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\begin{document}

\title{EXPLORING COLORANT PRODUCTION BY AMAZONIAN FILAMENTOUS FUNGI: STABILITY
AND APPLICATIONS}



\author[1]{João Vicente Braga de Souza}%
\author[2]{Luciana Oliveira}%
\author[2]{Michele Sanches}%
\author[2]{Walter Filho}%
\author[3]{Paulo Santiago}%
\author[4]{Rodrigo Lima}%
\author[1]{Ana Cortez}%
\author[2]{Érica Souza}%
\author[1]{Maria Lima}%
\author[3]{Emerson Silva}%
\author[2]{Hector Koolen}%
\author[5]{Laurent Dufossé}%
\affil[1]{National Institute of Amazonian Research}%
\affil[2]{Universidade do Estado do Amazonas}%
\affil[3]{Universidade Federal do Amazonas}%
\affil[4]{Centro Universitario do Norte}%
\affil[5]{Universite de La Reunion}%


\vspace{-1em}



  
  \date{May 23, 2024}


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\selectlanguage{english}
\begin{abstract}
The aim of this study was to investigate the production, stability and
applicability of colorants produced by filamentous fungi isolated from
soil samples from the Amazon. Initially, the isolates were evaluated in
a screening for the production of colorants. The influences of
cultivation and nutritional conditions on the production of colorants by
fungal isolates were investigated. The colorants produced by selected
fungal isolates were chemically characterized using the LC-MS technique.
The antimicrobial and cytotoxic activities, stability evaluation and
applicability of the colorants were investigated. As results, we
observed that the isolates Penicillium sclerotiorum P3SO224,
Clonostachys rosea P2SO329 and Penicillium gravinicasei P3SO332 stood
out since they produced the most intense colorants. Compounds produced
by Penicillium sclerotiorum P3SO224 and Clonostachys rosea P2SO329 were
identified as sclerotiorin and penicillic acid. The colorant fraction
(EtOAc) produced by these species has antimicrobial activity, stability
at temperature and at different pHs, stability when exposure to light
and UV, and when exposed to different concentrations of salts, as well
as being non-toxic and having the ability to dye fabrics and be used as
a pigment in creams and soap. Considering the results found in this
study, it was concluded that fungi from the soil in the Amazon have the
potential to produce colorants with applications in the textile and
pharmaceutical industries.%
\end{abstract}%



\sloppy


\textbf{1. INTRODUCTION}

A colorant is any substance added to a product for the purpose of
changing its color, enhancing the existing color or restoring lost
color. Natural colors are pigments made by living organisms (plants,
lichens, algae, insects, bacteria and fungi) {[}1--3{]}. There is low
consumer interest for the use of the synthetic colorants due to the fact
that they are toxic to human health (mutagenic and carcinogenic
potential) {[}4,5{]}. The current legislation of the European Union, the
United States, Brazil, Norway and Sweden also limits the use of
synthetic colorants as food additives, and the process used to produce
synthetic colorants is toxic to the environment~{[}6,7{]}.

However, natural colorants extracted from plants have a drawback, i.e.,
they depend on seasonal availability{[}8{]}. As a potential bioprocess
for the new bioindustry of colorants, the production of colorants using
microbial fermentation is rising, since it is possible to reduce
production costs by choosing the best growing conditions; in addition,
unlike plants microorganisms do not depend on climatic conditions
{[}9--11{]}. As well as these advantages, many fungi-based colorants
have important biological properties, such as antimicrobial,
antioxidant, immunosuppressive, antiviral, anticancer and
cholesterol-lowering properties, which make them important compounds for
numerous industries. As a result, there is growing worldwide interest
for the use of natural colorants{[}12--14{]} .

Studies have shown the potential of Amazonian fungi for colorant
production. One study that can be highlighted isolated fungi-producing
pigments {[}15{]} and obtained fractions with colors ranging from yellow
to red. \emph{Penicillium sclerotiorum} 2AV2 produced intensely colored
pigments and the pigment was identified as sclerotiorin, which has
specific biological activities such as the inhibition of aldose
reductase enzymes, lipases, integrins and proteases of the human
immunodeficiency virus HIV-1{[}16{]}. Teixeira et al. {[}17{]} evaluated
the antibacterial activity and toxicity of organic extracts. The authors
identified antimicrobial activity and lack of toxic action of the
colorants produced by strains of the genus \emph{Aspergillus}
and\emph{Penicillium}.

The Amazon Rainforest contains a rich diversity of microorganisms.
Despite the richness of biological diversity in the Amazon, few works
have been dedicated to researching fungal colorants. Therefore, the
present study aimed to identify fungal isolates with the potential for
production of colorants.

\textbf{2. MATERIALS AND METHODS}

\textbf{2.1 Collection of samples and isolation of fungi}

Superficial soil and litter were collected in September 2018 in a forest
zone located in the Campina Biological Reserve (2\selectlanguage{ngerman}°35'19.19'' S,
60°1'59.91'' W) Manaus, Brazil. The samples were collected at the
following three points: point 1 (-2.5891030, -60.0319530), point 2
(-2.589249, -60.031893) and point 3 (-2.589368, -60.031712).
Approximately 5 g of superficial soil and litter were collected with
clean, dry and sterile polythene bags and a sterile spatula. From the
collected samples, 1 g was added to 9 mL of sterile distilled water and
serial dilution was performed to dilute the sample. The suspension (0.1
mL) was added to sterile Petri dishes in duplicates containing sterile
Sabouraud agar and the spread plate technique was used. Plates were
incubated at 28 ºC±2 for 5-7 days {[}18{]}.

