Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199

Contents lists available at ScienceDirect

Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Penicillium brevicompactum as a novel source of

natural pigments with potential for food
applications

Carla S. Fonseca a,b , Nuno R. da Silva a,b,c , Lina F. Ballesteros a,b ,

Bruna Basto a,b, Luís Abrunhosa a,b, José A. Teixeira a,b, Sara C. Silvério a,b,∗
a CEB-Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
b LABBELS–Associate Laboratory, Braga, Guimarães, Portugal
c DWI-Leibniz Institute for Interactive Materials, Forckenbeckstrasse 50, 52074 Aachen, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Synthetic colorants have gradually been replaced by natural pigments, mainly in the food
Received 12 November 2021 industry. Among the several natural sources of pigments, filamentous fungi have gained
Received in revised form 5 January special attention. However, the eventual biosynthesis of mycotoxins is a limitation for the
2022 practical use of this kind of microorganisms as pigment producers. In this work, Penicil-
Accepted 19 January 2022 lium brevicompactum was studied for the first time as a potential pigment producer using
Available online 21 January 2022 submerged fermentation. The effect of several experimental parameters (culture medium
composition, agitation speed, type of carbon source, temperature, concentration of supple-
Keywords: ments and carbon source, natural light and initial pH) in the production of pigments was
Food colorants evaluated. Under the optimal conditions (culture medium I containing lactose (20 g/L) and
Phenolic compounds peptone/yeast extract (8/8 g/L), 23 ◦ C, initial pH 7.0, 150 rpm, and natural light), a mixture
Antioxidant activity of pigments (yellow, orange, and red) was obtained. This mixture is mycotoxin-free (ochra-
Colour stability toxin A and mycophenolic acid) and presented promising antioxidant activity (FRAP: 58.58
Mycotoxins ± 4.58 ␮mol Fe(II)/g; DPPH IC50 : 18.48 ± 0.26 ␮mol TE/g and ABTS IC50 : 28.38 ± 3.79 ␮mol
TE/g). Furthermore, these pigments were proved to be very stable for a wide range of pH and
temperatures. Overall, it was demonstrated that P. brevicompactum can be a safe source of
natural pigments with interesting properties for the food industry.
© 2022 The Authors. Published by Elsevier Ltd on behalf of Institution of Chemical
Engineers. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Colour is considered a crucial feature in marketing and advertising
since it can have a remarkable psychological impact on people, pro-
moting a wide range of reactions and emotions (You et al., 2019). Visual
appearance, which mainly includes colour and form, is recognized as a
key property for consumers and it can determine the acceptance or not
of a novel commercial product (MacDougall, 2003). In the food indus-
try, the use of colorants has generally three main purposes: to make
products more attractive, to control colour loss during processing and
storage, or to provide colour to colourless products (Lehto et al., 2017).
The use of synthetic pigments is well-established and regulated in the
food industry. Nevertheless, its production commonly presents recog-
nized environmental issues. Furthermore, some synthetic pigments
have been connected to carcinogenicity and toxicological effects on
human health. Therefore, the gradual substitution of these compounds
by alternative pigments obtained from natural sources has gained spe-
cial attention in the last decade. The increased interest for natural

Abbreviations: ABTS, 2,2 -azino-bis3-ethylbenzthiazoline-6-sulfonic acid diammonium salt; DPPH, 2,2-diphenyl-1-picrylhydrazyl;

