Unveiling the fungal color palette: pigment analysis of Fusarium solani species complex and Curvularia verruculosa clinical isolates.

Fungal species in the Nectriaceae, such as Fusarium spp. (Hypocreales: Nectriaceae), are etiologic agents of hyalohyphomycosis capable of producing violaceous or yellowish pigments under certain conditions, while Curvularia spp. (Pleosporales: Pleosporaceae) are agents of phaeohyphomycosis and typically produce melanin in their cell walls. In nectriaceous and pleosporaceous fungi, these pigments are mainly constituted by polyketides (e.g., azaphilones, naphthoquinones, and hydroxyanthraquinones). Considering the importance of pigments synthesized by these genera, this work focused on the selective extraction of pigments produced by eight Fusarium solani species complex and one Curvularia verruculosa isolate recovered from dermatomycosis specimens, their separation, purification, and posterior chemical analysis. The pigments were characterized through spectral and acid-base analysis, and their maximum production time was determined. Moreover, spectral identification of isolates was carried out to approach the taxonomic specificity of pigment production. Herein we describe the isolation and characterization of three acidic pigments, yellowish and pinkish azaphilones (i.e., coaherin A and sclerotiorin), and a purplish xanthone, reported for the first time in the Nectriaceae and Pleosporaceae, which appear to be synthesized in a species-independent manner, in the case of fusaria.

Highlighting the Phototherapeutical Potential of Fungal Pigments in Various Fruiting Body Extracts with Informed Feature-Based Molecular Networking.

Fungal pigments are characterized by a diverse set of chemical backbones, some of which present photosensitizer-like structures. From the genus Cortinarius, for example, several biologically active photosensitizers have been identified leading to the hypothesis that photoactivity might be a more general phenomenon in the kingdom Fungi. This paper aims at testing the hypothesis. Forty-eight fruiting body-forming species producing pigments from all four major biosynthetic pathways (i.e., shikimate-chorismate, acetate-malonate, mevalonate, and nitrogen heterocycles) were selected and submitted to a workflow combining in vitro chemical and biological experiments with state-of-the-art metabolomics. Fungal extracts were profiled by high-resolution mass spectrometry and subsequently explored by spectral organization through feature-based molecular networking (FBMN), including advanced metabolite dereplication techniques. Additionally, the photochemical properties (i.e., light-dependent production of singlet oxygen), the phenolic content, and the (photo)cytotoxic activity of the extracts were studied. Different levels of photoactivity were found in species from all four metabolic groups, indicating that light-dependent effects are common among fungal pigments. In particular, extracts containing pigments from the acetate-malonate pathway, e.g., extracts from Bulgaria inquinans, Daldinia concentrica, and Cortinarius spp., were not only efficient producers of singlet oxygen but also exhibited photocytotoxicity against three different cancer cell lines. This study explores the distribution of photobiological traits in fruiting body forming fungi and highlights new sources for phototherapeutics.

Chemical characterization and microencapsulation of extracellular fungal pigments.

In this work, extracellular colored metabolites obtained from the filamentous fungi Talaromyces australis and Penicillium murcianum, isolated in the Andean-Patagonian native forests of Chile, were studied as prospect compounds to increase the sustainability of cosmetic products. The chemical and antioxidant properties of these natural pigments were characterized and strategies for their microencapsulation were also studied. UHPLC/MS-MS analyses indicated that the predominant metabolites detected in the cultures of P. murcianum were monascin (m/z = 411.15) and monashexenone (m/z = 319.10), while athrorosin H (m/z = 458.20) and damnacanthal (m/z = 281.05) were detected in cultures of T. australis. ORAC tests revealed that P. murcianum's metabolites had the greatest antioxidant properties with values higher than 2000 µmol of trolox equivalents/g. The fungal metabolites were successfully microencapsulated by ionic gelation into structures made of 1.3% sodium alginate, 0.2% chitosan, and 0.07% hyaluronic acid. The microencapsulation process generated structures of 543.57 ± 0.13 µm of mean diameter (d50) with an efficiency of 30% for P. murcianum, and 329.59 ± 0.15 µm of mean diameter (d50) and 40% efficiency, for T. australis. The chemical and biological characterization show the biotechnological potential of these fungal species to obtain pigments with antioxidant activity that could be useful in the cosmetic industry. The encapsulation process enables the production of easy-to-handle dry powder from the fungal metabolites, which could be potentially marketed as a functional cosmetic ingredient. KEY POINTS: • The predominant fungal pigments were of azaphilone and anthraquinoid classes. • The fungal pigments showed high antioxidant activity by ORAC assay. • Fungal pigment microcapsules obtained by ionic gelation were characterized.

