Recent Findings in Azaphilone Pigments

Filamentous fungi are known to biosynthesize an extraordinary range of azaphilones pigments with structural diversity and advantages over vegetal-derived colored natural products such agile and simple cultivation in the lab, acceptance of low-cost substrates, speed yield improvement, and ease of downstream processing. Modern genetic engineering allows industrial production, providing pigments with higher thermostability, water-solubility, and promising bioactivities combined with ecological functions. This review, covering the literature from 2020 onwards, focuses on the state-of-the-art of azaphilone dyes, the global market scenario, new compounds isolated in the period with respective biological activities, and biosynthetic pathways. Furthermore, we discussed the innovations of azaphilone cultivation and extraction techniques, as well as in yield improvement and scale-up. Potential applications in the food, cosmetic, pharmaceutical, and textile industries were also explored.


Introduction
Color has been used by mankind since the Neolithic period and has been associated to different people such as purple to the Phoenicians, yellow (annatto) to the Mayans, and to different purposes as henna pigments for body and hair coloring in India. In human history, color gained a powerful status in many daily experiences and key decisions. Some studies show, for example, that preference for blues and reds (at the expense of yellowish and greenish hues) influenced auction prices, as reported for Mark Rothko's rectangular paintings [1].
Color is also naturally associated with chemosensory perceptions regarding flavor, quality and freshness, highly interfering in product choice [2]. In this way, consumers expect some foods to have specific colors. However, variation and heterogeneousness of natural color in foods initiated the process of adding pigments to maintain color uniformity while granting high coloring power, as well as stability in aqueous phase and in different pH [3].
Vegetal-derived natural products are source of pigments very important to the food industry. However, the production is limited by yield issues since the gross amounts of vegetal pigments recovered, even from improved cultivars is not sufficiently competitive to fulfill modern industrial demand. Yield improvement is surely the major problem which have been addressed by developing and breeding modified cultivars and new largescale processes were developed to the production of natural pigments [4]. Insect-derived coloring compounds such as carmine have been introduced in the market, but despite their natural origin, they are not accepted by many countries' regulatory agencies due to ethical
In this section, it is presented a summary of the new compounds reported after December 2019 classified according to the fungal genera source. Despite the great number of 100 new compounds reported from January 2020 to March 2021, the azaphilones were isolated only from nine fungal genera (Aspergillus, Chaetomium, Hypoxylon, Monascus, Muycopron, Penicillium, Phomopsis, Pleosporales, and Talaromyces). The genus Phomopsis was not cited in the latest review and now appeared as fungal endophytic sources of chlorinated azaphilone pigments. At this time, it will be presented the new compounds isolated from each genus (Figures 2-9) displayed according to the species (Table 1).

Azaphilones from Chaetomium Genus
Chaetomium is a large genus presenting more than 300 species worldwide. Chaetomium globosum represents one of the most studied species and is known as a rich source of azaphilones. Since the last two years, this species has still been contributing with new metabolites. The arthropod-associated endophytic fungus C. globosum TW1-1 was investigated considering whether the presence of 1-methyl-l-tryptophan into the growth medium would activate a biosynthetic pathway to produce novel alkaloids [37]. However, instead of nitrogenated metabolites, the authors isolated and identified two chlorinated azaphilones, chaephilone C and D (25)(26) with anti-inflammatory activity. Their stereostructures were unequivocally confirmed by X-ray analyses. Nevertheless, chaephilone C was also previously reported from the deep sea-derived fungus Chaetomium sp. NA-S01-R1 with the same planar structure of 25 but with different stereochemistry, suggesting that its structure should be revised [38]. Two months after the report of chaephilone C (25), a new chlorinated azaphilone from C. globosum, endophytic of Polygonatum sibiricum, was reported and also called chaephilone C (27) [39]. However, this latter compound displayed a chemical structure similar to (26), but completely different from the former (25).
From the wild-type strain C. globosum, a new dimeric azaphilone called cochliodone J (28) was identified in the same medium which cochliodone A had been isolated before [40]. The deep-sea C. globosum MP4-S01-7 provided eight new structurally correlated nitrogenated azaphilones 29-36 ( Figure 3 and Table 1) [41]. The azaphilone core is the same in all compounds with differences only in the lactone acyl substituents and the N-alquil groups. Seco-chaetomugilin (37) was isolated for the first time from the ethyl acetate extract of Chaetomium cupreum in a bio-guided fractionation for activities against human breast adenocarcinoma cell lines [42]. Although the authors named the compound isolated as seco-chaetomugilin, it presented the same structure of seco-chaetomugilin D, previously isolated from C. globosum [43]. A screening by LC-MS/MS-GNPS data base of a strain of an endophytic plant fungus Chaetomium sp. g1 resulted in the isolation of chaetolactam A (38), a unique 9-oxa-7-azabicyclo[4.2.1]octan-8-onering system with two new compounds chaetoviridins derivatives, 11-epi-chaetomugilide B (39), and chaetomugilide D (40) [44]. Another plant endophytic fungus C. globosum isolated from the desert Asteraceae species, Artemisia desterorum, yielded globosumone (41), a new stereoisomer of the known chaetoviridin E [45].

Azaphilones from Muyocopron Genus
The chemical investigation of the endophyte Muyocopron laterale ECN279 isolated from a health leaf of Conavalia lineata led to the isolation of the two new azaphilones muyocopronones A and B (54)(55) [50]. An endophyte fungus F53 from the traditional Chinese medicine plant Taxus yunnanensis had its genome sequenced and mined, and the multilocus phylogeny of F53 allowed its placement within the genus Muyocopron with its closest relative being Muyocopron atromaculans (MUCL 34983) [51]. Moreover, a new azaphilone lijiquinone 1 (56) with activities against human myeloma cells and the yeast Candida albicans and Cryptococcus albidus was isolated from its ethyl acetate extract ( Figure 5)

Azaphilones from Penicillium Genus
The Penicillium genus produces a great number of azaphilone metabolites [31]. Penicillium citrinum WK-P9 was isolated as an associated fungus from the sponge Suberea sp., displaying antibacterial activity. The bio-guided chemical investigation of its ethyl acetate extract led to the isolation of a new citrinin derivative called penicitrinone G (57) [52]. Genome mining, epigenetic regulation, optimization of culture conditions, and one-strainmany-compounds (OSMAC) were investigated as a possible way to prioritize the production of other polyketide metabolites different than the rubratoxins in Penicillium dangeardii [53]. Only the metabolic shunting strategy, based on the deletion of the key gene rbtJ encoding PKS for rubratoxins biosynthesis, and the optimization of culture conditions successfully led to the production of 35 azaphilones, from which 23 were new ones. They were identified as nine monomers named dangelones A-G (58-64), dangeloside A-B (65-66), eight dimers, didangelones A-G (67)(68)(69)(70)(71)(72)(73)(74), and five trimers, tridangelones A-E (75)(76)(77)(78)(79) [53] ( Figure 6). Dangelones A-G (58-64) have the same planar structure and the distinctions among them lay on the side chains at C-3. The differences at C-3 side chain are also present in the dimers. Still regarding Penicillium endophytic fungi, a strain of Penicillium sp. T2-11 isolated from the rhizomes of the underground portion of Gastrodia elata produced a citrinin dimer, named penctrimertone (80) [54].