FRAP, ferric reducing antioxidant power; HPLC, high-performance liquid chromatography; IC50 , inhibition concentration at 50%; MPA,
mycophenolic acid; MUM, Mycology collection of University of Minho; OTA, ochratoxin A; PEC, pigments crude extract; TLC, thin layer
chromatography; UHPLC, ultra-high-performance liquid chromatography.
∗
Corresponding author.
E-mail address: sarasilverio@deb.uminho.pt (S.C. Silvério).
https://doi.org/10.1016/j.fbp.2022.01.007
0960-3085/© 2022 The Authors. Published by Elsevier Ltd on behalf of Institution of Chemical Engineers. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199
pigments is mainly based on sustainability, green economy policies,
and changes in the consumers’ demand. Additionally, natural pigments
can also present advantageous biological properties such as antioxi-
dant, anti-inflammatory, anticancer, and antimicrobial activities (Tuli
et al., 2015). In 2017, the global market for natural pigments was valued
at 1180 million USD, and it is estimated to increase up to 1620 mil-
lion USD in 2023. Food and beverages are among the most important
applications for natural pigments (MarketWarch, 2020).
Plants are important sources of natural pigments. However, they
are also very dependent on seasonality, geographical conditions, and
climate issues (Babitha, 2009). Microorganisms are considered better
sources of natural pigments due to their non-seasonality, high avail-
ability, and easy scalability. Some microbial pigments obtained through
fermentative processes involving fungi are already available in the
market and allowed for food applications. Several Monascus pigments,
riboflavin from Ashbya gossypii, Arpink redTM from Penicillium oxalicum
or ␤-carotene from Blakeslea trispora can be found among the com-
mercial fungal pigments (Copetti, 2019). Different colours are usually
reported for fungal pigments, depending on the chemical structure of
the molecules. Nevertheless, the most common pigments are typically
red, orange or yellow (Caro et al., 2015). The disadvantages gener-
ally reported for microbial pigments include unsatisfactory production
levels and potential contamination with secondary toxic metabolites.
Optimization of the culture medium composition and other experi-
mental conditions is generally carried out to improve the production
yields (Hernández et al., 2019). On the other hand, to avoid the unde-
sirable production of toxins, a careful search and selection of safe
microorganism strains should be performed. Penicillium strains have
been recently identified as suitable producers of well-known Monascus-
like pigments without the detrimental synthesis of the toxin citrinin
(Afshari et al., 2015; Pattanagul et al., 2008). In this work, Penicillium bre-
vicompactum was studied as a novel source of safe and stable natural
pigments for food applications. A detailed study involving a conven-
tional one-at-a-time variation of experimental factors was carried out
to identify the parameters with significant impact and improve the
production of pigments under submerged fermentation conditions.
Additionally, the obtained crude extract containing the pigments was
partially characterized.
2. Materials and methods
2.1. Chemicals
All the chemicals and media components used in this work
were of analytical grade and obtained from Sigma-Aldrich.
2.2. Fungal strain
P. brevicompactum (MUM 02.07) was obtained from MUM
(Mycology collection of University of Minho). Spore suspen-
sions for stock cultures were prepared in semi-solid agar
medium (0.2% w/v) and stored at room temperature. Before
each fermentation, the microorganism was grown at 25 ◦ C for
7–10 days on agar plates containing (% w/v): malt extract (2),
glucose (2), peptone (0.1), and agar (2).
2.3. Pigment production
Spore suspensions for inoculum were prepared in sterile
saline solution 0.85% (w/v) NaCl containing 0.01% (w/v) Tween
80. The conidia density was adjusted to 106 conidia/mL. All
fermentations were performed in triplicate using a 250 mL
flask containing 50 mL of culture medium. The effect of several
fermentation conditions on pigments production was studied
for 12 days, namely culture medium composition, agitation
speed, type and concentration of carbon source, temperature,
initial pH, supplements concentration, and light. The detailed
189
conditions used in each study are described in Table 1. The pro-
duction of pigments was spectrophotometrically monitored
after 3, 5, 7, 10, and 12 days of fermentation, using a microplate
reader (Cytation3, BIOTECH). The absorbance was measure-
ment at 400, 470, and 500 nm, corresponding to the yellow,
orange, and red regions of the visible spectrum, respectively
(Pattanagul et al., 2008; Srianta et al., 2017). To determine the
best conditions for pigments production, both the sum of the
absorbances obtained for the three wavelengths and the time
(days) associated with the first observation of colour in the
fermentation medium were considered. The pigments crude
extract (PCE) obtained after 12 days of fermentation under the
optimal conditions (medium I, lactose (20 g/L), peptone/yeast
extract (8/8 g/L), 23 ◦ C, initial pH 7.0, 150 rpm, and natural
light) was filtered (0.2 ␮m membrane) to remove the biomass
and used for further characterization.
2.4. Biomass quantification
The fermentation broths were filtered through pre-weighed
filter paper (0.45 ␮m), and the biomass was conveniently
washed with distilled water. The mycelium retained by the
filter was dried to a constant weight (Saxena et al., 2015). The
concentration of biomass (Cbiomass , g/L) was obtained using Eq.
(1):
W(dried mycelium+filter paper) − W(filter paper)
Cbiomass (1)
V(fermentation broth)
where W(dried mycelium + filter paper) represents the total
weight of the paper filter containing the dried mycelium,
W(filter paper) corresponds to the pre-weighed filter paper
and V(fermentation broth) represents the volume of the filtered
fermentation broth.
2.5. Sugars quantification
Sugar concentration in the fermentation media was measured
by high-performance liquid chromatography (HPLC) according
to Cardoso et al. (2017). A calibration curve was previously pre-
pared with lactose, glucose, or sucrose standards in the range
of 0.5−20 g/L.
2.6. Analysis of mycotoxins
The production of mycotoxins, namely ochratoxin A (OTA) and
mycophenolic acid (MPA), was evaluated through HPLC. For
OTA analysis, the methodology described by Cardoso et al.
(2017) was used. A calibration curve was previously prepared
with OTA standards between 1−50 ng/mL.
For MPA analysis, ultra-high-performance liquid chro-
matography (UHPLC) was used according to Jesus et al. (2017).
A calibration curve was previously prepared with MPA stan-
dards (10−400 ␮g/mL).
2.7. Pigments analysis by thin layer chromatography
(TLC)
PCE was analysed by TLC according to (Hailei et al., 2011) with
some modifications. Briefly, PCE (20 ␮L) was loaded on a silica
plate (DC-Alufolien Kieselgel 60, Merck) and it was allowed
to separate with a mobile phase composed of water/ethanol
(50/50, v/v). After elution, the silica plate was visualized under
visible and UV light (254 and 366 nm).
190 Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199

Table 1 – Experimental conditions (fixed and variable) used for pigment production when different effects were evaluated.
Fixed conditions Variable conditions
◦
Lactose (20 g/L), 28 C, 150 rpm, Effect: culture medium composition
natural light Medium I1 (g/L): peptone Medium II2 (g/L): NaNO3 (3), Medium III (g/L):
(4), yeast extract (4), KH2 PO4 K2 HPO4 (1), MgSO4 ·7H2 O malt extract (20) and
(2), Na2 HPO4 ·12H2 O (8) and (0.5), KCl (0.5), FeSO4 ·7H2 O peptone (1)
MgSO4 ·7H2 O (0.25);
Medium I, lactose (20 g/L), 28 ◦ C, Effect: agitation speed (rpm)
initial pH = 7.0, natural light 0 150 200
Medium I, 28 ◦ C, initial pH = 7.0, 150 Effect: carbon source (20 g/L)
rpm, natural light Glucose Sucrose Lactose
Medium I, 28 ◦ C, initial pH = 7.0, 150 Effect: carbon source (20 g/L)
rpm, natural light Glucose Sucrose Lactose
o
Medium I, lactose (20 g/L), initial pH = Effect: temperature ( C)
7.0, 150 rpm, natural light 23 25 28
Medium I, lactose (20 g/L), 23 ◦ C, Effect: supplements (peptone/yeast extract (g/L))
initial pH = 7.0, 150 rpm, natural light 0/16 0/8 8/0 8/8 6/2 2/6 4/4 2/2
Medium I, lactose, peptone/yeast Effect: lactose concentration (g/L)
extract 8/8 (g/L), 23 ◦ C, initial pH = 7.0, 10 20 30
150 rpm, natural light
Medium I, lactose (20 g/L), Effect: natural light
peptone/yeast extract 8/8 (g/L), 23 ◦ C, Presence Absence
initial pH = 7.0, 150 rpm
Medium I, lactose (20 g/L), Effect: initial pH
peptone/yeast extract 8/8 (g/L), 23 ◦ C, 4.5 7.0 9.5
150 rpm, natural light

Fermentation media described by 1 (Nagy et al., 2001) and 2 (Méndez et al., 2011).