Talaromyces australis and Penicillium murcianum pigment production in optimized liquid cultures and evaluation of their cytotoxicity in textile applications.

In this work Talaromyces australis and Penicillium murcianum pigment production in liquid cultures and the cytotoxic effect of such pigments on skin model cells were studied. Response surface methodology (RSM) was used to optimize culture conditions aiming to increase pigment production in malt extract and peptone-glucose-yeast extract medium. Cytotoxicity of fungal pigments and also from lixiviates of wool fabrics dyed with T. australis and P. murcianum pigment was evaluated on mammalian cell lines HEK293 and NIH/3T3. Results showed that variations on initial pH, NaCl and peptone, resulted in increments up to 188.2% for red pigment of T. australis and 107.4% for yellow pigment of P. murcianum, regarding non-optimized conditions. Tested fungi also showed great differences in culture conditions for the maximum pigment production, with P. murcianum requiring an alkaline medium (initial pH 9) supplemented with NaCl and T. australis an acidic medium (initial pH 5) without addition of salt. The cytotoxicity assays provided evidences on the safe nature of these natural pigments when used for textile applications. The cytotoxicity assay showed that the threshold of toxicity, given by the lowest IC50 value (0.21 g L-1) was more than double of the concentration of pigment required to dye the wool samples. In addition, cytotoxicity of lixiviates depicted no toxic effect over tested cells.

Chemical characterization and toxicity evaluation of fungal pigments for potential application in food, phamarceutical and agricultural industries.

Concerns associated with the use of synthetic colourants backs the demand for natural colourants. Thus, the current study aimed at characterizing crude fungal pigments produced by Penicillium multicolour, P. canescens, Talaromyces verruculosus, Fusarium solani and P. herquie. This included their antioxidant and antimicrobial properties together with acute toxicity evaluation on zebrafish embryos. The identification of pigment compounds was achieved through MS and IR data. The study demonstrated a substantial radical scavenging activity of extracts ranging from 65.49 to 74.46%, close to that of ascorbic acid (89.21%). Penicillium canescens and F. solani exhibited a strong antimicrobial activity against Escherichia coli and Enterococcus aerogenes and Salmonella typhi, Staphylococcus aureus and Bacillus cereus at MIC values ranging from 1.5 to 2.5 mg/mL. However, some levels of toxicity were observed for all extracts at a concentration range of 3-5 mg/mL. Pigment by P. multicolour, T. verruculosus and F. solani were tentatively identified through IR and MS data as sclerotiorin (yellow), rubropunctamine (red) and bostrycoidin (red). In conclusion, the study demonstrates a market potential of filamentous fungi pigments due to their antioxidant, antimicrobial activities, and prominent colours. Although there are some toxicity concerns, further tests must be done using molecular docking, albino mice and cell linings.

Stability of the Fungal Pigment from Scytalidium cuboideum Carried in Food-Grade Natural Oils.

