Pigment production by a newly isolated strain Pycnoporus sanguineus SYBC-L7 in solid-state fermentation

Abstract

Natural pigments are playing important roles in our daily lives. They not only make products colorful but also provide various health benefits for humans. In addition, Pycnoporus genus, listed as food- and cosmetic-grade microorganism, is one of the promising organisms for developing natural pigments. In this study, a new fungal strain with high efficiency in producing intense orange pigments was isolated and identified as Pycnoporus sanguineus SYBC-L7. Different agro-industrial wastes were applied to evaluate the growth and pigment production of strain SYBC-L7. SYBC-L7 can grow rapidly and effectively produce pigments using wood chips as substrate in solid-state fermentation (SSF). Culture conditions were also optimized for value-added pigments production and the optimum production conditions were glucose as carbon source, ammonium tartrate as nitrogen source, initial pH 6.0, and relative humidity of 65%. Pigment components, cinnabarinic acid, tramesanguin, and 2-amino-9-formylphenoxazone-1-carbonic acid were confirmed by liquid chromatography–mass spectrometry. Meanwhile, an agar plate diffusion assay was performed to evaluate the antimicrobial activity of the pigment. These pigments showed more significant inhibition of Gram-positive than Gram-negative bacteria. The results showed that Pycnoporus sanguineus SYBC-L7 was able to cost-effectively produce intense natural orange pigments with antibacterial activity in SSF, which is the basis of their large-scale production and application.

Full text

Frontiers in Microbiology 01 frontiersin.org
Pigment production by a newly
isolated strain Pycnoporus
sanguineus SYBC-L7in
solid-state fermentation
DiMeng
1, XuanShao
1, Shou-PengLuo
2, Qiao-PengTian
3 and
Xiang-RuLiao
3*
1 Henan Provincial Engineering Research Center for Development and Application of Characteristic
Microorganism Resources, Shangqiu Normal University, Shangqiu, China, 2 Hua Tian Engineering &
Technology Corporation, MCC, Nanjing, China, 3 Key Laboratory of Industrial Biotechnology,
Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China
Natural pigments are playing important roles in our daily lives. They not only
make products colorful but also provide various health benefits for humans. In
addition, Pycnoporus genus, listed as food- and cosmetic-grade microorganism,
is one of the promising organisms for developing natural pigments. In this study,
a new fungal strain with high eciency in producing intense orange pigments
was isolated and identified as Pycnoporus sanguineus SYBC-L7. Dierent agro-
industrial wastes were applied to evaluate the growth and pigment production of
strain SYBC-L7. SYBC-L7 can grow rapidly and eectively produce pigments using
wood chips as substrate in solid-state fermentation (SSF). Culture conditions were
also optimized for value-added pigments production and the optimum production
conditions were glucose as carbon source, ammonium tartrate as nitrogen source,
initial pH 6.0, and relative humidity of 65%. Pigment components, cinnabarinic
acid, tramesanguin, and 2-amino-9-formylphenoxazone-1-carbonic acid were
confirmed by liquid chromatography–mass spectrometry. Meanwhile, an agar
plate diusion assay was performed to evaluate the antimicrobial activity of the
pigment. These pigments showed more significant inhibition of Gram-positive
than Gram-negative bacteria. The results showed that Pycnoporus sanguineus
SYBC-L7 was able to cost-eectively produce intense natural orange pigments
with antibacterial activity in SSF, which is the basis of their large-scale production
and application.
KEYWORDS
natural pigment, food- and cosmetic-grade microorganism, Pycnoporus
sanguineus, agro-industrial wastes, cinnabarinic acid
Introduction
Pigments have become an important part of cosmetics, food, textiles, and other
industrial elds (Meruvu and dos Santos, 2021). Some of them not only endow
products with dierent colors but also have antibacterial and antioxidant activity to
provide various health benets for humans (Tudor etal., 2013; Srivastava etal., 2022).