Azaphilones from Muyocopron Genus
The chemical investigation of the endophyte Muyocopron laterale ECN279 isolated from a health leaf of Conavalia lineata led to the isolation of the two new azaphilones muyocopronones A and B (54-55) [50]. An endophyte fungus F53 from the traditional Chinese medicine plant Taxus yunnanensis had its genome sequenced and mined, and the multi-locus phylogeny of F53 allowed its placement within the genus Muyocopron with its closest relative being Muyocopron atromaculans (MUCL 34983) [51]. Moreover, a new azaphilone lijiquinone 1 (56) with activities against human myeloma cells and the yeast Candida albicans and Cryptococcus albidus was isolated from its ethyl acetate extract ( Figure 5).

Azaphilones from Penicillium Genus
The Penicillium genus produces a great number of azaphilone metabolites [31]. Penicillium citrinum WK-P9 was isolated as an associated fungus from the sponge Suberea sp., displaying antibacterial activity. The bio-guided chemical investigation of its ethyl acetate extract led to the isolation of a new citrinin derivative called penicitrinone G (57) [52]. Genome mining, epigenetic regulation, optimization of culture conditions, and one-strain-many-compounds (OSMAC) were investigated as a possible way to prioritize the production of other polyketide metabolites different than the rubratoxins in Penicillium dangeardii [53]. Only the metabolic shunting strategy, based on the deletion of the key gene rbtJ encoding PKS for rubratoxins biosynthesis, and the optimization of culture conditions successfully led to the production of 35 azaphilones, from which 23 were new ones. They were identified as nine monomers named dangelones A-G (58-64), dangeloside A-B (65-66), eight dimers, didangelones A-G (67-74), and five trimers, tridangelones A-E (75-79) [53] (Figure 6). Dangelones A-G (58-64) have the same planar structure and the distinctions among them lay on the side chains at C-3. The differences at C-3 side chain are also present in the dimers. Still regarding Penicillium endophytic fungi, a strain of Penicillium sp. T2-11 isolated from the rhizomes of the underground portion of Gastrodia elata produced a citrinin dimer, named penctrimertone (80) [54].

Azaphilones from Talaromyces Genus
Most strains previously referred to as Penicillium sp. are now classified in the Talaromyces species, and some of them have been found to produce yellow and red azaphilone pigments. Two new pigments from T. atroroseus were described. The first belongs to the

Azaphilones from Talaromyces Genus
Most strains previously referred to as Penicillium sp. are now classified in the Talaromyces species, and some of them have been found to produce yellow and red azaphilone pigments. Two new pigments from T. atroroseus were described. The first belongs to the

Azaphilones from Talaromyces Genus
Most strains previously referred to as Penicillium sp. are now classified in the Talaromyces species, and some of them have been found to produce yellow and red azaphilone pigments. Two new pigments from T. atroroseus were described. The first belongs to the series of known Monascus orange azaphilone PP-O pigments, and it was unequivocally elucidated as the isomer trans-PP-O (91) [32] (Figure 9). The second was the unique azaphilone atrosin S, which presented the incorporation of a serine moiety into the isochromene/isoquinoline system. The fungus cultivation in medium enriched with a specific amino acid as sole source of nitrogen could allow seven atrorosin derivatives (atrorosin D, E, H, L, M, Q, and T, depending on the amino acid incorporated) (92)(93)(94)(95)(96)(97)(98)(99), which were identified by dereplication using HPLC-DAD-MS/HRMS analysis [32]. From the fungus Talaromyces albobiverticillius associated with the isopod Armadillidium vulgare, two interesting azaphilone pigments talaralbols A and B (100-101) was reported [58]. However, talaralbol B presents the same planar structure of trans-PP-O, early described in T. atroroseus [32], in which the C-9 stereochemistry was not reported.  (Figure 9). The second was the unique azaphilone atrosin S, which presented the incorporation of a serine moiety into the isochromene/isoquinoline system. The fungus cultivation in medium enriched with a specific amino acid as sole source of nitrogen could allow seven atrorosin derivatives (atrorosin D, E, H, L, M, Q, and T, depending on the amino acid incorporated) (92)(93)(94)(95)(96)(97)(98)(99), which were identified by dereplication using HPLC-DAD-MS/HRMS analysis [32]. From the fungus Talaromyces albobiverticillius associated with the isopod Armadillidium vulgare, two interesting azaphilone pigments talaralbols A and B (100-101) was reported [58]. However, talaralbol B presents the same planar structure of trans-PP-O, early described in T. atroroseus [32], in which the C-9 stereochemistry was not reported.

Biological Activities of Azaphilones
Azaphilones, besides being good compounds to replace synthetic pigments, aggregate valuable pharmacological properties. The wide broad range of biological activities that has been reported for azaphilones such as cytotoxic, anti-inflammatory, antimicrobial, antitumoral, antiviral and antioxidant is exemplified in Table 1.
Concerning the activities regarded to the new 101 azaphilones reported, the cytotoxic and antitumor potential are the most evaluated. Remarkably, compounds (29), (30), and (33) showed the most effective anti-gastric cancer activities (MGC803 and AGS cell lines) with IC50 values less than 1 μM, being more active than the positive control paclitaxel (3.8 μM) [41]. Additionally, (29) and (30) induced apoptosis in a concentration-dependent manner and (30) inhibited cell cycle progression. The authors also claim that 3,7-dimethyl-2,6-octadienyl group attached to N-2 contributed to the potent cytotoxic activities against MGC803 and AGS gastric cancer cell lines what can induce new investigations with semisynthetic azaphilone derivatives possessing this group [11]. The azaphilones (39) and (40) showed moderate activity against leukemia HL-60 and human breast cancer. However, (39) exhibited potent apoptosis induction activity by mediating caspase-3 activation and