\textbf{2.2 Purification and identification of fungi}

The isolated colonies were transferred to tubes containing Sabouraud
agar plus chloranphenicol. The isolated colonies were purified on Petri
plates containing potato dextrose agar (PDA) incubated at 28 ºC±2 for 7
days. Pure cultures were transferred into tubes containing PDA. These
pure cultures were stored in a refrigerator at 4 °C, with mineral oil
and sterile distilled water. The isolated fungi were identified to the
genus level by assessing the characteristics micro morphological
characteristics using slide cultures {[}19{]}.

The species of interest were identified via sequencing of the rDNA ITS
region. Fungal DNA was extracted from mycelium the
phenol:chloroform:isoamyl-alcohol method {[}20{]}. PCR reactions (3 \selectlanguage{greek}µ\selectlanguage{english}L
of buffer (1X), 1.2 \selectlanguage{greek}µ\selectlanguage{english}L of MgCl\textsubscript{2} (2.5 mM), 3 \selectlanguage{greek}µ\selectlanguage{english}L of DNTPs
(200 \selectlanguage{greek}µ\selectlanguage{english}M), 1.5 \selectlanguage{greek}µ\selectlanguage{english}L ITS1 (5'\textbf{-} TCCGTAGGTGAACCTGCGG\textbf{-} 3')
and ITS4 (5'\textbf{-} TCCTCCGCTTATTGATATGC\textbf{-} 3') {[}21{]} and
ultrapure water to a final volume of 30 \selectlanguage{greek}μ\selectlanguage{english}L) were carried out in a
thermocycler (SuperCycler\textsuperscript{TM} SC-200, Kyratec) using the
following amplification conditions: 94 \selectlanguage{ngerman}ºC/5 min, 30 cycles of 94 ºC/30
s, 53 ºC/30 s, 72 ºC/1 min and 72 ºC/10 min. PCR products (8 \selectlanguage{greek}µ\selectlanguage{english}L) were
visualized under UV light after electrophoresis (3 \selectlanguage{greek}μ\selectlanguage{english}L of
SYBR\selectlanguage{ngerman}\textsuperscript{®} Safe (Invitrogen), 2 \selectlanguage{greek}μ\selectlanguage{english}L of Orange 6X
(Fermentas), 10 \selectlanguage{greek}µ\selectlanguage{english}L 100-bp DNA Ladder, 40 min/100 V/100 A) on a 1.5\%
agarose gel in 1x TBE buffer. PCR products were purified via
precipitation with polyethylene glycol (20\% w/v PEG, 2.5 M NaCl). The
sequencing reaction was performed using a commercial kit
(BigColorant\selectlanguage{ngerman}\textsuperscript{®}, Applied Biosystems). Sequencing was
carried out in a genetic analyzer (AB3130, Applied Biosystems). The
sequences obtained were analyzed using BioEdit Sequence Alignment Editor
and compared to those in the GenBank database. A phylogenetic tree was
constructed using the program MEGA X v10.2.4 to verify the genetic and
evolutionary relationship among the sequences.

\textbf{2.3 Screening for colorant production}

To identify which fungi isolated from the soil had the potential for
extracellular colorant production, a visual evaluation of the colorants
produced by the fungi in the culture medium was carried out. The fungus
strains were transferred to tubes containing Czapeck-Dox-agar and
incubated at room temperature for 14 days. After colony growth, the
colorants present in the culture medium and soluble in ethyl acetate
were observed. This solvent was transferred to tubes and shaken for 30
seconds in a tube shaker and maintained at room temperature for 30 min.
Afterwards, the solvent was removed and the presence of color in the
solvent was evaluated {[}15{]}. The eight fungal strains that showed the
most intense colorants were submitted to a submerged bioprocess.

\textbf{2.4 Submerged bioprocesses for colorant production}

Submerged bioprocesses were performed based on the protocol described by
Celestino et al.{[}15{]} with modifications. The fungal strains were
transferred to tubes containing PDA and incubated at room temperature
for 72 h. The fungal spores of each strain were suspended in 5 mL of
sterile distilled water and the density of the spore suspension was
determined using a hemocytometer (Neubauer, Germany) under an optical
microscope. This spore suspension (1x10\textsuperscript{4} cell/mL) was
inoculated into Erlenmeyer flasks (125 mL) containing 50 mL Czapeck
broth plus yeast extract (3.0 g/L sodium nitrate, 1.0 g/L potassium
phosphate, 0.5 g/L magnesium sulfate, 0.5 g/L potassium chloride, 0.01
g/L ferrous sulfate, 30.0 g/L of sucrose and 0.5 g/L yeast extract), at
an initial pH of 5.0. The flasks were incubated in static conditions, in
a dark place, and at room temperature (28 ºC) for 14 days.

\textbf{2.5 Extraction and evaluation of the solubility of the
colorants}

After 14 days of fermentation, each culture broth was separated from the
mycelial mass by filtering through ordinary filter paper. The mycelial
mass was washed with hexane (20 mL), ethyl acetate (20 mL) and methanol
(20 mL). Solvents were added sequentially to the cultures and vigorously
shaken in an orbital shaker (100 rpm/60 min). Extracts were filtered
through filter paper for the removal of cell debris. The culture
filtrates were subjected to successive extractions. The culture filtrate
and solvent (hexane and ethyl acetate) were placed in a separating
funnel (1:1, v/v) and mixed well. After separation, the solvent fraction
was collected.

\textbf{2.6 Effect of nutritional parameters on colorants production}

To study the influence of nutritional factors in the production of
colorants via the three selected fungi, univariate experiments were
carried out to verify the influence of five carbon sources (sucrose,
glucose, maize starch, maltose and carboxymethylcellulose - CMC) and
four nitrogen sources (sodium nitrate, ammonium sulfate, peptone and
yeast extract). In each experiment, only the carbon source (30 g/L) or
nitrogen source (3 g/L) were modified separately. The production of
colorants was estimated by analyzing the maximum absorbance (\selectlanguage{greek}λ\selectlanguage{english}max) in
ethyl acetate via scanning (400 nm to 700 nm) in a UV/VIS
spectrophotometer.

