Monascus Purpureus: A Potential Source For Natural Pigment Production

Journal of Microbiology and Biotechnology Research
Scholars Research Library

J. Microbiol. Biotech. Res., 2011, 1 (4): 164-174
(http://scholarsresearchlibrary.com/archive.html)
ISSN : 2231 3168

CODEN (USA) : JMBRB4
Monascus purpureus : A potential source for natural pigment production

Rashmi Dikshit and Padmavathi Tallapragada *
Department of Microbiology, Centre for PG Studies, Jain University, Bangalore, Karnataka India
__________________________________________________________________
ABSTRACT
In this study, growth pattern, temperature & pH on pigment production and mycelial growth of
Monascus purpureus was investigated in submerged culture. It has been observed that maximum
red pigment was produced at the 16th day of incubation (11 CVU/ml at 510 nm). The favourable
temperature for microbial growth and pigment production was found to be 30C among the
experimented temperatures. Also, the maximum pigment was observed at a pH value of 5.5.
Effect of different solid substrate on pigment production and optimised by variable Carbon and
Nitrogen content has also been investigated. Local unpolished rice (11.5 CVU/ml) was found to
be the best source as a solid substrate and there was no impact on pigment production when the
substrates were supplemented with glucose. For variable nitrogen sources, a positive increase
in pigment yield was observed. Maximum pigment yield was observed with rice supplemented
with 5% yeast extract (29.9 CVU/gds) followed by 1% yeast extract (25 CVU/gds) and for 5%
MSG (25.2 CVU/gds) followed by 1% MSG (18.1 CVU/gds). There was no considerable impact
of peptone-supplemented substrates on pigment production. The presence of citrinin was
examined by LC-MS.
Key words: Monascus, pigment, mycelial growth, submerged culture, citrinin.
______________________________________________________________________________
INTRODUCTION
Angkak or red fermented rice is a product produced from the fermentation of rice substrates by
Monascus sp. Solid-state fermentation of Monascus on rice has a long tradition in East Asian
countries, dating back to at least the first century A.D. For centuries angkak has been consumed
extensively in Asia as a natural food coloring of fish, Chinese cheese, red wine and sausages
[11,7].
Monascus species are known to produce well-known azaphilone pigments like monascorubrin
rubropunctatin [20] and more recently, monascusones from a yellow Monascus mutant have
been identified [28]. Monascorubrin and rubropunctatin have a unique structure responsible for
their high-affinity for compounds with primary amino groups (so-called aminophiles). Reactions
with amino acids yield the water-soluble red pigments, monascorubramine and rubropunctamine.
In fact, various pigment derivatives with improved functional properties in the colour range of
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Padmavathi Tallapragada et al
J. Microbiol. Biotech. Res., 2011, 1 (4):164-174
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orange-red to violet-red can be produced by Monascus fermentations in the presence of different
amino acid [14]. However, Monascus -fermented rice has been found to contain the mycotoxin
citrinin [5]. The production of citrinin together with pigments clearly limits the use of Monascus
as a producer of natural food colourants [1].
Direct estimation of biomass or mold mycelium to elucidate its growth in solid-state
fermentation is usually impossible, because it is homogeneously adsorbed onto the substrate
matrix. Chitin, a fungal cell wall component is a polymer of N-acetylglucosamine could be
hydrolysed by acid into N-acetylglucosamine (aldehydes+amino sugars) monomers followed by
the aldehyde group (C2H2O) in N-acetylglucosamine, which is separated out of the amino sugar
group (C6H13NO5) to release glucosamine, amolecular weight of 179.0 which is directly
measured as a biomass indicator [9, 16].
At the same time, Monascus spp. has been reported to co-produce the mycotoxin citrinin, a
hepato-nephrotoxic compound in humans [22]. In addition to citrinin, other potential toxic
metabolites, such as monascopyridines [10,3] have also been reported in Monascus fermented
red rice [2].
MATERIALS AND METHODS
Culture
Monascus purpureus MTCC 410 was procured from the Microbial Type Culture collection,
IMTECH, Chandigarh, India. The strain was maintained on Potato Dextrose Agar (PDA)
medium and incubated at 28-30 C for 7 days, preserved at 4 C, and sub-cultured once every 4
weeks.
Inoculum preparation
Inoculum preparation for solid-state fermentation was performed as described by Babitha et al.