Wood-staining fungal pigments have shown potential use as colorants for wood and textiles, with organic solvents as the pigment carrier. Natural oils have been suggested as an environmentally friendly and more available carrier; however, oils promoted color degradation. The current study examined the mechanism of said degradation and tested therapeutic and food-grade oils (instead of finishing oils) for their potential to carry draconin red, the pigment from Scytalidium cuboideum, without color loss over time. FTIR analysis from finishing oils indicated that oxidation was not likely the cause of color loss as the pigment could not be distinguished from the oils in the IR spectra. SEM was employed to determine if crystal degradation was contributing to color loss and indicated, surprisingly, that the crystals of draconin red formed rather than degraded over time. This suggested crystal breakdown was also not likely the cause of color loss. The pigment did not show degradation in hemp oil, flaxseed oil, and cold-pressed linseed oil when treated with ß-carotene. Further in-depth chemical studies are needed to determine the mechanism of color loss in pigmented natural oils; however, food-grade oils appear to be a promising alternative to carry draconin red, without degradation of the color.

Safety Evaluation of Fungal Pigments for Food Applications.

Pigments play a major role in many industries. Natural colors are usually much safer when compared to synthetic colors and may even possess some medicinal benefits. Synthetic colors are economical and can easily be produced compared to natural colors. In addition, raw plant materials for natural colors are limited and season dependent. Microorganisms provide an alternative source for natural colors and, among them, fungi provide a wide range of natural colorants that could easily be produced cheaply and with high yield. Along with pigment, some microbial strains are also capable of producing a number of mycotoxins. The commercial use of microbial pigments relies on the safety of colorants. This review provides a toxicity evaluation of pigments from fungal origins for food application.

Feature-Based Molecular Networking-An Exciting Tool to Spot Species of the Genus Cortinarius with Hidden Photosensitizers.

Fungi have developed a wide array of defense strategies to overcome mechanical injuries and pathogen infections. Recently, photoactivity has been discovered by showing that pigments isolated from Cortinarius uliginosus produce singlet oxygen under irradiation. To test if this phenomenon is limited to dermocyboid Cortinarii, six colourful Cortinarius species belonging to different classical subgenera (i.e., Dermocybe, Leprocybe, Myxacium, Phlegmacium, and Telamonia) were investigated. Fungal extracts were explored by the combination of in vitro photobiological methods, UHPLC coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS2), feature-based molecular networking (FBMN), and metabolite dereplication techniques. The fungi C. rubrophyllus (Dermocybe) and C. xanthophyllus (Phlegmacium) exhibited promising photobiological activity in a low concentration range (1-7 µg/mL). Using UHPLC-HRMS2-based metabolomic tools, the underlying photoactive principle was investigated. Several monomeric and dimeric anthraquinones were annotated as compounds responsible for the photoactivity. Furthermore, the results showed that light-induced activity is not restricted to a single subgenus, but rather is a trait of Cortinarius species of different phylogenetic lineages and is linked to the presence of fungal anthraquinones. This study highlights the genus Cortinarius as a promising source for novel photopharmaceuticals. Additionally, we showed that putative dereplication of natural photosensitizers can be done by FBMN.

Fungal Melanins and Applications in Healthcare, Bioremediation and Industry.

Melanin is a complex multifunctional pigment found in all kingdoms of life, including fungi. The complex chemical structure of fungal melanins, yet to be fully elucidated, lends them multiple unique functions ranging from radioprotection and antioxidant activity to heavy metal chelation and organic compound absorption. Given their many biological functions, fungal melanins present many possibilities as natural compounds that could be exploited for human use. This review summarizes the current discourse and attempts to apply fungal melanin to enhance human health, remove pollutants from ecosystems, and streamline industrial processes. While the potential applications of fungal melanins are often discussed in the scientific community, they are successfully executed less often. Some of the challenges in the applications of fungal melanin to technology include the knowledge gap about their detailed structure, difficulties in isolating melanotic fungi, challenges in extracting melanin from isolated species, and the pathogenicity concerns that accompany working with live melanotic fungi. With proper acknowledgment of these challenges, fungal melanin holds great potential for societal benefit in the coming years.

Fungal Pigments and Their Roles Associated with Human Health.