2.8. Stability studies at different pH and temperatures 2.9.3. Antioxidant activity

Mixtures of PCE and a buffer solution with the desired pH (1/1
v/v) were used for all the stability tests (Santos-Ebinuma et al.,
2013). The pH effect was studied in the range 3.0–10.0 by incu-
bating the mixtures at room temperature in the dark. For the
temperature studies, the mixtures were incubated between
5−85 ◦ C in the dark at pH 7.0. For both studies, the absorbance
was read at 400, 470, and 500 nm before and after 24 h of
incubation. All experiments were performed in triplicate.
Three different assays were used to determine the antioxi-
dant activity of PCE following the methodologies described by
Ballesteros et al. (2014, 2015).
2.9.3.1. Ferric reducing antioxidant power (FRAP assay).
According to Ballesteros et al. (2014), a calibration curve was
constructed using an aqueous solution of ferrous sulphate
(200−1000 ␮M), and the FRAP values were expressed as micro-
moles of ferrous equivalent per gram of lyophilised PCE (␮mol
Fe(II)/g).

2.9. Functional properties
After PCE lyophilisation, a 20 mg/mL filtrated solution (0.22
␮m membrane) was prepared in ultrapure water and used
for analysing the total phenolic compounds, flavonoids, and
antioxidant activity. Commercial curcumin was used as stan-
dard.
2.9.1. Phenolic compounds
Total phenolic compounds were determined using the Folin-
Ciocalteu reagent according to Ballesteros et al. (2014). A
calibration curve was previously prepared using gallic acid as
standard (5−500 mg/L). The obtained results were expressed
in milligram gallic acid equivalent per gram of lyophilised PCE
(mg GAE/g).
2.9.3.2. Free radical scavenging activity (DPPH assay). The
radical scavenging activity was calculated using Eq. (2), as
described by Ballesteros et al. (2015):
%inhibition= (1−(As−Ab)/Ac)×100 (2)
where Ac, Ab, and As represent the absorbances of the con-
trol (methanol instead of PCE), the blank (PCE with methanol
instead of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution),
and the PCE solutions, respectively. The blank solution value
was used to eliminate any influence of sample colour on the
measurements. A calibration curve was prepared with a Trolox
standard solution in methanol (50−500 ␮M). DDPH percent
inhibition data were plotted as a function of antioxidant con-
centration to obtain DPPH inhibition concentration at 50%
(IC50 ). The IC50 values were expressed as micromoles of Trolox
equivalent per gram of lyophilised PCE (␮mol TE/g).

