4. DISCUSSION
The findings of this study suggest that we can isolate fungal strains from the Campina Biological Reserve that are able to produce colorant. We highlight that the strains isolated in the present work (Penicillium gravinicasei P3SO332, Clonostachys roseaP2SO329 and Penicillium sclerotiorum P3SO224) were able to produce colorants via submerged fermentation and the colorant presented good stability, significant antibacterial activity and low toxicity. In addition, we observed that the colored extracts of the fungi were able to dye fabric and stain pharmaceutical formulations.
The current study demonstrates the investigation of fungi able to produce colorants from the Campina Biological Reserve. Campina Biological Reserve is a Neotropical ecoregion in the Amazon that contains vegetation that is adapted to the extremely poor soil found in the region [31]. The fungi in this biome are poorly studied; however, we believe that fungi play an important role in ecology for nutrient reutilization. In this environment, we found eight isolates from which we were able to synthesize colorants that resulted in yellow, orange and red-colored fractions.
In this study, the identification of the microorganisms present in the samples revealed a predominance of filamentous fungi of the following genera: Penicillium , Clonostachys , Phoma andMonascus . The fungi of these genera reveal the potential of species isolated from the soil of forest fragments to produce substances of biotechnological interest, such as colorants [12,15,32–34].
The present work highlights Penicillium gravinicasei P3SO332,Clonostachys rosea P2SO329 and Penicillium sclerotiorumP3SO224 as the main producers of colorants. P.sclerotiorum is known to produce colored compounds ranging from orange to yellow including pencolide, sclerotiorin and isochromophylone VI [12,35]. P. gravinicasei is known to produce an abundant yellowish exudate, which corroborates our findings [36]. The soil-derived fungus Clonostachys rosea (synonymousGliocadium roseum ) has been extensively studied for the production of secondary metabolites, including nitrogen-containing metabolites, polyketides, triterpentoids, and fatty acids that exhibit biological activities (cytotoxic, phytotoxic and antibacterial activity) [37]. However, in these occurrences, there are no reports of studies with this species as a producer of colorants, and the present study is also the first to record an Amazonian strain of this species as a producer of colored compounds, including one of a yellow color.
The metabolite sclerotiorin was identified in the EtOAc extract of Penicillium sclerotiorumP3SO224. This metabolite is known to have been originally isolated from this species, but it can be produced by other fungi such asCephalotheca faveolate , Penicillium frequentans andPenicillium mallochii [38–40].
Penicillic acid was identified in the EtOAc extract of Clonostachys rosea P2SO329. Penicillic acid is a substance that is produced as a defense mechanism by a large number of fungi (48 documented species), including members of the generaAspergillus and Penicillium [41].
Under the experimental conditions, colored extracts of the fungiPenicillium gravinicasei P3SO332, Clonostachys roseaP2SO329 and Penicillium sclerotiorum P3SO224 showed good stability, significant antibacterial activity and low toxicity. These result agree with previous works from the literature: a) The red-orange colorant from Penicillium mallochii BAU-TACB-16 showed no color changes to the naked eye after 1 h of exposure to different temperatures and pH values [42]​​; b) The antibacterial activity of the crude extract obtained from the soil-derived fungus Clonostachys roseaYRS-06 exhibited a MIC of 25 μg/mL against E. coli [43], c) Pigments produced by Penicillium mallochii ARA1 andPenicillium purpurogenum Li-3 demonstrated activity againstStaphylococcus aureus using the disk diffusion method with the zone of inhibition ranging from 6 to 13 mm [44,45]; d) The dark-yellow pigment produced by Penicillium sclerotiorum AK-1 has no cytotoxic effect against the yeast Saccharomyces cerevisiae[46] and Penicillium murcianum yellow pigment had no toxic effect on HEK-293 ATCC® CRL-1573™ and NIH/3T3 ATCC® CRL-1658™ cells [47].
In the present work, the colored extracts of Penicillium gravinicasei P3SO332, Clonostachys rosea P2SO329 andPenicillium sclerotiorum P3SO224 were able to be used to dye fabrics and stain pharmaceutical formulations. The natural colorant isolated from Penicillium sclerotiorum AK-1 revealed a high potential for dyeing cotton fabrics on an industrial scale [46]. The colorant obtained from Penicillium minioluteum was used in the dyeing of wet blue goat nappa skin [48]. The dyeing of wool fabric samples by the yellow pigment produced by Penicillium murcianumwas considered promising [47]. Red and yellow pigments from fungi were evaluated to enhance sun protection factor (SPF) of commercial sunscreens and Aloe vera extract [49].
Among the limitations in the study, one of them was that it was not possible to carry out the purification step of the colored extracts, which limited the chemical characterization of these substances via nuclear magnetic resonance (NMR), which would have permitted the isolation and elucidation of all the pigmented and non-pigmented molecules of these extracts.
The present work presents the screening of colorant production by fungi from forest soil and litter and our findings contribute to the development of technologies for production, isolation, chemical characterization, stability and application of pigments due to the few existing works in the literature that have investigated the production of these compounds using fungi. Our findings may contribute to the process of including colorants of microbial origin in all industrial sectors, whether as a food additive or a nutritional supplement or in textile and cosmetic pigmentation, which benefits both the producer and the consumer due to the various promising characteristics of these compounds. The study of compounds produced by fungi found in Amazonian soils is also of great contribution to the knowledge and development of the region.
Considering the results found in this study, it was concluded that: a) the Campinas Biological Reserve could be a source of colorant-producing fungi; b) the genera of the colorant-producing isolates werePenicillium , Clonostachys , Phoma andMonascus ; c) Penicillium gravinicasei P3SO332,Clonostachys rosea P2SO329 and Penicillium sclerotiorumP3SO224 were the main colorant producers; d) the sclerothiorin metabolite was identified in the EtOAc extract of Penicillium sclerotiorum P3SO224; e) the metabolite penicillic acid was identified in the EtOAc extract of Clonostachys rosea P2SO329; f) under the experimental conditions, colored extracts of the fungi showed good stability, significant antibacterial activity and low toxicity; g) under the experimental conditions, the colored extracts of the fungi were able to dye fabrics and stain pharmaceutical formulations.