EEP Modulates Melanin Production in C. neoformans

C. neoformans was treated with EEP at concentrations of 0.5, 1 and 2 mg/mL for 2 h at 37 °C [18 (link)]. Yeasts were then cultured in the L-3,4-dihydroxyphenylalanine (L-DOPA) minimal medium (15 mM glucose, 10 mM MgSO₄, 29.4 mM KH₂PO₄, 13 mM glycine and 3 mM thiamine; pH 5.5), with 1 mM L-DOPA (Sigma-Aldrich, St. Louis, MO, USA) and incubated in a dark chamber at 30 °C and in a shaking incubator for 5 days. The melanin pigment production was observed daily. To confirm the viability of yeast cells, the cells were diluted, adjusted to 5 × 10⁵, 5 × 10⁴ and 5 × 10³ cells/mL, and dropped on a SDA plate. After 48 h of incubation at 37 °C, the number of colony-forming units (CFUs) were counted [19 (link)].