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Chiang Mai J. Sci. 2014; 41(5.1)
Chiang Mai J. Sci. 2014; 41(5.1) : 1032-1043
http://epg.science.cmu.ac.th/ejournal/
Contributed Paper
Effect of Ammonium Salts on Pigments Production
by Monascus anka Mutant in 5L Bioreactor
Bo Zhou* [a,b,c], Yan Wang [a,b,c], Huamin Lu [a,b,c] and Yanqing Zhou [a,b,c]
[a] School of Food Science and Engineering, Central South University of Forestry and Technology,
Changsha, 410004, P. R China.
[b] National Engineering Laboratory for Rice and By-products Processing, Changsha, 410004, P. R China.
[c] Hunan Province Key Laboratory of Grain, Oil Processing and Quality Control, Changsha, 410004,
P. R China.
*Author for correspondence; e-mail: [email protected]
Received: 4 July 2013
Accepted: 6 September 2013
ABSTRACT
This work characterized the fermentation and Monascus pigments production of
Monascus anka mutant in a 5L bioreactor under the different ammonium salt conditions:
ammonium chloride, ammonium sulfate or ammonium nitrate. All the experimental results
obtained in this work suggest that not the pH environment established by ammonium salts
but ammonium salts itself played a critical role on the production of Monascus pigments and
the growth of Monascus anka mutant. ammonium ion could inhibits the activity of some key
enzyme for Monascus pigments synthesis and the inhibition effect on some key enzyme for
Monascus red pigments synthesis is stronger than on some key enzyme for Monascus yellow
pigments synthesis. Key enzyme for inhibited Monascus pigments production would be
researched clearly by experiments in our future researching.
Keywords: Monascus anka mutant, ammonium salts, pH, yellow pigments, red pigments
1. INTRODUCTION
In food industrial nowadays, yellow
pigments production has two main
approaches: chemical synthesis and plant
extraction. The former, with products such
as tartrazine [1], due to its safety risk, has
withered in use. Meanwhile, the latter, with
extracts such as gardenia yellow
[2]ÿsafflower yellow [3], is natural and low
in toxicity, but is subject to the season
change, resulting in an unstable industrial
production. Compared with these two
approaches, microbiological fermentation
for yellow pigments production, because
of its few safety hazards, low cost, and wide
variety of resource choices [4-7], has
received increasing attention as a promising
alternative. As far as industrial production
is concerned, Monascus is one of the
microbial resources now in use to produce
pigments.
Thousands years before ancient
Chinese had been using Monascusfermented red rice as a food colorant to
make red rice wine, red soybean cheese,
Chiang Mai J. Sci. 2014; 41(5.1)
meat and fish products and so on [8-10].
The genus Monascus produces a mixture of
three categories of pigments, orange, red,
and yellow pigments. These three pigments
have two components of polyketide origin
in common, which are secondary
metabolites possessing an azaphilone
skeleton [8, 11-13]. The red pigments
produced by Monascus are applied widely
due to their safety with the characteristics
of high protein adhesion, thermal stability,
and wide-pH stability [14]. But with
citrinin production by Monascus was
discovered [15], which is nephrotoxic (kidney
damaging) and possibly carcinogenic
[16-18] to human beings, the safety of
Monascus products has been paid more and
more attention to.
The production of Monascus red
pigments has been researched a lot. Some
parameter factors affecting the growth of
Monascus and production of red pigments,
such as gas environment [19], agitation and
aeration [20], carbon and nitrogen sources
[21] and pH [22-24] were reported, and the
perstractive fermentastion of intracellular
Monascus pigments in nonionic surfactant
Triton X-100 micelle aqueous solution
was also reported [25-27]. However, due to
unavailability of microbial species, researches
on production of Monascus yellow pigments
(Monascusone A and B) is very limited
[28, 29]. One Monascus anka mutant able
to produce high yield of yellow pigments
(Ankaflavin and monascin) screened by
physical and chemical combination
mutagenesis was obtained in our
laboratory, the yellow pigments produced
by Monascus anka mutant have maximum
absorption at 410 nm [30]. After incubating
for 3 days on MEA medium (Per Liter
contains malt extract 20 g, peptone 1 g,
glucose 20 g, and agar 15 g), the colonies of
Monascus anka mutant showed yellowish
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in color with the size of 25-30 mm. After
incubating for 5 days, the aerial mycelium
was white; the colonies showed yellow
orange in color with the size of 29-30 mm,
and the air-borne mycelium was white.