\textbf{2.7 Kinetics of colorant production}

In order to describe the kinetics of colorant production by the three
selected isolates, submerged bioprocesses were performed
(See\emph{Submerged bioprocesses for colorant production} ). Samples
were collected in triplicate at intervals of 5 days for 20 days with the
view to determination of dry cell weight (g/L) and extracellular
colorants (Abs \textsubscript{extra}). To determine the biomass, the
mycelial masses were collected and filtered with filter paper previously
dried in a desiccator and then weighed. After filtration, both were
dried in an oven at 100 \selectlanguage{ngerman}ºC for 24 h. The biomass concentration was
expressed by the dry weight of the mycelial mass using the difference
between the final weight and the initial weight. The production of
colorants was estimated by analyzing the maximum absorbance (400 nm) in
ethyl acetate using a UV/VIS spectrophotometer.

\textbf{2.8 Analysis of the colorants using liquid chromatography
coupled to mass spectrometry (LC/MS-MS)}

The extracted colorants were diluted in methanol (HPLC-Honeywell) to a
concentration of 1 mg/mL. Then, the samples were analyzed using a liquid
chromatograph (Prominence UFLC, Shimadzu), which was equipped with a C18
Shim-pack CLC-ODS(M)\textsuperscript{®} column (5 \selectlanguage{greek}µ\selectlanguage{english}m, 250 x 4.6 mm), and
coupled to a mass spectrometer (Amazon Speed Ion trap, Brucker
Daltonics) with an ESI source operating in positive and negative modes.
Samples were eluted in gradient of 20-100\% methanol over 24 minutes at
a flow rate of 1 mL/min. The data obtained were processed using the
DataAnalysis program (Bruker Daltonics).

\textbf{2.9 Stability of the Colorants}

The investigation of the stability of the colorants and their resistance
to degradation by temperature, as well as the pH value in the color of
the colorants and salt concentrations was carried out as described
{[}22{]} with modifications, whereby the colorants (0.1 mg/mL of DMSO)
were incubated in a water bath for 1 hour at 40 \selectlanguage{ngerman}°C, 60 °C, 80 °C, 100 °C
or frozen at -20 °C before analysis. In another set of tubes, extracts
were adjusted to pH 2, 4, 6, 8, 10 and incubated for 1 hour at room
temperature. Test tubes containing the extracts maintained in 0.2\%,
0.4\%, 0.6\%, 0.8\% and 1\% (v/v) NaCl solutions for 6 h of exposure.

For the investigation of their resistance to photodegradation, the
colorants were left in a dark place (and covered with aluminum foil),
exposed to white light (distance of 30 cm, 28W Cold White 5000K Tubular
Fluorescent Lamp Brand/Manufacturer: Osram, Barueri-SP, Brazil) and
ultraviolet light (distance of 30 cm, Ultraviolet Lamp 24W Pl Osram
Germicidal Reactor, Barueri-SP, Brazil), for 6 h. Changes in absorbance
were determined using a spectrophotometer at the maximum wavelength of
the analyzed colorants (\selectlanguage{greek}λ\selectlanguage{english}max).

\textbf{2.10 Antimicrobial activity}

The ethyl acetate extracts from of the three isolates were subjected to
determination of the minimum inhibitory concentration (MIC) using the
broth microdilution method in 96-well microtiter plates, as described by
the Clinical and Laboratory Standards Institute - CLSI (2012) {[}23{]},
USA: standard M7-A9 (methodology of the antimicrobial dilution
sensitivity tests for aerobic growth bacteria), with the modifications,
and M27-A4 (susceptibility test for yeasts). Extracts were prepared at
the initial concentration of 3.2 mg/mL and dissolved in 10\% DMSO. Tests
were performed against \emph{Escherichia coli} ATCC
25922,\emph{Staphylococcus aureus} ATCC 25923 and \emph{Candida
albicans} ATCC 60193. Each microtiter plate was inoculated with a
previously prepared standard bacterial (10\textsuperscript{7} CFU/mL)
and fungal suspension (2.5 x 10\textsuperscript{3} CFU/mL). The
microtiter plates were incubated at 35-37 \selectlanguage{ngerman}°C for 16 to 20 hours for E.
\emph{coli} ATCC 25922 and S. \emph{aureus} ATCC 25923 and 35 °C for 24
h for C.\emph{albicans} ATCC 60193. The lowest concentration of extract
that inhibited visible growth was recorded as the MIC. Amoxicillin and
oxacillin were used as standard antibacterial agents and fluconazole was
used as the antifungal standard for positive inhibitory controls.

\textbf{2.11 Toxicity of the colorants}

The cytotoxicity of the colorants was evaluated in front of MRC-5 cells
(human fibroblast){[}24{]} grown in DMEM medium (Dulbecco's Modified
Eagle's Medium), which was supplemented with 10\% fetal bovine serum and
kept in a CO\textsubscript{2} incubator at 37 ºC and an atmosphere
containing 5\% CO\textsubscript{2}. Cells were seeded in 96-well plates
(0.5 x 10\textsuperscript{4} cells per well). After 24 hours, extracts
were dissolved in DMSO and added to each well (10 \selectlanguage{greek}μ\selectlanguage{english}g/mL) and incubated
for 48 hours. Doxorubicin (5 \selectlanguage{greek}µ\selectlanguage{english}g/mL) was used as a positive control.
Negative controls (blanks) received the same amount of DMSO and had the
same final DMSO concentrations as the samples (0.1\%). Two hours before
the end of the incubation, 10 \selectlanguage{greek}μ\selectlanguage{english}L of alamarBlue\textsuperscript{TM} were
added to each well. The fluorescence signal was monitored with a
multiplate reader using a range of 530-560 nm excitation wavelength and
590 nm emission wavelength. The fluorescence signal generated from the
assay was proportional to the number of live cells in the sample.

