel plate (20 × 20 cm: Merck, Darmstadt, Germany). A mixture of chloroform, methanol, and acetic acid (7:2.5:0.5, v/v) was used as an eluent. Spots in the silica gel plate were visualized and marked under a fluorescence analysis cabinet (Spectroline, CM-10, Westbury, NY, USA).

Each bacterial strain was cultured separately in 1 L of TSB at 28°C for 48 h. The cultured broth was centrifuged at 10,000 × g for 20 min at 4°C to obtain supernatant which was then subjected to fractionation. Briefly, the same volume (1 L) of hexane was mixed with the supernatant to separate organic and aqueous fractions. The resulting aqueous fraction was combined with the same volume of ethyl acetate. These processes were sequentially repeated for chloroform and butanol organic solvents. Resulting organic extracts [hexane extract (HEX), ethyl acetate extract (EAX), chloroform extract (CX), and butanol extract (BX)] containing bacterial metabolites were dried with a rotary evaporator (Eyela N-1110, Rikakikai, Tokyo, Japan) at 20°C for HEX, 25°C for CX, 30°C for EAX, and 40°C for BX. After weighing dried metabolites, each extract was resuspended with 5 mL of methanol. Resulting metabolites were subjected to TLC using silica gel plates (20 cm × 20 cm; Merck, Darmstadt, Germany). After developing with chloroform:methanol:acetic acid (7:2.5:0.5, v/v) as an eluent, silica gel plates were incubated with a mixture (19:1, g/g) of sea sand (Merck) and iodine (Duksan, Ansan, Korea). Spots were then visualized in a fluorescence analysis cabinet (Spectroline, CM-10, Westbury, NY, United States).

X. hominickii was cultured in 1 L of TSB at 28°C for 48 h. After centrifuging cultured broth at 10,000 × g for 20 min at 4°C, the resulting supernatant was used for organic extraction as described by Mollah et al. [48 (link)]. Briefly, the supernatant was mixed with the same volume of hexane. After 30 min of incubation 4°C, the hexane extract (HEX) was separated from the aqueous fraction. The same procedure was sequentially used to obtain chloroform (CX), ethylacetate (EAX), and butanol (BX) extracts. Resulting organic extracts containing bacterial metabolites were dried with a rotary evaporator (Eyela N-1110, Rikakikai, Tokyo, Japan). After weighing, extracts were resuspended in methanol. TLC was performed for resulting extracts to obtain metabolites on a silica gel plate (20×20 cm; Merck, Darmstadt, Germany). Different compositions of chloroform and methanol (v/v) were used as eluents. The developed silica gel plate was incubated with a mixture (19:1, g/g) of sea sand (Merck) and iodine (Duksan, Ansan, Korea). Spots were visualized and marked in a fluorescence analysis cabinet (Spectroline, CM-10, Westbury, NY, USA).