104
Chiang Mai J. Sci. 2009; 36(1)
Chiang Mai J. Sci. 2009; 36(1) : 104-109
www.science.cmu.ac.th/journal-science/josci.html
Contributed Paper
Screening of Nutrient Parameters for Red Pigment
Production by Monascus purpureus MTCC 369
Under Submerged Fermentation Using PlackettBurman Design
Md. Makhmur Ahmad, Md. Shivli Nomani, and Bibhu P. Panda*
Pharmaceutical Biotechnology Laboratory, Faculty of Pharmacy, Jamia Hamdard (Hamdard University),
Hamdard Nagar, New Delhi-110062 India.
*Author for correspondence; e-mail: [email protected]
Received: 7 August 2008
Accepted: 11 January 2009.
ABSTRACT
Plackett-Burman experimental design was used to screen important nutrient parameters
influencing the production of red pigments by Monascus purpureus MTCC 369 under submerged
fermentation. Nine nutrient parameters such as dextrose, NH4Cl, (NH4)2.SO4, NaCl, KH2PO4,
MgSO4.7H2O, CaCl2.2H2O, MnSO4.H2O and FeSO4.7H2O were screened along with two
dummy factors, in twelve experimental runs as per the design. From standard PlackettBurman data analysis it was conformed that, NH4Cl, NaCl, KH2PO4, MgSO4.7H2O and
MnSO4.H2O had contributed to a large extent, dextrose, CaCl2.2H2O and FeSO4.7H2O had
little impact, while, (NH4)2.SO4 contributes moderately for red pigment production by Monascus
purpureus MTCC 369 under submerged fermentation.
Keywords: red pigment, Plackett-Burman design, Monascus purpureus, submerged fermentation.
1. INTRODUCTION
Pigments, which are either natural or
synthetic, play an important role in food and
pharmaceutical industry as colorants. Synthetic
red pigments such as azorubin or tartrazin
causes allergic reactions [1] and C-red having
carcinogenic and teratogenic effects [2],
researchers from all over the world intensely
looking for natural occurring red pigments
from different natural sources. Monascus species
are well known to produce pigments like
monas-corubrine, rubropunctatine [3,4] and
more recently monascusones from a Monascus
mutant [5]. The use of Monascus pigments in
food has been carried out traditionally in the
orient for hundreds of years.
Numerous fungi namely Monascus
purpureus [6, 7] M. ruber [8], M. paxi [9], M.
anka [6] have been reported for biopigment
production.
Designing a fermentation medium is a
critical and important process as the medium
composition can significantly affect the
product yield [10,11]. An optimally balanced
culture medium was mandatory for maximal
production for the secondary metabolites.
Important medium variables are screened by
Chiang Mai J. Sci. 2009; 36(1)
105
Plackett-Burman experimental design [12]. It
is a partial factorial design; here large numbers
of independent variables (N) are studied in
small number of experiments (N+1) [13].
In the present study, screening and analysis
of important nutrient constituents was carried
out using Plackett-Burman experimental
design for the production of red pigments
by Monascus pur pureus MTCC 369 under
submerged fermentation.
actively growing slants in sterile water and
diluted to a concentration 6 103 spores per
ml. Fifteen percent spore suspension was
inoculated to conical flasks containing the basal
medium (100g dextrose, 10g peptone, 2g
KNO3, 2g NH4H2PO4, 0.5g MgSO4.7H2O,
0.1g CaCl2 in 1000 ml distilled water; adjusted
to pH 6.0). These cultures were incubated at
30 oC for 48 hrs in a shaker incubator at 110
rpm [6,14].
2. MATERIALS AND METHODS
2.3 Plackett-Burman Experimental
Design
Dextrose, NH4Cl, (NH 4)2.SO4, NaCl,
KH 2 PO 4 , MgSO 4 .7H 2 O, CaCl 2. 2H 2 O,
MnSO4.H2O and FeSO4.7H2O were the nine
medium constituents selected for the study.
The selection of nutrient for Plackett-Burman
experimental design was performed by
borrowing methodology of fermentation
medium optimization [10]. The PlackettBurman experimental design [12] for eleven
variables: nine nutritional components
2.1 Microorganism
Cultures of Monascus purpureus MTCC 369
was obtained from Institute of Microbial
Technology (IMTECH), Chandigarh, India.