TYPE Original Research
PUBLISHED 19 October 2022
DOI 10.3389/fmicb.2022.1015913
OPEN ACCESS
EDITED BY
Laurent Dufossé,
Université de la Réunion, France
REVIEWED BY
Ramesh Chatragadda,
Council of Scientific and Industrial
Research (CSIR), India
Pardeep Kumar,
Chaudhary Devi Lal University, India
Lourdes Morales-Oyervides,
Universidad Autónoma de Coahuila,
Mexico
*CORRESPONDENCE
Xiang-Ru Liao
liaoxiangru@163.com
SPECIALTY SECTION
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
RECEIVED 10 August 2022
ACCEPTED 12 September 2022
PUBLISHED 19 October 2022
CITATION
Meng D, Shao X, Luo S-P, Tian Q-P and
Liao X-R (2022) Pigment production by a
newly isolated strain Pycnoporus
sanguineus SYBC-L7in solid-state
fermentation.
Front. Microbiol. 13:1015913.
doi: 10.3389/fmicb.2022.1015913
COPYRIGHT
© 2022 Meng, Shao, Luo, Tian and Liao.
This is an open-access article distributed
under the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.

Meng et al. 10.3389/fmicb.2022.1015913
Frontiers in Microbiology 02 frontiersin.org
According to the source, pigments can bedivided into synthetic
pigments and natural pigments. Since synthetic pigments are
found to display potential toxicity, carcinogenicity, and
undesirable side eects on human health and the environment,
natural pigments (derived from plants, animals, or
microorganisms) are getting more attention due to their
biodegradability, no side eects, and biological activities
(Chatragadda etal., 2019; Darwesh etal., 2020; Chatragadda
and Dufosse, 2021). Among natural pigment producers,
microorganisms are noteworthy for their all-seasonal
production of stable and low-cost with high yield (Meruvu and
dos Santos, 2021). To date, many microorganisms have been
reported to produce pigments, including Monascus sp. (Chen
etal., 2021), Penicillium sp. (Meruvu and dos Santos, 2021),
Pycnoporus sp. (Tellez-Tellez etal., 2016; Zhang etal., 2019),
Rhodotorula sp., and Bacillus sp. (Usmani etal., 2020). Among
them, Pycnoporus genus is a white-rot fungus, listed as food-
and cosmetic-grade microorganism, and one of the promising
organisms for the development of natural pigments (Lomascolo
etal., 2011; Tellez-Tellez etal., 2016). It has been reported that
the major Pycnoporus pigments (cinnabarin, cinnabarinic acid,
and tramesanguin), possessing antiviral, antibacterial, and
anti-inammatory properties, are derived from a phenoxazine-
3-one type structure, which is the central core of many natural
active compounds (Tellez-Tellez et al., 2016; Zhang et al.,
2019). Furthermore, Pycnoporus strains also can produce
various useful enzymes for industry, including hydrolases and
oxidases mainly laccases, which make them easier to use agro-
industrial wastes (Tellez-Tellez etal., 2016).
Solid-state fermentation (SSF) is a traditional fermentation
method of fungi to produce pigments. SSF provides attachment
sites for the growth of strains, sucient nutrients, and oxygen
supply, making the yield of pigments produced much higher than
that of liquid submerged fermentation (Palma etal., 2016; Feng
etal., 2022). At present, various agro-industrial wastes (sugarcane
bagasse, sawdust, rice straw, wheat straw, orange peel, and so on)
are used as substrates for SSF, which can help to reduce the
production cost, reduce the pollution load from the environment,
and create maximum value (Sadh etal., 2018). In addition, some
fermentation conditions like pH, temperature, relative humidity,
and media nutrients also aect the production of pigment.
erefore, exploiting agro-industrial wastes as substrates and
optimizing fermentation conditions will beimportant for value-
added metabolite production.
Although Pycnoporus genus has been used to produce high
value-added metabolites through solid-state fermentation, few
studies on agro-industrial wastes as substrates for pigment
production have been reported at present. erefore, the aims of
this paper were to (1) isolate and identify a Pycnoporus fungus
with high-yield pigment production capacity; (2) improve
pigment production by controlling culture conditions, including
substrates, carbon and nitrogen sources, initial pH, and relative
humidity; (3) analyze and identify the pigments; and (4) evaluate
the antimicrobial activity of pigments.
Materials and methods
Materials
Cinnabarinic acid (CA, ≥98%, CAS: 606–59-7), Ehrlich
reagent (CAS: 100–10-7), and N-acetylglucosamine (CAS: 7512-
17-6) were purchased from Sigma-Aldrich (Shanghai, China).