\textbf{2.12 Textile dyeing}We conducted preliminary tests to evaluate
the potential of crude colorant extracts for dyeing textiles. To prepare
the coloring solutions, we dissolved the extracts from three studied
isolates (\emph{Clonostachys rosea} P2SO329, \emph{Penicillium
gravinicasei}P3SO332, and \emph{Penicillium sclerotiorum} P3SO224) in
10\% methanol, resulting in a concentration of 0.1 mg/mL. A control
solution without added colorant was also prepared. For the dyeing
process, we used polyester, silk, and cotton fabric fragments measuring
5 x 5 cm. The textile fragments were immersed in beakers containing 50
mL of the colorant solutions and left at 35 \selectlanguage{ngerman}°C for 1 hour, following the
method previous described {[}25,26{]}. After dyeing, the samples were
washed with warm distilled water at 35 °C to remove any unfixed
colorants. Next, the fabric fragments were soaked in a 0.1\%
CuSO\textsubscript{4} mordant solution for 10 minutes. Finally, the
samples were rinsed with water and air dried at room temperature.

Stability was visually assessed by observing the loss of color after
exposing the fabric fragments to natural light for a period of five
days.

\textbf{2.13 Evaluation of fungal and commercial colorants in cream and
cosmetic formulations}A cream with a water-in-oil (W/O) emulsion was
prepared using the following formulation. The oil phase consisted of 4 g
of mineral oil and 10 g of a commercial base containing vegetal oil and
an emulsifier, sourced from ``Essências da Amazônia'' (Manaus,
AM-Brazil). The aqueous phase was prepared by combining 79 g of
distilled water, 4 g of glycerin, and 2 mL of a commercial Aloe Vera
extract, also obtained from Essências da Amazônia. To achieve
emulsification, the oil phase was heated separately until it reached a
temperature of 80 ºC. Subsequently, the water phase was gradually added
to the oil phase under constant stirring, while cooling the mixture to
approximately 40 °C. This process resulted in the formation of a
homogeneous cream. Fungal and commercial colorants were incorporated
into the cream at a concentration of 0.05 mg per gram of the
formulation. The cream was fortified with 1mL of a preservative solution
(methyl paraben, propylparaben and propylene glycol){[}27,28{]}.

Colorant 1, from the \emph{Penicillium sclerotiorum} P3SO224 strain,
showed satisfactory results in the incorporation of the cream base a
more attractive coloring, and was thus selected for the soap development
test.

The soap formulation consisted of 250 g of glycerin soap base (Nossa
Terra; composition: sodium babassuate, sodium tallow, propylene glycol,
glycerin, sodium laureth sulfate, alcohol, water, sucrose, tetrasodium
EDTA, dipropylene glycol), 20 mL of lauryl, 2-5 mL of essence, 5 mL of
glycolic extract and the respective fungal colorant and commercial
colorant in a final content of 0.05 mg of colorant per g of soup. All
components were placed a hot plate for the complete casting of the base.
The formulation was added to a silicone mold and cooled to -20 °C for
solidification.

The developed cream formulation was subjected to a centrifugation test
(3,000 rpm/30 min, Cosmetic products stability guide) in order to assess
whether the product would maintain the miscible water and oil emulsion
phases. The pH, appearance, color and odor parameters were evaluated for
a period of 7 days. A 1:10 preparation (0.5 g cream/4.5 mL distilled
water) was used for pH determination with analysis at time zero and
every 48 hours for 7 days.

\textbf{3. RESULTS}

\textbf{3.1 Isolation and identification of fungi}

Samples of soil and litter were collected from the Campina Biological
Reserve in order to isolate fungal strains to be screened for colorant
production. The samples were subjected to the serial dilution method and
plated in Sabouraud agar medium to obtain isolated colonies. We obtained
184 colonies of filamentous fungi (55 from litter and 129 from
superficial soil). The micromorphological characteristics of the
isolates were analyzed and a predominance of unidentified isolates was
observed. However, among those that could be identified, the
genera\emph{Acremonium} , \emph{Aspergillus} and \emph{Penicillium} were
the most frequent.

\textbf{3.2 Screening for colorant production}

Based on the visual evaluation of the colorants produced by fungi in the
culture medium, eight isolates stood out for producing the most intense
colorants and were submitted to submerged bioprocesses, extraction, and
evaluation of the solubility of the colorant. In Fig. 1, more colorful
fractions were obtained from the extraction with different solvents from
the mycelial mass, and the fraction of the culture filtrate, which has a
greater polarity, showed the most intense extracellular colorants.

The isolates \emph{Penicillium} sp. P3SO332, \emph{Clonostachys} sp.
P2SO329 and \emph{Penicillium} sp. P3SO224 were selected to evaluate the
absorptivity of the colored extracts, which was obtained in the UV/VIS
region. The colored fractions were scanned at 400--700 nm to determine
the maximum absorption wavelength (\selectlanguage{greek}λ\selectlanguage{english}max) in ethyl acetate. Scanning
spectrophotometry showed that the three colorants evaluated have a
maximum absorbance around 400 nm.

The four fungi highlighted in the production of colorants
(\emph{Penicillium} sp. P3SO332,\emph{Clonostachys} sp.
P2SO329,\emph{Penicillium} sp. P3SO224,\emph{Monascus} sp. C02172R) were
subjected to DNA sequencing of the targets ITS1-5.8S rDNA-ITS2 to
confirm their genus and possible DNA identification. The phylogenetic
tree constructed is shown in Fig. 2. The isolate \emph{Penicillium}sp.
P3SO332 was identified as \emph{Penicillium gravinicasei} P3SO332 (99\%
accession: MG600581.1). \emph{Clonostachys} sp. P2SO329 was identified
as \emph{Clonostachys rosea} P2SO329 (99\% accession: MN452057.1).
\emph{Penicillium} sp. P3SO224 was identified as\emph{Penicillium
sclerotiorum} P3SO224 (99\% accession: MN639705.1).\emph{Monascus} sp.
C02172R was identified as \emph{Monascus purpureus}C02172R (99\%
accession: MT355839.1). The nucleotide sequences obtained after
amplification and sequencing were submitted to NCBI GenBank and the
accession numbers OP563023, OP563026, OP563024 and OP563023,
respectively, were obtained.