2.9.2. Flavonoids 2.9.3.3. Radical cation decolorization (ABTS assay). For

Total flavonoids were estimated by the microplate colorimet-
ric assay as described by Ballesteros et al. (2014). A calibration
curve was previously prepared using quercetin as standard
(25−200 mg/L). The obtained results were expressed in mil-
ligram quercetin equivalent per gram of lyophilised PCE (mg
QE/g).
2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
diammonium salt (ABTS) assay, a calibration curve was
constructed using a standard solution of Trolox diluted in
ethanol (50−600 ␮M) according to Ballesteros et al. (2015).
The percent inhibition of ABTS radical cation was calculated
using Eq. (2), where Ac, Ab and As represent the absorbances
 Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199
of the control (distilled water instead of PCE), the blank (PCE
with distilled water instead of ABTS radical cation solution),
and the PCE solutions, respectively. The IC50 values were
expressed as micromoles of Trolox equivalent per gram of
lyophilised PCE (␮mol TE/g).
2.10. Statistical analysis
GraphPad Prism (version 7.00) was used to perform: one-way
ANOVA followed by Tukey’s multiple comparisons test; two-
way ANOVA followed by Sidak’s multiple comparisons test;
multiple t-test; and unpaired t-test. The same letters represent
no significant differences for a 95% confidence level.
3. Results and discussion
The potential of P. brevicompactum to synthesize soluble extra-
cellular pigments was firstly noticed during a screening study
carried out to identify novel fungi with the ability to produce
␤-galactosidase (Silvério et al., 2018). Under submerged con-
ditions, the culture medium became slightly red after 5 days
of fermentation using lactose as a carbon source. Reddish-
brown pigments are mentioned in the literature as extrolites
generally observed in P. brevicompactum, which could be useful
in fungal classification (Frisvad et al., 2004; Pitt and Hocking,
2009). However, as far as we know, the practical application
of P. brevicompactum in pigment production under submerged
fermentation conditions was never explored. Therefore, a
detailed study to investigate the effect of several experimental
parameters on the production of pigments by this fungus was
performed.
3.1. Pigment production
The experimental parameters evaluated in pigments pro-
duction included the culture medium composition, agitation
speed, type of carbon source, temperature, supplements and
carbon source concentration, natural light and initial pH
(Fig. 1).
3.1.1. Culture medium
Culture medium I corresponds to the medium previously
used when pigment production by P. brevicompactum was first
noticed (Silvério et al., 2018). This medium containing mineral
salts and organic nitrogen sources was compared with two
typical media reported for fungal growth: modified Czapek-
Dox broth (medium II) and malt extract broth (medium III). For
the three media, lactose (20 g/L) was used as a carbon source.
Czapek-Dox broth is a salt-rich medium containing an inor-
ganic source of nitrogen, and it has been successfully used for
pigment production by several fungal strains, including Peni-
cillium spp. (Celestino et al., 2014; Méndez et al., 2011; Pandey
et al., 2018; Unković et al., 2018). Medium III corresponds to the
medium (without agar) frequently used for P. brevicompactum
propagation on Petri dish. This medium was already reported
for fungal pigment production under submerged fermentation
conditions by Monascus spp. (Kumar et al., 2017) and Fusarium
spp. (Pradeep et al., 2013). The production of pigments by P.
brevicompactum was visually detected after 5 days of fermen-
tation using medium I (culture medium became reddish) and
II (culture medium became slightly pinkish). When grown in
medium III, the fungus was not able to produce pigments. The
sum of the absorbances obtained for the three wavelengths
191
(400, 470, and 500 nm) after 12 days of fermentation in each
culture medium is represented in Fig. 1A.
Additionally, it was verified for both culture media I and
II that pigment production was noticed when lactose con-
sumption was detected (5 days of fermentation, according to
HPLC analysis). However, after 12 days of fermentation, total
lactose consumption was only achieved in medium I, which
provided the highest production of pigments (Fig. 1A). For
medium II, a considerable amount of lactose (13.75 ± 0.25 g/L)
remained in the culture medium, and no lactose consumption
was detected in medium III. Morales-Oyervides et al. (2015)
also reported a significant increase in pigment production
by Penicillium purpurogenum GH2 when a condition of almost
depletion of the carbon source was achieved.
The highest biomass concentrations were obtained with
medium I (3.54 ± 0.11 g/L) and III (2.71 ± 0.12 g/L), while in
medium II only a reduced amount of biomass was produced
(0.492 ± 0.091 g/L). Furthermore, the biomass aspect was differ-
ent in the three media: i) regular yellowish pellets in medium
I, ii) tiny black pellets in medium II, and iii) irregular greenish
pellets in medium III.
The presence of mineral salts in the culture medium has
been reported as an important issue for fungal growth and
metabolic synthesis (Akilandeswari and Pradeep, 2017). Some
cations such as K+ , Na+ , and Mg2+ proved to have a positive
effect on pigment production by Penicillium spp. (Hernández
et al., 2019; Pandey et al., 2018). This fact may explain the
absence of pigments in culture medium III since this medium
has no mineral salts in its composition. On the other hand, the
significantly higher production of pigments in medium I may
be associated with the presence of organic sources of nitro-
gen in the culture broth. In general, culture media containing
organic nitrogen sources lead to a higher production of micro-
bial pigments since they are also additional sources of other
important nutrients such as sulphur, amino acids, vitamins,
or coenzymes (Pradeep et al., 2013; Sun et al., 2016). In fact,
peptone and/or yeast extract were already described as prefer-
ential sources of nitrogen for pigment production by Penicillium
sp. (GBPI P155) Talaromyces purpureogenus F and Penicillium scle-
rotiourum when compared to sodium nitrate (Celestino et al.,
2014; Pandey et al., 2018; Parul et al., 2020), the nitrogen source
commonly used in Czapek-Dox broth (medium II). Based on
these results, the culture medium I was selected for further
studies.
3.1.2. Agitation speed
Pigments production was evaluated under different agitation
speeds, including static-state fermentation. The most com-
mon agitation conditions reported in the literature for fungal
pigment production are 150 rpm or no stirring. For exam-
ple, 150 rpm was successfully used for the production of red
pigments by Isaria farinose (Velmurugan et al., 2010a) and P.
purpurogenum (Santos-Ebinuma et al., 2013), and also yellow
pigments by Penicillium aculeatum (Afshari et al., 2015). On the
other hand, static fermentation was reported as a suitable con-
dition, closer to the natural conditions, for the production of
pigments by Penicillium sclerotiorum (Celestino et al., 2014), Peni-
cillium sp. GBPI P155 (Pandey et al., 2018) or Aspergillus terreus
(Akilandeswari and Pradeep, 2017). Besides these two agitation
speeds, we also evaluated 200 rpm, successfully described by
Méndez et al. (2011) to produce a red pigment from P. purpuro-
genum GH2.
The results obtained for the agitation speed are presented
in Fig. 1B. A reduced production of pigments was achieved
192 Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199

Fig. 1 – Sum of absorbances obtained for the three wavelengths (400 nm — striped bar; 470 nm — dotted bar; and 500 nm —
full bar) after 12 days of fermentation using different conditions: culture medium (A); agitation speed (B); carbon source (C);
temperature (D); supplements (E); lactose concentration (F); natural light (G); initial pH (H). Values are the mean ± SD (n = 3).
Statistical analysis was performed by one-way ANOVA using Tukey’s multiple comparisons test. The same letters represent
no significant differences at the 95% confidence level.

