Fungal Pigments: Their Diversity, Chemistry, Food and Non-Food Applications

: Colorants have many applications in food, cosmetics, pharmaceutics, textile, paints, plastics, paper, ink and photographic industries. Colorants are classiﬁed according to their solubility into dyes and pigments. Those of natural origin have many advantages over synthetic ones, as natural colorants usually do not induce allergies or other health problems. In addition, their consumption in the food and drug industries is fortiﬁed with nutritional and health beneﬁts as the majority of them possess antioxidant activity or can be used to produce some vitamins. Plants, animals, insects and microorganisms are rich sources of colorants. However, microbial pigments are favored over other natural pigments due to their higher yield, stability, economical production. Therefore, we focus in this review on fungal pigments, the history of their use, their chemistry and their applications in food and non-food ﬁelds. Additionally, the ability of the fungal genus, Epicoccum , to produce pigments is discussed. Moreover, the challenges and future prospects concerning fungal pigment production are highlighted in detail.


Introduction
Colorants are the compounds used to add or change the color of different substrates and are involved in different industries including food, cosmetics, pharmaceutics, textile, paints, plastics paper, ink and photographic industries.The global dye market was valued at USD 31.97 billion in 2019 and is expected to reach USD 50.38 billion by 2023 [1].According to their solubility, colorants are classified into two classes, dyes and pigments.The main differences are that dyes are soluble in organic solvents and water, while pigments are insoluble in them.The mechanism of coloring differs between dyes and pigments.Dyes color substrates to which they have affinity.In contrast, pigments color any polymeric substrate at the surface level unless the pigment is mixed with the polymer in earlier stages of its formation [2].Unlike the majority of organic compounds, dyes have color due to many reasons.First, they absorb light in the visible spectrum (400-700 nm) and have at least one color-bearing group (chromophore) or sometimes certain groups known as color helpers (auxochromes) such as hydroxyl groups, sulfo, carboxylic and amino groups.Such groups are not responsible for color appearance, but they affect dye solubility and contribute to shifting the color of a colorant.The structure of dyes carries a conjugated system (the double and single bonds alter their location).Their structure shows electron resonance that is considered as a stabilizing force [3].
Nature is the main source of colorants, but extraction of natural colors and their instability represent problems that are associated with the synthesis of artificial (synthetic) colors, which started with Perkin's mauve pigment in 1856 [4].The application of such synthetic colors in different industries in general and in food and pharmaceutical industries in particular has raised several concerns and compulsory warnings by the FDA, World Health Organization and European Union regulators, especially for children's food, drugs and cosmetics [5].Hence, due to various health problems associated with the overuse of synthetic colorants and the increasing customer and consumer awareness and demand for synthetic colorant-free products, scientific research has been oriented toward screening for natural colorants and improving their production and stability [6][7][8][9][10].The majority of natural colorants also have nutritional and health benefits, especially as antioxidant agents [11,12].Examples of well-known natural colorants that provide color and are considered safe are carotenoids, betalains, anthocyanins, curcumin and chlorophylls [5,13].Natural colorants originate from different sources, such as insects, animals, plants and microorganisms.However, pigments originating from microorganisms have many advantages over those produced by animals or plants, such as supply sustainability, higher yield, significant cost efficiency and considerable stability [5].Among different microbial genera, fungi and algae are ranked first in the production of a variety of water-soluble natural pigments [14,15].Still, the low pigment yields of algal cultures represents an obstacle to commercial production [16].Fungi were used centuries ago in dying textiles, especially silk and wool [17].Hence, the aim of this review is to elucidate the history of fungal pigments and their chemical structure.Well-known fungal producers are discussed, and some of the food and non-food applications of fungal pigments are highlighted.