\textbf{3.3 Effect of nutritional parameters on colorant production}

To study the effect of the alterations in the nutritional parameters on
colorant production, the strains \emph{Penicillium}
\emph{gravinicasei}P3SO332, \emph{Clonostachys rosea} P2SO329 and
\emph{Penicilliumsclerotiorum} P3SO224 were grown in Czapeck medium base
that was modified with different sources of carbon or nitrogen (Fig. 3).

Colorant production was achieved when maize starch was used as a carbon
source and peptone as a nitrogen source in the submerged bioprocess
for\emph{Penicillium} \emph{gravinicasei} P3SO332 (Fig. 3 A, B,).
Glucose and maize starch were the carbon sources that allowed the
highest absorbances and yeast extract was the best nitrogen source
for\emph{Clonostachys rosea} P2SO329 at the analyzed wavelength (Fig. 3
C, D). Maximum absorbance was observed in the submerged bioprocess that
contained sucrose as a source of carbon and yeast extract as a source of
nitrogen in \emph{Penicillium} \emph{sclerotiorum} P3SO224 (Fig. 3 E,
F).

Based on the above results, glucose (\emph{Clonostachys rosea} P2SO329),
sucrose (\emph{Penicillium} \emph{sclerotiorum} P3SO224) and maize
starch (\emph{Penicillium} \emph{gravinicasei} P3SO332) were selected as
carbon sources and, as nitrogen sources, yeast extract
(\emph{Clonostachys rosea} P2SO329, \emph{Penicilliumsclerotiorum}
P3SO224) and peptone (\emph{Penicilliumgravinicasei} P3SO332) were
selected. The optimal production conditions for the three selected
isolates were evaluated for 20 days.

\emph{Clonostachys rosea} P2SO329, \emph{Penicillium
sclerotiorum}P3SO224 and \emph{Penicillium gravinicasei} P3SO332
presented their maximum production of colorants at 15, 10 and 20 days,
respectively. The maximum production of biomass was observed at 20 days
by\emph{Clonostachys rosea} P2SO329 (5.14 g/L), \emph{Penicillium
sclerotiorum} P3SO224 (8.62 g/L) and \emph{Penicillium
gravinicasei}P3SO332 (6.30 g/L).

\textbf{3.4 Chemical characteristics of the color fractions
of\emph{Penicillium gravinicasei} P3SO332, \emph{Clonostachys
rosea}P2SO329 and \emph{Penicillium gravinicasei} P3SO332}

Fermentation was carried out in 125 mL Erlenmeyer flasks containing 50
mL of broth medium with the different sources of carbon or nitrogen that
were selected in the previous results. This procedure was repeated 20
times and resulted in 1,000 mL of fungal cultivation. Broth containing
plugs of mycelium was mixed with ethyl acetate (1:1 v/v) and shaken in
an orbital shaker (100 rpm for 24 h). The extraction produced colorant
yields of 105 mg/L by \emph{Penicillium} \emph{sclerotiorum} P3SO224, 83
mg/L by \emph{Clonostachys rosea} P2SO329 and 142 mg/L
by\emph{Penicillium} \emph{gravinicasei} P3SO332, and their metabolites
were analyzed using LC/MS-MS.

The EtOAc extract of \emph{P.} \emph{sclerotiorum} P3SO224 showed a main
peak eluting at 15 min (See Supplementary Material) and a molecular ion
of \emph{m/z} 391 {[}M+H{]}\textsuperscript{+} with competitive losses
of -42 (C\textsubscript{2}H\textsubscript{2}O) and -60 Da
C\textsubscript{2}H\textsubscript{4}O\textsubscript{2}), related to
neutral losses on the acetyl group. Moreover, after the loss of 60 Da, a
carbon monoxide loss (- 28 Da) yielding the ion \emph{m/z} 303. These
characteristics allowed us to propose that the this compound is the
chlorinated azaphilone named sclerotiorin (Peres et al. 2023) (Fig.
4,\textbf{1} ).

The EtOAc extract of the fungus \emph{Clonostachys rosea} P2SO329 showed
four main chromatographic peaks eluting at 3.6, 5.5, 7.8 and 8.5 min.
From these, only the peak eluting at 5.5 min was possible to annotate.
The positive and negative modes displayed m/z of 171
{[}M+H{]}\textsuperscript{+} and 169 {[}M-H{]}\textsuperscript{-}, both
with consecutive losses of - 18 Da (water), - 15 Da (methyl) and -- 28
Da (CO), all consistent with the annotation of the compound is
penicillic acid \textbf{(} Fig. 4, \textbf{2)} {[}30{]} .

\textbf{3.5 Stability of the colorants}

The stability of the colorants was thoroughly assessed in this study,
considering various environmental factors. The resistance of the
colorants to temperature variations (-20\selectlanguage{ngerman}°C, 40°C, 60°C, 80°C, and 100°C)
for a duration of 1 hour revealed no noticeable color reduction.
Additionally, the colorants displayed remarkable resistance to different
pH levels (2, 4, 6, 8, and 10) during the same timeframe. However, out
of the fungi-derived colorants, only those obtained from \emph{P.
sclerotiorum} P3SO224 exhibited 100\% stability when incubated for 6
hours at different salt concentrations (0.2\%, 0.4\%, 0.6\%, 0.8\%, and
1\% v/v). The other fungal colorants showed a significant reduction
(50\% color loss) starting from 0.2\% NaCl. Furthermore, no significant
color fading was observed in the fungal colorants after a 6-hour
incubation under both white light and UV light exposure.