Sugarcane bagasse, wheat straw, rice straw, water hyacinth, and
wood chip were purchased from a local market and dried to
constant weight at 60°C before use. Other chemical reagents,
unless otherwise specied, were all analytical grade.
Indicator strains, including Bacillus subtilis SYBC-hb1, Bacillus
subtilis SYBC-hb5, Bacillus licheniformis SYBC-hb2, Bacillus
pumilus SYBC-hb4, Bacillus thuringiensis SYBC-hb7, Bacillus
amyloliquefaciens Y1-A3, Bacillus megaterium H021-A1, Bacillus
cereus SYBC-hb8, Lysinibacillus sp. H021-S8, Staphylococcus kloosii
H008-B4, Staphylococcus aureus, Escherichia coli J159, Serratia sp.
L1, Serratia sp. L2, and Serratia sp. L3, were obtained from
Biocatalysis and Transformation Biology lab at Jiangnan University
(Wuxi, China).
Isolation samples
Ten fungal fruit body samples were collected from dierent
locations in a local forest in Wuxi, China (31°32′24′′N,
120°12′24′′E).
Cultivation medium
Potato dextrose agar (PDA) was prepared for purication
and preservation.
Seed medium was prepared as described by Zeng etal. (2011)
with certain modications: the seed medium consisted of 30 g·L
−1
potato starch, 4.5 g·L−1 yeast extract, and 10.5 g·L−1 peptone.
Solid-state fermentation (SSF) medium was prepared based
on Zeng etal. (2011), Liu etal. (2013), and Liu and Liao (2015)
with certain modications: the SSF medium consisted of 3 g dry
wood chips and 4.5 ml nutrient solution in 250 ml conical ask,
and the nutrient solution contained the following: glucose,
30 g·L
−1
; ammonium tartrate, 15 g·L
−1
; KH
2
PO
4
, 1 g·L
−1
; Na
2
HPO
4
,
0.2 g·L−1; MgSO4, 0.5 g·L−1; MnSO4, 0.034 g·L−1.
Isolation and identification of
pigment-producing fungi
Fragments of the basidiomata were treated as described in
Zeng etal. (2011) and cultured on PDA plate at 30°C for 12 days.
e strains with good growth and noticeable color change were
selected for shaker screening.
For shaker screening, 10ml of sterile normal saline (0.9% w·v
−1) were added to PDA plates covered with mycelium. e spores

Meng et al. 10.3389/fmicb.2022.1015913
Frontiers in Microbiology 03 frontiersin.org
were scraped o and suspended in sterile normal saline to
approximately10
8
spores·mL
−1
. Each 1 ml of spore suspension was
inoculated into 250 conical asks containing 50 ml seed medium
and cultured at 30°C, 200 r·min −1 for 1 days in the dark. en,
1 ml of seed medium was transferred into the SSF medium at 30°C
for 12 days in the dark. e strain with high pigment yield was
selected for subsequent studies.
e morphological characteristics were evaluated using a
scanning electron microscopy (Quanta-200, FET, Netherlands)
aer 5 days of incubation on PDA plates (Wang etal., 2021). e
molecular identication process was performed as follows: (1) e
isolated strain was cultured for 5 days and harvested by sterilized
spear tips; (2) aer drying and grinding of the collected mycelium
using liquid nitrogen, total genomic DNA was extracted using the
CTAB protocol; (3) PCR amplication was performed as described
by Saravanan etal. (2020), and the primers used were ITS1 and
ITS4; (4) e ITS-5.8S rRNA gene sequence of the isolated strain
was compared with the sequences deposited in the GenBank
database, and phylogenetic tree was constructed by neighbor-
joining method using MEGA 6.0 based on a bootstrap test of
1,000 replicates (Zeng etal., 2011; Huang etal., 2020).
Substrate and solid-state fermentation
Here, ve common agro-industrial wastes, such as sugarcane
bagasse, wheat straw, rice straw, water hyacinth, and wood chip, were
chosen to evaluate the growth and pigment production of isolated
strain. Each of these ve common agro-industrial wastes was used
instead of the substrate (dry wood chips) in the original SSF medium,
respectively, and the other components of the original SSF medium
were unchanged. Additionally, inoculum preparation, inoculum size,
and culture conditions were consistent with shaker screening of
section “Isolation and Identication of Pigment-Producing Fungi.”