Biological Activities of Azaphilones
Azaphilones, besides being good compounds to replace synthetic pigments, aggregate valuable pharmacological properties. The wide broad range of biological activities that has been reported for azaphilones such as cytotoxic, anti-inflammatory, antimicrobial, antitumoral, antiviral and antioxidant is exemplified in Table 1.
Concerning the activities regarded to the new 101 azaphilones reported, the cytotoxic and antitumor potential are the most evaluated. Remarkably, compounds (29), (30), and (33) showed the most effective anti-gastric cancer activities (MGC803 and AGS cell lines) with IC 50 values less than 1 µM, being more active than the positive control paclitaxel (3.8 µM) [41]. Additionally, (29) and (30) induced apoptosis in a concentration-dependent manner and (30) inhibited cell cycle progression. The authors also claim that 3,7-dimethyl-2,6-octadienyl group attached to N-2 contributed to the potent cytotoxic activities against MGC803 and AGS gastric cancer cell lines what can induce new investigations with semisynthetic azaphilone derivatives possessing this group [11]. The azaphilones (39) and (40) showed moderate activity against leukemia HL-60 and human breast cancer. However, (39) exhibited potent apoptosis induction activity by mediating caspase-3 activation and PARP degradation at 3 µM in leukemic cells HL-60 [44]. Another interesting result was the potent cytotoxic activity showed by the dimeric azaphilones (89) and (90) against five different human cell lines. (89) showed more potent cytotoxicity against MGC-803 than cisplatin and possessed a unique 6/4/6 ring system suggesting the new ring may play an important role in cytotoxicity [57].
In vitro antiviral activity against HIV-1 was detected for phomopsones B and C (82-83) (7.6 and 0.5 µM, respectively [52]). Research in antiviral potential of azaphilones may be strengthened as they have been focused as possible drug leads for the development of effective antiviral agents against SARS-CoV-2 [60,61]. This worldwide impact-generated virus draws attention to the difficulty in developing new non-toxic antiviral drugs, as viruses use cell host metabolism for replication. This is corroborated by previous reports of antiviral activity of azaphilone metabolites, such as chermisinone B, isolated from the endophytic fungus Nigrospora sp. YE3033, and active against A/Puerto Rico/8/34 (H1N1) in CPE assay (IC 50 0.80 µg/mL) with low cellular toxicity on MDCK cells (CC 50 184.75 µg/mL) [62]. In vitro HIV-1 replication inhibitory effects in C8166 cells were demonstrated for Helotialins A and B (EC 50 8.01 and 27.9 nM, respectively) [63]. In 2019, comazaphilone D was reported as a non-competitive inhibitor of neuraminidase from recombinant rvH1N1 (IC 50 30.9 µM) while rubiginosin A was active against H5N1 (IC 50 29.9 µM) [64]. The previous knowledge of the antiviral potential of azaphilone derivatives is an advantageous background for the development of new drugs to inhibit SARS-CoV-2. Table 1. Azaphilones fungal sources and reported biological activities.

Recent Insights in the Biosynthesis of Azaphilones
The biosynthesis of azaphilones has been reviewed by Pavesi et al. [65] and was also considered in the two latest reviews [31]. Five biosynthetic pathways were exhaustively discussed, which highlighted the comprehensive study of Monascus and Aspergillus pathways [65]. Furthermore, a thorough study performed about the precise role of ammonium nitrate in the production of Monascus pigments showed that some biosynthetic pathways can present changes due to the regulation and expression of several key genes involved [66]. The expression of the gene mppG (MrPigF), responsible for orange pigments, was significantly downregulated with ammonium nitrate addition, and an improvement in yellow pigment production was followed by an upregulated mppE expression. Additionally, ammonium nitrate increased the NH 3 content in the fermentation broth resulting in the increased red pigments yield [66].
Dimeric azaphilones have been described in the Chaetonium genus, and the fungal laccase-like multi-copper oxidase gene encoded by CcdJ (CHGG_10025) is believed to dimerize the cochliodones [65]. Cochliodone J (28), a new dimeric azaphilone containing a spirotetrahydropyran moiety, was reported, but the mechanism of the spiro ring formation still remains to be determined [40]. Moreover, the unusual fusion between an eightmembered lactam and a six-membered lactone, presented in the structure of chaetolactama A (38), has not been investigated yet.
The biosynthetic gene cluster responsible for the sequential and convergent production of azaphilones in Chaetonium sp. might count with a hidden gene allegedly responsible for the epimerization of the 7-OH group in chaetoviridin E as well as the oxidation/epoxidation leading to OH groups in C-8a and C-1 positions, followed by methylation of the latter, as in (41) [45]. Based on studies with Monascus, Aspergillus, and Talaromyces, two biosynthetic gene clusters were postulated to drive the diverse azaphilones in H. fragiforme. However, the biosynthetic dimerizations which led to the compounds (42)-(49) demand more investigations. This represents a challenge because Hypoxylaceae azaphilones are exclusively formed during stromata development, which cannot be induced under laboratory conditions [46]. A reasonable proposal consists on a spontaneous aldol condensation responsible for the heterodimerization of different azaphilones derivatives [46].
The biosynthesis of three different azaphilone skeletons was reported for P. tersa FS441. The tersaphilone B (85) showed the unique 6/6-6 carbon skeleton with a cleaved tetrahydrofuranyl ring, and the diastereomers tersaphilones D and E (87-88) displayed a unique five-membered furan ring open and an epoxide ring in C-8a and C-1 positions [56]. A remarkably biosynthetic proposal was provided to penctrimertone (80), which presented a 6/6/6/6 tetracyclic ring system with an unusual aldehyde group in one of the rings [54]. It is supposed to be a citrinin dimer furnished by a citrinin monomer that suffered hydration, oxidation, and reduction affording an orthoquinone methide susceptible to an unusual intermolecular hetero-Diels-Alder reaction with another citrinin molecule [57].
Another interesting observation is the presence of a six-membered ring at the C-3 position of the azaphilones core reported in the Muyocopron genus, which is present in less than 10% of the hundreds of azaphilones isolated to date. Regarding the compounds (54-56), the gene cluster lij. was proposed to control a convergent biosynthetic pathway. The LijE would be responsible for the formation of the aromatic ring with a carbon chain attached to the cyclohexanone ring. Reduction of the acyl ester followed by cyclization and dehydration afforded the azaphilone core. This core would be attached by the C-7 OH group to the acyl derivative formed by previous condensation of acetyl-CoA/malonyl-CoA and C-methylation controlled by the LijA gene. The compounds (54-55) also presented a 2,4-dimethyl-3-hydroxyhexanoate moiety that was reported in only eight compounds in this genus. The cyclohexanone ring and 2,4-dimethyl-3-hydroxyhexanoate moiety might be biomarkers of the Dothideomycetes class and constitute a noteworthy point to be more investigated [50].

Processing and Innovations in Azaphilones Production
Over the last decade, many studies have focused attention on optimizing production of pigments and growth of different fungal species. Many variables that affect the production, as fermentation process (submerged fermentation, solid-state fermentation, larger scale), culture media composition (carbon and nitrogen source, C/N ratio, co-factors, surfactants, tricarboxylic acid intermediates), inoculum type and age (spores and mycelium), temperature, pH, oxygen level and agitation; light, humidity, pigment recovery, extraction, and isolation have been critically discussed by recent reviews [11,31,[67][68][69]. Some related aspects of production, processing and innovations in azaphilones production published in 2020 and up to March 2021 are highlighted below.