3.6 Antimicrobial activity

In order to investigate whether the colored fractions from the
fungi\emph{Penicillium gravinicasei} P3SO332, \emph{Clonostachys
rosea}P2SO329 and \emph{Penicillium sclerotiorum} P3SO224 showed
antimicrobial activity, using the broth microdilution method, the ethyl
acetate extract was analyzed for MIC determination. The color fractions
demonstrated antimicrobial activity against the tested microorganisms
(Table 1). The ethyl acetate extract from \emph{Clonostachys
rosea}P2SO329 had MIC value of 6.25 \selectlanguage{greek}μ\selectlanguage{english}g/mL against the strain \emph{E.
coli}ATCC 25922 and a MIC of 12.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL against \emph{S. aureus} ATCC
25923. Ethyl acetate extracts of the three isolates presented activity
against\emph{C. albicans} ATCC 60193 (400 \selectlanguage{greek}µ\selectlanguage{english}g/mL).

\textbf{3.7 Cytotoxic activity of colorants}

The cytotoxicity of the colorants was evaluated using the
AlamarBlue\textsuperscript{TM} assay against MRC-5 cells. The results
obtained show the percentage of the number of live cells in the sample.
The cytotoxicity of \emph{Clonostachys rosea} P2SO329, \emph{Penicillium
gravinicasei} P3SO332 and \emph{Penicillium sclerotiorum} P3SO224 ethyl
acetate extracts show an IC\textsubscript{50} \textgreater{} 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL.

\textbf{3.8 Textile dyeing}

The ability of fungal colorants to dye materials, specifically fabrics,
was evaluated. The results revealed that the colorant produced
by\emph{Penicillium sclerotiorum} P3SO224 was the most promising for
this purpose (Fig. 5) due to the orange pigmentation that remained after
washing the three types of fabrics used (polyester, silk and cotton).
Polyester and silk fabrics dyed with the colorant produced
by\emph{Penicillium gravinicasei} P3SO332 showed a slightly pink hue.
The dyeing of fabrics with the colorant produced by \emph{Clonostachys
rosea} P2SO329 was not effective. It was found that all fabrics
presented lighter shades after washing as well as after the period of
exposure to light. Our study also indicated that the orange colorant
produced by \emph{Penicillium sclerotiorum} P3SO224 has a higher degree
of pigmentation in polyester fabrics than in the other fabrics tested.

\textbf{3.9 Formulation of fungal cosmetic colorants}

The formulations were developed with the aim of evaluating whether the
fungal extracts, in addition to presenting antimicrobial activity and
low toxicity, could potentially be incorporated into products for
cosmetic and pharmaceutical purposes. Five cream formulations were
tested. The organoleptic characteristics (appearance, color, and odor)
and pH were analyzed, and samples were also submitted to the
pre-stability test.

When incorporated with the fungal extracts, they appeared as a cream
with a shiny, homogeneous appearance, without lumps or fine
granulations, and had an attractive color and light texture (Fig. 6).
The formulation remained stable after the centrifugation test and
presented no changes in color or odor during the period evaluated. The
pH of the formulations did not change significantly during the period
studied, and remained within a slightly acidic pH range, which is
compatible with the pH of human skin.

Colorant 1, which was obtained from the \emph{Penicillium
sclerotiorum}P3SO224 strain, showed satisfactory results when
incorporated into the cream base as it had a more attractive color and
an inhibitory effect against E. \emph{coli} ATCC 25922, S. \emph{aureus}
ATCC 25923 and C.\emph{albicans} ATCC 60193 strains. As a result, it was
selected for a soap development test (Fig. 7) in order to assess its
potential for incorporation into skin care products. The results of the
tests involving the formulation showed to be promising with the
incorporation of the colorant in a glycerin-base soap that had a
homogeneous appearance, no lumps and production of adequate amounts of
foam.

\textbf{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
(\emph{Penicillium gravinicasei} P3SO332, \emph{Clonostachys
rosea}P2SO329 and \emph{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: \emph{Penicillium} , \emph{Clonostachys} , \emph{Phoma}
and\emph{Monascus} . 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 \emph{Penicillium gravinicasei}
P3SO332,\emph{Clonostachys rosea} P2SO329 and \emph{Penicillium
sclerotiorum}P3SO224 as the main producers of colorants.
\emph{P.sclerotiorum} is known to produce colored compounds ranging from
orange to yellow including pencolide, sclerotiorin and isochromophylone
VI {[}12,35{]}. \emph{P.} \emph{gravinicasei} is known to produce an
abundant yellowish exudate, which corroborates our findings {[}36{]}.
The soil-derived fungus \emph{Clonostachys rosea}
(synonymous\emph{Gliocadium} \emph{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
\emph{Penicillium sclerotiorum}P3SO224. This metabolite is known to have
been originally isolated from this species, but it can be produced by
other fungi such as\emph{Cephalotheca faveolate} , \emph{Penicillium
frequentans} and\emph{Penicillium mallochii} {[}38--40{]}.

Penicillic acid was identified in the EtOAc extract of
\emph{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 genera\emph{Aspergillus}
and \emph{Penicillium} {[}41{]}.

Under the experimental conditions, colored extracts of the
fungi\emph{Penicillium gravinicasei} P3SO332, \emph{Clonostachys
rosea}P2SO329 and \emph{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 \emph{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 \emph{Clonostachys
rosea}YRS-06 exhibited a MIC of 25 \selectlanguage{greek}μ\selectlanguage{english}g/mL against E. \emph{coli}
{[}43{]}, c) Pigments produced by \emph{Penicillium mallochii} ARA1
and\emph{Penicillium purpurogenum} Li-3 demonstrated activity
against\emph{Staphylococcus 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 \emph{Penicillium sclerotiorum} AK-1 has
no cytotoxic effect against the yeast \emph{Saccharomyces
cerevisiae}{[}46{]} and \emph{Penicillium murcianum} yellow pigment had
no toxic effect on HEK-293 ATCC\selectlanguage{ngerman}\textsuperscript{®} CRL-1573\selectlanguage{english} and NIH/3T3
ATCC\textsuperscript{(r)} CRL-1658\selectlanguage{english} cells {[}47{]}.