Optimization of pigment production
According to the method described in Darwesh etal. (2020)
and Chen etal. (2021), the “one factor at a time” design was used
to assess the eect of dierent cultural conditions. ese variables
were dierent extra carbon sources (Monosaccharide: glucose and
fructose; Disaccharide: sucrose and maltose; Polysaccharide:
starch and β-cyclodextrin), nitrogen sources (Organic nitrogen:
peptone, yeast extract, and carbamide; Inorganic nitrogen:
ammonium nitrate, potassium nitrate, ammonium chloride, and
ammonium tartrate), initial pH values (3, 4, 5, 6, 7, 8, 9, and 10),
and relative humidity values (40, 45, 50, 55, 60, 65, 70, 75, and
80%). All the optimization experiments were performed in 250 ml
conical asks, and the SSF medium was prepared based on the
section “Substrate and solid-state fermentation.” Unless otherwise
indicated, the inoculum preparation, inoculum size, and culture
conditions were consistent with shaker screening of section
“Isolation and Identication of Pigment-Producing Fungi.”
Biomass estimation
e biomass was estimated by measuring the
N-acetylglucosamine released by the acid hydrolysis of the chitin,
present in the cell walls of fungi. Sample handling and detection
processes were according to the method described by Velmurugan
etal. (2011). In brief, 0.5 g of dry fermented solid-state powder
was rst mixed with 1 ml of concentrated H2SO4. en, 1 ml of
acetylacetone reagent was added to the mixture and placed in a
water bath at 100°C for 20 min. Aer cooling, 6 ml of ethanol and
1 ml of Ehrlich reagent were added successively and incubated at
65°C for 10 min. Aer cooling to room temperature, optical
density (OD) was measured at 530 nm against the reagent blank
using N-acetylglucosamine as the external standard.
Pigment identification and estimation
Pigment extraction: Aer 10 d of incubation, the fermented
solid substrate was dried to constant weight at 60°C in a cabinet
and ground to a ne powder using a muller. 0.3 g of dry fermented
solid-state powder was mixed with 15 ml of methanol and placed
in a water bath at 35°C for 1 h, and this extraction was repeated
twice (Luo etal., 2013). en the extracts were ltered through
Whatman lter paper Grade No. 1 at constant volume of 30 ml.
UV–visible spectroscopy: e production of pigments was
estimated by detecting λmax of pigment extract (Chen et al.,
2021). e maximum absorption peak of pigment extract was
performed on the UV–visible U-3000 Spectrophotometer
(Hitachi, Japan) with a scanning wavelength near 350 nm to
550 nm (Cruz-Munoz etal., 2015).
Chromatography: e method was according to that
described by Dias and Urban (2009) and Kakoti etal. (2022)
with certain modications. In brief, chromatographic separation
was performed on a BEH AMIDE column (1.7 μm,
2.1 mm × 100 mm; Waters, UnitedStates) using the ACQUITY
Ultra Performance Liquid Chromatography system (Waters,
UnitedStates). e column was maintained at 45°C and eluted
with the gradient % A (solvent A): T (time, min) 5:0; 60:7; 100:9;
100:12; 5:15 at a ow rate of 0.3 ml·min
−1
. Solvent A was 100%
acetonitrile, and B was 0.1% formic acid. e detection
wavelength was 254 nm, and 5 μl of testing samples (methanol
extraction and cinnabarinic acid standard) were injected into the
column. All testing samples were ltered with nylon membrane
(0.22 μm) before chromatographic separation.
Mass spectrometry: e mass spectrometry method was
referred to that described by Xu et al. (2020) with certain
modications: mass spectrometry was performed on a Waters Maldi
Synapt Q-TOF MS (Waters, US) operating in ESI
+
ion modes. e
desolvation gas was set to 300 l·h−1 at 300°C. e cone gas was set to
500 l·h−1 and the source temperature was set to 100°C. e capillary
voltage and cone voltage were set to 3.5 kV and 30 V, respectively. e
collision energy was set to 6 eV. e detector voltage was set to 1.7 kV
and the scan range was from 50 to 2000 m·z−1.