It was maintained on slants of PotatoDextrose Agar (PDA) medium at 4 oC and
subcultured every 30 days.
2.2 Preparation of Seed Culture
Ascospore suspension of Monascus
purpureus MTCC 369 was prepared from
Table 1. Concentrations of variables at different levels in Plackett-Burman design for red
pigment production in submerged culture.
No. Designation
Variable
Low
level(-) g/l
High
level(+) g/l
1
X1
Dextrose
60
80
2
X2
NH4Cl
2
6
3
X3
(NH4)2.SO4
2
6
4
X4
NaCl
2
6
5
X5
KH2PO4
2
6
6
X6
MgSO4.7H2O
0.1
1
7
X7
Dummy 1
-
-
8
X8
CaCl2.2H2O
0.0
0.6
9
X9
MnSO4.H2O
0.0
0.6
10
X10
FeSO4.7H2O
0.0
0.6
11
X11
Dummy 2
-
-
106
Chiang Mai J. Sci. 2009; 36(1)
(independent variables) and two dummy
variables (Table 1) were used to evaluate the
relative importance of various nutrients for
bio pigment production in submerged culture
and experimental design was prepared with
the help of software Design Expert 7.1.3 (Stat
Ease Inc USA). In Table 2, each row represents
an experiment and each column represents a
different variables. For each nutrient variable
two different concentrations high (+) and low
(-) was tested (Table 1).
2.4 Submerged Fermentation
All experiments have been carried out in
duplicates in 250 ml Erlenmeyer flasks
containing 50 ml media as per experimental
designs. The medium pH was adjusted to 6.0
with 0.1 M HCl or 0.1 M NaOH and flasks
were autoclaved at 15 psi and 121 oC for
15 min. Finally each flask were inoculated with
10 % seed culture and incubated at 30 oC for
14 days on a rotary shaker at 110 rpm [6].
2.5 Bio-pigment Extraction and Estimation
Extraction of water-soluble Monascus red
pigments was carried out by cold centrifugation (1500 X G) for 10 min to separate the
fungal biomass and followed by filtration of
supernatant. Estimation of extracted red
pigment was carried out at 500 nm by spectrophotometer (Shimadzu, Japan) [15, 16].
2.6 Experimental Data Analysis
Experimental data were analyzed by the
standard methods of Plackett-Burman [12]
and software Design Expert 7.1.3 (Stat Ease
Inc USA).
The effect of each variable was determined
with the following equation.
Exi = 2 (ΣHxi - ΣLxi) / N
Where, Exi is the concentration effect of the
tested variable, Hxi and Lxi are the concentration of biopigments at high level and low level
of the same variable, and N is the number of
Table 2. Plackett - Burman experimental design of 12 trials for eleven variables in high level
(+), Low level (-) along with observed concentration of red pigment in fermented broth.
Trial
X1
X2
X3
X4
X5
X6
X7
X8
X9
X10
X11
Red
pigment
(mg l-1)
1
+
+
-
+
+
+
-
-
-
+
-
32.114
2
-
+
+
-
+
+
+
-
-
-
+
8.938
3
+
-
+
+
-
+
+
+
-
-
-
9.649
4
-
+
-
+
+
-
+
+
+
-
-
12.908
5
-
-
+
-
+
+
-
+
+
+
-
5.632
6
-
-
-
+
-
+
+
-
+
+
+
2.668
7
+
-
-
-
+
-
+
+
-
+
+
0.193
8
+
+
-
-
-
+
-
+
+
-
+
0.840
9
+
+
+
-
-
-
+
-
+
+
-
0.600
10
-
+
+
+
-
-
-
+
-
+
+
5.992
11
+
-
+
+
+
-
-
-
+
-
+
0.249
12
-
-
-
-
-
-
-
-
-
-
-
0.858
Chiang Mai J. Sci. 2009; 36(1)
107
trials. When the sign is positive, the influence
of variable upon biopigment production is
greater at high concentration, and when the
negative, the influence of variable is greater at
a low concentration.
Mean squares of each variable (the
variance of effect) were calculated as follows
Vxi = (ΣHxi - ΣLxi)2 / N
The experimental error was calculated by
averaging the mean squares of the dummy
variables.
R = ΣVxd / n
Factor showing larger effects were
identified using F-test.
