DOI: 10.1002/cbdv.

201800300 FULL PAPER

Chemical Composition, Security and Bioactivity of the Red Pigment

from Penicillium purpurogenum Li-3
Hong-Jie Jin,a, b Xin Zhang,c Hong Cao,*a Yu-Jing Niu,c Chun Li,d and Hong Liub
a
College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing 314001, P. R. China,
e-mail: chyf_ch@126.com
b
College of Life Science, Shihezi University, Shihezi 832000, P. R. China
c
College of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832000, P. R. China
d
College of Life Science, Beijing Institute of Technology, Beijing 100081, P. R. China

The chemical composition, security and bioactivity of pigments from Penicillium purpurogenum Li-3 strain
screened by our group were firstly studied in this work. DPPH and the filter disc diffusion method were used to
determine the biological activities of the red pigments. The pigment was characterized by UV/VIS, FT-IR, NMR
and UPLC-Q-TOF-MS. HPLC/MS was used to detect mycotoxins (citrinin) in fermentation broth. An acute toxicity
was detected in the embryos of zebrafish. As a consequence, the crude red pigment from the AcOEt fraction
showed better DPPH scavenge capacity and antibacterial activity. Spectroscopic (UV, FT-IR, 13C-NMR) and UPLC-
Q-TOF-MS analysis revealed that the Penicillium purpurogenum Li-3 red pigment (RPs) was monascus-like
pigment and its molecular weight was 439.1997. Moreover, the red pigment was shown to be weak cytotoxic
against the zebrafish embryos. The yield of the red pigment increased 69 % under optimized culture conditions.
These outstanding properties will enlarge the application of RPs for natural food additives, new antioxidant and
antibacterial drug development.

Keywords: Penicillium purpurogenum, monascus-like pigments, antioxidant activity, antibacterial activity,

biological activity, zebrafish.

Introduction
Pigments play an important role in many industries.
Natural pigments are widely used as additives, color
intensifiers, antioxidants and antibiotics. Most of the
natural pigments are extracted from plants, insects,
animals and microbes.[1] Biopigments are renewable
biological resources and environmental friendly, be-
cause they do not create any environmental problems
and maintains ecological balance. Natural pigments
with the characteristic of chemical stability, chemically
diverse and rich colors are produced by filamentous
fungi.[2] Moreover, natural pigments such as, anthra-
quinone, flavonoid, carotenoids, etc., produced from
microorganism, have pharmaceutical applications and
exhibit several beneficial biological activities, e. g.,
antioxidant,[3] antimicrobial,[4] anticarcinogenic,[5] anti-
inflammatory,[6] antiobesity, anti-angiogenic and neu-
roprotective.[7] Therefore, the utilization of natural
pigments with medicinal values in foodstuff has
aroused people’s concern in recent years.
Pigments produced by Monascus spp. were demon-
strated to have physiological activities and have been
legally used as food colorants in South East Asia for
more than thousand years.[8] However, monascus pig-
ments are not approved as food colorants both in the
European Union (EU) and the United States (US),[9]
mainly due to the risk of the possible contamination
by the nephrotoxic and hepatotoxic metabolite cit-
rinin.[10] In the literature, Penicillium purpurogenum has
been found to produce monascus pigment homologs
in culture with a specific medium. Ogihara reported
that Penicillium purpurogenum IAM15392 produced PP-
O ((10Z)-12-carboxymonascorubrin), PP-Y ((10Z)-mon-
ascorubrin),[11] PP-V ((10Z)-12-carboxymonascoru-
bramine) and PP-R ((10Z)-7-(2-hydroxyethyl)monasco-
rubramine) in the medium under specific conditions,