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Pigment estimation: Following the method of Velmurugan
etal. (2011), the extracted pigment was quantied by measuring
optical density (OD) at λmax and pigment yield was calculated by
OD/gdfs. e gdfs represents per gram of dry fermented substrate.
Methanol extract of the unfermented sample was used as the
blank for pigment analysis.
Assessment of antimicrobial activity
Agar plate diusion assay was used to determine the
antibacterial activity of the pigment produced by strain SYBC-L7
using Bacillus subtilis SYBC-hb1, Bacillus subtilis SYBC-hb5, Bacillus
licheniformis SYBC-hb2, Bacillus pumilus SYBC-hb4, Bacillus
thuringiensis SYBC-hb7, Bacillus amyloliquefaciens Y1-A3, Bacillus
megaterium H021-A1, Bacillus cereus SYBC-hb8, Lysinibacillus sp.
H021-S8, Staphylococcus kloosii H008-B4, and Staphylococcus aureus
(Gram-positive bacteria) and Escherichia coli J159, Serratia sp. L1,
Serratia sp. L2, and Serratia sp. L3 (Gram-negative bacteria). e
indicator strains were incubated at 37°C in Luria-Bertani (LB) broth.
Aer 18 h of incubation, the cultures were diluted to 107 cfu·mL−1
and “ood-inoculated” onto the surface of LB plates. 9 mm diameter
wells were cut using a punching bear. en, 100 μl of methanol
extract of pigment with dierent concentrations (pigment
extraction:5, 10, 15, 20 OD·gdfs−1, containing CA concentrations of
21.6, 43.2, 64., and 86.3 mg·L−1, respectively) were delivered into the
wells with pure methanol as blank and dierent concentrations (50,
100, 150, 200, 300, 400, 500 mg·L−1) of ampicillin as reference. Aer
incubation at 37°C for 18 h, plates were examined for any zones of
growth inhibition and measured the diameter of each zone
(subtracted the diameter of well).
Statistical analysis
All the experiments were performed in triplicate, and the
error bar represents the standard deviations (SD). OriginPro
2022b soware was used to calculate the mean value/SD and to
plot gures of the collected data.
Results and discussion
Isolation and Identification of
Pigment-Producing Fungi
Ten fungal fruit body samples collected from dierent
locations were subjected to plate screening and shaker screening
successively to select the most ecient strain in terms of pigment
production. A fungal strain designated SYBC-L7 was clearly
observed to rapidly produce an intense orange pigment with an
absorption maximum at a wavelength of 430 nm (Figures1A,B).
us, strain SYBC-L7 was selected for the subsequent studies.
Strain SYBC-L7 was identied based on its morphological and
molecular properties (Figure1). e basidiocarp of strain SYBC-L7
was smooth, acute margin, smooth to wavy thin, pileus shortly, and
sessile or sometimes overlapping with characteristic color of bright
orange-red (Figure1C). Aer inoculation to PDA plate and culture
at 30°C, the mycelium of strain SYBC-L7 grew faster and gradually
broke o to form a large number of spores, which are rectangular in
shape, smooth in surface, and varied in size (Figure1D); By the 9th
day of culture, the mycelium covered full of the plate and was
orange-red (Figure1A). ese characteristics were consistent with
Pycnoporus genus (Tellez-Tellez et al., 2016). Moreover, the
amplication of the genomic DNA of strain SYBC-L7 by primers
ITS1 and ITS4 produced a single amplication product of
approximately 639 bp. When comparing the sequence to the
GenBank database and constructing the phylogenetic tree, it was
observed that the sequence (GenBank ID: HQ891291.1) was
clustering to Pycnoporus sanguineus genus (Figure 1E). As the
sequence similarity to the most closely reference strain (GenBank
ID: KC525202.1) was only 68%, strain SYBC-L7 may bea new
strain of Pycnoporus sanguineus.