under static-state fermentation, and no lactose consumption
was detected during the fermentation time. The maximum
pigments production was obtained for 200 rpm. However, this
production was not significantly different from that obtained
using 150 rpm (Fig. 1B). Furthermore, for both agitation speeds,
the presence of pigments in the culture medium was visually
noticed after 5 days of fermentation, and total consumption
of lactose was verified at the end of fermentation (12 days).
The colour of the culture broth was the most significant dif-
ference observed: dark red for 150 rpm and brownish-red for
200 rpm. The occurrence of this brownish-red colour could be
associated with the increased aeration rate under 200 rpm,
which in turn could lead to some oxidation of the pigments
(Mantzouridou et al., 2002). Also, the aspect and concentra-
tion of biomass were very similar for 150 and 200 rpm (3.78
± 0.08 and 3.54 ± 0.11 g/L, respectively). However, for static-
state conditions, small white pellets were formed, and a lower
biomass concentration was obtained (0.617 ± 0.142 g/L). In
order to avoid potential oxidation issues, the agitation speed
of 150 rpm was selected for further studies.
3.1.3. Carbon source
The carbon source is generally described as one of the most
important parameters in pigments production. Therefore, two
additional sugars (glucose and sucrose) were evaluated and
compared with lactose. Glucose and sucrose were already
 Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199
Fig. 2 – Sum of absorbances obtained for the three
wavelengths (400, 470 and 500 nm) during the
fermentation time (12 days) using lactose and glucose as
carbon sources. Values are the mean ± SD (n = 3).
reported in the literature as carbon sources commonly used
to produce microbial pigments (Akilandeswari and Pradeep,
2017; Celestino et al., 2014; Santos-Ebinuma et al., 2013;
Velmurugan et al., 2010a). The results obtained for the three
carbon sources are presented in Fig. 1C. In all cases, pig-
ments production was noticed. However, sucrose provided
lower production than the other sugars. No significant dif-
ferences were observed for lactose and glucose regarding the
sum of absorbances after 12 days of fermentation. In part,
these results disagree with the literature since glucose and
sucrose are generally reported as better carbon sources for
fungal pigment production than lactose (Celestino et al., 2014;
Gunasekaran and Poorniammal, 2008; Kumar et al., 2017;
Pandey et al., 2018; Pisareva and Kujumdzieva, 2010). Never-
theless, for few fungi such as P. aculeatum, Auricularia auricular,
and Monascus ruber, lactose proved to be a suitable alterna-
tive for the synthesis of secondary metabolites like pigments
(Afshari et al., 2015), providing, in some cases, higher produc-
tion than sucrose or glucose (da Costa and Vendruscolo, 2017;
Sun et al., 2016).
The consumption of the 3 carbon sources was analysed
by HPLC. It was observed that total glucose was rapidly con-
sumed (7 days) while sucrose was completely hydrolysed in
3 days, and its respective monomers (glucose and fructose)
were both consumed after 10 days. Lactose was also totally
consumed after 12 days, and no monomers (glucose or galac-
tose) were detected in the culture broth. Curiously, lactose, the
most slowly consumed carbon source, originated a biomass
concentration similar to that obtained with sucrose (3.78 ±
0.08 and 3.88 ± 0.14 g/L, respectively). Using glucose, only 3.08
± 0.40 g/L of biomass was produced.
The final colour of the fermentation broth was also visually
different: dark red for lactose and sucrose; and pinkish-orange
for glucose. Another dissimilarity noticed for the medium con-
taining glucose was the formation of smaller pellets and some
turbidness, probably due to some biomass disintegration.
Although no significant differences were observed for lac-
tose and glucose regarding the sum of absorbances after 12
days of fermentation (Fig. 1C), the production of pigments
along the fermentation time was slightly different (Fig. 2).
After 5 days, a considerable increase in pigment production
was observed using lactose, surpassing the values obtained
with glucose and remaining lightly superior up to the end of
fermentation. For this reason, lactose was selected for fur-
ther studies. Furthermore, lactose is present in considerable
193
amounts in cheese whey, an abundant by-product from the
dairy industry, which may allow reducing the production costs
if this alternative carbon source is used in future studies of
pigments production (da Costa and Vendruscolo, 2017).
3.1.4. Temperature
According to the literature, the temperature is also a param-
eter that can strongly influence the production of pigments.
Therefore, a higher and a lower temperature (33 and 23 ◦ C,
respectively) considered compatible with the fungal growth
were included in the study. The results obtained for these tem-
peratures were compared with that achieved at 28 ◦ C (Fig. 1D).
Pigments production was higher at 23 ◦ C. However, no signif-
icant differences were found when it was compared to 28 ◦ C.
Furthermore, at 23 ◦ C pigments production was also visually
detected after 5 days. On the other hand, when using 33 ◦ C, pig-
ments were detected later (after 10 days), and consequently,
the sum of absorbances obtained at the end of fermentation
was considerably lower.
Different temperatures have been studied for fungal pig-
ment production, being the optimal range generally reported
as 25−30 ◦ C (Afshari et al., 2015; Ahn et al., 2006; Akilandeswari
and Pradeep, 2017; Gunasekaran and Poorniammal, 2008).
It is known that temperature plays a crucial role in cell
metabolism. For this reason, temperatures distant from those
usually present in natural habitats are generally not suitable
either for the fungal growth or the regulation of the enzymatic
processes involved in the synthesis of pigments (Afshari et al.,
2015). This fact may explain the lower concentration of pig-
ments (Fig. 1D) and biomass obtained at 33 ◦ C (2.22 ± 0.13 g/L)
when compared to those obtained at 23 or 28 ◦ C (3.57 ± 0.18
and 3.78 ± 0.08 g/L, respectively). However, the appearance of
biomass was similar for all the tested temperatures. Addition-
ally, total consumption of lactose was detected after 10 days
of fermentation at 23 ◦ C (similar to 28 ◦ C after 12 days), while
at 33 ◦ C, a considerable amount of lactose (11.07 ± 0.04 g/L)
remained in the culture medium at the end of fermentation.
Although similar results were obtained at 23 and 28 ◦ C, the
lowest temperature was selected for further studies since it
can significantly reduce the contamination risk in the long-
term fermentations required for fungal pigments production.
3.1.5. Supplements
The effect of organic sources of nitrogen on the production
of pigments was evaluated using different ratios and concen-
trations of peptone and yeast extract. The results obtained
are shown in Fig. 1E. In all cases, pigments production was
observed after 5 days, except when using the ratio 16/0 of
yeast extract/peptone (7 days of fermentation). Nevertheless,
the final colour of the fermentation broth was not the same
for all the conditions. It was found that for the conditions with
the lowest concentrations of yeast extract (0/8, 2/2, and 2/6
g/L of yeast extract/peptone), the fermentation broth became
orange. On the other hand, when higher concentrations of
yeast extract were used, the fermentation broth exhibited a
reddish colour. Similar behaviour was reported for pigments
production by Monascus anka (Shi et al., 2015). Furthermore,
the final pH of the fermentation broth decreased to acid val-
ues (around 6.5) when peptone was used as the major nitrogen
source. On the contrary, a pH rising to alkaline values (7.3–8.4)