Both conidiospores and ascocarpes were
colorless. The conidiospores were 40-50 μm
in size and the ascospores were oval-shaped
with the size of 30-35 μm.
As the use of available microbial source
of nitrogen, it was key inhibition effect
factors for antibiotics formation by high
concentrations of nitrogen sources, such
as 14-membered macrolide antibiotic
erythromycin [31]and the 16-membered
macrolide antibiotics leucomycin [32].
But in our previous study, ammonium salts
played a critical role on Monascus yellow
pigments production by Monascus anka
mutant in the shaker cultivation [30].
In present study, we characterized the
fermentation and Monascus pigments
production of Monascus anka mutant in the
bioreactor under different ammonium
salt conditions, ammonium chloride,
ammonium sulfate or ammonium nitrate,
and then made an analysis about ammonium
salts affects on Monascus yellow pigments
production by Monascus anka mutant.
2. MATERIALS AND METHODS
2.1 Organism and Cultivation
The microorganism used in this work is
Monascus anka mutant able to produce
comparatively high-production of yellow
pigments, which was screened by
combination of physical and chemical
mutagenesis in our laboratory. The original
strain of Monascus anka obtained from
China Center of Industrial Culture
Collection(CICC 5013).
Stock cultures of the mutant were
maintained and periodically subcultivated
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on wort agar slants, which contains 15°
wort (provided by Guangzhou Zhujiang Beer
Co., Ltd, China) and 20g/L agar (Difco
Labatory, Loveton Circle, USA). Cultures
were reactivated by being transferred onto
fresh wort agar slants. After cultivation of
2-3d at 31 °C, spores were collected with
5 mL of sterilized water, and the spore
suspension corrected was used as inoculum
preparation. 1 mL of spores suspension
was inoculated in 100 mL of seed medium
(30g/L corn powder, 3g/L NaNO3, 0.01g/
L FeSO4.7H2O, and 4g/L KH2PO4, pH6.0),
and cultured at 31 °C, 200rpm for 2d in a
rotatory shaker as the seeds cultures.
The seed cultures were inoculated in
the submerged culture medium at volume
ratio of 1:9, and fermented in a 5L
bioreactor (BIOFLO 3000 Batch/Continuous
Bioreactor, New Brunswick Scientific Edison,
N.J., USA) at 300 rpm, 31 and aeration of
0.15 m3.h-1 for 6-7d. Submerged culture
medium contains ammonium salts, 12g/L
corn steep liquor, 5g/L KH2PO4 , 20g/L
glucose, 40g/L starch and 0.1g/L CaCl2,
pH4.0. The type of ammonium salts was
various in different experiments, and the
dosage of ammonium salts was determined
by NH4+, 0.3moL/L of which was used for
each fermentation. The 5L bioreactor loaded
3 L of submerged culture medium.
2.2 Measurement of Pigments
5 mL of the culture broth was
extracted with 5 mL of 70% (v/v) ethanol
at room temperature for 1h, and then
centrifuged at 4000rpm for 20min. The
supernatant obtained was filtered with filter
paper. The filtrate contained two pigments:
yellow pigments and red pigments, whose
concentrations were determined by measuring
the optical density of the filtrate using a
2802SUV/VIS spectrophotometer (Unicosh
Scientific Instrument Co., Ltd, Shanghai,
China) at 410nm and 510nm respectively
Chiang Mai J. Sci. 2014; 41(5.1)
(Yoshimura et al. 1975). Results were
expressed with OD units per mL of
fermented broth. The linearity equation
between absorbance and diluting
proportions is y = -0.0054x + 1.5462 (R2
= 0.9907). In where, y is absorbance and x
is dilution proportions (in the range from
100 to 300).
2.3 Measurement of Dry Cell Weight
(DCW)
5mL of cultures broth was filtered
through fibre-glass membranes (Whatman
GF/C), the retained biomass was rinsed twice
with distilled water, vacuum dried completely
at 60!, and weighed.
2.4 Data Calculation and Analysis
The specific growth rate, m(h-1), was
calculated with the equation:
where
X is the cell concentration (g/L) at time t (h).