[6] with some modification. One full loop of sporulated (6-day old) agar slope culture was
diluted in distilled water. The spores were scraped off under aseptic conditions to produce
homogenous spore suspension to be used as the inoculum.
Evaluation of growth pattern, temperature and pH on mycelial growth & pigment
production of Monascus purpureus
Growth pattern of Monascus purpureus was investigated in potato dextrose broth (PDB) media.
Media was inoculated with M. purpureus and incubated for 16 days in static condition. Biomass
and pigment were estimated every 4th, 8th, 12th and 16th days after inoculation.
To study the effect of temperature on mycelial growth and pigment production PDB media was
inoculated with M. purpureus and incubated at different temperature viz. 16C, 30, C 37C and
50C for 15 days in static condition
Varying range of pH was also investigated. pH of the medium was adjusted in the steps of 4.5,
5.5, 6.5, 7.5 and 8.5. Media was inoculated with M. purpureus and incubated for 15 days in
static condition and the effect of pH on mycelial growth and pigment production was studied
[27].
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Dry cell weight
The mycelia separated from broth by filtration (Whatmann No. 1) were weighed on an analytical
scale, vacuum filtered through pre-weighed membrane filters, washed with distilled water, dried
in an oven at 50C. The results were expressed in grams per litre [13].
Pigment Estimation
Pigment estimation in submerged culture was carried out using culture filtrate. The mycelia
were separated from broth by filtration (Whatmann No. 1). Filtrate was centrifuged at 10000xg
for 15 minutes. The supernatant was decanted and the pigment concentration was determined at
510 nm. The absorbance values were converted into pigment units using by the following
formula:
Colour value = O.D. x dilution x volume of extracts / Amount of sample (ml) [28].
Substrate Selection and Solid-State Fermentation
For solid-state fermentation four substrates had been chosen viz. Oryza spp. (local unpolished
rice), Fagopyrum spp. (Buckwheat) flour, Colocasia spp. (arbi), and Manihot spp. (tapioca).
These were purchased from a local market of Bangalore, India, and were used as the basic solid
substrate for pigment production by means of solid-state fermentation. Effect of solid substrate
on pigment production has been investigated on strain Monascus purpureus. Initially, twenty
gram of substrate was placed in a 250 ml conical flask to which distilled water was added, pH of
the medium was adjusted to 6.0 and autoclaved at 121C for 20 minutes. After cooling, the
substrate-based medium was inoculated with 10% of the seed culture of the Monascus purpureus
and incubated at 28C- 30C for 20 days [19]. Moisture content was maintained between 56-60%
and was calculated based on the following formula,
Moisture content of substrate (%)=100*(wet weight dry weight) /wet weight [20]
Screening of Nitrogen source on pigment production
Nitrogen sources such as peptone, yeast extract, monosodium glutamate at 1%, 5%, 10%
concentration were used for pigment production. Twenty gram of substrate supplemented with
above concentration of N sources was autoclaved and then inoculated with 10% of the seed
culture of the Monascus purpureus and incubated at 28- 30C and 56-60% relative humidity for
20 days [23].
Screening of carbon source on pigment production
Effect of carbon source has been investigated in solid- state fermentation. Glucose and Xylitol as
a carbon source were used in the concentration of 4%, 8% & 12%. Twenty gram of substrate
supplemented with above concentration of carbon source was autoclaved and then inoculated
with 10% of the seed culture of the Monascus purpureus and incubated at 28C- 30C and 5660% relative humidity for 20 days [19].
Pigment extraction and estimation
For solid substrate the culture medium was dried at 50C for 48 hours. One gram of fermented
solid substrate was taken for pigment extraction using 10 ml of 95% ethanol and shaking it on a
rotary shaker at 200 rpm for 24 hrs. The extracts were allowed to settle at room temperature and
then filtered through Whatmann filter paper. Ethanol extracts of unfermented substrates were
kept as blanks. Analysis of pigment production was done by measuring absorbance at 510 nm,
near the absorbance peak of red pigments.
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The absorbance values were converted into pigment units using by the following formula:
Colour value = O.D. x dilution x volume of extracts / Amount of sample (g) [15].
Analysis of Citrinin
Qualitative analysis of citrinin has been done by LC-MS. The Instrument used for citrinin
