Iodine

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).

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).

Chitosan from shrimp shells (75–85% degree of deacetylation and 1747.5 g/mol of molecular weight), 3-chloro-2-hydroxypropyl trimethylammonium chloride (CHPTAC) solution, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid, sodium carbonate, methyl alcohol, sodium hydroxide, and iodine were purchased from Duksan Pure Chemicals Co., Ltd. (Ansan, Gyeonggi-do, Korea).
Tryptic soy broth (TSB), tryptic soy agar (TSA), yeast malt broth (YMB), and yeast malt agar (YMA) were purchased from KisanBio Co., Ltd. (Seoul, Korea). Potato dextrose broth (PDB), potato dextrose agar (PDA), and peptone water were gained from Difco (Becton Dickinson, Sparks, MD, USA). YM 3M Petri film was purchased from 3M Corporation (St. Paul, MN, USA). Test microbial strains, Staphylococcus aureus (ATCC 13565, enterotoxin A), Escherichia coli O157: H7 (ATCC 11234), Candida albicans (ATCC 18804), and Aspergillus flavus (ATCC 22546), were obtained from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea). Human keratinocyte HaCaT cells were provided by Dr S.J. Kim (CHA University, Seoul, Korea). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and 1% penicillin/streptomycin were purchased from Gibco (ThermoFisher Scientific, Waltham, MA, USA).