The specific production rate of yellow
pigments, qY(OD units /h), was calculated
with the equation:
where Y is
the yellow pigments value (OD units) at time
t (h). The specific production rate of red
pigments, qR(OD units /h), was calculated
with the equation:
where R is the
red pigments value(OD units) at time t (h).
2.5 Data Analysis
All the data shown in Figures were
expressed as mean ± standard deviation of
the three replicates. The statistical
evaluation of all data was done by
OriginPro8.0.
3. RESULTS AND DISCUSSION
3.1 Effect of Ammonium Salts on pH of
Cultures in Fermentation of Monascus
anka Mutant
When using ammonium chloride
or ammonium sulfate as nitrogen source
for production of pigments, the pH
Chiang Mai J. Sci. 2014; 41(5.1)
value of cultures changed in downtrend
during the whole fermentation and the
final pH value of cultures was 1.90 and
2.30, respectively (Figure 1). When using
ammonium nitrate for production
of pigments, pH value of the cultures
changed in downtrend within first
54hrs and pH value reached 2.97 at 54h,
however, afterwards pH value of
the cultures increased gradually and got
to 6.00 in the end of fermentation
(Figure 1).
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Three kinds of ammonium salts used
in this work are all strong-acidity and
weak-alkalescence salts. Ammonium
chloride is the strongest in acidity, and
followed by ammonium sulfate and
ammonium nitrate successively. Along with
NH4+ consumed as nitrogen sources for
Monascus growth, the acidity of
fermentation broths rises and the pH of
cultures declines. The lowest pH (pH<2)
environment was established because of
Figure 1. Effect of ammonium salts on culture pH by Monascus anka mutant fermentation in
a 5L bioreactor.
strong acidity of ammonium chloride.
When using ammonium sulfate for
production of Monascus pigments, the final
pH value could maintain within the range
from 2 to 2.5. But the different situation
occurred while using ammonium nitrate
for production of Monascus pigments,
because Monascus can not only use NH4+
for Monascus growth but also use NO3- for
production of Monascus pigments [21]. In
the beginning, NH4+ was used for Monascus
growth and the pH of cultures declined,
but 54hrs after fermentation, the pH of
cultures rose due to a great deal of NO3consumption for production of red
pigments.
3.2 Effect of Ammonium Salts on Growth
of Monascus anka Mutant
Figure 2 indicates that ammonium salts
used in this work have no significant effects
on the maximum dry cell weight (DCW), but
bear significant relationship with the growth
process. The trend of growth is similar
between the fermentations using ammonium
chloride and ammonium sulfate, but different
from the fermentation using ammonium
nitrate. The time to reach maximum DCW in
the fermentation using ammonium nitrate is
earlier than those using ammonium chloride
and ammonium sulfate. The maximum
DCW could arrive at 27.86 g/L, 27.46 g/L
and 27.78 g/L at 84h, 102h and 102h,
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respectively (Figure 2).
However, ammonium salts have effects
on specific growth rate ( μ ) (Figure 2).
The trend of is similar between the
fermentations using ammonium chloride
and ammonium sulfate. When using
Chiang Mai J. Sci. 2014; 41(5.1)
ammonium nitrate, however, the value is
higher than that using ammonium chloride
and ammonium sulfate before 40h. Their
maximum value can get to 0.048 h-1 at 24h,
0.041 h -1 at 12h and 0.045 h -1 at 12h,
respectively.
Figure 2. Effect of ammonium salts on dry cell weight (DCW) and specific growth rate (μ)
of Manascus anka mutant.
In a way, ammonium salts little
influence the yield but affect the specific
growth rate of Monascus anka mutant.
Effect of ammonium salts on Monascus
pigments production by Monascus anka mutant
As far as yield of yellow pigments, the
maximum amounts of yellow pigments has
no significant difference between
ammonium chloride, ammonium sulfate
and ammonium nitrate, the maximum
amounts of yellow pigments got to 54.87
OD units at 96h, 51.12 OD units at 96h,
49.56 OD units at 120h, respectively
(Figure 3). But it has obviously difference
for yield of red pigments, the maximum
amounts of red pigments got to 11.26 OD
units at 80h, 34.08 OD units at 96h, 42.06
OD units at 132h, respectively (Figure 3).