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 Chem. Biodiversity 2018, 15, e1800300

and these homologs of monascorubramine were health and pharmaceutical applications. In short, a
synthesized via the biosynthetic pathway common to potential source of nature pigment, which is biode-
a series of Monascus pigments, and their biological gradable and ecofriendly/non-toxic, has antimicrobial
activities were not studied.[12] In a study performed by and antioxidant properties and meets the various
Teixeira, P. purpurogenum DPUA 1275 showed poten- needs of food additive, cosmetics and pharmaceutical
tial to produce natural colorants with the antimicrobial drug applications, is worth investigating.[19]
activities and absence of toxicity to brine shrimp
Artemia salina.[13] Furthermore, the studies found in
the literature confirmed that Penicillium spp. had no Results and Discussion
toxic effects and their pigments were biodegrad-
UPLC Q-TOF-MS Analysis
able.[14] Recently, Penicillium purpurogenum and re-
lated species, such as P. citrinum, P. expansum and P. The LC/MS technique can quickly afford the molecular
verrucosum, have also been reported to produce weight and even the molecular formula of unknown
citrinin.[15] Chai isolated four kinds of secondary complex compounds. The compound at 6.09 min in
metabolites from two mutant Penicillium purpuroge- the UPLC chromatography of the red pigment (Fig-
num G59 strains, including citrinin.[16] ure 1A) corresponded to the peak in the total ion
Currently, the toxicity evaluation of food pigment
was mostly dominated by the acute toxicity in mice
and less use of zebrafish as a model to evaluate the
safety of natural pigments.[17] Zebrafish is a small
freshwater fish and has become one of the most
popular model organisms in toxicity assessment.
Owing to the transparency and rapid development of
the eggs, and several observable lethal and sub-lethal
endpoints during the exposure, zebrafish embryos
have become the promising models for testing
chemicals. In addition, zebrafish and human genome
has a high homology, this means that the test results
are applicable to humans. To understand the toxic
effects of nitrite on human heart, Li exposed zebrafish
embryos to different concentrations of sodium nitrite,
their results showed that sodium nitrite caused the
developmental defects of zebrafish heart dose-de-
pendently.[18]
In recent years, Penicillium spp. pigments have
attracted the attention of researchers. Many literatures
reported the optimization fermentation condition, pig-
ment purification, structural identification and stability
of Penicillium spp. red pigments, little research had
been published about the security, antimicrobial and
antioxidant activity of Penicillium spp. pigments. In this
study, HPLC/MS was used to check the safety of the
pigment extract. The zebrafish embryo test was used
to assess the toxicity of P. purpurogenum Li-3 pig-
ments. Fertilized zebrafish eggs were exposed to both
the Penicillium spp. pigments and carmine in order to
Figure 1. A) UPLC chromatogram (210–600 nm), B) total ion
evaluate its safety. Herein, the objective of this work chromatogram (m/z 50– 1500, ESI (pos.)), C) ESI-MS (pos.)
was not only to introduce the chemical composition of spectrum and D) UV/VIS spectrum of the red pigment (PP-Z).
the red pigments but also to evaluate the security and
biological activity of them. These results will provide
theoretical guidance for the P. purpurogenum strains
pigments for potential applications in cosmetics,