F = Vxi / R
Where, R is experimental error (mean
square for error), Vxd is mean square of
dummy variable; Vxi is mean square of variable
and n is number of dummy variables.
3. RESULTS AND DISCUSSION
In the present study screening of important nutrients influencing red pigment
production by M. purpureus MTCC 369 was
analyzed by Plackett-Burman experimental
design. The purpose of was to select important nutritional factors of fermentation
medium contribution to Monascus red pigment
production under submerged fermentation.
Maximum red pigment production was
found in 1 st experimental trial, whereas
minimum in 7 th trial under submerged
fermentation using M. purpureus MTCC 369
(Table 2). Effect of dummy 1 and dummy 2
were close to zero (Table 3), indicates successful experimental work. Experimental error
was calculated and found to be 0.018. Among
the nine nutrient components used in study,
NH4Cl, NaCl, KH2PO4, MgSO4.7H2O and
MnSO4.H2O had contributed to a large extent
for biopigment production. Dextrose,
CaCl 2. 2H 2O and FeSO 4 .7H 2 O had little
impact, while, (NH 4 ) 2 .SO 4 contributes
moderately in production of red pigment.
Between MnSO 4.H 2O and FeSO 4.7H 2 O,
contribution of MnSO4.H2O for pigment
synthesis found to be more with F value of
1.243 than the FeSO4.7H2O with F value 0.193
Table 3. Influence of medium variables on red pigment production.
Designation
Variable
ΣH
ΣL
Mean
Experimental
Effect F-value
Square
error
X1
Dextrose
43.645
39.996
0.102
1.108
0.045
X2
NH4Cl
61.392
19.249
4.110
7.023
1.818
X3
(NH4)2.SO4
31.060
49.581
0.793
-3.086
0.350
X4
NaCl
63.580
17.061
5.009
7.753
2.216
X5
KH2PO4
60.034
20.607
3.598
6.571
1.592
X6
MgSO4.7H2O 59.841
20.800
3.527
6.506
1.560
X7
Dummy 1
34.956
45.685
0.266
-0.010
0.117
X8
CaCl2.2H2O
35.214
45.427
0.241
-1.702
0.160
X9
MnSO4.H2O
22.897
57.744
2.180
-5.807
1.243
X10
FeSO4.7H2O
47.199
33.442
0.437
2.292
0.193
X11
Dummy 2
18.880
61.761
4.255
0.008
1.882
0.018
108
(Table 3). Dextrose, NH4Cl, NaCl, KH2PO4,
MgSO4.7H2O and FeSO4.7H2O were influences
the production in their higher concentration,
whereas (NH 4 ) 2 .SO 4 , CaCl 2. 2H 2 O and
MnSO4.H2O were effective in lower levels,
with in the initial search levels.
It was found that dextrose is useful as a
carbon source towards red pigment production.
Among all the nitrogen sources NH4Cl was
found to be better than (NH4)2.SO4 for biopigment production by Monascus purpureus
MTCC 369. This might due the growth of
M. purpureus was largely depending on one
type of nitrogen source [17]. Moreover,
NH4Cl contribute highly to wards lovastatin
biosynthesis by as reported earlier by us [18].
This may concludes that M. purpureus MTCC
369 secondary metabolites synthesis depends
on NH4Cl concentration. In fungal nutrition
magnesium and calcium are noted as
macronutrients and manganese, iron, copper
and zinc as micronutrient [19] but in case of
red pigment production by Monascus purpureus,
magnesium contribution was higher than
calcium, iron and manganese. However,
manganese contribution was found to more
then calcium and iron. This may be due to
manganese, acting as cofactor for different
enzyme required for pigment biosynthesis
[19].
Designing the medium is an open ended,
time-consuming and laborious process
involving large number of experiments. The
Plackett-Burman experimental design is the
preliminary technique for rapid illustration of
the effects of various medium constituents. It
tests each variable at two levels only; hence it
can not give exact idea regarding the optimum
level of constituents required in the medium.
Therefore further optimization of selected
nutrients such as dextrose, NH4Cl, KH2PO4,
MgSO 4 .7H 2 O and MnSO 4 .H 2 O for red
pigment production by M. purpureus MTCC
369 is necessary.
