The generic term "melanin" describes a black pigment of biological origin, although some melanins can be brown or even yellow. The pigment is characterized as a heterogenic polymer of phenolic or indolic nature, and the classification of eu-, pheo- and allo- melanin is broadly accepted. This classification is based on the chemical composition of the monomer subunit structure of the pigment. Due to the high heterogeneity of melanins, their analytical characterization can be a challenging task. In the present work, we synthesized the current information about the analytical methods which can be applied in melanin analysis workflow, from extraction and purification to high-throughput methods, such as matrix-assisted laser desorption/ionization mass-spectrometry or pyrolysis gas chromatography. Our thorough comparative evaluation of analytical data published so far on melanin analysis has proven to be a difficult task in terms of finding equivalent results, even when the same matrix was used. Moreover, we emphasize the importance of prior knowledge of melanin types and properties in order to select a valid experimental design using analytical methods that are able to deliver reliable results and draw consistent conclusions.

From Extraction to Advanced Analytical Methods: The Challenges of Melanin Analysis

Melanins are high molecular weight hydrophobic pigments that have been studied for their role in the virulence of fungal pathogens. We investigated the amount and type of melanin in 20 isolates of Aspergillus spp.; A. niger (n = 3), A. flavus (n = 5), A. tamarii (n = 3), A. terreus (n = 3), A. tubingensis (n = 3), A. sydowii (n = 3). Aspergillus spp. were identified by sequencing the internal transcribed spacer (ITS) region. Extraction of melanin from culture filtrate and fungal biomass was done and followed by qualitative and quantitative analysis of melanin pigment. Ultraviolet (UV), Fourier transformed infrared (FT-IR), and electron paramagnetic resonance (EPR) spectra analyses confirmed the presence of melanin. The melanin pathway was studied by analyzing the effects of inhibitors; kojic acid, tropolone, phthalide, and tricyclazole. The results indicate that in A. niger and A. tubingensis melanin was found in both culture filtrate and fungal biomass. For A. tamarii and A. flavus melanin was extracted from biomass only, whereas melanin was found only in culture filtrate for A. terreus. A negligible amount of melanin was found in A. sydowii. The maximum amount of melanin from culture filtrate and fungal biomass was found in A. niger and A. tamarrii, respectively. The DOPA (3,4-dihydroxyphenylalanine) pathway produces melanin in A. niger, A. tamarii and A. flavus, whereas the DHN (1,8-dihydroxynaphthalene) pathway produces melanin in A. tubingensis and A. terreus. It can be concluded that the amount and type of melanin in aspergilli largely differ from species to species.

DOPA and DHN pathway orchestrate melanin synthesis in Aspergillus species.

Nanobiotechnology applies the capabilities of biological systems in generating a variety of nano-sized structures. Plants, algae, fungi and bacteria are some systems mediating such reactions. In fungi, the synthesis of melanin is an important strategy for cell-survival under metal-stressed conditions. Yarrowia lipolytica, the biotechnologically significant yeast also produces melanin that sequesters heavy metal ions. The content of this cell-associated melanin is often low and precursors such as L-tyrosine or 3, 4-dihydroxy-L-phenylalanine (L-DOPA) can enhance its production. The induced melanin has not been exploited for the synthesis of nanostructures. In this investigation, we have employed L-DOPA-melanin for the facile synthesis of silver and gold nanostructures. The former have been used for the development of anti-fungal paints. Yarrowia lipolytica NCIM 3590 cells were incubated with L-DOPA for 18 h and the resultant dark pigment was subjected to physical and chemical analysis. This biopolymer was used as a reducing and stabilizing agent for the synthesis of silver and gold nanostructures. These nanoparticles were characterized by UV-Visible spectra, X-ray diffraction (XRD) studies, and electron microscopy. Silver nanoparticles were evaluated for anti-fungal activity. The pigment isolated from Y. lipolytica was identified as melanin. The induced pigment reduced silver nitrate and chloroauric acid to silver and gold nanostructures, respectively. The silver nanoparticles were smaller in size (7 nm) and displayed excellent anti-fungal properties towards an Aspergillus sp. isolated from a wall surface. An application of these nanoparticles as effective paint-additives has been demonstrated. The yeast mediated enhanced production of the metal-ion-reducing pigment, melanin. A simple and rapid method for the extracellular synthesis of nanoparticles with paint-additive-application was developed.