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 Chem. Biodiversity 2018, 15, e1800300
chromatogram (TIC; Figure 1B) at 6.14 min. The red
pigment (PP-Z, cf. Figure 3) was detected at m/z
440.2077 ([M + H] +) and confirmed by the adduct ion
peak at m/z 472.2326 ([M + H + MeOH] +). The accurate
mass of PP-Z (439.1997 [M + H] +) was acquired by Q-
TOF-MS, from which the elemental composition could
be deduced (C25H30NO6).
The UV/VIS spectrum of PP-Z was illustrated in
Figure 1D, and the main peaks (λmax) were observed at
223, 259, 322, 398 and 502 nm. There was a strong
absorption peak beyond 300 nm, which indicated that
the structure of PP-Z contains a large conjugated
system, and by comparing with the monascus pig-
ment, they have the same characteristic of UV
absorption.[20] Polyketides contain undelocalized neg-
ative δ electrons due to the unsaturated functional
groups, for example, COOH, COO and CONH, which
showed the specific UV/VIS spectra.[21] Moreover, three
absorbance peaks (223, 259, 322 nm) of PP-Z in the UV
spectrum were very similar to those of the N pigment
monascorubrin (200, 260, 320 nm),[22] indicating that
the chromophore of PP-Z was same as that of the N
pigment monascorubrin. It can be deduced that the
red pigment produced by this P. purpurogenum Li-3
was either likely to be the monascus pigments or had
the similar chromophores as the monascus pigments.
FT-IR Analysis
The FT-IR spectroscopy provides the information on
the main functional groups in the pigment structure.
The main functional groups in the structure of PP-Z
produced by P. purpurogenum Li-3 could be illustrated
from Figure 2. The peak of N H stretching vibration
Figure 2. FT-IR spectrum of the red pigment (PP-Z).
was at 3451 cm 1.[23] The absorption peaks at
1469 cm 1, 2847 cm 1 and 2920 cm 1 indicated a long
CH2 chain in the molecular structure.[24] The absorp-
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Figure 3. Structures of PP-R and PP-Z.
tion peaks at 1021 cm 1, 1632 cm 1 and 1658 cm 1
were attributed to the stretching vibrations of C O,
C=C and C=O,[25] respectively. Therefore, a long CH2
chain and the moieties of C=O, C O, C=C and N H
were indicated in the structure of PP-Z (PP-Z).
NMR Analysis
The NMR spectra of PP-Z, integrated with the UV/VIS,
IR, and UPLC Q-TOF/MS data, implied that the
structure of PP-Z was similar to that of PP-R.
PP-R is one of the homolog of major monascus
pigments. Compared with the 13C-NMR data of PP-R
(Table 1), those of PP-Z showed that the carbon atoms
of C2 C12 had the similar chemical shifts to those of
PP-R. The long-chains and the heterocyclic moieties of
the two compounds are almost identical, so it could
be inferred that they have the same skeletal structure.
By comparing their 1H-NMR data, it could be also
found that PP-Z had two less CH2 signals and one less
OH signal than PP-R, but had an additional MeO signal
(δ(H) 4.21 (s, 3 H)). For the 13C-NMR data, the signal at
δ(C) 60.7 ppm should be attributed to the MeO group,
and that at δ(C) 170.1 ppm should be attributed to the
C(1’) = O group. Therefore, it could be inferred that PP-
Z had an ester group. On the basis of the data
mentioned above, the structure of the red pigment
was determined as shown in Figure 3, and named PP-
Z.
DPPH Radical Scavenging Capacity of the Pigments
In this work, the antioxidant properties of the red
pigments were evaluated by the DPPH assay, a widely
used method to estimate the antioxidant activities in a
relatively short time compared with other methods.
Figure 4 showed that the free radical scavenging
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 Chem. Biodiversity 2018, 15, e1800300

Table 1. 1H- and 13C-NMR data for PP-Z and PP-R (600 MHz, CDCl3, δ in ppm, J in Hz).
Position PP-Z PP-R
δ(H) δ(C) δ(H) δ(C)
2 171.6 171.4
3 100.1 104.5
3a 166.7 173.9
4 6.50 (s, 1 H) 96.1 6.66 (s, 1 H) 97.2
4a 148.4 151.0
5 6.69 (s, 1 H) 117.2 6.80 (s, 1 H) 120.9
6 147.3 147.9
8 8.19 (s, 1 H) 142.8 8.15 (s, 1 H) 144.0
8a 111.3 117.8
9 202.1 195.3
9a 86.1 85.9
9a-Me 1.69 (s, 3 H) 31.8 1.61 (s, 3 H) 30.5
10 6.37 (d, J = 7.5, 1 H) 115.5 6.50 (d, J = 11.6, 1 H) 122.6
11 6.24 (d, J = 7.5, 1 H) 129.9 6.30 (qd, J = 11.6, 7.1, 1 H) 137.1
12 1.98–2.01 (m, 3 H) 17.2 1.92–1.95 (m, 3 H) 15.2
13 192.9 195.9
14 2.75 (t, J = 4.0, 2 H) 43.0 2.80 (t, J = 7.4, 2 H) 41.0
15 1.60–1.63 (m, 2 H) 22.6 1.56 (quint., J = 7.4, 2 H) 25.3
16 1.02–1.41 (m, 8 H) 29.5 1.28–1.33 (m, 8 H) 30.2
17 1.02–1.41 (m, 8 H) 27.2 1.28–1.33 (m, 8 H) 30.0
18 1.02–1.41 (m, 8 H) 35.1 1.28–1.33 (m, 8 H) 32.5
19 1.02–1.41 (m, 8 H) 20.1 1.28–1.33 (m, 8 H) 23.3
20 0.85 (t, J = 8.1, 3 H) 14.1 0.88 (t, J = 7.1, 3 H) 14.3
1’ 170.1 4.26–4.28 (m, 2 H) 57.2
2’ 60.7 3.90 (q, J = 5.5, 2 H) 61.1
OH 4.42 (t, J = 5.5, 1 H)
MeO 4.21 (s, 3 H)