In the present work, the colored extracts of \emph{Penicillium
gravinicasei} P3SO332, \emph{Clonostachys rosea} P2SO329
and\emph{Penicillium sclerotiorum} P3SO224 were able to be used to dye
fabrics and stain pharmaceutical formulations. The natural colorant
isolated from \emph{Penicillium sclerotiorum} AK-1 revealed a high
potential for dyeing cotton fabrics on an industrial scale {[}46{]}. The
colorant obtained from \emph{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 \emph{Penicillium
murcianum}was considered promising {[}47{]}. Red and yellow pigments
from fungi were evaluated to enhance sun protection factor (SPF) of
commercial sunscreens and \emph{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
were\emph{Penicillium} , \emph{Clonostachys} , \emph{Phoma}
and\emph{Monascus} ; c) \emph{Penicillium gravinicasei}
P3SO332,\emph{Clonostachys rosea} P2SO329 and \emph{Penicillium
sclerotiorum}P3SO224 were the main colorant producers; d) the
sclerothiorin metabolite was identified in the EtOAc extract of
\emph{Penicillium sclerotiorum} P3SO224; e) the metabolite penicillic
acid was identified in the EtOAc extract of \emph{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.

\textbf{Authors' contributions:}

L.A.O was responsible for conceptualization, methodology, investigation,
data curation, and writing in both the original draft and review/editing
stages. M.A.S contributed to the investigation phase. W.O.P.F.S was
involved in investigation, data curation, and writing review/editing.
P.A.L.S focused on data curation and writing review/editing. R.Q.L
contributed to investigation and data curation. A.C.A.C was responsible
for investigation. E.S.S was involved in writing review/editing. M.P.L
also contributed to writing review/editing. E.S.L handled investigation
and writing review/editing. H.H.F.K was responsible for writing
review/editing. L.D. was involved in writing review/editing. Finally,
J.V.B.S took on multiple responsibilities including conceptualization,
methodology, supervision, writing review/editing, and project
administration.

\textbf{Funding}

We would like to thank Fundacao de Amparo a Pesquisa do Estado do
Amazonas (FAPEAM) for the funding of the research by Luciana Aires de
Oliveira under the Universal Call FAPEAM-006/2019 and for the POSGRAD
2018 grant.

This work was funded by Coordenacao de Aperfeicoamento de Pessoal de
Nivel Superior (Finance code 001) and Fundacao de Amparo a Pesquisa do
Estado do Amazonas (FAPEAM) via the project POSGRAD 2021/2022.

\textbf{Statements and Declarations}

The authors declare that they have no conflict of interest. This
research was conducted independently, and the views and opinions
expressed in this article are solely those of the authors and do not
represent those of any institution or organization.

\textbf{Data Availability Statement:}

The data supporting the findings of this article are available within
the article itself, as well as in the National Center for Biotechnology
Information (NCBI) website and the supplementary material referenced in
the text of the article.

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, 1--9.

{[}48{]} Sudha, Gupta, C., Aggarwal, S., 2016. Dyeing wet blue goat
nappa skin with a microbial colorant obtained from Penicillium
minioluteum. J. Clean. Prod., 2016, \textbf{127} , 585--590.

{[}49{]} Koli, S.H., Suryawanshi, R.K., Mohite, B. V., Patil, S. V.,
2019. Prospective of Monascus Pigments as an Additive to Commercial
Sunscreens. Nat. Prod. Commun., 2019, \textbf{14} .

\textbf{TABLES}

\textbf{Table 1}

Minimum inhibitory concentration (MIC in \selectlanguage{greek}μ\selectlanguage{english}g/mL) of ethyl acetate extract
from three isolates using the broth dilution method.

\selectlanguage{greek}\textbf{Minimum Inhibitory Concentration (µg/mL)}\selectlanguage{english}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Microorganisms}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Microorganisms}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Microorganisms}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\textbf{Microorganisms}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Fungi\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{E. coli} ATCC 25922\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{S. aureus} ATCC 25923\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{C. albicans} ATCC 60193\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\emph{Penicillium} \emph{sclerotiorum} P3SO224\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
25 \selectlanguage{greek}µ\selectlanguage{english}g/mL\textsuperscript{c}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
25 \selectlanguage{greek}µ\selectlanguage{english}g/mL\textsuperscript{t}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
400 \selectlanguage{greek}µ\selectlanguage{english}g/mL\textsuperscript{ft}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
\emph{Penicillium} \emph{gravinicasei} P3SO332\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
12.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL\textsuperscript{c}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
100 \selectlanguage{greek}µ\selectlanguage{english}g/mL\textsuperscript{t}\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
400 \selectlanguage{greek}µ\selectlanguage{english}g/mL\textsuperscript{ft}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
\emph{Clonostachys rosea} P2SO329\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
6.25 \selectlanguage{greek}µ\selectlanguage{english}g/mL\textsuperscript{c}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
12.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL\textsuperscript{c}\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
400 \selectlanguage{greek}µ\selectlanguage{english}g/mL\textsuperscript{ft}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Positive control\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Amoxilin (3.125 \selectlanguage{greek}µ\selectlanguage{english}g/mL)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Oxacillin (0.39 \selectlanguage{greek}µ\selectlanguage{english}g/mL)\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Fluconazole (0.125 \selectlanguage{greek}µ\selectlanguage{english}g/mL)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

c: bactericidal t: bacteriostatic ft: fungistatic

\textbf{FIGURES LEGENDS}

\textbf{Fig. 1.} Phases of the extracts of different polarities (hexane,
ethyl acetate, methanol) using the mycelial mass and broth of the eight
isolates that produce the most intense colorants (\emph{Penicillium} sp.
P3SO332\emph{, Clonostachys} sp. P2SO329, \emph{Penicillium}
sp\emph{.}P3SO224, \emph{Penicillium} sp. P3SO322, \emph{Phoma} sp.
P2SO127,\emph{Penicillium} sp. P3SO133, \emph{Penicillium} sp.
P2SO334,\emph{Monascus} sp. C02172R).