Overcoming Mycotoxin Issues
The consensual approval of color additives for food industry by international regulatory bodies is of great importance for commercial transactions, so that in-house products can be exported to other markets without alterations to remove or replace pigments regularized only in the exporting country. US and EU are good examples. Sixteen color additives allowed in the EU are not accepted by US regulatory agency, while four color additives allowed in the US are not permitted in the EU [70]. The ancient knowledge about Monascus pigments and utilization of Monascus by Asian people for hundreds of years has motivated the search for beneficial and healthy metabolites of Monascus azaphilones. Despite the isolation of many Monascus metabolites, these pigments were not approved by regulatory agencies in the US and UE so far, due to concerns over co-production of the hepatonephrotoxic mycotoxin citrinin (102, Figure 10). Co-production of azaphilones and citrinin is a major issue on this point and optimization of azaphilones production on industrial scale must assure no production of toxic metabolites [71]. For this purpose, genetic techniques have been used, such as depletion of ctnE gen, responsible for the production of citrinin (102), successfully performed in Monascus aurantiacus Li AS3.4384 [72]. The medicinal properties reported for azaphilones are a catalyst in the search for fermentative processes suitable for the production of these pigments from safe biosynthetic routes, obtained by deletion of citrinin gene.
M. purpureus has also been studied with the aim of inhibiting citrinin (102) production without negative change in pigments biosynthesis. Hong et al. [71] used transcriptome sequencing to explore citrinin gene expression in experiments comparing the effect of inorganic (ammonium chloride and ammonium nitrate) with organic nitrogen (peptone group) sources in M. purpureus M3103 metabolism. It was found that biosynthesis of amino acids was up-regulated by ammonium chloride and ammonium nitrate, enhancing the producing of biosynthetic precursors of pigments while essential genes and transcription factors involved in the biosynthesis pathway of citrinin (102) were down-regulated by these inorganic nitrogen sources. Therefore, inorganic nitrogen proved to be more favorable for the biosynthesis of citrinin-free pigments (especially orange and red pigments) by M. purpureus M3103.
Industry Research and Development Institute in Taiwan is dedicated to investigating new ways to obtain azaphilone pigments using genetic manipulation and optimization of a fermentative process, aiming to avoid the production of citrinin (102) (Figure 10). They successfully developed some citrinin-free Monascus strains, including the strain M. pilosus BCRC 38072, previously mentioned for its production of azaphilones 51-53 [49].
Other mycotoxins are also of concern. Talaromyces genus have species reported to produce both, red colorants and mycotoxins (T. atroroseus [32], Talaromyces purpureogenus [73] and T. albobiverticillius [58]) while other species of this genus are not reported to produce known mycotoxins [11,74,75]. Mycotoxins reported from T. purpureogenus are rubratoxins A (103) and B (104), rugulovasins (105) and luteoskyrin (106), (Figure 10) therefore limiting the use of this species for biotechnological production of food pigments [73]. T. purpureogenus CFRM0 produces higher yield of pigments in Potato Dextrose Agar (PDA) and Charcoal Yeast Extract (CYE) rather than in Malt Extract Agar (MEA) and Yeast Extract with Supplements (YES) media (30 • C, 3-4 days), although the growth rate was similar in all conditions [73]. The pigments produced by T. purpureogenus CFRM0 were not toxic to female Wistar rats. No alterations related to toxicity were found, including no biochemical, hematological and histological modifications, indicating the safety of this pigment even when administrated in successive days [73].
J. Fungi 2021, 7, x FOR PEER REVIEW 18 of 31 by these inorganic nitrogen sources. Therefore, inorganic nitrogen proved to be more favorable for the biosynthesis of citrinin-free pigments (especially orange and red pigments) by M. purpureus M3103. Industry Research and Development Institute in Taiwan is dedicated to investigating new ways to obtain azaphilone pigments using genetic manipulation and optimization of a fermentative process, aiming to avoid the production of citrinin (102) (Figure 10). They successfully developed some citrinin-free Monascus strains, including the strain M. pilosus BCRC 38072, previously mentioned for its production of azaphilones 51-53 [49].
Other mycotoxins are also of concern. Talaromyces genus have species reported to produce both, red colorants and mycotoxins (T. atroroseus [32], Talaromyces purpureogenus [73] and T. albobiverticillius [58]) while other species of this genus are not reported to produce known mycotoxins [11,74,75]. Mycotoxins reported from T. purpureogenus are rubratoxins A (103) and B (104), rugulovasins (105) and luteoskyrin (106), (Figure 10) therefore limiting the use of this species for biotechnological production of food pigments [73]. T. purpureogenus CFRM0 produces higher yield of pigments in Potato Dextrose Agar (PDA) and Charcoal Yeast Extract (CYE) rather than in Malt Extract Agar (MEA) and Yeast Extract with Supplements (YES) media (30 °C, 3-4 days), although the growth rate was similar in all conditions [73]. The pigments produced by T. purpureogenus CFRM0 were not toxic to female Wistar rats. No alterations related to toxicity were found, including no biochemical, hematological and histological modifications, indicating the safety of this pigment even when administrated in successive days [73].

Color-Directed Production of Pigments
Fungi from Monascus genus are the oldest source of azaphilone pigments and this genus is still considered as one of the most proliferous sources of pigments nowadays [76]. Azaphilones produced by Monascus species are usually refered as MonAzPs (Monascus azaphilone pigments) and are incorporated in many food products as a natural colorant in China, where MonAzPs exceed 20 thousand tons per year. It is estimated that the number of consumers that eat food containing MonAzPs daily is over one billion people [77]. Monascus pigments have predominantly three colors, yellow (monascin (107) and ankaflavin (108)), orange (rubropunctatin (109) and monascorubrin (110)) and red (rubropunctamine (111) and monascorubramine (112)) [78]. The structures of the mentioned substances and their chromophores, the part of the molecule responsible for their color, are shown in Figure 11. Several works focus M. purpureus metabolism [66,79,80]. Literature is also rich in reports presenting conditions to drive the metabolism of other fungal species to biosynthesize or to improve the production of pigments.

