Biocatalysis and Agricultural Biotechnology 32 (2021) 101931

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Biocatalysis and Agricultural Biotechnology

journal homepage: http://www.elsevier.com/locate/bab

Pigment bioproduction by Monascus purpureus using corn bran, a byproduct

of the corn industry
Adrielle Borges de Almeida a, Nathalia Horrana Santos b, Thayanara Mayara de Lima a,
Railany Vieira Santana a, Josemar Gonçalves de Oliveira Filho c, Daiane Sousa Peres a,
Mariana Buranelo Egea a, *
a
Goiano Federal Institute of Education, Science, and Technology, Campus Rio Verde, Rod. Sul Goiana, Km 01, Cx Postal 66, Rio Verde, GO, CEP: 75901-970, Brazil
b
Department of Chemical Engineering and Food Engineering - Federal University of Santa Catarina, Campus Trindade, Florianópolis, SC, CEP: 88040900, Brazil
c
São Paulo State University (UNESP), School of Pharmaceutical Sciences, Rod. Araraquara Jaú, Km 01, S/n - Campos Ville, CEP 14800-903, Araraquara, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Monascus purpureus is an ascomycete known for decades for producing several metabolites of commercial in­
Natural red pigment terest. The objective of this work was to optimize the production of pigments of M. purpureus using submerged
Waste valorization fermentation with corn bran. An experimental design containing 11 tests was realized twice to determine the best
Byproduct
concentration of corn bran and peptone in submerged fermentation to produce higher red pigment concentration
Bioprocess
(AU500). After the best substrate concentration was chosen, pigment production was evaluated up to 20 days of
submerged fermentation. Antioxidant and antibacterial activities and thermal and pH stabilities of the pigment
extract were also evaluated. The optimized concentration (42.5 g/L of corn bran and 30 g/L of peptone) was used
in submerged fermentation for 6 days to produce the pigment extract. The pigment extract showed high anti­
oxidant activity, twice as much as the substrate (corn bran) used in its production. The pigment extract produced
by M. purpureus presented thermal (sterilization and 40–80 ◦ C in hot dry) and pH (4–7) stabilities. Thus, the
production of pigment from M. purpureus can be an alternative use for corn bran utilization.

Statement of novelty animal feed (Yadav et al., 2016).

Monascus purpureus is a fungus widely known in popular cuisine for
its application. However, few studies are focused on pigment production
and extraction, as well as determining its characteristics for application
in the food industry. In this work, we produce and evaluate the red
pigment extract produced by M. purpureus using a byproduct of the
agribusiness. In this way, this technology can also be used to increase the
value-added in corn bran, which is produced in large quantities and
underused.
1. Introduction
Corn (Zea mays), a conventional cereal in Brazil, is a raw material for
the production of various products intended for human consumption
(grits, cornmeal, and oil, among others) and can also be consumed in
natura. Corn bran is generated in large quantities from industrial pro­
cessing of corn grinding into food products and is generally intended for
Corn bran consists mainly of insoluble fiber, cellulose, hemicellulose,
and xylooligosaccharide, which are partially indigestible (Rose et al.,
2010). Thus, corn bran cannot be directly digested by enzymes in
humans and monogastric animals, but it can be degraded by colon
bacterial communities (Sonnenburg and Sonnenburg, 2014). For this
reason, only a small amount is used in human and animal nutrition, and
the disposal of corn bran is one of the main concerns of the food in­
dustries in conforming to environmental regulations (ElMekawy et al.,
2013). The waste management requires that much of what is considered
as waste be seen as valuable raw material for the production of a
value-added product. This approach is probable to lead to
ever-decreasing volumes of waste meant for ultimate discarding (Gar­
cia-Garcia et al., 2017).
The filamentous fungus Monascus has been used for decades in Asia
and, more recently, has been the topic of studies because it can be a
natural source of pigment to be used in the food industry (Haque et al.,
2016). This compound belongs to the group of azafilones, which react

* Corresponding author.
E-mail address: mariana.egea@ifgoiano.edu.br (M.B. Egea).