Fungi as a Source of Pigments
The oldest record of using fungal pigments was the use of Monascus pigments in producing red mold rice (ang-kak).Fungi are an excellent source of natural pigments with significant advantages over plants.For example, fungi provide season-independent pigment production, and their growth is easier and faster and requires a cheap culture medium [18].Moreover, fungal pigments are of different color hues and possess higher stability and solubility [19].The members of specific fungal families are known as promising pigment producers, including Chaetomiaceae, Chlorociboriaceae, Cordycipitaceae, Herpotrichiellaceae, Hypocreaceae, Hyaloscyphaceae, Hymenochaetaceae, Monascaceae, Nectriaceae, Ophiostomataceae, Pleosporaceae, Polyporaceae, Sordariaceae, Tremellaceae, Trichocomaceae, Tuberaceae and Xylariaceae [18,20].
Fungal pigments are considered secondary metabolites that are produced by mycelium under certain conditions as the shortage in nutrients or under some unfavorable environmental stresses [21].Many fungal genera, such as Aspergillus, Trichoderma, Fusarium and Penicillium, produce intermediate metabolites (pigments) during their growth [22].Production of pigments such as melanin has a role in fungi protection, helping the microorganism to survive under severe environmental stresses and protecting from UV light.However, a single fungal species can produce different pigments with different properties.

Application of Fungal Pigments in Different Industries
Natural pigments have different applications (Figure 1) in food-and non-food-related fields.This came as a result of scientific research, which has warned of the health hazards accompanying the use of artificial synthetic pigments.Hence, we highlight here some of the applications of fungal pigments.

Application of Fungal Pigments in Different Industries
Natural pigments have different applications (Figure 1) in food-and non-food-related fields.This came as a result of scientific research, which has warned of the health hazards accompanying the use of artificial synthetic pigments.Hence, we highlight here some of the applications of fungal pigments.

Fungal Pigment Applications in Food Industry
As mentioned previously, there are continuous demands of consumers for less synthetic and artificial colors in their food and dairy products due to the health hazards and adverse effects caused by these artificial colors.In the middle of the 1980s, tartrazine was nominated as potential cause of sleep disturbance, hyperactivity and irritability in children [58].Furthermore, azo-dyes, which are commonly used as additives in the food industry, were regarded as potential carcinogens after being transformed by the gut microbiota [59].Artificial colors such as tartrazine, sunset yellow and ponceau are also capable