Color-Directed Production of Pigments
Fungi from Monascus genus are the oldest source of azaphilone pigments and this genus is still considered as one of the most proliferous sources of pigments nowadays [76]. Azaphilones produced by Monascus species are usually refered as MonAzPs (Monascus azaphilone pigments) and are incorporated in many food products as a natural colorant in China, where MonAzPs exceed 20 thousand tons per year. It is estimated that the number of consumers that eat food containing MonAzPs daily is over one billion people [77]. Monascus pigments have predominantly three colors, yellow (monascin (107) and ankaflavin (108)), orange (rubropunctatin (109) and monascorubrin (110)) and red (rubropunctamine (111) and monascorubramine (112)) [78]. The structures of the mentioned substances and their chromophores, the part of the molecule responsible for their color, are shown in Figure 11. Several works focus M. purpureus metabolism [66,79,80]. Literature is also rich in reports presenting conditions to drive the metabolism of other fungal species to biosynthesize or to improve the production of pigments. Color-directed production of pigments is advantageous as this approach would eliminate purification steps slowing down the processing by adding a separation step, to purify or concentrate pigments of the desired color. Therefore, a big challenge in pigments production is to obtain pure extracts, containing fewer substances and, preferably, with only one color [19]. Figure 12 presents some fungal species and associated fermentative parameters that resulted in the production of yellow [66,[80][81][82][83][84], orange [14,85] or red [14,79,[85][86][87][88][89] pigments. However, in most of the works, yellow, orange and red azaphilones are produced simultaneously (cocktail pigments phenomenon) in different proportions.
Regarding Monascus species, M. ruber CCT 3802 has been studied in terms of colony morphology and biomass production during pigments production utilizing cheese whey as substrate [90]. Strain M. ruber M7 showed different response to the addition of acetic acid, sodium acetate and ammonium acetate to PDA culture medium. The original big orange fleecy colony morphology turned into small compact reddish or tightly-packed orange colony upon increase of acetic acid or acetate. Pigment production, in turn, was enhanced by addition of acetate to the culture medium [91]. Yang et al. [16] reported that the expression of key genes for Monascus pigment biosynthesis was significantly up regulated in the presence of sodium nitrate. Increase in total pigment production and yellow pigment proportion was reported for a M. purpureus strain (LQ-6), after adding exogenous cofactor methyl viologen and rotenone (1.0 mg/L) to the submerged batch-fermentation [84].
The color of pigments produced by Talaromyces amestolkiae DPUA 1275 was shown to be pH-dependent. Low pH (2.59 and 3) directed to small production of yellow pigments while red ones were not detected [86]. On a further study, T. amestolkiae DPUA 1275 was grown in MSG-glucose medium supplemented with three individual complex nitrogen sources (yeast extract, meat extract and meat peptone), six individual amino acids (glutamic acid, threonine, tyrosine, glycine, cysteine and tryptophan), and two vitamins (biotin and thiamine) [92]. Complex nitrogen and amino acid supplementation did not favor red pigments production but small improvement (1.3 times) was detected after thiamine supplementation. Color-directed production of pigments is advantageous as this approach would eliminate purification steps slowing down the processing by adding a separation step, to purify or concentrate pigments of the desired color. Therefore, a big challenge in pigments production is to obtain pure extracts, containing fewer substances and, preferably, with only one color [19]. Figure 12 presents some fungal species and associated fermentative parameters that resulted in the production of yellow [66,[80][81][82][83][84], orange [14,85] or red [14,79,[85][86][87][88][89] pigments. However, in most of the works, yellow, orange and red azaphilones are produced simultaneously (cocktail pigments phenomenon) in different proportions.
Regarding Monascus species, M. ruber CCT 3802 has been studied in terms of colony morphology and biomass production during pigments production utilizing cheese whey as substrate [90]. Strain M. ruber M7 showed different response to the addition of acetic acid, sodium acetate and ammonium acetate to PDA culture medium. The original big orange fleecy colony morphology turned into small compact reddish or tightly-packed orange colony upon increase of acetic acid or acetate. Pigment production, in turn, was enhanced by addition of acetate to the culture medium [91]. Yang et al. [16] reported that the expression of key genes for Monascus pigment biosynthesis was significantly up regulated in the presence of sodium nitrate. Increase in total pigment production and yellow pigment proportion was reported for a M. purpureus strain (LQ-6), after adding exogenous cofactor methyl viologen and rotenone (1.0 mg/L) to the submerged batch-fermentation [84].
The color of pigments produced by Talaromyces amestolkiae DPUA 1275 was shown to be pH-dependent. Low pH (2.59 and 3) directed to small production of yellow pigments while red ones were not detected [86]. On a further study, T. amestolkiae DPUA 1275 was grown in MSG-glucose medium supplemented with three individual complex nitrogen sources (yeast extract, meat extract and meat peptone), six individual amino acids (glutamic acid, threonine, tyrosine, glycine, cysteine and tryptophan), and two vitamins (biotin and thiamine) [92]. Complex nitrogen and amino acid supplementation did not favor red pigments production but small improvement (1.3 times) was detected after thiamine supplementation.
On the other side, the production of yellow and orange colorants was increased adding yeast extract as nitrogen source in the medium in pH above 5.0. In this condition, conidiation and biomass production were enhanced. The higher yield of colorants in the monosodium glutamic acid (MSG) glucose medium was attributed to the metabolic stress caused by poor nutrition provided by this medium [92]. The production process was scaled-up to a 4 L stirred-tank bioreactor. In another study, the same group [87] evaluated the effect of pH and agitation (100 to 600 rpm) in the improvement of pigments production. They reported near 4-fold increase in orange and red pigments production at 500 rpm, under the pH-shift strategy from 4.5 to 8.0, after 96 h of cultivation at 2.0 vvm at 30 • C. Moreover, the aforementionated work also demonstrated the possibility of using T. amestolkiae colorants in the preparation of cassava starch-based biodegradable films for food packaging, resulting in enhancement of protection against butter oxidation, reducing peroxide amount. •Mutant strains of Monascus anka produces yellow azaphilones in aerated submerged fermentation bioreactor containing corn steep liquor, ammonium chloride, potassium dihydrogen phosphate, glucose and starch. Agitation at 300 rpm/min improved yellow pigment production [82].
•High osmotic concentration (NaCl 3.5%) increase the production of yellow intracellular metabolites by M. ruber CGMCC 10910 by 40%, due to the up-regulation of specific genes [83]. •T. purpureogenus was reported to have a pH-related biosynthesis of yellow piments in Czapeck yeast medium [84]. •Increase in total pigment production and yellow pigment proportion by M. purpureus strain (LQ-6) can be achieved adding the exogenous cofactor methyl viologen and rotenone (1.0 mg/L), in submerged batch-fermentation [85]. •Expression level of key genes of M. purpureus ZH106-E metabolic pathway can be significantly improved by addition of 3% ethanol in submerged culture medium after 168 h, increasing the production of yellow pigments [81] •M. purpureus M9, grown in rice supplemented with ammonium nitrate (10 g/L), improves production of yellow (monascin and ankafavin) and red (rubropunctamine and monascorubramine) azaphilones and reduce the yields of rubropunctatin or monascorubrin (both orange) [67].