BuOH extracts and the crude red pigment were 0.44,

0.50 and 1.17 mg/mL, respectively. These data had no
statistically significant difference. The results showed
that the AcOEt and BuOH extracts might have some
constituents which possessed better DPPH-scavenging
activity than the crude red pigment. However, the
DPPH radical scavenging effect of the AcOEt fraction
was slightly weaker than that of ascorbic acid (Vc) at
the same concentrations. The above results suggested
that the compounds with relatively high antioxidant
activities might be present in the AcOEt fraction.

Figure 4. Free radical scavenging activity of various fractions.

Data points represent the mean � standard deviation for three
replicates.
activity increased with the increasing concentration of
various fractions, indicating that the components
present in the broth were electron donors and could
react with the free radicals and convert them into
more stable compound to terminate the radical chain
interaction. The EC50 values of the AcOEt extract, the
Antimicrobial Activity of the Pigments
With respect to the antimicrobial activity of RPs, the
filter paper method was used to test the effects on
bacteria based on the diameter of inhibition zones,
and the results are listed in Table 2.
It was observed that various fractions showed the
antibacterial activity against Staphylococcus aureus
(Table 2). However, they showed no inhibitory effect
on E. coli. It was apparent that the pigments were toxic
against the Gram-positive strains, while the maximum

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Table 2. Antimicrobial activity of different fractions.
Bacteria Zone of inhibition
BuOH AcOEt Crude Chloramphenicol
Fraction Fraction pigment
E. coli – – – +++
(27 mm)[a]
S. aur- ++ +++ +++ +++
eus (6 mm)[a] (11 mm)[a] (9 mm)[a] (25 mm)[a]
[a]
Excluding the diameter of the well which is 6 mm.
inhibition (11 mm) was showed against Staphylococcus
aureus. This result was consistent with that of
Mukherjee et al., who reported that the purified red
fraction was active only against the Gram-positive
bacteria tested.[23] These results might suggest that
the RPs could be developed as antibiotic for Staph-
ylococcus aureus. The selective antibacterial activity
might be due to several factors,[26] including the
charge density, the structure of lipopolysaccharides
and the lipid composition of the cytoplasmic mem-
brane in Gram-negative and Gram-positive bacteria.[27]
At the same time, this kind of red pigment might be
effective on the surface of microbial cells, which could
destroy the cell walls and achieve to inhibit bacteria.[28]
The antibacterial activity of RPs could broaden the
potential applications of RPs.
HPLC Analysis of Citrinin
The samples of citrinin standard solution and the
fermented broth were analyzed with HPLC/MS. The
TICs of citrinin standard solution and the broth were
recorded, and citrinin could be determined by compar-
ing the extracted ion chromatograms (EICs) and the
corresponding retention times of the two solutions.
The EIC showed the relationship between the ionic
strength of mass (or m/z) and the retention time.
When the sample concentration was very low or the
sample was very complex, it was difficult to find the
target peak in the TIC. Therefore, based on the target
molecular weight and the M + 1 values, the EIC could
be observed and the target was determined if there
was a peak and its retention time was consistent with
that of the citrinin standard solution. EIC is the most
useful way to analyze complicated mixtures and trace
complex.
As shown in Figure 5E, the peak at tR 6.70 min was
attributed to citrinin. The MS spectrum of citrinin
(Figure 5E1) showed the [M + H] + peak at m/z 251.10,
indicating the molecular weight (Mr 250) of citrinin. In
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Figure 5G, the TIC of the purple broth was complicated,
and most of the peaks were not totally separated, and
thus, EIC was necessary. In Figure 5H, there was a peak
at m/z 251.10 with tR 5.12 min. Because there was no
peak at tR = 6.70 min, citrinin was not found in the P.
purpurogenum Li-3 broth.
Acute Toxicity Effect of PP-Z
To evaluate the possible toxicity of PP-Z and carmine
to the zebrafish embryos, the mortality was deter-
mined at 96 hpf (hours post-fertilization). During the
experimental period, the zebrafish embryos developed
normally and the mortality was about 0 –5.0 % in two
controls, i. e., reconstituted water and 0.3 % ethanol.
There was no significant difference in the mortality at
these concentrations. As shown in Figure 6, the mortal-
ity of sample (PP-Z and carmine) after 96 hpf exposure
was 12.8 % and 17.9 %, respectively. They displayed
very weak toxicity toward the zebrafish embryos with
lethality below 50 %.
Carmine, one kind of synthetic pigments, belong-
ing to single azo compounds, is currently most widely
used in China in food, beverages, pharmaceuticals,
cosmetics, tobacco, toys, food packaging materials
and in many more applications. According to GB2760-
19966 in China, the limit of usage of carmine in food is
lower than 0.05 g/kg. By comparing the LC50 values, it
was found that both PP-Z and carmine were weakly
toxic to the zebra fish embryos and PP-Z was safer
than carmine, so the maximum usage of PP-Z could be
referred to that of carmine in food, which indicated
that the P. purpurogenum Li-3 red pigment could be a
potential alternative pigment in commerce.
Pigment Optimization
The UV/VIS spectrum of the crude red pigment was
illustrated in Figure 7. It can be seen that the main
absorption of the red pigment was at 500 nm. There-
fore, the yield of the red pigment can be determined
by measuring the absorbance value at 500 nm.
As shown in Figure 8, different media had a huge
impact on the production of the red pigment. In the
CZ (Czapek Dox Liquid) medium, there was a little
production of the red pigment, but in the PDB
medium, the yield of the red pigment was high and
much higher than those under the YPD (Yeast Extract
Peptone Dextrose) and SDAY culture conditions. So,
the PDB medium was chosen as the fermentation
medium.
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 Chem. Biodiversity 2018, 15, e1800300