was detected for yeast extract concentrations ≥ 4 g/L.
Although the highest concentration of biomass (5.18
± 0.19 g/L) was achieved when using 16/0 (g/L) of yeast
extract/peptone, the maximal production of pigments was
194 Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199
obtained for 8/8 (g/L) yeast extract/peptone (4.07 ± 0.48 g/L of
biomass). Additionally, the ratio 0/8 (g/L) yeast extract/peptone
provided the lowest biomass concentration (1.84 ± 0.43 g/L),
reduced production of pigments, and a considerable amount
of lactose (13.09 ± 0.02 g/L) remained in the fermentation
broth. For all the other conditions, total consumption of lac-
tose occurred during the fermentation time. According to the
results, it seems that yeast extract has a more noticeable effect
on both fungal growth and pigments production. Neverthe-
less, the combination of yeast extract and peptone in the ratio
8/8 (g/L) proved to be the most suitable condition for pigments
production, suggesting that peptone may also play a relevant
role in the process. Gunasekaran and Poorniammal (2008) also
reported a significant increase in red pigment production by
Penicillium sp. when using simultaneously yeast extract and
peptone (comparatively to simply yeast extract). Based on
these results, a ratio of 8/8 (g/L) of yeast extract/peptone was
selected for further studies.
3.1.6. Lactose concentration
since we previously found that pigments production by P. bre-
vicompactum seems to be connected with the consumption of
lactose (both detected after the 5th day), we decided to study
the effect of a lower and a higher concentration of the car-
bon source (10 and 30 g/L, respectively). The results obtained
for these concentrations were compared with that achieved
using 20 g/L, as illustrated in Fig. 1F. The highest pigments pro-
duction was obtained for the concentration of 20 g/L, and no
significant differences were detected for 10 and 30 g/L. Differ-
ent behaviours have been reported in the literature concerning
the influence of the carbon source concentration on pigment
production, depending on the fungal source. The increase of
glucose concentration from 15 up to 150 g/L promoted the pro-
duction of yellow pigments by M. ruber due to the phenomenon
of high sugar stress (Wang et al., 2017). Similarly, the increase
of glucose concentration up to 30 g/L favoured the produc-
tion of a yellow pigment by Metarhizium anisopliae. However, for
higher glucose concentrations (40−60 g/L), the pigment con-
tent remained almost constant (Yan et al., 2019). On the other
hand, the progressive increase of the glucose concentration
from 10 up to 30 g/L had a clear inhibitory effect on the produc-
tion of a red pigment by Monascus purpureus under solid-state
culture (Lee et al., 2002). The maximal production of a red pig-
ment by Gibberella fujikuroi immobilized on alginate beads was
obtained for glucose concentration in the range 10−30 g/L,
while lower (5 g/L) and higher (50 g/L) concentrations had a
negative effect (Roisin et al., 1996).
Some differences were also detected for the colour of the
culture medium after 12 days of fermentation. When 10 and 20
g/L of lactose were used, the culture media became dark red,
while for 30 g/L of lactose, the detected colour was orangish-
red. For the higher concentration of lactose, the final pH of the
culture medium was also different, being in the acidic zone
(around 6.6), while for the lower concentrations, the pH was
in the range 7.6−8.1. Additionally, the presence of pigments in
the culture medium was noticed after 5 days for the lower con-
centrations of lactose and later (after 7 days) for 30 g/L. These
results seem to agree with the consumption of lactose. When
10 and 20 g/L of lactose were used, the consumption of this
sugar was detected after 5 days and reached 0 g/L at the end
of the fermentation. On the other hand, for 30 g/L of lactose,
the consumption was detected only after 7 days, and the sugar
was not totally consumed during the fermentation time (2.98 ±
0.02 g/L after 12 days). The concentration of biomass obtained
in each culture medium followed the order: 30 g/L > 20 g/L >
10 g/L of lactose. This fact corroborates the general idea that
the energy source in the culture medium directly contributes
to microbial growth. However, it was demonstrated once more
that the production of pigments by P. brevicompactum seems to
be not exclusively related to the amount of biomass. According
to the obtained results, the concentration of 20 g/L of lactose
was selected for further studies.
3.1.7. Natural light
Light is described as an essential stimulus for all organisms
with recognized impact in the regulation of numerous physi-
ological processes (Velmurugan et al., 2010b). The absence of
light frequently favours the production of fungal pigments.
In fact, several fungal pigments have been produced under
dark conditions with increased yields (Babitha et al., 2007;
Bühler et al., 2015; Velmurugan et al., 2010a, 2010b). In this
work, the presence and absence of natural light were eval-
uated, and the results obtained for pigment production are
presented in Fig. 1G. No significant difference in the sum of
absorbances after 12 days was observed between the tested
conditions. Also, the visualization of pigments in the fer-
mentation broth was simultaneously detected (5 days), and a
similar final colour (dark red) was achieved. The consumption
of lactose and the concentration of biomass were also very
comparable. Overall, these results suggest that light has no
significant effect on the growth and production of pigments
by P. brevicompactum. Since the condition with natural light
is more practical and easier to perform, it was selected for
further studies.
3.1.8. Initial pH
One of the most studied parameters in the production of fun-
gal pigments is the pH. Both the production and final colour
of the pigments can depend on the pH of the culture medium.
In this work, the initial pH of the culture medium (pH = 7.0)
was changed to acidic (4.5) and alkaline (9.5) values, and the
production of pigments was evaluated under these conditions.
The obtained results are presented in Fig. 1H. The highest pro-
duction of pigments was achieved when using a neutral initial
pH. However, no significant differences were found when com-
pared with the acidic pH. During the fermentation time, the
microorganism produced metabolites that naturally changed
the pH of the culture medium. Thus, after 12 days of fermen-
tation, a pH of 7.2 ± 0.1 was measured in the acidic condition,
and a pH of 8.3 ± 0.2 was obtained in the alkaline condition.
On the other hand, for the condition with initial pH of 7.0, the
final pH measured in the fermentation broth was 7.7 ± 0.1.
Although the detection of pigments in the fermentation
broth coincided (5 days), the final colour was not similar for
all the tested conditions. For neutral and alkaline initial pH,
the fermentation broth became dark red. The fermentation
broth became dark red for neutral and alkaline initial pH, and
under the acidic condition, it became orangish. This observa-
tion seems to agree with the literature. Mukherjee and Singh
(2011) reported that acidic pH values (below 5.0) favoured the
predominance of yellow pigments from M. purpureus, while
the red pigments dominated for higher pH values (above
6.0). It was also reported for some microorganisms that pH
can strongly affect the activity of specific enzymes directly
involved in the biosynthesis of pigments (Méndez et al., 2011).
In this work, the production of pigments by P. brevicompactum
was clearly favoured by acidic to neutral pH values. Similar
results were obtained for the production of red pigments by
 Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199 195