https://doi.org/10.1016/j.bcab.2021.101931
Received 7 September 2020; Received in revised form 20 January 2021; Accepted 21 January 2021
Available online 27 January 2021
1878-8181/© 2021 Elsevier Ltd. All rights reserved.
A.B. Almeida et al. Biocatalysis and Agricultural Biotechnology 32 (2021) 101931

with amines of proteins, amino acids, and nucleic acids, replacing oxy­
gen with nitrogen (Sen et al., 2019).
Among these secondary metabolism pigments produced during the
growth of Monascus, the red pigment (monascorubramine and rubro­
punctamine) has higher commercial value due to the interest of the food
and pharmaceutical industries (Kraboun et al., 2019). Pigments pro­
duced by microorganisms are more attractive because they do not pre­
sent seasonal impediments and have the possibility of being provided for
high demand (De Carvalho et al., 2006; Kraboun et al., 2019).
An economically viable alternative appears to be the use of
byproducts in submerged fermentation processes as nutrients for mi­
croorganisms. Submerged fermentation uses suspension in a liquid
medium to grow the microorganism (Braga et al., 2018). Thus, the
objective of this work was to optimize the production of pigments of
Monascus purpureus using submerged fermentation with corn bran.
2. Material and methods
same volume (1.9 mL) of a sodium carbonate solution (60 g/L) was used
for neutralizing the mixture. After a 120-min reaction in the dark at
room temperature, mixture absorbance was measured at 725 nm. Gallic
acid was used as the standard, and the results were expressed as mg
gallic acid equivalent (GAE) per 100 g.
Antioxidant capacity was evaluated by three measures as follows: i)
reducing ability of antioxidants toward DPPH• as according to Brand-­
Williams et al. (1995), ii) scavenging ability of antioxidants to the
long-life radical anion ABTS•+ as according to Miller et al. (1993)
modified by Rufino et al. (2010), and iii) reducing antioxidant power of
ferric 2,4,6-tripyridyl-s-triazine (TPTZ) as according to Benzie and
Strain (1999) modified by Pulido et al. (2000). In the DPPH and ABTS
methods, the results were expressed as the percentage of discoloration
(%). In the Ferric Reducing Antioxidant Power Assay (FRAP), an ethanol
solution was used for calibration and the results were expressed in μg
ferrous sulfate (FS) equivalent per g of sample.