Fungal Pigment Applications in Food Industry
As mentioned previously, there are continuous demands of consumers for less synthetic and artificial colors in their food and dairy products due to the health hazards and adverse effects caused by these artificial colors.In the middle of the 1980s, tartrazine was nominated as potential cause of sleep disturbance, hyperactivity and irritability in children [58].Furthermore, azo-dyes, which are commonly used as additives in the food industry, were regarded as potential carcinogens after being transformed by the gut microbiota [59].Artificial colors such as tartrazine, sunset yellow and ponceau are also capable of inducing allergic reactions in many individuals even when consumed at low concentrations [60].In addition, glossitis was linked to ponceau 4R consumption at high concentrations [61].Hence, natural colors are favored; however, the safety of colors from microbial origins must be firstly checked.It is known that the majority of fungi produce mycotoxins, which can restrict their application in food and pharmaceutics.Some fungal species, such as Aspergillus carbonarius, produce a yellow pigment without producing any mycotoxins [62]. A. carbonarius produces polygalacturonase, which tolerates UV irradia-tion, and during its growth phase, a safe yellow pigment is accumulated in its biomass which can be used in food industry [63].Thermomyces sp.produces a thermophilic yellow pigment that has antioxidant activity [60].Pigmentation varies from yellow to red, according to many growth conditions such as temperature, age of the fungus and the used substrate.Food and beverages fortified by the yellow pigment recorded high antioxidant properties, antimicrobial properties and color stability [64].Blackslea trispora is a promising source of safe β-carotene as this fungus does not produce mycotoxins [65].The β-carotene from this fungus was the first approved microbial food colorant in the European Union.Different Fusarium species are capable of producing a wide range of diverse pigments, especially F. graminearum, which produces rubrofusarin, a red naphthoquinone pigment [66]; F. fujikuroi, which produces fusarubin, an orange carotenoid pigment [67]; and F. oxysporum, which secretes bikaverin, a red naphthoquinone [68].However, there are safety concerns about such pigments because the metabolites produced by Fusarium species contain different mycotoxins such as fumonisins, fusaric acid, fusarins, zearalenone and beauvericins [57].On the contrary, Monascus species are generous producers of safe pigments with strong contributions in food industry, such as M. purpureus, which produces monascorubramine and rubropunctamine; M. anka, which secretes ankaflavin and monascin; and M. ruber, which produces monascorubrin and rubropunctatin [69][70][71].Monascus pigments are produced on rice using solid-state microbial fermentation, and red mold rice was used centuries ago as a food colorant in traditional oriental medicine [72].Many Penicillium species produce antibiotics and pigments and are used in manufacturing cheese [73].P. purpurogenum secretes an azaphilone-like, brick-red pigment.Additionally, violet and orange pigments were obtained by modifying conditions of culturing [74].Pigment production using Penicillium as a source is more efficient and highly preferred because it secretes water-soluble and stable pigments extracellularly, so it can be easily purified [75].Talaromyces purpureogenus (previously known as Penicillium purpureogenum), secretes safe yellow and red pigments under submerged fermentation conditions, while other strains of Talaromyces as T. aculeatus, T. funiculosum, T. purpurogenum and T. pinophilus produce Monascus-like polyketide azaphilone pigments [76].Trichoderma viride secretes a brown pigment and can synthesize the yellow pigment emodin [60].Neurospora crassa produces safe yellow to orange-red polyketide and carotenoid fungal pigments that are used as food colorants.Mushrooms are known for producing pigments, especially members of family Cordycipitaceae such as Cordyceps, Torrubiella, Hyperdermium, Beauveria and Lecanicillium.Beauveria bassiana produces tenellin, pyridomacrolidin and pyridovericin, which is blood-red dibenzoquinone, while B. brongniartii secretes bassianin, and Torubiella produces torrubiellones [60].

Non-Food Applications of Fungal Pigments
Safe fungal pigments can be used for various industrial applications such as dyes for textiles, paper, paints, leather and cosmetics.Many fungal pigments originating from genera such as Monascus, Talaromyces, Penicillium, Trichoderma and Aspergillus showed antibacterial activity against many pathogenic bacteria [77][78][79][80].Such antimicrobial potential leads to application of these pigments in different fabrics, with promising results that suggest their potential use in suture threads, bandages, face masks and other related medical applications [81,82].Some fungal pigments such as naphthoquinones, melanin, violacein and carotenoids showed antioxidant activity [83,84].Interestingly, some Monascus fungal pigments such as monascin, monapurone A-C, ankaflavin, monasphilone A-B, monaphilone A-B and monapilol A-D show anticancer or antitumor activity against different cancer cells such as mouse skin carcinoma, human colon adenocarcinoma, pulmonary adenocarcinoma, human laryngeal carcinoma and human hepatocellular carcinoma [85,86].Fungal pigments are utilized in the cosmetics industry due to their reported biological activities.Carotenoids, melanin and lycopene are already applied in cosmetics, sunscreens, sunblocks, sun lotions, anti-ageing creams, face creams, skin conditioning and lipsticks.[18,87].Fungal pigments, as natural pigments with different advantages (eco-friendly, easy degradation, safe and high staining capability) over synthetic pigments, represent a good alternative to the synthetic dyes in the textiles industry.Pigments of the fungal genera Monascus, Curvularia, Aspergillus, Penicillium, Talaromyces, Trichoderma, Alternaria, Cordyceps, Bisporomyces and Cunninghamella were applied to different fabrics such as cotton yarn, wool, silk, nylon and polyester [21,88].