When using ammonium chloride as
nitrogen source for production of Monascus
pigments, compared to yellow pigments,
the production of red pigments was
remaining at a very low level during the
whole fermentation. (Figure 3). Before
48hrs, the trend of production of yellow
pigments was similar to that of red pigments
when using ammonium sulfate for
production of pigments, but the production
of yellow pigments was obviously higher
than that of red pigments after 48hrs, and
the pH value of cultures was 2.56 at this
time (Figure 1). The pH of cultures
declined to 2.97 after 54hrs of fermentation
when using ammonium nitrate as nitrogen
Chiang Mai J. Sci. 2014; 41(5.1)
source for production of Monascus
pigments (Figure 1). Compared to yellow
pigments, the production of red pigments
was obviously retarded before 54hrs and
then accelerated, and the yield of yellow
pigments was 4.5 times as much as that of
red pigments before 54hrs. In the end of
fermentation, the yield was not of
significant difference between yellow and
red pigments (Figure 3). The maximum
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production of red pigments using
ammonium chloride or ammonium sulfate is
just 26.77% and 81.10% as much as that
using ammonium nitrate, respectively
(Figure 3). The maximum yield of yellow
pigments using ammonium chloride,
ammonium sulfate, ammonium nitrate, was
4.87 times, 1.50 times, 1.18 times,
respectively, as much as that of red
pigments (Figure 3).
Figure 3. Effect of ammonium salts on red and yellow pigments production by Monascus
anka mutant in a 5L bioreactor.
The specific production rate of yellow
pigments (qY) can maintain at a low level
(the maximum qY can get to 0.032 OD
units /h at 72h) when using ammonium
nitrate for fermentation of Monascus
pigments. When using ammonium chloride
and ammonium sulfate as nitrogen sources,
from 36h to 60h, the value of qY can increase
to 0.08 OD units /h from 0.05 OD units /
h. The maximum value of qY can reach 0.076
OD units /h and 0.072 OD units /h at 54h
and 36h, respectively (Figure 4).
1038
Chiang Mai J. Sci. 2014; 41(5.1)
Figure 4. Effect of ammonium salts on specific production rate of red pigments(qR) and
yellow pigments (qY) of Manascus anka mutant.
Compared with using ammonium nitrate
and ammonium sulfate, the production of
red pigments was inhibited more obviously
when ammonium chloride was used, but the
trend is similar to that when using ammonium
sulfate and only the inhibitory degree is
different, the specific production rate of
red pigments (qR) can be up to 0.037 OD
units/h for ammonium chloride and 0.048
OD units /h for ammonium sulfate at 36h.
But when using ammonium nitrate as nitrogen
source, at 84h, the can get to 0.043 OD
units/h, and the can be kept at this value or
up for longer than that when using ammonium
chloride and ammonium sulfate (Figure 4).
Despite the trend of production of red
pigments using ammonium chloride as
nitrogen source is similar to that using
ammonium sulfate, the descending speed of
using ammonium chloride is faster than
that using ammonium sulfate. Thus, the yield
of red pigments using ammonium sulfate
is higher than that using ammonium
chloride (Figure 3). When ammonium nitrate
was used for production of red pigments,
not only the can keep at a low value (low to
0.016 OD units/h ) for a long time, but also
the time to reach maximum is later, and
the dropping range is narrower than those using
ammonium chloride and ammonium sulfate
(Figure 4). The trend of production of
yellow pigments is similar to that using
ammonium chloride or ammonium sulfate.
In the case of ammonium chloride, both the
rising and dropping slopes of are steep. As a
result, the yield of yellow pigments was of no
significant difference between using ammonium
chloride and ammonium sulfate (Figure 3).
When using ammonium nitrate for production
of yellow pigments, not only the can keep
at a low level (from 0.01 OD units/h to
0.03 OD units/h) for a long time, but also the
time to reach maximum is later than that
using ammonium chloride and ammonium
sulfate(Figure 4). So, the yield of yellow
pigments using ammonium nitrate was of no
significant difference from using ammonium
chloride and ammonium sulfate (Figure 3).
Chiang Mai J. Sci. 2014; 41(5.1)
Kurono et al. reported that the
ammonium salts were bad for production
of yellow pigments and also had no
significant influence on production of red
pigments [14], Chen et al. observed that
low pH was good for production of yellow
pigments [24]. Our results obtained in the
present work indicate that although
ammonium salts did obviously influence
not only yellow pigments production but
also red pigments production by Monascus
anka mutant, they did influence the specific
production rate of yellow pigments (Fig.