Fungi can produce myriad secondary metabolites, including pigments. Some of these pigments play a positive role in human welfare while others are detrimental. This paper reviews the types and biosynthesis of fungal pigments, their relevance to human health, including their interactions with host immunity, and recent progresses in their structure-activity relationships. Fungal pigments are grouped into carotenoids, melanin, polyketides, and azaphilones, etc. These pigments are phylogenetically broadly distributed. While the biosynthetic pathways for some fungal pigments are known, the majority remain to be elucidated. Understanding the genes and metabolic pathways involved in fungal pigment synthesis is essential to genetically manipulate the production of both the types and quantities of specific pigments. A variety of fungal pigments have shown wide-spectrum biological activities, including promising pharmacophores/lead molecules to be developed into health-promoting drugs to treat cancers, cardiovascular disorders, infectious diseases, Alzheimer's diseases, and so on. In addition, the mechanistic elucidation of the interaction of fungal pigments with the host immune system provides valuable clues for fighting fungal infections. The great potential of fungal pigments have opened the avenues for academia and industries ranging from fundamental biology to pharmaceutical development, shedding light on our endeavors for disease prevention and treatment.

Biotechnological approaches for the production of natural colorants by Talaromyces/Penicillium: A review.

There has been an increased interest in replacing synthetic colorants by colorants obtained from natural sources, especially microbial pigments. Monascus pigments have been used as natural colorings and food additives in Asia for centuries but have raised toxicity issues. Recently, Talaromyces/Penicillium species have been recognized as potential strains to produce natural pigments similar to those produced by Monascus species. To date, it has not been published a literature compilation about the research and development activity of Talaromyces/Penicillium pigments. Developing a new bioprocess requires several steps, from an initial concept to a practical and feasible application. Industrial applications of fungal pigments will depend on: (i) characterization of the molecules to assure a safe consumption, (ii) stability of the pigments to the processing conditions required by the products where they will be incorporated, (iii) optimizing process conditions to achieve high yields, iv) implementing an efficient product recovery and (v) scale-up of the bioprocess. The above aspects have been reviewed in detail to evaluate the feasibility of reaching a commercial scale of the pigments produced by Talaromyces/Penicillium. Finally, the biological activities of the pigments and their potential applications are discussed.

Fungal Pigments: Potential Coloring Compounds for Wide Ranging Applications in Textile Dyeing.

Synthetic pigments/non-renewable coloring sources used normally in the textile industry release toxic substances into the environment, causing perilous ecological challenges. To be safer from such challenges of synthetic colorants, academia and industries have explored the use of natural colorants such as microbial pigments. Such explorations have created a fervent interest among textile stakeholders to undertake the dyeing of textile fabrics, especially with fungal pigments. The biodegradable and sustainable production of natural colorants from fungal sources stand as being comparatively advantageous to synthetic dyes. The prospective scope of fungal pigments has emerged in the opening of many new avenues in textile colorants for wide ranging applications. Applying the biotechnological processes, fungal pigments like carotenoids, melanins, flavins, phenazines, quinones, monascins, violacein, indigo, etc. could be extracted on an industrial scale. This review appraises the studies and applications of various fungal pigments in dyeing textile fabrics and is furthermore shedding light on the importance of toxicity testing, genetic manipulations of fungal pigments, and their future perspectives under biotechnological approaches.

Alternative Extraction and Characterization of Nitrogen-Containing Azaphilone Red Pigments and Ergosterol Derivatives from the Marine-Derived Fungal Talaromyces sp. 30570 Strain with Industrial Relevance.