Epicoccum as an Example of Pigment-Producing Fungi
Many studies have discussed the promising potential of different fungal genera to secrete pigments.However, few reports have described pigments secreted by the genus Epicoccum.Epicoccum is an endophytic dematiaceous ascomycetous fungus that is known for its application as biocontrol agent against many phytopathogens [89].Colonies of Epicoccum are fast-growing, suede-like to downy, showing a deep yellow to brownish orange diffusible pigment.During sporulation, many black conidiophore aggregates can be observed.Conidia of Epicoccum appear singularly on densely compacted, determinant and faintly pigmented conidiophores.Conidia are characterized by being globose to pyriform, with a characteristic funnel-like base and broad attachment scar, frequently seceding with a protuberant basal cell.Conidia are multicellular, deeply pigmented and have a surface that appears verrucose externally.Epicoccum sp.secretes different metabolites from different chemical classes such as polyketides, diketopiperazines, polyketide hybrids, carotenoids and siderophores.Some of these metabolites showed promising biological activities, such as antimicrobial, antioxidant and anticancer properties, and there are many promising and important metabolites with potent biological activities such as the anticancer drug taxol, D8646-2-6 orsellinic acid and curvularin.Additionally, many Epicoccum species produce pigments that have potential industrial applications, such as E. nigrum (Tables 2 and 3), for example epicocconone, which is commercially known as fluorophore and is used in cell staining and in gel electrophoresis for protein detection [40,41].Other reported Epicoccum-derived pigments are listed in Table 3. Foppen and Gribanovski-Sassu [100] γ-Carotene Carotenoid Orange pigment Foppen and Gribanovski-Sassu [100] Rhodoxanthin Carotenoid Red pigment Foppen and Gribanovski-Sassu [100] Torularhodin Carotenoid Violet pigment Foppen and Gribanovski-Sassu [100] As shown in Table 3, the pigments secreted by Epicoccum species are of polyketide and/or carotenoid origin [101,102] with pigment color shades ranging in yellow, orange and red spectra.

Extraction and Optimization of Pigment Production
Extraction process of metabolites such as pigments from microbial sources is conducted using different solvents depending on various variables, for example the type of used solvent, temperature used during the extraction process, the time of extraction process and the solid/liquid ratio [103].Nevertheless, application of this extracted pigment in the industrial field will be economical only if high yield (efficient extraction) is achieved.Many approaches were applied in order to optimize fungal pigment production, such as optimization of production medium components, culturing conditions and using statistical optimization methods [104].Some statistical optimization methods are based on applying one factor at a time, which is less efficient, consumes time, is relatively expensive and fails to determine the relationship between different variables [105].Meanwhile, other statistical optimization methods such as the response surface model can investigate variable independent parameters and their interactive relationship and subsequently can be utilized to develop relevant mathematical models that can predict the whole process.In other words, applying such statistical optimization models produces substantial results from a small number of experiments [106].Hence, it is critically important to use such efficient methods for optimization of pigment production for industrial application.The main obstacle in the fungal pigment production process is the difficulty in optimizing both biomass yield and pigment yield, since both are indirectly proportional.Therefore, such a relationship (between pigment yield and biomass) must be studied.The use of genetic engineering approaches can solve such a problem and control pigment production through employing recombinant DNA, for example, to change the activity of enzymes responsible for carotenoid biosynthesis [22].