analysis was HPLC Thermo Finnigan Surveyor and MS Thermo LCQ Deca XP MAX. The
software used in this experiment was Xcalibur.
The following conditions of the HPLC experiment were followed:
The column used was BDS HYPERSIL C18 with a length of 250 mm, I.D. of 4.6 mm and
particle size of 5 m. The UV @ 280 nm (Channel C) was used for the detection of Monascus
purpureus.
The detectors used were HPLC PDA / UV detector and the temperature were ambient with 10L
as the volume injected. The eluent was C18 at 0 mins and the eluent mixture was 40 / 40 / 20:
Acetonitrile / Methanol / water.
MS Experimental Conditions used in the experiment were with Probe/ source voltage of 4.5kV,
Sheath gas flow of 40.00 and Auxiliary/Sweep gas flow of 26.00. The Source type was Electro
Spray Ionisation (ESI) with Capillary temperature of 275C and Capillary voltage of 16V. The
nebulization gas flow was with Helium at approx 1mL/min and the Helium in the mass analyzer
cavity was maintained at 0.1Pa (10-3) [24].
Statistical analysis
The data was analysed using MS Excel and ANOVA. All the experimental data were analysed
with SPSS 15.0.1 program.
RESULTS AND DISCUSSION
Evaluation of growth pattern, temperature and pH on mycelial growth & pigment
production of Monascus purpureus
The growth pattern of the organism was measured on dry weight basis and pigment production.
The pattern was observed to be more or less linear in nature with biomass showing a steady
increase with the period of incubation. Maximum biomass was observed on 12th (2.46gm/l).
Since, day 4 of incubation, sign of pigment production appeared and continued to accumulate
through out the fermentation period but yield reached to its maximum concentration at 16th day
of inoculation (11 CVU/ml). (Fig.1).
Pigment production was observed on 3rd day after incubation and continued to accumulate
throughout the fermentation period. Maximum pigment yield was observed on the 10th day after
incubation. Mycelial growth increased with time and reached to its maximum on 6th day of
incubation [13].
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12
10
2.5
1.5
0.5
Dry Weight (gm/l)
CVU/ml at 510 nm
Fig 1: Growth Pattern of Monascus purpureus
0
96
192
288
384
Time in hours
Pigment Yield of M purpureus
Biomass of M purpureus
Vertical bars represent the mean value standard error
Monascus purpureus was subjected to temperature variation (16 C, 30 C 37C and 50 C) and
the results showed that this strain was not able to grow on temperature 16 C, and 50 C.
Maximum red pigment was observed at 30 C and there was reduction in pigment yield at 37 C.
Fig 2: Spectral Analysis on pigment production
60
50
CVU/ml
40
30
20
10
0
450 nm
470 nm
510 nm
520 nm
540 nm
570 nm
600 nm
Wavelength
30 deg C
37 deg C
The enzymatic activity might be optimum for red pigmentation at 28C to 32C, which is the
reason for maximum red pigments observed at these temperatures. Beyond 35C, there is a shift
of absorbance spectra at 470 nm, which was the range for yellow-orange pigments absorbance
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(Fig. 2). At 37C, there was a reduction in pigment yield. Hence temperature plays a pivotal role
in cell metabolism thus influencing pigment yield [27].
It was found that maximum absorbance at 510 nm (red pigment) was obtained around 32C to 35
C but beyond 40 C, there was drastic reduction in red pigment [6]. Carvatho et al. [16]
reported a shift in absorbance maxima of pigment extract at different incubation temperature.
Optimum cultural conditions for Monascus spp. could be incubation period, 8 days, temperature
32C and pH 6.0.
The growth of Monascus purpureus was observed at entire tested range of pH (4.5 to 8.5),
though it showed a downward trend. Maximum biomass was observed at pH 4.5 (5.5 CVU/ml)
and maximum pigment at pH 5.5 (16.5 CVU/ml). Yellow pigment was dominating at pH 4.5
and red pigment at pH 6.5. (Fig.3)
pH influenced the physiology of fungi, conidial development and pigment synthesis. At acidic
range of pH 4.5, conidiation was found to be increasing whereas the red pigment synthesis
showed reduction [13].
The red pigment at 500nm was more pronounced at pH 6, but acidic pH supported yellow
pigment production [27]. It has been reported that at lower pH, there was predominance of
yellow pigments and at a higher pH; there was predominance of red pigments [4]
Fig 3: Effect of pH on pigment production and mycelial growth of Monascus purpureus
18
16
5
12
10
3
8
2
Dry weight (gm/l)
CVU/ml at 510 nm
14
4
1
2
0
0
4.5
5.5
6.5
7.5
8.5
pH variation
Pigment Yield of M purpureus
Biomass of M purpureus
Screening of substrates for Monascus purpureus