/pdf/3-4-dihydroxy-l-phenylalanine-derived-melanin-from-yarrowia-1e6b3yi0wj.pdf

3, 4-dihydroxy-L-phenylalanine-derived melanin from Yarrowia lipolytica mediates the synthesis of silver and gold nanostructures

Melanin pigments are frequently used in medicine, food and cosmetic preparations. In this study, three strains among 21 Actinomycetes sp isolates produced a diffusible dark pigment on starch casein an agar medium which was water soluble. The pigment production was estimated using L-tyrosine as substrate. Among the strains, 3 (F1, F2, F3) were used for further analysis. Optimized culture condition for melanin production were; 1% of starch- best carbon source, 0.2% of soyabean- best nitrogen source, salinity- 15 ppt, temperature- 35°C, pH-7.0 and incubation time- 168 h. Sugarcane waste was found to be the cheaper and best source for melanin production. Melanin also showed activity against potential pathogens. The maximum antimicrobial activity was observed with Escherichia coli (20 mm) and Lactobacilus vulgaris (20 mm). F1 strain producing melanin was taken for FT-IR analysis. The FT-IR result confirmed that it was melanin pigment. Therefore, this study proved that sugarcane waste can be used for the production of melanin and it (melanin) has potential anti-bacterial activity.

	 

	Key words: Melanin, sugarcane waste, antibacterial, FT-IR.

Melanin production from marine Streptomyces

Water-soluble squid melanin fractions were firstly prepared using ultrasound-assistant degradation method under alkaline condition, which is optimized by response surface methodology. The processing melanin fractions were divided into different molecular weight (Mw) fractions by membrane separation (below 10 kDa, among 10–50 kDa and over 50 kDa). The AFM image and particle-size analysis showed monomer units of the melanin were destroyed, and huge polymers were degraded into smaller soluble particles after ultrasound. While, UV, IR and solid 13C NMR spectra indicated that the basic structure of melanin fraction was still retained after ultrasound process. Further analysis showed soluble melanin fractions obtained in 0.5 and 1 M NaOH, with Mw above 10 kDa exhibited much higher in vitro antioxidant potency. The IC50 of these fractions (IC50 among 19-80 μg) on scavenging O
 2
 ∙¯ is more efficient than carnosine (IC50 = 355 μg/ml.), a commercialized antioxidant. They (IC50 mong 115–180 μg/ml) are as efficient as carnosine (IC50 = 110 μg/ml) on scavenging ∙OH. Our research has reported a novel method for preparation of water-soluble melanin fractions from squid ink, which could be a promising free radical scavenger from nature resource.

/pdf/preparation-of-water-soluble-melanin-from-squid-ink-using-324ruqdgd3.pdf

Preparation of water-soluble melanin from squid ink using ultrasound-assisted degradation and its anti-oxidant activity

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of bacteria and fungi. For more than 40 years, fungi have been known to produce pigments called melanins. Melanin pigment production by mushrooms was not intensively studied. The present study was carried out on isolation and characterization of melanin from an edible mushroom Pleurotus
 cystidiosus var. formosensis. The mushroom produced dark mucous mass of hyaline arthrospores on mycelium. The coremia exclusively produced dikaryotic arthrospores with the remnant of a clamp connection. Continuous cell extension and division in the coremium stipe supplied cells for arthroconidiation at the coremium apex, which is surrounded by a liquid droplet (coremioliquid). The black coloured coremea (conidia) were produced by Antromycopsis macrocarpa (anamorph of P. cystidiosus) when cultured on potato dextrose agar medium. The agar plate was incubated at continuous light illumination for high amount of pigment (coremea) production. The slimy layer of the coremea was extracted and partially purified by alkaline and acid treatment. The black pigment was confirmed as melanin based on UV, IR and EPR spectra apart from chemical analysis. This is the first report on characterization of melanin obtained from Pleurotus
 cystidiosus var. formosensis.

K. Periasamy

Papers

Isolation and characterization of melanin pigment from Pleurotus cystidiosus (telomorph of Antromycopsis macrocarpa)