Solid substrate chosen for solid-state
fermentation
In the solid-state fermentation process, the fermentation
substrate has a great inuence not only on the growth, attachment,
and extension of the mycelium but also on factors such as heat
dissipation and oxygen supply (Mussatto etal., 2012). Here, ve
common agro-industrial wastes (sugarcane bagasse, wheat straw,
rice straw, water hyacinth, and wood chip) were selected to evaluate
the growth and pigment production of strain SYBC-L7. As shown
in Figure2, the growth and pigment production of strain SYBC-L7
were greatly inuenced by the used agro-industrial waste. Among
the tested wastes, the wood chip was found to bethe best substrate
not only giving a maximum pigment yield but also greatly
promoting the growth of strain SYBC-L7. In the literature, most
Pycnoporus species have been isolated and used as enzyme
producers (Tellez-Tellez etal., 2016), but few of them were used to
produce pigment, which was shown in (Supplementary Table S1;
Eggert, 1997; Smania etal., 1997; Cruz-Munoz etal., 2015; Sutthisa
and Sanoamuang, 2017; Zhang etal., 2019). However, our study is
the rst report, to our knowledge, on using agro-industrial waste,
especially wood chips as a culture substrate for pigment production
by Pycnoporus sanguineus SYBC-L7.
Optimization of pigment production by
strain SYBC-L7
Biomass growth and pigment productivity can beinuenced
by nutrient fermentation conditions (Darwesh etal., 2020). Here,
environmental and cultural conditions were studied to improve
the yield of pigment produced by strain SYBC-L7. Carbon is not
only an essential nutrient for the biosynthesis of cellular
components but also an energy resource for cells (Feng etal.,
2018). For example, carbon plays a critical role in cell growth,

Meng et al. 10.3389/fmicb.2022.1015913
Frontiers in Microbiology 05 frontiersin.org
metabolism, and pigment production of Monascus spp. (Darwesh
etal., 2020; Long etal., 2020). In the present study, dierent extra
carbon sources (Monosaccharide: glucose and fructose;
Disaccharide: sucrose and maltose; Polysaccharide: starch and
β-cyclodextrin) were used to improve the biomass and pigment
production of strain SYBC-L7. e results presented in Figure3A
showed that under the experimental conditions, monosaccharide
was easier to beutilized for growth and pigment production than
disaccharide and polysaccharide. Glucose was better for pigment
synthesis, fructose was better for fungal growth, while, lower
pigment production and biomass were obtained with
β-cyclodextrin and starch. In accordance with our results, Zhao
etal. (2019) and Shahin etal. (2022) stated that glucose was the
optimum carbon source for pigment production.
Nitrogen source, another essential building component, can
inuence microbial growth and the production of bioactive
metabolites (Tudzynski, 2014; Landi etal., 2019; Darwesh etal.,
2020). To improve the pigment yield, dierent extra nitrogen
sources (Organic nitrogen: peptone, yeast extract, and carbamide;
inorganic nitrogen: ammonium nitrate, potassium nitrate,
ammonium chloride, and ammonium tartrate) were added to the
solid-state fermentation culture. As shown in Figure3B, strain
SYBC-L7 grew better and produced the most pigments with
ammonium tartrate, and it grew better and produced more
pigments with peptone. While it grew best but produced fewer
pigments with yeast extract as an extra nitrogen source.
e initial pH of the culture medium is one of the most critical
environmental and culture parameters determining microbial
AB
DE
C
FIGURE1
Orange pigment-producing fungal isolate (strain SYBC-L7) with the highest potency. (A) The mycelia of strain SYBC-L7 cultured on PDA medium
for 9 d; (B) Spectral analysis of extract samples obtained from strain SYBC-L7 with wood chip for 10 d, and 0 d represent the unfermented solid
substrate sample; (C) The fruit body of strain SYBC-L7; (D) Microscopic characteristics of the mycelia of strain SYBC-L7 cultured on PDA medium
for 5 d; (E) Phylogenetic trees based on ITS-5.8S rRNA sequences of strain SYBC-L7 and others downloaded from NCBI.
FIGURE2
Eect of solid substrate on biomass and pigment production.
Data correspond to the mean ± SD of three replicates.

Meng et al. 10.3389/fmicb.2022.1015913
Frontiers in Microbiology 06 frontiersin.org
growth and metabolic activity (Ouyang etal., 2020). As dierent
strains have dierent optimum pH, here, an experiment was
conducted to determine the eect of dierent initial pH values
(3–10) on the biomass and pigment yield of strain SYBC-L7. As
shown in Figure3C, high pigment production was obtained at pH
5 to 9, and the maximum production was received at pH 6. Low
or high pH (like 3, 4, and 10) inhibited both biomass and pigment
production. e results were similar to those of Cruz-Munoz etal.