The screening of solid substrate for pigment production by Monascus purpureus was done by
using cheap and abundant agricultural products like Oryza spp. (local unpolished rice),
Colocasia spp. (arbi), Manihot spp. (tapioca) and Fagopyrum spp (Buckwheat flour). It was
169
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found Monascus purpureus grew on all experimented substrates though pigment yield varied.
Local unpolished rice with 12 CVU/gds at 510 nm, Fagopyrum spp. with pigment yield of 6.2
CVU/gds at 510 nm showed maximum pigment yield followed by Manihot spp with 5.9
CVU/gds at 510 nm. It was also observed that Colocasia was not suitable substrate for Monascus
purpureus because of less pigment yield (2 CVU/gds). Monascus purpureus produced maximum
pigment with local unpolished rice. (Fig.4)
Steinkraus [17] established that pigment can be produced from several rice strains with high
amylose (25-33%) and more pigment production will be obtained using rice with low
amylopectin. Also it was suggested that the use of sticky rice would inhibit the growth of
Monascus purpureus [12].
To select the best natural substrate for bio pigment by Monascus spp., four different substrate
with greater carbohydrate concentration were evaluated. It was expected that substrate with
higher carbohydrate content could behave a better fermentation media for Monascus spp.
however the results revealed that some of the substrates (Colocasia spp.) had shown decreased
fungal growth. This might be due to moisture content as it is observed that 56-60% water content
is not adequate for all substrates [8].
Fig 4: Screening of substrates for pigment production by Monascus purpureus
14
Pigment Yield (CVU/gds)
12
10
8
450 nm
510 nm
6
4
2
0
Colocasia spp.
Manihot spp.
Fagopyrum spp.
Oryza spp.
Solid Substrates
Effect of Carbon Source on pigment production by Monascus purpureus

