Biocatalysis and Agricultural Biotechnology 3 (2014) 140–145

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Biocatalysis and Agricultural Biotechnology

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Original Research Paper

An enhancement of red pigment production by submerged culture

of Monascus purpureus MTCC 410 employing statistical methodology
Vimal S. Prajapati, Nidhi Soni, Ujjval B. Trivedi, Kamlesh C. Patel n
B.R.D. School of Biosciences, Sardar Patel University, Sardar Patel Maidan, Vadtal Road, Vallabh Vidyanagar 388120, Gujarat, India

art ic l e i nf o a b s t r a c t

Article history: Pigments produced by Monascus spp. can be used as food grade biocolorant and are preferred over the
Received 1 August 2013 synthetic variants which elicit various adverse effects. Monascus purpureus MTCC 410 has been
Received in revised form investigated in the present study for red pigment production employing submerged fermentation. The
25 August 2013
medium components influencing the pigment production were identified using Plackett–Burman design.
Accepted 26 August 2013
Available online 7 September 2013
Among various variables screened, glucose, tryptone and pH were found to be highly significant. The
optimum concentrations of these significant parameters were determined employing the response
Keywords: surface central composite design. Glucose (28 g/L), tryptone (1 g/L) and pH 8.0 showed highest pigment
Red pigment production.
Plackett–Burman design
& 2013 Elsevier Ltd. All rights reserved.
Central composite design
Monascus purpureus
Medium optimization

1. Introduction Studies on red pigment synthesis by various strains of Mon-

The scrutiny and negative assessment of synthetic food dyes by
the modern consumer has resulted into a strong interest in natural
coloring alternatives. Monascus pigments produced by various
species of Monascus have been used as natural colorants and as
traditional food additives in East Asia (Carels and Shepherd, 1977).
Monascus spp. produces a complex mixture of three categories of
pigments such as orange, red and yellow. Among these pigments,
the red pigment (monascorubramine and rubropunctamine)
is having high market potential for its use in meat products
(Fabre et al., 1993). Monascus metabolites have been evaluated
for their various biological activities such as embryotoxicity,
teratogenicity, immunosuppressive properties, antioxidant proper-
ties, antibiotic and cytotoxic activity (Loret and Morel, 2010).
Monascus pigment fermentations have been performed mainly in
solid cultures; however production yield is too low to compensate
its economical viability and hence recent research efforts have
focused on submerged fermentation to increase the pigment yield
(Mukherjee and Singh, 2011). Moreover, solid state fermentation is
labor-intensive, time consuming and requires large cultivation
areas, and hence submerged culture technique for Monascus
pigments has been studied to overcome the problems of space,
scale up and process control of solid culture (Lin, 1973).
n
Corresponding author. Tel.: þ 91 2692 231041; fax: þ91 2692 231042.
E-mail address: comless@yahoo.com (K.C. Patel).
ascus purpureus in submerged culture have shown that the yield is
affected by medium composition, pH and agitation (Hamdi et al.,
1996). Composition of the pigments synthesized varies signifi-
cantly depending on the types of nutrients available, such as
nitrogen sources and the strain used (Miyake et al., 2008).
Majority studies on pigment production have been carried out
using conventional one-factor-at-a-time method which frequently
fails to locate optimal parameters and is also unable to show
possible interaction effects between the selected parameters (Kalil
et al., 2000). Silveira et al. (2008) have reported pigment produc-
tion by M. purpureus in grape waste using factorial design.
Factorial design and response surface techniques are important
tools to determine the optimal process conditions. This methodol-
ogy has been successfully used in many areas of biotechnology,
particularly to optimize the production of bioactive molecules
(Cladera-Olivera et al., 2004; Thys et al., 2006).
Conventional approaches for increased microbial metabolite
production usually employ manipulation of nutritional require-
ments, physical parameters and genetic makeup of the producing
strain. Development of economical medium requires selection of
carbon, nitrogen, phosphorous, potassium and trace element
sources. Nutritional requirement can be manipulated by the
conventional or statistical methods. Conventional method involves
changing one independent variable at a time while keeping the
others at fixed level. However, statistical method offers several
advantages over conventional method being rapid and reliable,
short lists significant nutrients, helps understanding the interac-
tions among the nutrients at various concentrations and reduces