(2015), who reported that the maxima pigment synthesis was
obtained at pH 7 for Pycnoporus sanguineus strain H1 and H2.
e relative humidity of the incubator is also one of the most
critical environmental parameters of the SSF process. It can
prevent accelerated drying of the fermentation substrate, and it is
directly related to water activity, which is a critical factor for fungal
metabolism performance during the fermentation process (Osorio
etal., 2020). As shown in Figure3D, maximum pigment yield and
biomass were observed at 65% of relative humidity, and a decrease
in pigment yield was observed below or above 65%. e humidity
gradient occurred between the exterior surface and inner surface
of substrate (He et al., 2019). Low relative humidity could
accelerate the formation of this humidity gradient, promote liquid
water movement and evaporation from the interior to the surface
of substrate, and lead to low nutrient availability and less ecient
heat exchange, causing poor pigment yield (Babitha etal., 2007;
He etal., 2019). On the contrary, the higher relative humidity
could promote moisture in the air movement from the surface to
the interior of substrate, and reduce the mass transfer process, air
transfer, and extension of mycelium in SSF, leading to suboptimal
pigment formation (Babitha etal., 2007).
Identification of pigment from strain
SYBC-L7
Pycnoporus sanguineus is one of the promising organisms for
developing natural pigments, and it could produce various shades
of red, orange, yellow, and brown color (Zhang etal., 2019). ese
primary or secondary metabolites produced by Pycnoporus genus
are dierent and depend on the species and culture conditions
(Cruz-Munoz etal., 2015; Tellez-Tellez etal., 2016). Nevertheless,
AB
CD
FIGURE3
Eect of carbon sources (A), nitrogen sources (B), initial pH (C), and relative humidity (D) on biomass and pigment production strain SYBC-L7in
solid-state fermentation. Data correspond to the mean ± SD of three replicates.

Meng et al. 10.3389/fmicb.2022.1015913
Frontiers in Microbiology 07 frontiersin.org
previous studies have demonstrated that cinnabarin and
cinnabarinic acid (CA) are the main pigment components of
Pycnoporus strains, which have antiviral, antibacterial, and anti-
inammatory activity (Tellez-Tellez etal., 2016). In this study, CA
was taken as a standard sample and used for pigment component
analysis. In Figure4, three obvious strong peaks (marked as Pk1,
Pk2, and Pk3) appeared at the retention time (RT) of 3.89 min,
4.40 min, and 4.69 min, respectively. e mass spectrum of
Pk2 showed a molecular ion at m/z 301 [M + H]+
(Supplementary Figure S1), which was not only the same as
described in some previous studies (Dias and Urban, 2009; Tellez-
Tellez etal., 2016), but also was consistent with the mass spectrum
of CA standard sample (data was not shown). us, Pk2 was
identied as CA. e protonated ions of Pk1 and Pk3 were both at
m/z 285 [M + H]+ (Supplementary Figures S1), and wavelength
scanning results showed that their characteristic absorption peaks
were both between 430 and 450nm (Supplementary Figure S2);
ese results are in fair agreement with the results reported for
tramesanguin (Dias and Urban, 2009). As Pk1 and Pk3 are
dierent compounds, Pk1 and Pk3 maybe tramesanguin and its
isomer, 2-amino-9-formylphenoxazone-1-carbonic acid. e
structures of these compounds are shown in Table1.
Antibacterial activity analysis of pigment
extract
e antibacterial activity of pigment extract was evaluated by
agar plate diusion assay. In agar plate diusion assay, pigment
extract exhibited signicant antibacterial activity on Gram-positive
A
B
FIGURE4
HPLC analysis of pigment extracts of strain SYBC-L7. (A) HPLC chromatograms of pigment extracts of strain SYBC-L7; (B) HPLC chromatograms of
CA standard sample.
TABLE1 Identification of pigment from strain SYBC-L7.