2.1. Corn bran byproduct and microorganism 2.3. Pigment production maximization using submerged fermentation

A donation of corn bran was obtained from an agribusiness located in
Rio Verde (Goiás, Brazil), packed in plastic polypropylene bags, and
stored at 10 ◦ C. The Monascus purpureus ATCC 36928 was obtained from
the Centro de Culturas Tropicais (CCT) from Fundação Tropical de
Pesquisas “André Tosello” (Campinas, Brazil). The microorganism was
previously maintained for repetitions every 7 days on Potato Dextrose
Agar (PDA) and incubated at 30 ◦ C.
The inoculum was prepared by adding 20 mL of saline solution sterile
(NaCl, 0.85 g/100 mL) to the Petri plate containing the microorganism.
The plate scraping was aseptically performed and a 1 mL aliquot of this
suspension was diluted in 9 mL of saline solution. Spore counting was
performed using an aliquot (50 μL) of the suspension (1:10) in a Neu­
bauer chamber using a microscope (Zeiss, Primo Star, Germany) with
the 40× magnifying glass.
2.2. Chemical analysis
Official methods (AOAC, 2000) were used to estimate the corn bran
contents: moisture by evaporation of water at 105 ◦ C (no. 968.11); lipid
(Bligh & Dyer method); protein by determining total nitrogen using
Kjeldahl method (conversion factor = 5.75, no. 991.20); and ash using
muffle incineration at 550 ◦ C (no. 945.46). Carbohydrate content was
calculated by difference (100-moisture-protein-ash-lipid).
Glucose-reducing total sugar content was determined using the Fehling
method (no. 958.06). Three grams of corn bran were combined with
50 mL of water and the mixture was heated in a water bath (~90 ◦ C) for
5 min. The hot solution was transferred to a volumetric flask (100 mL)
and the volume was adjusted. This solution was filtered through
Whatman filter paper No. 1 and transferred to the burette to be used as a
titration agent. In a 250 mL Erlenmeyer, a mixture of 10 mL of Fehling A
solution (34.639 g of copper sulfate in 1000 mL of water), 10 mL of
Before the submerged fermentation, the corn bran was dried in an
oven at 60 ◦ C for 3 h, ground in a knife mill (Start FT 50, Fortinox,
Brasília, Brazil), and homogenized in a 50-mesh sieve.
The prepared culture media contained sources of carbon and nitro­
gen as follows (g/L): K2HPO4 (5.0), KH2PO4 (5.0), MgSO4⋅7H2O (0.01),
CaCl2 (0.01), and ZnSO4⋅7H2O (0.01). The initial pH of the medium was
adjusted to 6.0 ± 0.2 (HCl 1:10). Erlenmeyer flasks (125 mL) containing
25 mL of medium were inoculated with 250 μL of a suspension of
mycelium or spores (NaCl, 0.85 g/100 mL). Flasks were incubated at
30 ± 3 ◦ C for 9 days in a shaker at 100 rpm (Silveira et al., 2008).
The influence of the concentrations of corn bran and peptone on red
pigment production was evaluated using a factorial design (22) with
three replicates in the central points and with the inclusion of axial
points, which means a total of 11 runs (Myers et al., 2016). In the sta­
tistical model, the coded variables were defined as follows: corn bran
(X1) and peptone (X2), with the dependent variable being pigment
production (Y). In the first test, the determination of the maximum and
minimum values for X1 and X2 was based on the values used in Silveira
et al. (2008), while in the second test the displacement of the values was
performed based on the result of the first test. Table 1 shows the two
independent variables (corn bran (X1) and peptone (X2)) and their
concentrations at the different coded levels of the factorial design ex­
periments (Assay 1 and 2) as well as the response evaluated (Y, pigment
production).
The software Statistica version 5.0 (Statsoft Inc., USA) was used for
Table 1
Real values for corn bran (CB) and peptone (g/L) and coded values (in paren­
theses) for factorial design in submerged fermentation using Monascus purpureus
and its pigment production (Y) in AU500.
Assay 1 Assay 2

Fehling B solution (173 g of sodium tartrate and potassium [KNa­ CB (X1) Peptone Y CB (X1) Peptone Y
C₄H₄O₆.4H₂O] dissolved in 250 mL of water was added of 250 mL 20% (X2) (X2)
NaOH and the final volume was adjusted to 1000 mL using volumetric 1 8.60 (− 1) 10.00 (− 1) 0.800 21.10 (− 1) 5.00 (− 1) 3.065
flask), and 40 mL of water was heated to a boil. The titration was done 2 8.60 (1) 10.00 (− 1) 0.939 38.90 (1) 5.00 (− 1) 2.567
with sample previously extracted thorough the mixture of Felling solu­ 3 26.40 (− 1) 35.00 (1) 3.549 21.10 (− 1) 30.00 (1) 1.969
4 26.40 (1) 35.00 (1) 2.398 38.90 (1) 30.00 (1) 3.814
tions under agitation, observing the change of color from blue to 5 5.00 22.50 (0) 1.112 17.50 17.50 (0) 2.759
colorless with a red residue on the bottom. Values of pH and soluble (− 1.41) (− 1.41)
solids (SS) were determined from a sample:water solution (1:10) using a 6 30.00 22.50 (0) 2.884 42.50 17.50 (0) 3.321
potentiometer and refractometer, respectively. (1.41) (1.41)
7 17.50 (0) 5.00 1.973 30.00 (0) 0.00 2.425
The crude extract of corn bran was obtained as described by Larrauri
(− 1.41) (− 1.41)
et al. (1997) and used to measure total phenolic content (TPC) and 8 17.50 (0) 40.00 1.613 30.00 (0) 35.00 2.082
antioxidant capacity. TPC of the crude extract was determined by the (1.41) (1.41)
Folin–Ciocalteu method (Singleton et al., 1999), with modifications, as 9 17.50 (0) 22.50 (0) 1.605 30.00 (0) 17.50 (0) 2.953
described by Li et al. (2009). Diluted crude extracts (200 mL) were 10 17.50 (0) 22.50 (0) 2.587 30.00 (0) 17.50 (0) 3.702
11 17.50 (0) 22.50 (0) 2.079 30.00 (0) 17.50 (0) 3.275
mixed into 1.9 mL 10-fold freshly diluted Folin-Ciocalteau reagent. The