The strain Monascus purpureus was able to assimilate the carbon source but there was a decrease
in the pigment yield when supplemented with 4%, 8% and 12% glucose as compared to the
control. Maximum red pigment yield in control was (11.5 CVU/gds) with local unpolished rice
followed by Fagopyrum spp. with (6.1 CVU/gds) and Manihot spp. with (5.6 CVU/gds) (Fig.5).
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The strain Monascus purpureus has not shown any growth with substrate supplemented with
xylitol.
Solid-state fermentation done with Jackfruit seed powder with a particle size between 0.4 and 0.6
mm by Monascus purpureus without any additional carbon source was found to be the best
substrate for pigment production [6]. The results of our study agree with the above findings.
Utilization of carbon sources for growth appears to be strain specific since for other stains of
Monascus glucose and its oligo-and polysaccharides were better than other carbon sources both
for growth and pigment production [18,30,31].
Fig 5: Effect of C Source on pigment production by Monascus purpureus
12
10
0
control
4% glucose(w/w)
Colocasia spp.
Manihot spp.
8% glucose(w/w)
Fagopyrum spp.
12% glucose(w/w)
Oryza spp.
Effect of N Source on pigment production by Monascus purpureus

This strain was not able to grow on peptone media and maximum pigment yield was observed
when substrates were supplemented with 1% and 5% MSG and yeast extract as a nitrogen
source. Maximum pigment yield was observed with rice supplemented with 5% yeast extract
(29.9 CVU/gds) followed by 1% yeast extract (25 CVU/gds) and for 5% mono sodium glutamate
(MSG) (25.2 CVU/gds) followed by 1% MSG (18.1 CVU/gds). Manihot spp and Fagopyrum
spp. have shown maximum pigment yield with 5% yeast extract (8.8 CVU/gds and 9.1 CVU/gds
respectively) (Fig.6). Whereas 10% Nitrogen content in all the substrates were found to be
inhibitory in nature.
The chemistry of the Monascus pigment suggests that the orange pigment forms the red pigment,
as the yellow pigment is unable to react with NH group to produce corresponding amine. This
also depends on the cultural conditions and C-N ratio [13]. Water-soluble pigments were
produced when jackfruit seed powder was supplemented with monosodium glutamate, soybean
171
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meal, and peptone or chitin powder. The addition of external nitrogenous compounds showed a
positive impact on water-soluble pigment production [26]. Vidyalakshmi et al., [23] determined
the pigment yield of Monascus ruber by the solid-state fermentation of rice supplemented with
different N sources and maximum pigment yield was observed when rice supplemented with
monosodium glutamate.
Fig 6: Effect of N Source on pigment production by Monascus purpureus
35
30
25
20
15
10
5
0
control
1%
5%
10 %
1%
5%
10%
peptone peptone peptone yeast ex yeast ex yeast ex
Colocasia
Manihot spp.
Fagopyrum spp.
1%
MSG
5%
MSG
10%
MSG
Oryza spp.
Citrinin analysis of Monascus purpureus by LC-MSCitrinin presence was identified by means of

LC-MS. Analysis describes an approach involving the recognition of pattern of mass spectral
analysis lines that are produced as result of LC separated analytes. The pattern recognition
provides a method for the prediction of chemical structure and can be applied to the sample that
has not been examined. The spectral data used where the values of m/z and their related
intensities. Frame A shows LC spectral analysis of Monascus purpureus detected as 5.95
minutes.
Frame B shows LC separated analytes detected by ESI mode (m/z of 104.78, 114.90, 212.14 and
214.14) (Fig.7).
A highly sensitive method for the detection of citrinin was developed. Chemical fingerprint
profile of Monascus and the product was studied by HPLC-RD-PAD-MS. The chromatogram
profile of sample could provide detailed information about the main significant metabolites and
citrinin [32]. A new method was developed for qualitative and quantitative analysis of citrinin in
Monascus by gas chromatography selected ion monitoring (SIM) mass spectroscopy. Using this
method, chemical derivatization of citrinin is not required [25].
172
______________________________________________________________________________
Fig 7: Mass fragmentation spectrum of ESI chromatogram of Monascus purpureus
Frame A
Frame B
CONCLUSION
Monascus purpureus has produced highest pigment yields and biomass at 28C - 30C and this
temperature range was found to be optimum for growth and pigment production. Monascus
purpureus can survive on a wide range of pH (4.5-8.5). Maximum biomass was observed at
acidic pH (4.5) and around this range yellow-orange pigment was dominated. Maximum red
pigment has been observed at room temperature and at pH 5.5. Local unpolished rice and
Fagopyrum spp were established to be the best substrates for Monascus purpureus. Maximum
red pigment was observed when substrates were supplemented with N sources. There was no
increase in pigment yield when substrates were supplemented with C source. Presence of citrinin
was confirmed by LC-MS. There is not much information available on pigment production by
Monascus spp.. Hence there is a need to explore Monascus spp. to make it more adaptable and
acceptable to the food industry for its industrial production
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Journal of Microbiology and Biotechnology Research

Scholars Research Library

ISSN : 2231 3168

Monascus purpureus : A potential source for natural pigment production

Dry Weight (gm/l)

Fig 1: Growth Pattern of Monascus purpureus

Vertical bars represent the mean value standard error

Dry weight (gm/l)

Vertical bars represent the mean value standard error

Screening of substrates for Monascus purpureus

Pigment Yield (CVU/gds)

Effect of Carbon Source on pigment production by Monascus purpureus

Pigment Yield (CVU/gds)

Vertical bars represent the mean value standard error

Effect of N Source on pigment production by Monascus purpureus

Pigment Yield (CVU/gds)

Vertical bars represent the mean value standard error

Citrinin analysis of Monascus purpureus by LC-MSCitrinin presence was identified by means of

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