Products RT
(min)
Measured mass
[M + H]+ (m/z)
Common name Proposed structure References
Pk2 4.40 301 Cinnabarinic acid Dias and Urban (2009),
Tellez-Tellez etal. (2016)
Pk1 3.89 285 Tramesanguin, 2-amino-9-
formylphenoxazone-1-carbonic
acid
Dias and Urban (2009)
Pk3 4.69 285

Meng et al. 10.3389/fmicb.2022.1015913
Frontiers in Microbiology 08 frontiersin.org
bacteria (Figure5A), and the zone of inhibition was increased with
the increase of pigment concentration. However, pigment extract
exerted no inhibitory eect on Gram-negative bacteria (data not
shown). ese ndings were similar to those of Tellez-Tellez etal.
(2016), who reported that the component from Pycnoporus
sanguineus showed more activity against Gram-positive than
Gram-negative bacteria. Kakoti etal. (2022) reported that CA
extracted from Trametes coccinea fruiting bodies showed high
inhibition for Gram-negative bacteria, and the minimal inhibitory
concentration of CA for Escherichia coli was 300 mg·L−1. e
pigment extract (containing CA concentrations of 21.6, 43.2, 64.7,
and 86.3 mg·L
−1
, respectively) in our study showed no inhibitory
eect on Gram-negative bacteria including Escherichia coli, which
may be because of the low concentration of pigment extract.
Additionally, when compared with the antibacterial activity of
dierent concentrations of ampicillin (50–400 mg·L
−1
) on Gram-
positive bacteria, the pigment extract had an inhibitory eect on
all 11 kinds of indicator bacteria, while ampicillin only had an
inhibitory eect on ve of them (like Bacillus subtilis SYBC-hb1,
Bacillus amyloliquefaciens Y1-A3, Bacillus megaterium H021-A1,
Staphylococcus kloosii H008-B4, Staphylococcus aureus; Figure5B).
Conclusion
A new fungal strain Pycnoporus sanguineus SYBC-L7 was
shown to eectively produce intense orange pigments in solid-
state fermentation. Pigment production processes could
bemore economic and high yield by controlling agro-industrial
wastes as substrates and fermentation conditions such as carbon
sources, nitrogen sources, initial pH, and relative humidity. e
pigment extract was identied as cinnabarinic acid,
tramesanguin, and 2-amino-9-formylphenoxazone-1-carbonic
acid by HPLC-MS. Additionally, antibacterial activity analysis
of pigment extract produced by strain SYBC-L7 showed
signicant antibacterial activity on dierent bacteria, and the
pigment was more sensitive to Gram-positive bacteria,
indicating its potential application for areas such as the food,
cosmetics, nutraceuticals, and textile industry that need color.
Further studies should beconducted to better understand the
biosafety of microbial pigments as promising alternatives to
hazardous articial colorants.
Data availability statement
e original contributions presented in the study are included
in the article/Supplementary material, further inquiries can be
directed to the corresponding authors. e ITS-5.8S rRNA gene
sequence of Pycnoporus sanguineus SYBC-L7 is available in the
NCBI database, accession number HQ891291.1.
Author contributions
DM and XS participated in data analysis and wrote the paper.
S-PL, Q-PT, and X-RL performed the research. All authors
contributed to the article and approved the submitted version.
AB
FIGURE5
Heatmaps of antibacterial activity of pigment extract (A) and ampicillin (B) for Gram-positive bacteria. Data correspond to the mean of three
replicates.

Meng et al. 10.3389/fmicb.2022.1015913
Frontiers in Microbiology 09 frontiersin.org
Funding
is work was nancially supported by startup funds from
Shangqiu Normal University (grant no. 7001/700219), horizontal
subject (grant no. 8001/801141), and the project fund from the
Program for Science and Technology Innovative Research Team
in University of Henan Province (grant no. 21IRTSTHN025).
Conflict of interest
S-PL was employed by Hua Tian Engineering & Technology
Corporation, MCC, Nanjing, China.
e remaining authors declare that the research was conducted
in the absence of any commercial or nancial relationships that
could beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those
of the authors and do not necessarily represent those
of their affiliated organizations, or those of the publisher,
the editors and the reviewers. Any product that may be
evaluated in this article, or claim that may be made by
its manufacturer, is not guaranteed or endorsed by
the publisher.
Supplementary material
e Supplementary material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.1015913/
full#supplementary-material
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