Figure 5. E), E1) TIC of citrinin standard, F) EIC of citrinin standard, G) TIC of the fermentation broth and H) EIC of the fermentation
broth.

Figure 6. The lethal toxicity at 96 h caused by carmine and PP- Figure 7. UV/VIS spectrum of the crude red pigment.
Z at various concentrations. Data points represent the mean �
standard deviation for three replicates.

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 Chem. Biodiversity 2018, 15, e1800300
Figure 8. Absorbance values (at 500 nm) of the red pigment in
different media. Data points represent the mean � standard
deviation for three replicates.
Both too low and too high speeds reduced the
yield of the red pigment, the highest yield of the red
pigment was at the speed of 150 r/min (Figure 9A). It
showed that the excessive shear force generated by
the excessive shaking speed of the shaker would cause
the damage to the hyphae and reduce the yield of the
pigment.
The previous work of the research group showed
that the suitable temperature for the growth of P.
purpurogenum was 32 °C.[29] Therefore, in the culture
process, under the premise that other conditions were
unchanged, the first stage was at 32 °C for 50 h to
allow the cells to grow rapidly, and the second stage
changed the culture temperatures to 16, 20, 24 and
28 °C to investigate the effect of temperature on the
yield of the red pigment. The results were shown in
Figure 8B and 8C, compared with the constant temper-
ature culture at 28 °C, the two-stage temperature-
controlled culture (the temperature of the first culture
stage was 32 °C, then the temperature was changed to
20 °C) afforded the higher yield of the red pigment.
Figure 9. Absorbance values (at 500 nm) of the red pigment A) at different speeds, B) at constant temperature or varying
temperature and C) at different temperatures. Data points represent the mean � standard deviation for three replicates.
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The optimal process conditions for the production
of the red pigment from P. purpurogenum Li-3 were
determined: PDB was used as the medium, the speed
was at 150 r/min, the culture was first cultured at 32 °C
for 50 h, and then, at 20 °C for 118 h. After the
repeated experiments, the yields of the red pigment
were stable. The final yield of the red pigment was
2.89 UA500 nm and was 69 % higher than that before.
Conclusions
The P. purpurogenum Li-3 red pigment and the
monascus pigment with the similar polyketide skel-
eton were a kind of polyketide-monascus-like pig-
ments. We believe that the production of the polyke-
tide azaphilone pigments from such potentially safe
hosts is advantageous over the traditional processes
that involve Monascus spp., which risks the co-
production of the mycotoxin citrinin. Thus, the
Penicillium spp. strains have a potential to substitute
Monascus spp. to produce a new generation of natural
functional food ingredients with bioactive properties.
These results provide the reference data for this
pigment that can be used in cosmetics, pharmaceut-
icals and food additives industries.
Experimental Section
Microorganisms
The P. purpurogenum Li-3 strains were obtained from
the Laboratory of Microecology and Biotransformation,
Beijing Institute of Technology, Beijing, China. The
cultures preserved in distilled water were reactivated
in Potato Dextrose Agar (PDA) and maintained at 32 °C
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 Chem. Biodiversity 2018, 15, e1800300
for 3 d. After this period, the cultures were kept in the