1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bcab.2013.08.008
 V.S. Prajapati et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 140–145 141

the total number of experiments tremendously resulting in saving
time, glassware, chemicals and manpower (Srinivas et al., 1994).
Initial screening of the ingredients is done using Plackett–Burman
design to understand the significance of their effect on the product
formation and then a few better ingredients are selected for further
level optimization employing response surface methodology.
In the present study statistical approach has been employed in
which a Plackett–Burman design is used for identifying significant
variables influencing red pigment production by M. purpureus MTCC
410 under submerged fermentation. The levels of the significant
variables such as glucose, tryptone and initial pH of the medium,
have been further optimized using response surface central composite
design to determine the optimum conditions for red pigment
production.
YM broth. The flasks were incubated at 30 1C and 120 rpm on a
rotary shaker for red pigment production.
2.3. Pigment estimation
After incubation, the mycelia were separated from broth by
filtration using Whatman #1 filter paper. Filtrate was subjected for
centrifugation at 8000g for 20 min. The supernatant was collected
and the pigment production was analyzed by measuring the
absorbance of the supernatant using UV–visible spectrophot-
ometer (Shimadzu) having un-inoculated medium as blank and
considering the dilution factor of the sample (Chiu and Poon, 1993;
Carvalho et al., 2003). Pigment yield was expressed as specific
absorbance (Amax) at 500 nm ml  1 of filtrate (OD U/ml).

2.4. Optimization of process parameters

2. Materials and methods
2.4.1. Identifying the significant variables using Plackett–Burman
2.1. Microorganism and maintenance design
The present study was aimed at screening of the important
The stock culture of M. purpureus (MTCC 410) procured from medium components with respect to their main effects by Plack-
Microbial Type Culture Collection and Gene Bank, Institute of ett–Burman design. This experimental design is a two factorial
Microbial Technology, Chandigarh, Punjab, India was maintained design and was used to identify the critical parameters required
on YM (Yeast and Mold medium) agar slants containing (g/l): for optimum red pigment production by screening n variables in
glucose 20, malt extract 3, peptone 5, yeast extract 3 and agar 1.5. n þ1 experiments (Plackett and Burman, 1946). The eight variables
The culture was maintained at 4 1C in the laboratory. selected on the basis of our previous study for the present
investigation were, amount of malt extract, sucrose, lactose,
2.2. Inoculum preparation and pigment production glucose, tryptone, yeast extract, peptone and salt solution (g/l:
K2HPO4 1, MgSO4 0.5, KCl 0.5 and FeSO4 0.01), as well as pH under
The plate containing 7 days old growth of the organism was submerged fermentation (Table 1). The experimental design for
used as inoculum. Two plugs of 8 mm size were taken from this the screening of the variables is presented in Table 2. The Plakett–
plate and inoculated in 250 ml Erlenmeyer flask containing 100 ml Burman design assumes that there are no interactions between
different medium components. All the variables were denoted as
Table 1
numerical factors and investigated at two widely spaced intervals
Medium components as variable and their values used in Plackett–Burman design designated as  1 (low level) and þ 1 (high level). The effects of
for red pigment production using M. purpureus MTCC 410. individual parameters on red pigment production were calculated
by the following equation:
Variable Medium component þ Values  Values
EðXiÞ ¼ 2ð∑M þ ∑M  Þ=N ð1Þ
X1 Malt extract 0.5 g 0.05 g þ 
X2 Sucrose 1.0 g 0.1 g where E is the effect of parameter under study and M and M are
X3 Lactose 1.0 g 0.1 g responses (red pigment production) of trials at which the para-
X4 Glucose 1.0 g 0.1 g meter was at its higher and lower levels, respectively and N is the
X5 Tryptone 0.5 g 0.05 g
total number of trials.
X6 Yeast extract 0.5 g 0.05 g
X7 Peptone 0.5 g 0.05 g Experimental error was estimated by calculating the variance
X8 Salt solution 10 ml 1 ml among the dummy variables as
X9 pH 8 3
Veff ¼ ðEdÞ2 =n ð2Þ

Table 2
Plackett–Burman design generated by fractional rotation of full factorial design where X1–X9 are independent variables and D1–D2 are dummy variables.

Run Components Red pigment

production (OD U/ml)
X1 X2 X3 X4 X5 X6 X7 X8 X9 D1 D2
w/v w/v w/v w/v w/v w/v w/v v/v

1 1 1 1 1 1 1 1 1 1 1 1 10.99 7 0.403
2 1 1 1 1 1 1 1 1 1 1 1 08.42 7 0.480
3 1 1 1 1 1 1 1 1 1 1 1 06.477 0.445
4 1 1 1 1 1 1 1 1 1 1 1 12.317 0.770
5 1 1 1 1 1 1 1 1 1 1 1 06.967 0.381
6 1 1 1 1 1 1 1 1 1 1 1 05.88 7 0.296
7 1 1 1 1 1 1 1 1 1 1 1 06.597 0.360
8 1 1 1 1 1 1 1 1 1 1 1 05.26 7 0.664
9 1 1 1 1 1 1 1 1 1 1 1 04.517 0.417
10 1 1 1 1 1 1 1 1 1 1 1 05.107 0.565
11 1 1 1 1 1 1 1 1 1 1 1 10.52 7 1.753
12 1 1 1 1 1 1 1 1 1 1 1 01.43 7 0.169
142 V.S. Prajapati et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 140–145

