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.