Many species of Talaromyces of marine origin could be considered as non-toxigenic fungal cell factory. Some strains could produce water-soluble active biopigments in submerged cultures. These fungal pigments are of interest due to their applications in the design of new pharmaceutical products. In this study, the azaphilone red pigments and ergosterol derivatives produced by a wild type of Talaromyces sp. 30570 (CBS 206.89 B) marine-derived fungal strain with industrial relevance were described. The strain was isolated from the coral reef of the Réunion island. An alternative extraction of the fungal pigments using high pressure with eco-friendly solvents was studied. Twelve different red pigments were detected, including two pigmented ergosterol derivatives. Nine metabolites were identified using HPLC-PDA-ESI/MS as Monascus-like azaphilone pigments. In particular, derivatives of nitrogen-containing azaphilone red pigment, like PP-R, 6-[(Z)-2-Carboxyvinyl]-N-GABA-PP-V, N-threonine-monascorubramin, N-glutaryl-rubropunctamin, monascorubramin, and presumed N-threonyl-rubropunctamin (or acid form of the pigment PP-R) were the major pigmented compounds produced. Interestingly, the bioproduction of these red pigments occurred only when complex organic nitrogen sources were present in the culture medium. These findings are important for the field of the selective production of Monascus-like azaphilone red pigments for the industries.

Influence of Environmental Growth Factors on the Biomass and Pigment Production of Chlorociboria aeruginascens.

The soft rot fungus Chlorociboria aeruginascens produces a blue-green pigment xylindein, which is of considerable interest for various applications such as in the veneer industry or in organic semiconductors. To understand the fungal growth as well as pigment production of C. aeruginascens, several studies were performed, the results of which are presented here. These studies investigated various growth conditions such as temperature, pH value, oxygen level and light intensity. It was observed that the formation of xylindein by C. aeruginascens decoupled from growth. In the primary metabolismus, the uncolored biomass is formed. Pigment production took place within the secondary metabolism, while biomass growth as well as pigment production depended on various growth conditions. It was also found that certain conditions encourage the switch in metabolism, leading to pigment production.

Fungal Pigments and Their Prospects in Different Industries.

The public's demand for natural, eco-friendly, and safe pigments is significantly increasing in the current era. Natural pigments, especially fungal pigments, are receiving more attention and seem to be in high demand worldwide. The immense advantages of fungal pigments over other natural or synthetic pigments have opened new avenues in the market for a wide range of applications in different industries. In addition to coloring properties, other beneficial attributes of fungal pigments, such as antimicrobial, anticancer, antioxidant, and cytotoxic activity, have expanded their use in different sectors. This review deals with the study of fungal pigments and their applications and sheds light on future prospects and challenges in the field of fungal pigments. Furthermore, the possible application of fungal pigments in the textile industry is also addressed.

Influence of the Nutrients on the Biomass and Pigment Production of Chlorociboria aeruginascens.

The blue-green pigment xylindein, produced by the soft rot fungus Chlorociboria aeruginascens, is of considerable interest for various applications such as the veneer industry or organic semiconductors. The studies presented were performed in order to understand the fungal growth as well as the pigment production of C. aeruginascens. Therefore, various nutrient compositions were investigated. As a result, observations of the formation of xylindein through C. aeruginascens decoupling from growth were made. In the primary metabolism the uncolored biomass is formed. Various carbohydrates were determined as nutrients for the fungus and as a nitrogen source it was observed that the fungus prefers the complex organic nitrogen source, that being yeast extract. Furthermore, it was discovered that the ratio between carbohydrate and nitrogen sources encourages the switch of the metabolism and therewith the production of the blue-green pigment xylindein.

Salinity and Temperature Influence Growth and Pigment Production in the Marine-Derived Fungal Strain Talaromyces albobiverticillius 30548.

Marine-derived fungi that inhabit severe changing environments have gained increasing interest for their ability to produce structurally unique natural products. Fungi belonging to the Talaromyces and the close Penicillium genera are among the most promising microbes for bioactive compound production, including colored metabolites. Coupling pigment producing capability with bioactive effectiveness would be a valuable challenge in some specific fields such as dyeing, cosmeceutical, or food industries. In this sense, Talaromyces albobiverticillius 30548, a red pigment producing strain, has been isolated from the marine environment of Reunion Island, Indian Ocean. In this research, we analyzed the effect of temperatures (21⁻27 °C) and salinity levels (0⁻9%) on fungal growth and pigment production. Maximum pigment yield was obtained in non-salted media, when cultured at 27 °C after 10 days of submerged fermentation in PDB. However, maximum dry biomass production was achieved at stressed condition with 9% sea salts concentrated media at the same temperature. The results indicate that salinity of the culture media positively influences the growth of the biomass. Inversely, pigment production decreases with increase in salinity over 6%. Color coordinates of secreted pigments were expressed in CIELAB color system. The hue angles (h°) ranged from red to yellow colors. This indicated that the color distribution of fungal pigments depends on the salinity in the culture media. This study emphasizes the impact of abiotic stress (salt and temperature) on the growth and metabolome of marine-derived fungal strains.