ORANGE AZAPHILONES
•Ammonium nitrate successfully increases the yield of orange pigments and inhibit production of red and yellow metabolites by Monascus sp. KCCM 10093 [14].
•Choe et al. [14] also report the possibility to manipulate the metabolism in suboptimal conditions. •Acidic condictions are effective for accumulation of orange pigments by Monascus ruber M7 [86].
•Stimulation of the synthesis of orange metabolites in low pH (2.5) is also effective for Monascus sp. KCCM 10093, since, under acidic conditions, conversion of orange to red metabolites is low due to greater expression of the genes MrpigA, B, F, and K, responsible for the production of orange pigments [86]. •Improvement of O 2 dispersion in the culture medium by aeration stimulates the production of orange pigments in Monascus sp. KCCM 10093, since O 2 is a substrate for the enzyme flavin adenine dinucleotide-dependent monooxygenase, essential in the production of these pigments [14].

RED AZAPHILONES
•High yield of water-soluble red colorants was achieved culturing T. amestolkiae DPUA 1275 using glucose, magnesium and iron sulfate, sodium chloride and monosodium glutamate for 14 days at pH 5.0 [87]. •Despite the low fungal biomass production, improvement of 31% in the red colorant production using MSG-glucose medium and high oxygen supply (8.0 Lmin -1 ) after 48 h (30 °C) in comparison to control conditions was reported for T. amestolkiae DPUA [88]. •Solid state fermentation using palm oil of M. purpureus FTC 5357, followed by ethanol extraction provided red pigments in expressive yield (60%) [80].
•Optimum biomass production, in addition to increasing the presence red pigments can be achieved growing T. purpureogenus strain F in Czapek yeast medium at pH 5 [84].
•Rice and peptone are optimum carbon and nitrogen sources, respectively, to produce citrinin-free red pigments by M. ruber OMNRC45 under submerged fermentation conditions [89].
•Using response surface methodology, an improvement by 38-fold in the yield of an antioxidant red pigment was related culturing T. purpurogenus KKP in a medium containing dextrose and peptone (2.25 and 1.10% respectively), pH 6.0 at 27 °C [90].

Yield Improvement
Yield is another key bottle neck in the way to produce fungal pigments to supply industrial demand. Yield improvement can start early in wet bench step, selecting promising species from under-studied niches. Marine environment has gained prominence in this area in recent decades. In terms of chemical structures, marine metabolites are frequently halogenated in comparison to metabolites biosynthesized by non-marine microorganisms. Halogenated fungal metabolites reach 59.2% of metabolites isolated from marine fungi and, among these metabolites, several halogenated pigments of the azaphyllone class have been reported, as penicilazaphilones D (113) and E (114) isolated from Penicillium sclerotiorum ( Figure 13) [38,93]. It is noteworthy that fungal species isolated from marine environment can also be isolated from terrestrial sources, such as P. sclerotiorum, that, despite being isolated from soil, was also reported of being capable of producing halogenated derivatives (115 and 116) (Figure 13) [94,95]. the effect of pH and agitation (100 to 600 rpm) in the improvement of pigments produc-tion. They reported near 4-fold increase in orange and red pigments production at 500 rpm, under the pH-shift strategy from 4.5 to 8.0, after 96 h of cultivation at 2.0 vvm at 30 °C. Moreover, the aforementionated work also demonstrated the possibility of using T. amestolkiae colorants in the preparation of cassava starch-based biodegradable films for food packaging, resulting in enhancement of protection against butter oxidation, reducing peroxide amount.