Chiang Mai J. Sci. 2009; 36(1)
REFERENCES
[1] Fabre C.E., Goma G., and Blanc P.J.,
Production and food applications of the
red pigments of Monascus ruber, J. Food
Sci., 1993; 58: 1099-1110.
[2] Merlin U., Gagel U., Popel O., Bernstein
S., and Rosenthal I., Thermal degradation
kinetics of prickly pear fruit red pigments,
Food Sci., 1987; 52: 485-486.
[3] Juzlova P., Martinkova L., and Kren, V.
Secondary metabolites of the fungus
Monascus: A review, J. Ind. Microbiol., 1996;
16: 163-170.
[4] Pattanagul P., Pinthong R., Phianmongkhol
A., and Leksawasdi N., Review of Angkak
Production (Monascus purpureus), Chiang
Mai J. Sci., 2007; 34: 319-328.
[5] Jongrungruangchok S., Kittakoop P.,
Yongsmith B., Bavovada R., Tanasupawat
S., Lartpornmatulee N., and Thebtaranonth
Y., Azaphilone pigments from a yellow
mutant of the fungus Monascus kaoliang,
Phytochemistry, 2004; 65: 2569-2575.
[6] Su Y.C., Wang J.J., Lin T.T., and Pan T.M.,
Production of secondary metabolites,
gamma amino butyric acid and monacolin
K by Monascus, J. Ind. Microbiol. Biotechnol.,
2003; 30: 41-46.
[7] Wang J.J., Lee C.L., and Pan T.M.,
Improvement of monacolin K, gammaamino butyric acid and citrinin production ratio as a function of environmental
conditions of Monascus purpureus NTU
601, J. Ind. Microbiol. Biotechnol., 2003; 30:
669-676.
[8] Endo A., Monacolin K, a new hypocholesterolemic agent produced by
Monascus species, J. Antibiot., 1979; 32:
852-854.
[9] Manzoni M., and Rollini M., Biosynthesis
and biotechnological production of statins
by filamentous fungi and application of
these cholesterol-lowering drugs, Applied.
Microbiol. Biotechnol., 2002; 58: 555-564.
Chiang Mai J. Sci. 2009; 36(1)
109
[10] Kennedy M., and Krouse D., Strategies
for improving fermentation medium
performance: A review, J. Ind. Microbiol.
Biotechnol., 1999; 23: 456-475.
[15] Lin T.F., and Demain A.L, Formation of
water soluble Monascus red pigments by
biological and semi synthetic processes,
J. Ind. Microbiol., 1992; 9: 173-179.
[11] Panda B.P., Javed S., and Ali M., Fermentation process optimization, Res. J.
Microbiol., 2007; 2: 201-208.
[16] Johns M.R., and Stuart D.M, Production
of pigments by Monascus purpureus in solid
culture, J. Ind. Microbiol., 1991; 8: 23-38.
[12] Plackett R.L., and Burman J.P, The design
of optimum multifactorial experiments,
Biometrika., 1946; 33: 305-325.
[17] Miyake T., Uchitomo K., Zhang M.Y.,
Kono I., Nozaki N., Sammoto H., and
Inagaki K., Effects of the principle
nutrients on lovastatin production by
Monascus pilosus, Biosci. Biotechnol. Biochem.,
2006; 70: 1154-1159.
[13] Naveena B.J., Altaf M., Bhadriah K., and
Reddy G., Selection of medium
components by Plackett-Burman design
for production of L (+) lactic acid by
Lactobacillus amylophilus GV6 in SSF using
wheat bran, Biores. Technol., 2005; 96: 485490.
[14] Sayyad S.A., Panda B.P., Javed S., and Ali
M., Optimization of nutrient parameters
for lovastatin production by Monascus
purpureus MTCC 369 under submerged
fermentation using response surface
methodology, Applied. Microbiol. Biotechnol.,
2007; 73: 1054-1058.
[18] Sayyad S.A., Panda B.P., Javed S., and Ali
M., Screening of nutrient parameters for
lovastatin production by Monascus
purpureus MTCC 369 under submerged
fermentation using Plackett-Burman
design, Res. J. Microbiol., 2007; 2: 601-605.
[19] Yu X., Hallett S.G., Sheppard J., and
Watson A.K. Application of PlackettBurman experimental design to evaluate
nutritional requirements for the production
of Collectotrichum coccodes spores, Appl
Microbiol Biotechnol., 1997; 47: 301-305.