Induction of pigment production through media composition, abiotic and biotic factors in two filamentous fungi.

In addition to plant-derived, fungal pigments have become an alternative in respect to synthetic ones. Besides Monascus sp., several pigment-producing fungi do not have culture conditions well-established yet. In this research, media composition, light wavelength and co-culture were evaluated, results were reported in Absorbance Units per gram of biomass (AU/Bgr). For Fusarium oxysporum a C:N ratio above 7 was advantageous, using both complex and defined media; blue LED light increased the AU/Bgr value from 18013 to 344; co-culture did not enhance pigment production. In Aspergillus chevalieri a high C:N ratio with glucose as carbon source was ideal. When exposing cultures to light, UV and red light gave the highest pigmentation; moreover, differential UV-VIS spectra in all wavelengths suggested production of additional pigments. Particularly a pigment observed when cultured in green light was also found in co-culture with yeast and there was an improvement of AU/Bgr value of 52549%. This is the first report regarding light effect and co-culture for these fungi, as well as C:N ratio for A. chevalieri.

Stimulating Production of Pigment-Type Secondary Metabolites from Soft Rotting Wood Decay Fungi ("Spalting" Fungi).

A small group of soft rotting wood decay fungi produce extracellular pigments as secondary metabolites in response to stress and as a means of resource capture. These fungi are collectively known as "spalting fungi" and have been used in wood art for centuries. The pigments produced by these fungi are finding increasing usage in industrial dye applications and green energy but remain problematic to grow in batch culture. Additionally problematic is that the pigments, especially the blue-green pigment known as xylindein, produced by Chlorociboria species, have yet to be fully synthesized. In order to further research development of these pigments and find success in areas such as textile and paint dyeing, wood UV protection, and organic photovoltaic cells, methods must be developed to mass produce the pigments. To date, three distinct methods have been developed, with varying degrees of success depending upon the fungal species (amended malt agar plates, shake liquid culture, and stationary liquid culture). This chapter details these three methods, their history, advantages and disadvantages, as well as their potential for industrial scale-up in the future. Graphical Abstract.

Alternative Carrier Solvents for Pigments Extracted from Spalting Fungi.

The use of both naturally occurring and synthetic pigmented wood has been prevalent in woodcraft for centuries. Modern manifestations generally involve either woodworkers' aniline dyes, or pigments derived from a special class of fungi known as spalting fungi. While fungal pigments are more renewable than anilines and pose less of an environmental risk, the carrier required for these pigments-dichloromethane (DCM)-is both problematic for humans and tends to only deposit the pigments on the surface of wood instead of evenly within the material. Internal coloration of wood is key to adoption of a pigmenting system by woodworkers. To address this issue, five solvents that had moderate solubility with the pigments extracted from Chlorociboria aeruginosa and Scytalidium cuboideum were identified, in the hopes that a reduction in solubility would result in a greater amount of the pigment deposited inside the wood. Of the tested solvents, acetonitrile was found to produce the highest internal color in ash, Douglas-fir, madrone, mountain hemlock, Port-Orford cedar, Pacific silver fir, red alder and sugar maple. While these carrier solvents are not ideal for extracting the pigments from the fungi, acetonitrile in particular does appear to allow for more pigment to be deposited within wood. The use of acetonitrile over DCM offers new opportunities for possible industrial spalting applications, in which larger pieces of wood could be uniformly pigmented and sold to the end user in larger quantities than are currently available with spalted wood.