4). Ammonium salts influenced the yield
as well as specific production rate of red
pigments (Fig. 4). Taken together with the
results that ammonium salts did not
influence the yield but affected the specific
growth rate of Monascus anka mutant and
with the results that ammonium salts
affected the pH of cultures (Figs. 1-2).
Three kinds of ammonium salts used in this
work are all strong-acidity and weakalkalescence salts. Ammonium chloride is
the strongest, ammonium sulfate the second
and ammonium nitrate the third in acidity.
Along with NH4+ consumed as nitrogen
sources for Monascus growth, the acidity
of fermentation broths rises and the pH
of cultures declines. There is still one
different point, however, in the case of
ammonium nitrate, it is clear as Pastrana
et al. reported that NH4+ as one nitrogen
source is mainly used for the growth of
Monascus [21], NO 3- is a good nitrogen
source able to be used not only for the
growth of Monascus but also for production
of red pigments. The pH of cultures using
ammonium nitrate first declines then
increases gradually along with the
consumption of NO 3 - . In the end of
fermentation, the pH of cultures using
ammonium chloride, ammonium sulfate or
ammonium nitrate was 1.90, 2.3 and 6.0,
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respectively (Figure 1). Low pH of cultures
(<2) like using ammonium chloride greatly
inhibits the production of red pigments and
high pH of cultures like using ammonium
nitrate is beneficial for the production of
red pigments. Lin et al. also reported the
similar results [22]. The maximum
production of red pigments using
ammonium chloride is just 26.77% as much
as that using ammonium nitrate in our
experiments. Taken together with the
results, we made an assumption that pH
of cultures of Monascus anka mutant
directly influenced the production of
Monascus pigments, and pH of cultures
influences the whole process of the
production of Monascus pigments including
yellow and red pigments.
In order to verified our assumption,
A two-stage pH control strategy was
carried out in an attempt to maximize
yellow pigment formation in this paper.
Firstly, an initial pH of 6.5 was kept
o
with 0.1 mol/ L NaOH at 31 C for 2 days
in order to obtain maximum biomass. In
the second stage, the pH was adjusted
to 2.5 with 0.1 mol/L H2SO4 to stimulate
the production of yellow pigment for
the next 4 days. However, yellow pigment
yield was no greater than with the single
stage fermentation (data not shown), the
results was the same as described by
Yongsmith [33]. Our results further
demonstrated that not the pH environments
established by ammonium salts but the
ammonium ion itself played a critical role
on the production of Monascus pigments
and the growth of Monascus in the Monascus
anka mutant fermentation.
As the use of available microbial source
of nitrogen, it was key inhibition effect
factors for antibiotics formation by high
concentrations of nitrogen sources [34, 35],
such as 14-membered macrolide antibiotic
1040
erythromycin [31] and the 16-membered
macrolide antibiotics leucomycin [32]
and tylosin [36] is inhibited by high
concentrations of ammonium. The
production of polysaccharides by
Aureobasidium pullulans was also suppressed
under high nitrogen levels [37]. The
inhibition effect was considered that the
key enzyme activity for antibiotics
synthesis has been inhibited, such as valine
dehydrogenase [38] and threonine
dehydratase [39] as key enzyme providing
the precursors for tylosin formation are
repressed by ammonium ion in batch
culture.
According to the results in this paper
and previously reported results,
ammonium ion maybe inhibits the activity
of some key enzyme for Monascus pigments
synthesis, and the inhibition effect on some
key enzyme for Monascus red pigments
synthesis is stronger than on some key
enzyme for Monascus yellow pigments
synthesis. This hypothesis should be
verified by experiments in our future
researching.
The production of mycotoxin citrinin
of Monascus is affected not only by the
growth conditions [39, 40] but also by
genus itself [15, 41]. Sandra et al. reported
that the high pH (about 6) was not good
for but low pH was good for the citrinin
production by Monascus [42]. In this study,
the citrinin was unable to be detected by
HPLC method as described by Xu [43],
namely, the Monascus anka mutant may
produce no or just a little citrinin (the
detection limit is 0.1mg/L).
ACKNOWLEDGEMENTS
This work was supported by the National
Natural Science Foundation of China
(Nos.: 31301550), the youth Scientific Research
Chiang Mai J. Sci. 2014; 41(5.1)
Fundation of Central South University of
Forestry & Technology (Nos: QJ2010009A).
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