2
A.B. Almeida et al.
the regression analysis of the experimental data obtained. The coeffi­
cient of determination R2 expressed the quality of fit of the model
equation, and F-test (p < 0.05) determined its statistical significance.
Three replicates at the center points were used to estimate the experi­
mental error and to allow for checking the adequacy of the model.
After choosing the best substrate concentration for the maximization
of red pigment production, the kinetic experiment was carried out. The
objective of evaluating pigment production during the time was to
decrease the pigment production time. The literature search revealed
that the production of pigments by M. purpureus could occur from 7 to 18
days (Silveira et al., 2008; Vendruscolo et al., 2016a; Zhang et al., 2015).
For this reason, the best treatment (previously chosen) was inoculated
with 106 spores/mL and incubated for 18 days at 30 ◦ C in a shaker at
100 rpm, with the determination of pigment production daily until the
9th day, and every 3 days until the 18th day.
2.4. Quantification and stability of the pigment extract obtained
The concentration of extracellular red pigment was estimated as
described by Silveira et al. (2008) with modifications. All fermented
solution was transferred to the Falcon tube (50 mL) and vortexed
(K40-1020, Kasvi, São José do Pinhais, Brazil) for 1 min and filtered
through Whatman filter paper No. 1 using vacuum. The volume of the
liquid was corrected to 50 mL with distilled water. The absorbance of
filtrates (extract) was measured at 500 nm (absorbance units, AU500),
considering the dilution factor.
The submerged fermentation using 42.5 g/L of corn bran and 30.0 g/
L of peptone (best assay with maximized response) was carried out (106
spores/mL) and incubated for 6 days at 30 ◦ C in a shaker at 100 rpm. The
fermented solution was filtered and pigment extract was used in thermal
and pH stability tests, as well as subjected to freeze-drying to be used for
FTIR analysis and antibacterial activity evaluation.
The Fourier transform-infrared absorption spectroscopy (FTIR)
analysis was performed on PerkinElmer model equipment (FTIR/IR
spectrometer, Frontier), following the operating conditions:
650–4000 cm− 1 region with 8 scans and 2 cm− 1 resolution. The graph
was generated using the Origin 8.1 software (OriginLab Corporation,
2009) and the peaks were compared with the literature.
Thermal stability was determined submitting the pigment extract to
the treatment in an autoclave (121 ◦ C for 15 min), and to oven in the
range from 40 to 80 ◦ C. The calculation of thermal degradation constant
(Dc), half-life (t1/2), and activation energy (Ae) was performed as
described by Jung et al. (2005). Dc (h− 1) was calculated using Equation
(1).
dA
= − Dc t (1)
dt
where A is absorbance (UA500nm) and t is time (h). Using conditions
where A = A0 (initial absorbance) and when t = 0 (initial time) and A is
absorbance in time t, resulting in Equation (2):
( )
A
ln = − Dc (2)
A0
The half-life (t1/2) for each condition was calculated from Dc values
using Equation (3):
t1=ln2 (3)
2 Dc
Arrhenius equation (Equation (4)) was used to calculate the activa­
tion energy:
(4)
− Ea
Dc = D0 e RTe
where Ea is the activation energy (Kcal/mol), D0 is the pre-exponential
factor (h), R is the universal gas constant (cal/mol/K), and Te is the
temperature (K), which after linearized resulted in Equation (5).
3
Biocatalysis and Agricultural Biotechnology 32 (2021) 101931
( )
Ea 1
lnDc = − + lnD0 (5)
R T
The pigment extract was evaluated for stability at pH (4.0–8.0). The
0.2 M sodium citrate-phosphate (pH 4.0, 5.0, and 6.0) and 0.2 mol/L
sodium phosphate solutions (pH 7.0 and 8.0) were used for the correc­
tion of the pH and the extract was subjected to incubation at 80 ◦ C for
180 min.
The quantification of the pigment at the end of the processes was
measured in the absorbance of filtrates (extract) at 500 nm (UA500nm),
considering the dilution factor and expressed as a percentage of absor­
bance observed at zero time (control - 100%) (Corradini and Peleg,
2004).
2.5. Evaluation of the antibacterial activity of the pigment extract
The evaluation of antibacterial activity was performed using the disc
and well agar diffusion methodology. Before carrying out the experi­
ments, the strains of Staphylococcus aureus ATCC-6538 and Escherichia
coli ATCC 700926 were recovered in Brain Heart Infusion (BHI) broth at
36 ◦ C for 18 h. Culture suspensions were prepared and diluted in saline
solution (0.85 g/100 mL) using the MacFarland 0.5 scale until approxi­
mately 1.5 × 108 Colony Forming Units (CFU/mL) were obtained.
Antibacterial susceptibility was evaluated according to the disk
diffusion method following the recommendations of CLSI (2009) with
adaptations. Sterile filter paper discs (6 mm) received an aliquot of 20 μL
of different concentrations ranging from 100 to 300 mg of freeze-dried
pigment extract/mL of sterile distilled water. Then, each disc was
applied to a Petri dish containing Mueller-Hinton agar (MH) previously
inoculated with the microorganism to be tested. For the disk diffusion
test, halos with a diameter ≥6 mm were considered as an inhibitory
activity.
The well-diffusion test was carried out according to CLSI (2009),
with adaptations, which differs from the disk test by making three 6 mm
diameter holes in the middle of Petri dishes containing MH agar. The
plates were inoculated on the surface by microorganisms using a swab,
and the wells were filled with 20 μL of different concentrations ranging
from 100 to 300 mg of freeze-dried pigment extract/mL of sterile
distilled water. In both tests, the plates were incubated at 35 ◦ C for 24 h.
The microbial growth inhibition halos were measured in millimeters,
with the aid of a millimeter rule.
2.6. Statistical analysis
After optimizing the experiment using the statistical tools described
in item 2.3, the statistical analysis of the other experiments was per­
formed using experiment repetition and analytical triplicate. The results
were evaluated using mean and standard deviation and submitted to
analysis of variance followed by the Tukey test and the difference in
means determined at the level of 5% probability. The data obtained in
the agar diffusion evaluations were subjected to analysis of variance
(ANOVA), with the averages of the halo measurements compared by the
Tukey test (5% significance).
3. Results and discussion
3.1. Effect of medium components on pigment production
The corn bran showed moisture, protein, lipid, carbohydrate, and
ash contents of 12.36, 8.13, 11.20, 44.93, and 3.10 g/100 g, respec­
tively, and also had the pH of 6.4, soluble solid of 1.13 ◦ Brix, and total
phenolic content of 174.84 mg/100 g (Supplementary file 1). Corn bran
is the byproduct of the extraction of the starch part (destined for the
brewer grits) and the literature has highlighted that the insoluble frac­
tion can contain ~70, 28, and 1 g/100 g of hemicellulose, cellulose, and
A.B. Almeida et al. Biocatalysis and Agricultural Biotechnology 32 (2021) 101931