refrigerator at 4 °C.
Pigment Fermentation
The experiments were conducted in 250 mL Erlen-
meyer flasks containing 100 mL PDB fermentation
culture medium (potato 200 g, glucose 20 g, per liter
of sterile water). The culture medium was inoculated
with 1 % (v/v) of the seed culture (glucose 2 g, NaNO3
3 g, K2HPO4 1 g, KCl 0.5 g, MgSO4 · 7 H2O 0.5 g,
FeSO4 · 7 H2O 0.01 g, per liter of sterile water) and
incubated up to 7 d with the agitator speed of
150 rpm.
Extraction of Pigments
After the termination of incubation, the biomass was
separated from the fermented broth by filtration with
a filter paper, and the bright red supernatant contain-
ing the pigment was separated. The crude red pig-
ment sample was obtained on a rotary evaporator at
50 °C. Furthermore, the crude red pigment was
extracted with AcOEt and BuOH. The fractions were
concentrated under reduced pressure at 50 °C to
remove the solvents. The yields of the AcOEt and
BuOH fraction were 0.231 g/L and 0.235 g/L, respec-
tively. The organic extracts (AcOEt and BuOH) and the
crude pigment were tested for biological activity.
Purification of Pigments
The fermentation broth obtained by filtration was
extracted three times with 150 mL (each) of AcOEt,
and then, the resulting extracts were concentrated by
using a vacuum rotary evaporator under reduced
pressure at 50 °C. The residue was chromatographed
by silica gel column chromatography (50 cm × 3 cm,
AcOEt/MeOH 1:0, 50 : 1, 30 : 1 (v/v)). The collected red
fractions were examined by preparative thin-layer
chromatography (TLC). The target band (Rf = 0.40) was
scrapped off, suspended in MeOH, and separated from
silica gel through filtration. The filtrate was concen-
trated to dryness at 50 °C under reduced pressure on a
rotary evaporator to afford the purified red pigment.
Chemical Composition and Structural Analysis
The LC/MS spectra were recorded on ACQUITYTM
UPLC-Q-TOF PremierTM (Waters, Milford, MA, USA)
equipped with an electrospray ionization ion source
(ESI). The analytical column was ACQUITY UPLCTM BEH
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C18 (100 mm × 2.1 mm, 1.7 μm, Waters, Milford, MA,
USA). The mobile phase consisted of A (0.1 % formic
acid in water) and B (acetonitrile), and the gradient
elution was programmed as follows: 0 –1.5 min, 5–
10 % B (v/v); 1.5 –3.5 min, 40 % B; 3.5 –5.0 min, 60 % B;
5.0 –7.0 min, 85 % B; 7.0 –11.0 min, 100 % B; 11.0 –
11.5 min, 5 % B; 11.5 –13.0 min, 5 % B. The flow rate
was 0.4 mL/min. The column was held at 45 °C and the
injection volume was 2 μL. The mass spectrometer was
operated in positive ion mode with the capillary
voltage of 2.5 kV, the sampling cone voltage of 35 V,
the cone gas flow of 50 L/h, the desolvation gas flow
of 800 L/h, the desolvation temperature of 350 °C, the
source temperature of 120 °C and the collision energy
of 5.0 V. The mass spectra were collected at a rate of 1
spectra/s and the inter-scan delay was 0.02 s. The Q-
TOF instrument was operated in full scan-survey
mode. The full scan spectra from 50 to 1500 Da were
acquired.
The UV/VIS spectrum was obtained from the UV/VIS
chromatogram after background subtraction. The IR
spectrum was recorded on a FT-IR spectrometer at
400 –4000 cm 1 in dry methanol solutions. The NMR
spectrum was recorded in deuterated chloroform. The
structure elucidation was carried out on the basis of
the UV/VIS, IR, NMR and mass spectra.
Antioxidant Activity of Pigments
The free radical scavenging activity of the extracts
were measured by 1,1-diphenyl-2-picrylhydrazyl