Yield Improvement
Yield is another key bottle neck in the way to produce fungal pigments to supply industrial demand. Yield improvement can start early in wet bench step, selecting promising species from under-studied niches. Marine environment has gained prominence in this area in recent decades. In terms of chemical structures, marine metabolites are frequently halogenated in comparison to metabolites biosynthesized by non-marine microorganisms. Halogenated fungal metabolites reach 59.2% of metabolites isolated from marine fungi and, among these metabolites, several halogenated pigments of the azaphyllone class have been reported, as penicilazaphilones D (113) and E (114) isolated from Penicillium sclerotiorum (Figure 13) [38,93]. It is noteworthy that fungal species isolated from marine environment can also be isolated from terrestrial sources, such as P. sclerotiorum, that, despite being isolated from soil, was also reported of being capable of producing halogenated derivatives (115 and 116) (Figure 13) [94,95]. Enhancement of metabolites yield can be achieved applying stressing conditions during fungal development, aiming at activating unconventional metabolic routes related to the production of substances linked to defense (biotic stress) or adaptation (abiotic stress). This technique is particularly interesting for the production of fungal pigments, since these metabolites are associated with defense against various types of abiotic stress [96]. Abiotic stress is usually caused by altering nutrients (carbon, nitrogen, minerals) and conditions (temperature, length, oxygen supply) in the culture medium, improving pigments production, although independently of directing to a single pigment color. Increase in biomass development is not a must to enhance pigments production, as optimized conditions for development of fungal biomass not necessarily guarantee maximum production of metabolite [82]. In general, in the search for better yields, both, biomass and metabolite yield should increase [97]. Enhancement of metabolites yield can be achieved applying stressing conditions during fungal development, aiming at activating unconventional metabolic routes related to the production of substances linked to defense (biotic stress) or adaptation (abiotic stress). This technique is particularly interesting for the production of fungal pigments, since these metabolites are associated with defense against various types of abiotic stress [96]. Abiotic stress is usually caused by altering nutrients (carbon, nitrogen, minerals) and conditions (temperature, length, oxygen supply) in the culture medium, improving pigments production, although independently of directing to a single pigment color. Increase in biomass development is not a must to enhance pigments production, as optimized conditions for development of fungal biomass not necessarily guarantee maximum production of metabolite [82]. In general, in the search for better yields, both, biomass and metabolite yield should increase [97].
The relationship between fungal development and pigments secretion was reported for T. albobiverticillius (IBT31667). When cultured on Czapek Yeast Agar (CYA), a malt-free extract, this species produced atrorosins, pigments already reported as metabolites of T. atroroseus IBT 11181 [32]. Production of atrorosins by T. atroroseus was accomplished on a complex culture medium containing metals solution supplemented with single amino acids as the sole nitrogen source in the range of pH 4-5. In sequence, Tolborg et al. (2019) [97] demonstrated that individual amino acids as the sole nitrogen source led to high biomass production but not necessarily to high amounts of red pigment in T. atroroseus. Tolborg's group also reported that some amino acids can avoid the cocktail pigments phenomenon directing T. atroroseus to produce single atrorosins. Corroborating their work, only atrorosin S (92) was detected in the fermentation broth when serine was used as the sole nitrogen source. Addition of glutamic acid as a second nitrogen source induced the production of atrorosin E (94). Interestingly, only some aminoacids induced atrorosins biosynthesis, since individual supplementation of proline, lysine, asparagine and tryptophan as the sole nitrogen source did not result in atrorosins production by T. atroroseus [32]. This strain produced two new azaphilone pigments, talaralbols A and B, along with five known azaphilone metabolites, when subjected to growth under submerged fermentation in malt extract medium (ME) (28 • C, 120 rpm) during 14 days [58].
Pigments production by T. atroroseus strain GH2 was studied in two different culture media (pH 5.0, 30 ± 2 • C, 200 rpm, 8 days) [98]. The first one was composed by synthetic Czapek-dox modified medium containing high levels of xylose, with and without nutrients supplementation and the second medium was composed by hydrolyzed corncob, a lignocellulosic waste. T. atroroseus GH2 demonstrated a significantly different response to the carbon and nitrogen composition of the culture media, with improved growth and enhanced pigments production in the hydrolyzed corncob medium without any nutrient supplementation. Therefore, T. atroroseus was pointed by the authors as a promising pigment-producing microorganism for economically competitive large-scale fermentation at lower cost [98].
Carbon source in the fermentation is a very major parameter to direct fungal metabolism. Parul et al. [83] demonstrated that mannitol is the best carbon source for reproduction and growth of T. purpureogenus strain F, but the growth is accompanied by low yield of pigment production, while sucrose causes the opposite effect. The authors correlate this fact to species and strain-specific capacity to produce specific enzymes that will dictate the fungus priorities. Under no stressing conditions and abundant carbon availability, primary metabolism is prioritized and the metabolism will be directed to biomass productions instead of secondary metabolites production [83]. In addition, the rate of carbon source depletion is also important. In large-scale industrial production, the rapid growth of the fungus occurs together with rapid decrease in the carbon source concentration. To avoid decrease of metabolite production rate, the carbon source must be constantly added to the fed-batch fermentation to guarantee a constant concentration of this substrate and, consequently, uninterrupted production of pigments [99]. In the same way, culture medium agitation and aeration ensure better distribution of nutrients and better growth, but at the expense of faster depletion of carbon sources. Therefore, agitation and aeration are factors that must be strictly controlled in industrial production [83].
Another tool to improve the yield of fungi metabolites is to create stress conditions during fungal development, thar results in activation and/or suppression of gene clusters to allow fungal adaptation and survival. Co-cultivation two fungal species is an example of stressing condition that generates metabolic responses to allow survival in multispecies environment. Oppong-Danquah et al. [100] described a specific co-cultivation gene cluster, when studying the co-culture of pigment producer fungus Plenodomus influorescens with Pyrenochaeta nobilis, where five polyketides were produced, including the yellow azaphilones spiciferinone (117) and 8a-hydroxy-spiciferinone (118) (Figure 14). The cultivation of Trichoderma guizhouense NJAU 4742 in the presence of Fusarium oxysporum cells also resulted in increase in azaphilone production, which was demonstrated experimentally by the increased activity of the gene cluster responsible for pigment production. This fungal response was drove to neutralize the high concentration of H 2 O 2 , produced as a defense mechanism during co-cultivation, since azaphilones are capable of neutralizing free radicals, especially the superoxide anion [101]. The same effect is observed in other oxidative stress conditions related to H 2 O 2 , such as fungal cultivation in the presence of the fungicides amphotericin B, miconazole and ciclopirox. The production of azaphilones increases as a survival mechanism directed to the neutralization of fungicide effects rather than a decrease in antifungal concentration [101]. also resulted in increase in azaphilone production, which was demonstrated experimentally by the increased activity of the gene cluster responsible for pigment production. This fungal response was drove to neutralize the high concentration of H2O2, produced as a defense mechanism during co-cultivation, since azaphilones are capable of neutralizing free radicals, especially the superoxide anion [101]. The same effect is observed in other oxidative stress conditions related to H2O2, such as fungal cultivation in the presence of the fungicides amphotericin B, miconazole and ciclopirox. The production of azaphilones increases as a survival mechanism directed to the neutralization of fungicide effects rather than a decrease in antifungal concentration [101]. Cost minimization for industrial production of azaphilones can be reduced by using agro-industrial waste as material for fungal growth, which also helps to solve the problem of pollution associated with the disposal of residues in the environment [19,98]. Liu et al. Cost minimization for industrial production of azaphilones can be reduced by using agro-industrial waste as material for fungal growth, which also helps to solve the problem of pollution associated with the disposal of residues in the environment [19,98]. Liu et al. [102] used rice straw hydrolysate for pigment production by M. purpureus M630 but reported that this substrate and does not have the ideal carbon content required by the fungus. Although supplementation may be necessary in some cases, the use of agroindustrial residues has been reported to be economically viable also adding sustainability to the process.
As aforementioned, another approach to achieve yield improvement and consequently increase the viability of industrial production of fungal metabolites is the use of mutant strains and genetic engineering [99]. The current knowledge of the metabolic pathways and secondary metabolism precursors allow to manipulate fungi as "real industrial cell factories" [103] and take advantage of the entire pigment gene cassette to improve pigment yield [104]. In this way, Liu et al. [99] managed to knock-out a cAMP phosphodiesterase gene in M. purpureus HJ11, which led to the accumulation of intracellular cAMP causing a stimulating effect in secondary metabolism that resulted in 2.3-fold increase in pigment production.