lignin, respectively (Rose et al., 2010). concentration of red pigment occurred at six days, and after that, there
In our previous study, M. purpureus modified corn bran (solid-phase was no significant change in the production of pigment concentration.
fermentation), increasing protein and lipid contents. However, solid- Thus, the submerged fermentation was conducted until the sixth day to
phase fermentation was not efficient in the production of red pigments produce the pigment extract. This result was satisfying because in
(Almeida et al., 2019). In the present study, we evaluated the influence addition to using a byproduct from the agribusiness, we were able to
of the concentrations of corn bran and peptone on red pigment pro­ reduce the time of the red pigment production compared to other works
duction using submerged fermentation. available in the literature (Kantifedaki et al., 2018; Mukherjee and
Table 1 shows the concentration of red pigment obtained for Assay 1 Singh, 2011; Silbir and Goksungur, 2019). In addition, AU500 values in
and 2 of the submerged fermentation using M. purpureus. Table 2 shows the present work were higher than reported by Zeng et al. (2019) for red
the regression coefficients for Assay 1 and 2, with L representing linear pigment produced using submerged fermentation using starch.
terms and Q representing quadratic terms. Considering the inherent
variability of bioprocesses that use microorganisms, coefficients were 3.2. Pigment extract characterization
disregarded when the parameters with p values (significance) were
higher than 5% (p < 0.05). The infrared absorption spectrum of the pigment extract (Fig. 2)
In Assay 1, the F-value in the analysis of variance was not significant, showed characteristic peaks of aromatic compounds (1589, C– – C),
and the percentage of variation explained for the model was low aliphatic compounds (2933, C–H), the ester bonds (1044, C–O), and
(R2 = 34.42%). Therefore, was possible to conclude that this model did carboxylic acids (1405, C–O). These bands indicate compounds present
not fit well with the experimental data. This behavior was confirmed for in the structure of red pigments: monascorubramine (C23H27NO4) and
the p-value of the regression coefficients of each variable tested. Even rubropuntamine (C21H23NO4) (Pattanagul et al., 2007). Similar results
without providing significant mathematical modeling, it was possible to were related by Srivastav et al. (2015), who used infrared spectroscopy
observe that a higher concentration of corn bran and peptone in the to characterize the chemical structure of red pigment and identified
submerged fermentation resulted in higher pigment concentration functional groups suggesting the presence of monascorubramine and
(Supplementary file 2). Thus, Assay 2 was carried with other concen­ rubropuntamine produced by Monascus purpureus using sweet potato
trations of corn bran and peptone in order to find better results in medium in submerged fermentation.
mathematical modeling.
In Assay 2, the variation of the explained experimental results found 3.2.1. Antioxidant activity of pigment extract
was 87.65%. The regression coefficients of the evaluated independent Natural compounds, including pigments obtained through fermen­
variables (concentration of corn bran and peptone), as well as their tation, have antioxidant activities. The ability to act as an antioxidant
interaction, were significant and it was possible to establish a mathe­ was tested for corn bran and pigment extract using three methods
matical model (Equation (6)). (ABTS, DPPH, and FRAP) (Fig. 3B). The percentage of discoloration
found in the present work, regardless of the method used, was close to