(DPPH) using the method of Musa.[30] One milliliter of
the samples was added to 4 mL of 0.1 mM DPPH
ethanol solution. The mixtures were shaken vigorously
and incubated at room temperature for 30 min. The
mixture of distilled water and the DPPH solution
served as a blank. A sample control was prepared by
replacing the DPPH solution with ethanol. Then, the
absorbance was measured on a UV/VIS spectropho-
tometer (Agilent carry 60) at 517 nm. The results were
expressed as the percentage of reduction of the initial
DPPH radical absorption by the pigments. L-Ascorbic
acid was used as a positive control, and the percent-
age of DPPH scavenging activity was calculated as
follows:
DPPHSCAV ð%Þ ¼ ½1 ðAsample Acontrol Þ=Ablank � � 100
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 Chem. Biodiversity 2018, 15, e1800300
Antimicrobial Activity of Pigments
The antibacterial activities were evaluated against two
pathogenic bacterial species, Escherichia coli
ATCC8739 and Staphylococcus aureus ATCC29213. The
bacterial strains were cultured in Luria Bertani (LB)
medium supplemented at 37 °C for 18– 24 h with a
load of 108 CFU mL 1. The culture (33 μL) was spread
on a nutrient agar plate by the spread plate method.
The crude extract of the red pigment, the AcOEt
extract of the red pigment and the BuOH extract of
the red pigment at a concentration of 3.5 mg/mL were
used in the antimicrobial activity test. The antibacterial
activity was checked by the filter paper method as
reported by Ma.[31] Sterile water and the same
concentration of chloramphenicol was used as a
control. After the incubation, the inhibition zone (mm)
around the filter was recorded.
HPLC/MS Condition for Citrinin Detection
Sample Pretreatment. The fermentation broth with
mycelium (10 mL) and ethanol (20 mL) was added in a
50 mL centrifuge tube, kept at 60 °C for 1 h in a water
bath, and then centrifuged for 15 min at 3000 r/min.
The fermented broth was obtained after filtering by a
filter membrane (0.45 μm). The citrinin methanol
standard solution of 25 mg/L was prepared for the
HPLC/MS analysis.
The HPLC/MS/MS spectra were recorded on Agilent
1260/Waters equipped with an electrospray ionization
ion source (ESI). The analytical column was Waters C18
(2.1 mm × 100 mm, 2.7 μm). The mobile phase con-
sisted of A (0.1 % formic acid in water) and B (0.4 %
formic acid in acetonitrile), and the gradient elution
was programmed as follows: 0–8 min, 95.0 %–0.0 % A;
8 –11 min, 0.0 % A; 11 –14 min, 0.0 –95.0 % A; 14–
15 min, 95.0 % A. The flow rate was 0.250 mL/min,
while the column temperature was set at 35 °C. High
purity nitrogen was used as the nebulizer and auxiliary
gas. The mass spectrometer was operated in positive
ion mode with the capillary voltage of 4 kV, the
desolvation temperature of 320 °C, the source temper-
ature of 300 °C, the collision voltage of 4.0 V, and the
capillary lens voltage of 65 V.
Acute Toxicity of Pigments
The control condition of zebrafish was as follows:
temperature (26.0 � 1.0 °C), hardness (250 mg/L), pH
(7.5 � 0.5), dissolved oxygen (10.5 � 0.5 mg/L = 95 %
www.cb.wiley.com (9 of 11) e1800300
saturation) and light/dark cycle (14 : 10 h).[32] The
spawning and fertilization took place within 30 min
after the lights were turned on in the morning. The
eggs were transferred onto Petri dishes and rinsed
several times with reconstituted water.[33] Then, the
healthy and normally developed eggs were selected
for the following exposure test.
The exposure experiment was initiated at one hour