Mukherjee and Singh (2011) using M. purpureus (optimal pH

6.0) and by Méndez et al. (2011) using P. purpurogenum (optimal
pH 5.0). However, other authors reported the maximal produc-
tion of red pigments at higher pH values, namely at pH 9.0,
using Penicillium sp. (Gunasekaran and Poorniammal, 2008).
Lactose was entirely consumed after 10 days for the acidic
and neutral conditions, while in the alkaline condition, it was
observed only after 12 days of fermentation. The production
of biomass in each culture medium followed the order: pH 9.5
> pH 7.0 > pH 4.5. Although no significant differences were
found between initial pH 4.5 and 7.0 (Fig. 1H), the neutral pH
was selected since it is the original pH of the culture medium.
Thus, there is no need for pH adjustments.
Overall, based on results obtained for each experimental
parameter under study, the optimal conditions for the pro-
duction of pigments by P. brevicompactum were defined as
follows: culture medium I containing lactose (20 g/L) and pep-
tone/yeast extract 8/8 (g/L), 23 ◦ C, 150 rpm, natural light and
initial pH of 7.0. PCE obtained after 12 days was subsequently
characterized.

3.2. Mycotoxin analysis

Fungal pigments can have practical applications as additives

in the food industry. Therefore, it is important to have infor-
mation on the eventual presence of mycotoxins in the culture
medium. Mycotoxins are also secondary metabolites whose
production is dependent on several external parameters such
as temperature, pH, water activity, substrate, or light (Touhami
et al., 2016). Two mycotoxins generally associated with P. bre-
vicompactum are OTA (Vega et al., 2006) and MPA (Ardestani
et al., 2010; Patel et al., 2016). Consequently, the presence of
OTA and MPA was investigated in PCE. Nevertheless, none of
these mycotoxins was detected in the range of concentrations
tested. Thus, if present, their concentrations are not suscep-
tible to cause any health concern. It seems that under the
optimized experimental conditions used for pigments produc-
Fig. 3 – Silica gel TLC showing the separation of the
tion, the synthesis of these two mycotoxins was not favoured.
pigments mixture at visible light (VIS) and exposed to UV
It is known that the production of OTA by some Penicillium is
light (UV) at 366 nm.
negatively affected by light (Schmidt-Heydt et al., 2010). Simi-
larly, the production of MPA is also dependent on the type and
intensity of light (Shu et al., 2010), and it was demonstrated
that the use of lactose and pH 7.0 can have a detrimental effect
on the synthesis of this mycotoxin (Patel et al., 2016).
stable in the pH range 3.0–10.0 (absorbance variation < 4%) and
3.3. Analysis of pigments by TLC also in the temperature range 5−85 ◦ C (absorbance variation
< 7%). However, it is important to mention that for pH val-
The PCE obtained under optimal conditions was also by TLC ues in the range 3.0–5.0, the mixture PCE/buffer was visually
(Fig. 3). After elution, three main spots were visualized under more yellowish, while in the pH range of 6.0–10.0, the mixture
visible light, namely a yellow spot in the front of the elution, PCE/buffer was clearly reddish. According to the literature,
followed by orange and red spots. The visualization under UV fungal pigment stability is generally limited to specific ranges
light intensified the spots, and the differences between the of pH and temperature. For example, the red pigments from P.
colours became clearer. These results demonstrated that PCE purpurogenum proved to be more stable at basic to neutral pH
comprises a mixture of pigments (yellow, orange, and red), and temperatures up to 50 ◦ C (Santos-Ebinuma et al., 2013).
which is the common profile for many fungi (Pisareva and The red pigments from I. farinose were stable in acid to neutral
Kujumdzieva, 2010). pH and temperature below 60 ◦ C (Velmurugan et al., 2010a). On
the other hand, the pigments from M. purpureus were more
3.4. Temperature and pH stability stable at near-neutrality pH and temperatures below 60 ◦ C
(Silveira et al., 2013). For M. ruber, the red pigment showed high
Stability at pH and temperature is an important technological stability at pH close to neutral, and the orange pigment proved
issue that can determine the successful application of pig- to be more stable at acid pH (Vendruscolo et al., 2013). There-
ments at the industrial level. The results obtained after 24 h fore, the results here obtained for P. brevicompactum suggest
of PCE incubation at different pH and temperatures are pre- that the mixture of pigments presents good colour stabil-
sented in Fig. 4. The mixture of pigments proved to be very ity.
196 Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199