Pigment production(AU500 ) = 3.309 + 0.268 x corn bran − 0.478 x peptone2
what had been reported for red pigment produced with submerged
(6) fermentation using orange waste (73–77%) (Kantifedaki et al., 2018)
As demonstrated in Assay 1, the increase in the concentration of corn and submerged fermentation using starch as carbon source (20–69%)
bran and peptone in the submerged fermentation increased the pro­ (Zeng et al., 2019).
duction of M. purpureus pigments. Using the desirability tool of the In the present work, the pigment extract showed twice of antioxidant
Statistica software, the maximized fermentation should contain 42.5 g/L activity of corn bran, independent of the methods used. This increase
of corn bran and 30 g/L of peptone (Fig. 1). The mathematical modeling with fermentation process in the antioxidant activity is directly related
approach was tested, and the experimental and theoretical results were to the production of pigments since their molecular structure has oxygen
compared. The experimental value found for pigment concentration was with free electrons that can bind to free radicals. This behavior was also
3013 UA500nm, which corresponds to 91.05% of the theoretical value, reported by Huang et al. (2013), who used M. purpureus in the fermen­
which validates the assay. These concentrations (42.5 g/L of corn bran tation of sorghum residue.
and 30 g/L of peptone) were used in the submerged fermentation with
M. purpureus to evaluate how many days was necessary to produce the 3.2.2. Thermal stability of pigment extract
highest concentration of pigment in the extract. Typical behavior for natural pigments is color degradation when
Fig. 3A shows the pigment production for 18 days. The higher they are exposed to high temperatures (Vendruscolo et al., 2016a,b). In
the present work, the pigment extract showed 98.88% of thermal sta­
bility to the sterilization process. This process, which uses high tem­
Table 2
Regression coefficients for pigment production in submerged fermentation by
perature and water vapor pressure, is used for the food industry to
Monascus purpureus. process canned products. Great difficulty is reported by canned industry
in maintaining the color of food products. Thus, the pigment extract of
Assay 1
M. purpureus could be an alternative for use in products that are sub­
Factor Regression coefficient Std. Err. t-value P-value jected to sterilization.
Mean 2.090 0.566 3.692 0.014 Table 3 shows the constant thermal degradation of the pigment
Corn bran (L) 0.186 0.694 0.537 0.614 extract. As expected, the increase in the temperature increased the
Corn bran(Q) − 0.040 0.829 − 0.095 0.928
thermal degradation sixfold (Dc = 0.043 to 0.273 h− 1 from 40 to 80 ◦ C).
Peptone (L) 0.464 0.694 1.336 0.239
Peptone (Q) − 0.143 0.829 − 0.344 0.745
Vendruscolo et al. (2013) reported a Dc value of 0.171 h− 1 at 90 ◦ C for
Corn bran x Peptone − 0.323 0.981 − 0.658 0.540 red pigments produced using Monascus ruber.
The experimental results obtained in the present work fit the
Assay 2
Factor Regression coefficient Std. Err. t-value P-value Arrhenius model (Supplementary file 3). When the angular coefficient
(-Ae/R) of the linear regression was multiplied by 8.31 (ideal gas con­
Mean 3.309 0.175 18.91 0.000
Corn bran (L) 0.268 0.215 2.50 0.055 stant in J/mol/K), the activation energy (Ae) of 42.67 kJ/mol was ob­
Corn bran (Q) − 0.082 0.256 − 0.64 0.549 tained. A value close to that found in the present study for Ae was
Peptone (L) − 0.042 0.215 − 0.39 0.714 described by Silveira et al. (2013) (40.81 kJ/mol) for pigments produced
Peptone (Q) − 0.478 0.256 − 3.73 0.014 by M. purpureus using sugarcane bagasse.
Corn bran x Peptone 0.586 0.303 3.87 0.012
The half-life was inversely proportional to the increase in

