Production and characterization of melanin pigment from black fungus Curvularia soli AS21 ON076460 assisted gamma rays for promising medical uses

Abstract

Owing to the growing need for natural materials in different fields, studying melanin production from biological sources is imperative. In the current study, the extracellular melanin pigment was produced by the fungus Curvularia soli AS21 ON076460. The factors that affect the production of melanin were optimized by the Plackett-Burman design (P-BD). The effect of gamma irradiation on melanin productivity was investigated. The maximum melanin yield (3.376 mg/L) was elicited by a stimulus of gamma irradiation at 1.0 kGy. The results evoked that, Curvularia soli AS21 ON076460 melanin exhibited excellent antimicrobial activity against all tested bacteria and fungi. Klebsiella pneumoniae ATCC 13883 and P. digitatum were mostly affected by melanin registering the inhibition zone diameters of 37.51 ± 0.012 and 44.25 ± 0.214 mm, respectively. Moreover, Curvularia soli AS21 ON076460 melanin indicated a significant antiviral efficacy (77% inhibition) of Herpes simplex virus (HSV1). The melanin pigment showed antioxidant activities with IC₅₀ of 42 ± 0.021 and 17 ± 0.02 µg/mL against DPPH and NO, respectively. Melanin had cytotoxic action against human breast cancer and skin cancer cell lines (Mcf7and A431) as well as exerting a low percentage of cell death against normal skin cell lines (Hfb4). Melanin was effective in wound management of human skin cells by 63.04 ± 1.83% compared with control (68.67 ± 1.10%). The novelty in the study is attributed to the possibility of using gamma rays as a safe method in small economic doses to stimulate melanin production from the fungi that have been isolated. In summary, melanin produced from fungi has significant biological activities that encourage its usage as a supportive medical route.

Keywords: Anticancer; Antimicrobial; Antioxidant; Curvularia soli; Gamma radiation; Melanin pigment.

Fig. 1

a The production of melanin by the isolated fungi. F10 was found to have the superior melanin yield than the other strains. Data are analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test that represented by the letters (a,b, c, d). The same letters in represented not significantly different in results at p < 0.05 (b) Effect of gamma irradiation doses, 0.25, 0.5, 1.0 and 2.0 kGy on the production of melanin by the isolated fungi. At 1.0 kGy, melanin was produced sufficiently for all isolates except F2 that represents the maximum melanin production at 0.5 kGy. The results were plotted as mean values. Error bars represent the SD. According to Duncan’s multiple range test, the same letters represented not significant difference at p < 0.05

Fig. 2

Phylogenetic relationship of Curvularia sp. AS 21 in a relation with related isolates. The proportion of replicated trees in which the linked taxa and grouped together is exhibited adjacent to the branches. The closest hits of Curvularia sp. AS 21 are Curvularia geniculata strain BRIP 72433 b and Curvularia senegalensis SC4.1

Fig. 3

Individual parameter effects on melanin production by Curvularia soli AS 21 ON076460 a normal plot and b Pareto chart for un-irradiated control, c normal plot and d Pareto chart for 1.0 kGy. The positive effect was represented by orange columns, while the negative effect was represented by blue columns. The extremely optimistic factors on the optimizing conditions for melanin production with the un-irradiated control are heavy metals, C-source, N-source, temperature, pH, incubation time, and L-tyrosine. With gamma irradiation at 1.0 kGy, shaking and medium type have positive effect on the melanin productivity

Fig. 4

a Effect of metals on the stability of melanin, Ca and Zn have positive effect on melanin production. Fe and Mn decreases melanin production produced by Curvularia soli AS21 ON076460 compared with control. The results were plotted as mean values. Error bars represent the SD. According to Duncan’s multiple range test, the same letters represented not significant difference at p < 0.05 (b) Fourier-transform infrared (FTIR) spectrum showing the functional groups of melanin produced by Curvularia soli AS21 ON076460. c XRD spectrum of the melanin sample

Fig. 5

SEM analysis of melanin particles produced by Curvularia soli AS21 ON076460 at different magnifications, 1000 and 500 X shown that melanin has average size 130–160 nm. SEM films revealed that crude melanin was free of cells and mycelium. EDX analysis revealed the elements constituents of melanin with their weight and atomic %. The most abundant elements are carbon and oxygen with weights of 66.32 and 25.57%, respectively

Fig. 6

a The antibacterial activity of melanin pigment produced by Curvularia soli AS21 ON076460 against the bacterial strains, E. coli ATCC 25922, S. aureus ATCC 25923, and K. pneumonia ATCC 13883 compared with the antibiotics, ampicillin used as a positive control. The most affected bacteria is K. pneumoniae ATCC 13883. b The antifungal activity of melanin against A. brasiliensis, A. niger, A. sydowii, A. candidus, A. parasiticus, P. notatum, F. oxysporum, and P. digitatum fungal strains compared with the antibiotics, nystatin used as a positive control. The greatest affected fungi are P. digitatum and F. oxysporum

Fig. 7

Bioassay of melanin produced by Curvularia soli AS21 ON076460. a The antioxidant activity by DPPH and NO. b The antiviral against HSVI and Vero cells rep Curvularia soli AS21 ON076460resented as cell viability (%) of viral cells. Melanin has considered cytotoxicity against the HSV1 and melanin has not cytotoxic action on normal Vero cells. (c) Cytotoxicity against Hfb4, McF, and A431 cells. Melanin does not apply cytotoxic effects on the tested cell lines. IC₅₀ value of melanin pigment against Hfb4, Mcf7 and A431 cell lines are 350 ± 0.23, 84.23 ± 0.094 and 89.96 ± 0.16 µg/mL, and d wound healing activity of melanin toward human skin epithelial cells. Cell migration efficiency decreased with increased melanin concentration. The wound edges were enclosed by 63.04 ± 1.83% compared with the control (68.67 ± 1.10%) within 48 h. The results were plotted as mean values. Error bars represent the SD. According to Duncan’s multiple range test, the same letters represented not significant difference at p < 0.05