Fig. 4 – Relative absorbance obtained for the pigments crude extract after incubation for 24 h at different pH (A) and
temperature (B).

extracts from M. purpureus (1.98 mg GAE/g) (Smith et al., 2015),

Table 2 – Antioxidant potential, total phenolic
compounds and flavonoid content determined for the
pigments crude extract (PCE).
Assay Values
Total phenolic compounds (mg GAE/g lyophilised) 46.33 ± 2.58
Flavonoid content (mg QE/g lyophilised) 6.04 ± 0.39
FRAP (␮mol Fe(II)/g lyophilised) 58.58 ± 4.58
DPPH IC50 (␮mol TE/g lyophilised) 18.48 ± 0.26
ABTS IC50 (␮mol TE/g lyophilised) 28.38 ± 3.79
Results are expressed as mean ± standard deviation; n = 6.
FRAP: antioxidant activity by the ferric reducing antioxidant
power assay; DPPH: antioxidant activity by the 2,2-diphenyl-1-
picrylhydrazyl assay; ABTS: antioxidant activity by 2,2 -azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) diammonium salt.
3.5. Functional properties
Natural antioxidant pigments such as canthaxanthin,
riboflavin, curcuminoids, and carotenoids are widely used
in the food industry, as they provide enormous human
health benefits (Narsing Rao et al., 2017). In this study, the
antioxidant potential of PCE was evaluated, and the obtained
results are shown in Table 2, together with the total phenolic
compounds and flavonoids.
The content of phenolic compounds (46.33 ± 2.58 mg
GAE/g) was higher than those reported for pigments-rich
Lecanicillium aphanocladii (15.8 mg GAE/g), Penicillium flavigenum
(18.8 ± 0.21 mg GAE/g), and Aspergillus keveii (18.6 ± 0.25
mg GAE/g) (Tavares et al., 2018). Moreover, the content of
flavonoids (6.04 ± 0.39 mg QE/g) was considerably higher than
that achieved by M. purpureus (0.22 mg QE/g) (Smith et al.,
2015). Since both types of compounds are directly related to
antioxidant capacity, the obtained results suggest the promis-
ing potential of PCE.
Incorporating natural pigments with antioxidant proper-
ties in food decreases the detrimental effect of free radicals
in the body, which are responsible for multiple diseases such
as cancer and diabetes. Antioxidant pigments can delay or
inhibit the cellular damage by donating electrons and neu-
tralizing such radicals due to their scavenging characteristics
(Lobo et al., 2010). In this work, FRAP, DPPH, and ABTS assays
were used to determine the antioxidant potential of PCE
since each one assesses different reactive functional groups.
The obtained values are shown in Table 2. Additionally, the
antioxidant activity of PCE was compared with a commer-
cial standard (Fig. 5). For the FRAP assay, a 3.4-fold higher
value was obtained for curcumin (commercial standard) when
compared to PCE. On the other hand, when PCE (20 mg/mL)
was evaluated by the ABTS and DPPH assays, the percent
inhibition values were closer to the standard (Fig. 5). It is
known that the scavenging activity of bioactive compounds
is straight associated with their concentration. Thus, the IC50

Fig. 5 – Antioxidant activity of the pigments crude extract (PCE) and the commercial antioxidant curcumin evaluated by
different methods: FRAP assay (A); and DPPH and ABTS assays (B). Values are the mean ± SD (n = 3). Statistical analysis was
performed by multiple t-tests test to FRAP data and by two-way ANOVA using Sidak’s multiple comparisons test to DPPH
and ABTS data. The same letters represent no significant differences at the 95% confidence level.
 Food and Bioproducts Processing 1 3 2 ( 2 0 2 2 ) 188–199
reported values for ABTS and DPPH (Table 2) were achieved
with 11.94 ± 1.60 and 12.33 ± 0.17 mg/mL of PCE, respec-
tively, while only 0.5 mg/mL of curcumin was needed to reach
50% of inhibition, regardless of the method used, highlighting
its stronger antioxidant potential. Comparing to the litera-
ture, PCE showed equivalent or higher free radical scavenging
activity than extracts obtained from Aspergillus aureolatus (IC50
= 21.44 and 25.41 mg/mL, for ABTS and DPPH, respectively),
and Epicoccum nigrum (IC50 = 17.75 mg/mL for DPPH) using
submerged fermentation (Tavares et al., 2018), and Monascus-
fermented adlay (IC50 = 12.45 mg/mL for DPPH)) (Srianta et al.,
2017). Therefore, all these results demonstrated that PCE is a
phenolic-rich extract with significant antioxidant properties.
4. Conclusions
The potential of P. brevicompactum to synthesize pigments was
clearly demonstrated. The production of pigments was signif-
icantly affected by agitation and type/concentration of both
carbon source and supplements. Lactose was proven to be an
adequate substrate for the synthesis of pigments, allowing the
use of a low-cost carbon source (i.e. cheese whey) as an alter-
native culture medium in future studies. Under the optimal
conditions, a mixture of yellow, orange, and red pigments was
obtained. The high stability, mycotoxin-free condition, and
antioxidant activity are interesting characteristics for applying
these pigments in the food industry. However, additional stud-
ies should be performed to separate and identify the chemical
structure of the pigments from P. brevicompactum.
Data availability
Data will be made available on request.
Declaration of Competing Interest
The authors declare no conflicts of interest.
Acknowledgment
This work was financially supported by the Portuguese Foun-
dation for Science and Technology (FCT) under the scope of
the strategic funding of UIDB/04469/2020 unit. Luís Abrun-
hosa acknowledges FCT for the assistant research contract
CEECIND/00728/2017, obtained under CEEC Individual 2017.
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