4
A.B. Almeida et al. Biocatalysis and Agricultural Biotechnology 32 (2021) 101931

Fig. 1. Maximization of pigment production as responses of desirability-related characteristics in submerged fermented with corn bran (CB) and peptone by
Monascus purpureus.

Fig. 2. Infrared absorption spectrum of the pigment extract obtained for sub­
merged fermentation by Monascus purpureus.

temperature, and the value found at 80 ◦ C (2.539 h− 1) was about 6 times

less than that obtained at 40 ◦ C (16.148 h− 1). Half-life values of
63.99 h− 1 at 40 ◦ C, 29.77 h− 1 at 60 ◦ C, and 10.23 h− 1 at 80 ◦ C had been
Fig. 3. (A) Pigment production during 18 days and (B) Antioxidant activity of
reported by Silveira et al. (2013). It can be seen that the red pigments corn bran and pigment extract produced by Monascus purpureus using sub­
degraded more quickly at higher temperatures, since the half-life, that merged fermentation.
is, the time in which the initial concentration was reduced by half,
decreased while the temperature studied was increased as occurred in
3.2.3. pH stability of the pigment extract
Ou et al. (2009).
Table 3 shows the results of pigment stability at different pH (4.0,
5.0, 6.0, and 7.0) as a function of time (0, 30, 60, 90, 120, 150, and

5
A.B. Almeida et al. Biocatalysis and Agricultural Biotechnology 32 (2021) 101931

Table 3
Thermal and pH stabilities of pigment extract produced for submerged fermentation by Monascus purpureus using corn bran.
Thermal stability

Temperature (◦ C) Degradation coefficient (h− 1) Half-life (h) Activation energy (KJ/mol)

40 0.0430 ± 0.002 16.1483 ± 0.659 42.67

60 0.1387 ± 0.031 5.2049 ± 0.031
80 0.2734 ± 0.013 2.5394 ± 0.013

pH stability
Time (min) 4.0 5.0 6.0 7.0

0 100.00 ± 0.0a 100.00 ± 0.0a 100.00 ± 0.0a 100.00 ± 0.0a

30 88.59 ± 5.02c 92.71 ± 5.01a 94.52 ± 1.67b 93.62 ± 3.61a
60 83.16 ± 4.60b 95.97 ± 5.55a 95.65 ± 1.30b 93.92 ± 3.49a
90 79.79 ± 7.21b 95.53 ± 5.58a 95.77 ± 1.25b 93.81 ± 3.35a
120 81.64 ± 5.50b 96.16 ± 5.61a 95.49 ± 1.22b 94.27 ± 3.60a
150 82.04 ± 4.98b 96.22 ± 5.69a 95.53 ± 1.20b 93.75 ± 3.51a
180 83.00 ± 4.01b 96.17 ± 5.56a 95.23 ± 1.67b 94.14 ± 3.34a