\textbf{Fig. 2.} The evolutionary history was inferred by using the
maximum-Likelihood likelihood method and the Tamura-Nei (1993) model.
The percentage of trees in which the associated taxa clustered together
in the bootstrap test (1,000 replicates) is shown next to the branches.
The tree is drawn to scale, with branch lengths measured in the number
of substitutions per site. This analysis involved 12 nucleotide
sequences. The green dots indicate the isolates from this study and the
others are ITS sequences of colorant-producing fungi. There was a total
of 738 positions in the final dataset. Evolutionary analyses were
conducted in MEGA X v10.2.4.

\textbf{Fig. 3.} Effect of different carbon and nitrogen sources in the
production of colorants with the three selected fungi.\emph{Penicillium}
\emph{gravinicasei} P3SO332 (A, B);\emph{Clonostachys rosea} P2SO329 (C,
D); \emph{Penicilliumsclerotiorum} P3SO224 (E, F). Different letters
represent significant differences according to the Student's t-test
(\emph{p\textless{}} 0\emph{.} 05).

\textbf{Fig. 4.} Structure of sclerotiorin present in the EtOAc extract
of the fungi \emph{P} . \emph{sclerotiorum} P3SO224 (1) and penicillic
acid (2) present in the EtOAc extract of \emph{Clonostachys
rosea}P2SO329. *Structures designed using Chemdraw 19.0

\textbf{Fig. 5.} Polyester, silk and cotton fabrics dyed with crude
colorants extracted from \emph{Clonostachys rosea} P2SO329
(CR9),\emph{Penicillium gravinicasei} P3SO332 (PG2) and
\emph{Penicillium sclerotiorum} P3SO224 (PS4).

\textbf{Fig. 6.} Color and aspect of cream formulations comprising the
colorant extracts (0.05 mg per gram of the formulation) produced
by\emph{Penicillium sclerotiorum} P3SO224 (PS4 10 mg),
\emph{Clonostachys rosea} P2SO329 (CR9 14 mg), \emph{Penicillium
gravinicasei} P3SO332 (PG2 14 mg) and commercial colorant (yolk-yellow
liquid food coloring - MIX).

\textbf{Fig. 7.} Soap formulation using colorant 1 (0.05 mg per gram)
obtained from \emph{Penicillium sclerotiorum} P3SO224. Commercial
colorant (yolk-yellow liquid food coloring - MIX).

\textbf{FIGURES}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig. 1.} Phases of the extracts of different polarities (hexane,
ethyl acetate, methanol) using the mycelial mass and broth of the eight
isolates that produce the most intense colorants (\emph{Penicillium} sp.
P3SO332\emph{, Clonostachys} sp. P2SO329, \emph{Penicillium}
sp\emph{.}P3SO224, \emph{Penicillium} sp. P3SO322, \emph{Phoma} sp.
P2SO127,\emph{Penicillium} sp. P3SO133, \emph{Penicillium} sp.
P2SO334,\emph{Monascus} sp. C02172R).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2.} The evolutionary history was inferred by using the
maximum-Likelihood likelihood method and the Tamura-Nei (1993) model.
The percentage of trees in which the associated taxa clustered together
in the bootstrap test (1,000 replicates) is shown next to the branches.
The tree is drawn to scale, with branch lengths measured in the number
of substitutions per site. This analysis involved 12 nucleotide
sequences. The green dots indicate the isolates from this study and the
others are ITS sequences of colorant-producing fungi. There was a total
of 738 positions in the final dataset. Evolutionary analyses were
conducted in MEGA X v10.2.4.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig. 3.} Effect of different carbon and nitrogen sources in the
production of colorants with the three selected fungi.\emph{Penicillium}
\emph{gravinicasei} P3SO332 (A, B);\emph{Clonostachys rosea} P2SO329 (C,
D); \emph{Penicilliumsclerotiorum} P3SO224 (E, F). Different letters
represent significant differences according to the Student's t-test
(\emph{p\textless{}} 0\emph{.} 05).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 4.} Structure of sclerotiorin present in the EtOAc extract
of the fungi \emph{P} . \emph{sclerotiorum} P3SO224 (1) and penicillic
acid (2) present in the EtOAc extract of \emph{Clonostachys
rosea}P2SO329. *Structures designed using Chemdraw 19.0\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig. 5.} Polyester, silk and cotton fabrics dyed with crude
colorants extracted from \emph{Clonostachys rosea} P2SO329
(CR9),\emph{Penicillium gravinicasei} P3SO332 (PG2) and
\emph{Penicillium sclerotiorum} P3SO224 (PS4).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Fig. 6.} Color and aspect of cream formulations comprising the
colorant extracts (0.05 mg per gram of the formulation) produced
by\emph{Penicillium sclerotiorum} P3SO224, \emph{Clonostachys
rosea}P2SO329, \emph{Penicillium gravinicasei} P3SO332 and commercial
colorant (yolk-yellow liquid food coloring - MIX).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Fig. 7.} Soap formulation using colorant 1 (0.05 mg per gram)
obtained from \emph{Penicillium sclerotiorum} P3SO224. Commercial
colorant (yolk-yellow liquid food coloring - MIX).

\selectlanguage{english}
\FloatBarrier
\end{document}