Conclusions and Future Directions
Fungal pigments are promising metabolites with a wide range of applications when compared to artificial pigments.Additionally, the elevated consumer awareness of the advantages of using natural pigments in general and fungal ones in particular has oriented researchers toward screening for other novel and safe pigments from fungi.Ascomycetous fungal genera are favorable in this field as they are characterized by the ease of their growth any time of the year under relatively simple laboratory conditions and therefore can be applied in large industrial production (in contrast to plant-derived pigments where their availability is affected by the producer plant's growing season).Many scientific studies have described the large-scale production of fungal pigment production in a bioreactor under controlled conditions.Industrial use of fungal-derived pigments has gradually progressed throughout history.In the beginning, natural food colorant from fungal sources was restricted to the semi-fermentative production of the yellow natural food colorant, riboflavin, which is secreted by the filamentous fungus Eremothecium gossypii (previously known as Ashbya gossypi) [107].However, the use of riboflavin as food colorant has some limitations because it is sensitive to light.After that, the fungus Blakeslea trispora was used to produce β-carotene as a food colorant.Previously, tomato was the sole source of lycopene, but nowadays Blakeslea trispora is approved as a promising source of lycopene [108,109].Cordyceps unilateralis (also known as Ophiocordyceps unilateralis) is capable of producing pigments that have similar structure to shikonin and alkanin (red pigments derived from plants) [110].
There are various color hues of polyketide pigments with anthraquinone, naphthoquinone, azaphilone and hydroxyanthraquinone structure.However, for centuries, polyketide pigments secreted by the fungus genus Monascus have been the most widely used in Asian countries such as Southern China, Southeast Asia and Japan for making anka, red soybean cheese and red rice wine [111].As described previously, Monascus sp.secretes many pigments such as monascin and ankaflavin (yellow in color), rubropunctatin and monascorubrin (orange in color) and rubropunctamine and monascorubramine (purple-red in color).Hence, different companies have filed many patents for Monascus-like pigments, and their products are now available in the market.It should be noted that Monascus secretes a hepato-nephrotoxic mycotoxin (citrinin); however, the literature does not describe any death due to consumption of red rice wine, red soybean cheese or anka made using Monoascus pigments.Hence, evaluation of toxicity of different fungal pigments is of critical importance before testing their potential and allowing them to enter the market as food colorants or as drugs, which explains why application of fungal pigments in food and medical fields is relatively difficult.Nowadays, researchers are focusing on screening of fungal diversity for pigment production with an emphasis on two major points, which are finding a non-mycotoxin producing strain and a pigment that is water-soluble [110].Challenges facing the industrial application of some Monascus pigments include their weak water solubility, poor pH stability and sensitivity to heat and light.One of the proposed solutions for such challenges is changing the pigment's chemical structure through substituting oxygen with nitrogen from the amino group in its structure [112].Moreover, another serious problem is the co-production of toxic metabolites with the pigment, which prevents its application, or production of a mixture of pigments, which represents a challenge when trying to produce a pigment with one color tone.This problem can be solved by optimizing culturing conditions such as changing the used substrates, temperature, pH and dissolved oxygen either in submerged or solid-state fermentation.However, it was reported that solid-state fermentation results in higher secondary metabolites yield because it resembles the natural habitat of fungi besides providing a solid support that fungal strain can attach to [113].Another critical point that affects the application of a pigment in industry is the stability of this pigment over time, but some approaches such as nanoencapsulation can address both the stability and solubility problems [5].A recent study has described the effect of ozone processing, which is used during the pasteurization process of some juices and foods on some fungal pigments, and fungal pigments showed higher stability compared with other tested natural pigments [114].Interestingly, biological activities of fungal pigments may open new opportunities for their use in the production of functional textiles with medical properties or functional colored food with nutritional and health-improving benefits.Finally, construction of an online database that carries all information about fungal producers of different pigments and their chemical characteristics will facilitate the work of scientists and save their time.

Figure 1 .
Figure 1.Examples of fungal pigments that are involved in some food and non-food applications.

Figure 1 .
Figure 1.Examples of fungal pigments that are involved in some food and non-food applications.

Table 1 .
Well-known chemical classes responsible for different colors.

Table 1 .
Well-known chemical classes responsible for different colors.

Table 2 .
Examples of well-known fungal pigments and their producers.

Table 2 .
Examples of well-known fungal pigments and their producers.

Table 2 .
Examples of well-known fungal pigments and their producers.

Table 2 .
Examples of well-known fungal pigments and their producers.

Table 3 .
Some of the pigments produced by Epicoccum species.