post-fertilization (hpf). Then, the P. purpurogenum Li-3
red pigment (RPs) and carmine were dissolved in
1.0 mL ethanol (i. e., 1000 mg/mL), and the samples
were stored at 4 °C until testing. The maximum
concentration of ethanol was 0.3 % (v/v) in the test
assay. The exposure concentrations of RPs and carmine
were set at 0, 1, 6.25, 12.5, 25, 50 and 100 mg/L. the
embryos were cultured in an incubator at 28 � 0.5 °C
during the 96 hpf exposure experiment. The death
rate of the zebrafish embryos at 96 hpf was recorded.
The photographs at each stage of development were
taken using a microscope (Nikon TS100, Japan).
Pigment Optimization
The red pigment metabolized by P. purpurogenum Li-
3 in PDB medium for 7 d was quantitatively deter-
mined using an Agilent Carry 60 UV/VIS spectropho-
tometer. After diluting the fermentation broth with
distilled water for a certain volume, the diluted
pigment fermentation broth was spectrally scanned at
390 –700 nm.
Four Different Media. YPD (yeast 5 g, glucose
20.0 g, K2HPO4 3.0 g, MgSO4 1.5 g, per liter of sterile
water); PDB (potato 200 g, glucose 20 g, per liter of
sterile water); CZ (NaNO3 3.0 g, K2HPO4 1.0 g,
MgSO4 · 7 H2O 0.5 g, KCl 0.5 g, FeSO4 · 7 H2O 0.01 g,
sucrose 30.0 g, per liter of sterile water); SDAY (glucose
40.0 g, yeast 10 g, peptone 10 g, per liter of sterile
water). After 7 d of incubation, the yield of the red
pigment was determined by a UV/VIS spectrophotom-
eter.
Effect of Rotation Speed on the Production of the
Red Pigment. The liquid fermentation was carried out
at a rotational speed of 0, 120, 150, 180 and 210 r/min.
The absorbance value of the red pigment was
measured after 7 d of fermentation, and each treat-
ment was repeated three times.
Effect of Temperature on the Production of the Red
Pigment. The experiment adopted a two-stage tem-
© 2018 Wiley-VHCA AG, Zurich, Switzerland
 Chem. Biodiversity 2018, 15, e1800300
perature-controlled culture method. In the first stage,
the cells were cultured for 50 h at 32 °C for the rapid
growth. The temperature was changed to 16, 20, 24
and 28 °C in the second stage. The absorbance value
of the red pigment was measured after 118 h of
fermentation, and each treatment was repeated three
times. At the same time, the effects of constant
temperature and varying temperature on the yield of
the red pigment of P. purpurogenum were compared.
Statistical Analyses
The data were analyzed using SPSS and presented as
means with standard errors (mean � SE). The differ-
ences between the control and the treatment groups
were determined using one-way analysis of variance
(ANOVA). A p value of < 0.05 was considered statisti-
cally significant.
Acknowledgments
This work was financially supported by the grants from
the National Natural Science Foundation of China
(21146012, 21266029) and the Natural Science Foun-
dation of Zhejiang Province (LY17B060011).
Author Contribution Statement
The authors state that all procedures were approved.
Hong Cao participated sufficiently in the study con-
ception and design, data analysis and interpretation,
drafting and revision of manuscript. Yu-Jing Niu, Xin
Zhang and Hong-Jie Jin, as post graduates, carried out
in the specific experimental study. Chun Li and Hong
Liu participated in the study conception and design,
revision of manuscript. Each author takes the responsi-
bility for the validity and objectivity of the entire
study.
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Received June 25, 2018
Accepted September 17, 2018
© 2018 Wiley-VHCA AG, Zurich, Switzerland