180 min). At pH 8.0 there was particle precipitation, which is when
molecules became insoluble, making it impossible to measure stability.
This precipitation may be due to the presence of monacolin, which is
more soluble at pH below 8.0 (Ou et al., 2009).
At pH 4.0, stability decreased up to 60 min and then showed no
significant change. At pH 5.0 and 7.0, the pigment extracts remained
stable during the 180 min in which they were monitored. At pH 6.0, the
pigments showed degradation in the first 30 min (94.52%) and then
remained stable until 180 min. This behavior demonstrates the stability
of the pigment extract produced using submerged fermentation by
M. purpureus for the pH range of 4–7. In this range, a large part of pro­
cessed foods needs coloring or red color correction. The pigment of
M. purpureus can be used more effectively as a coloring than anthocyanin
compounds, which could have the same color but which have low pH
stability (Srivastav et al., 2015). The high interest in this pigment is
mainly due to its pH stability range (2–10) and resistance to high tem­
perature (Mapari et al., 2005). Microbial pigments are a better alter­
native to synthetic and natural food colors of plants because of their
availability, non-seasonality, scalability, higher yield per hectare, and
straightforward down streaming processing (Sen et al., 2019).
3.2.3. Antibacterial activity of the pigment extract
The antibacterial activity of the pigment extract obtained from corn
bran fermented using Monascus purpureus was evaluated in vitro against
Staphylococcus aureus ATCC-6538 and Escherichia coli ATCC 700926
using two methods. Regardless of the method used and the microor­
ganism in the zone of inhibition at the concentrations tested, the
pigment extract showed no antibacterial activity.
M. purpureus KCCM 10093 was related to low antimicrobial activity
against gram-positive bacteria, gram-negative bacteria, and filamentous
fungi (Kim et al., 2006). Patel et al., 2016 observed no antibacterial
activity of orange pigments produced by Salinicoccus Sp. MKJ997975
against Pseudomonas aeruginosa, Sarcina lutea, Bacillus subtilus, or Proteus
vulgaris. However, the antimicrobial activity of the pigments obtained
through fermentation with Monascus purpureus has been reported in
several studies (Ferdes et al., 2009; Narsing Rao et al., 2017; Ven­
druscolo et al., 2010; Xu, 2011). The antimicrobial activity mechanisms
of the pigments are still unknown; however, findings suggest that anti­
microbial activity is significantly determined by cell growth conditions
and culture medium compositions (Narsing Rao et al., 2017; Xu, 2011).
In this sense, it is believed that the extract needs to be tested in higher
concentrations, or it must undergo a process of purification and con­
centration of pigments to analyze the antibacterial activity at higher
concentration levels.
4. Conclusion
After carrying out two factorial experiments, extract with red
pigment maximized concentration was produced with 42.5 g/L of corn
bran and 30 g/L of peptone in submerged fermentation using Monascus
purpureus for 6 days (time was studied for greater production of red
pigment in less time).
Submerged fermentation produced pigment extract with high anti­
oxidant activity (twice as much as the substrate). Thermal (sterilization
and 40–80 ◦ C in hot dry) and pH (4.0–7.0) stabilities were demonstrated
for the pigment extract produced by M. purpureus.
Thus, the production of pigments using microorganisms seems to be
a strategy to increase the use of corn bran, which is currently under­
valued and even neglected due to the lack of industrial application.
Author contributions
Adrielle Borges de Almeida, Investigation, Writing - Original Draft.
Nathalia Horrana Santos, Investigation.
Thayanara Mayara de Lima, Investigation, Writing - Original Draft.
Railany Vieira Santana, Investigation.
Josemar Gonçalves de Oliveira Filho, Investigation, Writing - Orig­
inal Draft.
Daiane Sousa Peres, Investigation.
Mariana Buranelo Egea: Conceptualization, Formal analysis, Writing
- Review & Editing, Supervision, Project administration, Funding
acquisition.
Acknowledgment
The authors received financial support from CNPq, IF Goiano, FAPEG
(003/2017), and Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior – Brasil (CAPES) – Finance Code 001.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bcab.2021.101931.
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