Extraction Approach
Another phase important in yield improvement consists of the extraction step, which helps in concentration and pre-purification of fungal pigments. Prior to the extraction, it is necessary to take into consideration where the pigments produced are deposited. Classically, the extraction procedure is usually accomplished by liquid-liquid extraction of the broth with medium polarity solvents such as ethyl acetate. This extraction works well to obtain extrolytes, i.e., extracellular metabolites present in the broth or linked to the external surface of fungal biomass. On some occasions, mycelial adhesion is verified, as reported for a water-soluble extracellular yellow pigment produced by a Monascus in submerged fermentation. This effect was reversed furnishing sodium and potassium nitrate as nitrogen source to the fungus. Sodium nitrate is suggested to reduce the total amount of extracellular polysaccharides, increase extracellular proteins, and diminish the viscosity of the fermentation broth, rising pigment recovery [16].
Although effective for extraction of metabolites produced in liquid cultures, ethyl acetate is not a choice solvent in terms of toxicity. Non-toxic and easily available ethanol is a better choice for the extraction step, although can only be applied to solid state fermentation, as it is water-miscible and cannot be utilized to extract aqueous liquid media. Ethanol was utilized for pigments extraction in the solid-state fermentation of M. purpureus M9 using durian seed as substrate. Extractions were carried out at two temperatures (30 and 60 o C) using a mixture of ethanol and water in different proportions (10:0; 9:1; 8:2; 7:3; 6:4 and 5:5). The most effective conditions for pigments recovery were achieved using the lowest ethanol:water ratios at 30 • C [105].
Occasionally the pigments remain inside the cells requiring disintegration and dissolution of the glucan-chitin complex of the wall cell to be recovered, therefore demanding alternative extraction procedures [106], while cellular lysis is necessary for recovering intracellular metabolites. In this way, T. amestolkiae DPUA 1275 was subjected to an alternative extraction procedure to recover red pigments. The procedure was conducted with aqueous solutions of imidazolium salt instead of organic solvents, together with ultrasound-temperature-assisted mechanical cell disruption to enhance the recovery of intracellular T. amestolkiae pigments [103].
Cell Pressurized Liquid Extraction technique was utilized to recover pigments produced by mycelial biomass of Talaromyces sp. 30570 (CBS 206.89 B) isolated from the coral reef of the Réunion island (France) and cultivated in PDB media containing complex organic nitrogen sources like amino acids and proteins. Eco-friendly solvents were chosen for the extraction (90 • C and 10 MPa) as water, methanol and/or ethanol. At the end, twelve nitrogen-containing azaphilone red pigments were identified while known mycotoxins were not produced [13]. Two-phase aqueous extraction [107] was successfully tested for the extraction of pigments from T. albobiverticillius. These organic solvents free techniques guarantee good extraction yields without structural damage in the extracted compounds. Cell disruption methods for improved extraction of pigments from microorganisms were recently reviewed [108].
For pigments production, submerged fermentation is preferable, as it produces better yields, has lower risk of contamination and is easier to monitor when compared to cultivation in solid medium [109]. In addition, using submerged fermentation, it is possible to separate intracellular and extracellular pigments, soluble in the culture medium [102]. However, it is known that not all species of pigment-producing fungi have the ability to diffuse these pigments into the culture medium [73]. Among the techniques to increase the production of extracellular pigments, the design of mutant strains of M. purpureus [102], the addition of glycerol to the cultivation medium of M. pilosus MS-1 [110] and the establishment of a hyperosmotic environment to M. ruber CGMCC 10910 [82] were successfully utilized. The last two methods are related to the regulation of metabolism and gene expression caused by environmental stress.

Potential Applications of Azaphilones outside Food Sector
As in the food industry, azo dyes represent the most widely used chemical class of dyes in textiles production, an industrial sector that requires high amounts of stable colorants/pigments [111]. Textiles dying quality is also highly important for market competitiveness and consumer identification and public opinion have been driving an increase demand for natural pigments to replace synthetic dyes. In addition, change is necessary to avoid chronic effects in workers exposed to hazardous synthetic dyes during industrial processes. Once present in clothes, aromatic amines can be biotransformed by skin bacteria into aromatic amines, many of which are carcinogenic and can be absorbed by human skin [112]. Non-regulated aromatic amines were detected in a substantial number of colored textiles in a survey done in Switzerland raising questions on genotoxicity, dyes purity, consumer health risks, release of dyestuffs and dermo absorption [113]. Last, but not least, environmental pollution by effluents from textile industry cause multiple environmental harms.
Textile market can absorb some microbial dyes excluded from food applications by regulatory agencies [19]. Toxicity issues and growing preference for natural goods reached clothing sector and many brands are adapting themselves to meet the expectations for sustainable products. This demand increased, especially in millennials and Z-Generation group, as statistics proved to be alarming in global scale in terms of gas emission by textile industry, water contamination and pollution with industrial dyes [114,115]. Modern demands have been raising integrated practices, as well as international networks and partnerships to address sustainability issues and to look for solutions in the textile and clothing industry [115]. This behavior applies to the low-income clothing producing/exporting countries as well as the buyers' international market. The latter can impose restrictions to imported products containing artificial dyes that either are rejected or avoided by consumers due to the awareness of the unsustainable effluents generated in producing countries.
Cosmetics sector is another market that may incorporate azaphilone compounds in the future. Development of new strategies for on line sales, digital advice, and decentralization of distribution centers helped some cosmetic chains to grow even with the world economic problems associated to the COVID-19 pandemic [116]. The global cosmetics industry was valuated in over USD 380 billion in 2019 and is projected to reach USD 463 billion by 2027 [117]. Several facts contributed to the massive growth of this segment in the last period, such as increase in sale of personal care products, conquering an expressive number of male consumers, increasing number of make-up tutorials in social media and the search for well-being taking into consideration the connection of cosmetics and self-esteem increase [118]. It is also noteworthy that a new type of cosmetics is increasingly growing, named cosmeceuticals. Although regulation of cosmeceuticals was not fully addressed, these products claim biological effects beyond cosmetic utility and many times are referred as cosmetic-pharmaceuticals hybrids.
This expansion in cosmetic market was accompanied by the aforementioned conscientious choice of safe, natural, and "not tested on animals" products [119]. Cosmetics and personal care products are usually directly applied to the skin in a daily basis, many times associated to active ingredients to facilitate fastening or product penetration over the skin. Therefore, allergy and long-term toxicity have also been driving huge efforts for modernization in this area. In this way, long-lasting innovative natural color sources are also an important goal of cosmetics industry. Azaphilone metabolites comprise an important part of the color pallet required by cosmetic industry and their reported biological effects make these compounds also good active components for cosmeceuticals formulations. Anti-inflammatory activity, related for some azaphilone [37,51] is a mechanism associated with anti-aging dermo-cosmetics [120], while antimicrobial activity [46] associated to color pigments can be helpful to extend shelf life of cosmetics.

Conclusions
The development of new pigments safe and effective to apply in foods, medicines, textile and cosmetic industries is essential and welcome. Natural pigments are a great alternative regarding not being related to toxic, allergic, and pollutant characteristics of the most common synthetic dyes. Fungi azaphilone pigments are recognized as promising candidates of colorants to substitute azo dyes in the food, cosmetics, and textile industrial sectors, as long as safety and production issues are overcome.
Azaphilone research is proliferous and at least 101 new compounds of this class were reported between December 2019 and March 2021 from nine fungal genera (Aspergillus, Chaetomium, Hypoxylon, Monascus, Muycopron, Penicillium, Phomopsis, Pleosporales, and Talaromyces). Some of the new azaphilones exhibit complex chemical structures, and their biosynthesis have been studied to understand nutrients requirements for biomass production and yield improvement. Also, several studies have been conducted to understand down-regulation of citrinin co-production.
Coloring properties and the natural origin are not the only features of azaphilones, since antimicrobial, antioxidant, anti-inflammatory, and other properties related to these molecules have been widely reported. This potential can be explored in food or cosmetic processing to avoid microbial contamination or to furnish functional properties to foods.
This review brought some strategies used to improve fermentation conditions, control pigment production, and issues related to different fungal strains that produce azaphilone pigments, reported in the last two years. Future perspectives include more research that could allow azaphilone dyes to be regularized by the EU, US and other regulatory agencies, so they can be plentiful incorporated in different technological innovative applications.