where Veff is the variance of the effect of level, Ed is the effect of
level for the dummy variables and n is the number of dummy
variables used in the experiment. The standard error (SE, Es) of
concentration effect was the square root of variance of an effect,
and the significance level (P-value) of each concentration effect
was determined using the student's t-test:
tðXiÞ ¼ EðXiÞ=Es
where E (Xi) is the effect of variable Xi.
2.4.2. Response surface methodology (RSM)
The levels of the significant parameters as revealed from
Plackett–Burman experiment and their interaction effects which
may influence the red pigment production significantly were
analyzed and optimized by response surface central composite
design (CCD). RSM is useful for small number of variables (up to
five) but is impractical for large number of variables, due to high
number of experimental runs required. The level of the three
major components glucose, tryptone and pH were optimized,
keeping temperature and inoculum size constant.
According to the design, the total number of treatment combi-
nations is 2k þ2k þno, where k is the number of independent
variables and no is the number of repetition of experiments at the
central point. Each factor in the design was studied at five different
levels (  α,  1, 0, þ1, þα) as shown in Table 3. This experimental
design comprises a two level fractional factorial points (  1 and
þ1), central point (0) and axial or star points encoded as –α
and þ α. All variables were set at a central coded value of zero.
The minimum and maximum ranges of variables were determined
on the basis of our previous experiments. The full experimental
plan with respect to their values in actual and coded form is listed
Table 3
Experimental range and levels of the independent variables of selected compo-
nents used for response surface central composite design.
Levels of variable studied
Variable Components Range
α 1 0 þ1
X1 Glucose 0.1–3.0 (g%)  0.89 0.1 1.55 3 3.99
X2 Tryptone 0.1–1.5 (g%)  0.88 0.1 0.88 1.5
X3 pH 3.0–10 0.61 3 6.5 10
in Table 4. Red pigment production (OD U/ml) was measured in
triplicate in 20 different experimental runs. The red pigment
production was analyzed using a second-order polynomial equa-
tion and the data were fitted into the equation by multiple
regression procedure. The model equation for analysis is given as
Y ¼ β0 þ ∑βiXi þ ∑βiiXi2 þ ∑βijXiXj ð4Þ
ð3Þ
where βo, βi, βii and βij represent the constant, linear, quadratic
effect of Xi and interaction effect between Xi and Xj, respectively
for the production of red pigment. Later, validation experiment
was performed and maximum production of red pigment was
confirmed using the optimum values for variables predicted by
response optimization.
2.5. Software and data analysis
The results of the experimental design were analyzed and
interpreted using Design Expert Version 8.0 (Stat-Ease Inc., Min-
neapolis, Minnesota, USA) statistical software.
3. Results and discussion
3.1. Screening of significant parameters for red pigment production
using Plackett–Burman design
The variables selected in the present investigation are on the
basis of our previous study in which different carbon and different
organic-inorganic nitrogen sources were evaluated for their effects
on the red pigment production using one factor-at-a-time approach
and shortlisted for the further (Plackett–Burman) experiment. We
observed that inorganic nitrogen sources have no effect on the red
pigment production while organic nitrogen sources showed promi-
nent effect. Many investigators have reported addition of mono-
sodium glutamate giving maximum red pigment production
(Pastrana et al., 1994; Babitha et al., 2006) but in our study we
observed that presence of tryptone as compared to monosodium
þα glutamate showed more red pigment production. Hence, tryptone
was selected as organic nitrogen source for further optimization
studies.
1.98
12.39
The pigments produced by Monascus sp. are soluble only in the
organic solvent but the addition of the monosodium glutamate,

Table 4
Full experimental central composite design with actual and coded level of variables and the response function.

A: Glucose (g%) B: Tryptone (g%) C: pH Red pigment production (OD U/ml)

Run no.
Actual Coded Actual Coded Actual Coded Observed Predicted

1 0.10 1 0.10 1 3.00 1 0.6 70.035 1.94

2 3.00 þ1 0.10 1 3.00 1 0.68 70.028 1.92
3 0.10 1 1.50 þ1 3.00 1 3.05 70.049 5.80
4 3.00 þ1 1.50 þ1 3.00 1 0.52 70.049 2.22
5 0.10 1 0.10 1 10.00 þ1 0.58 70.028 2.00
6 3.00 þ1 0.10 1 10.00 þ1 17.83 71.768 13.75
7 0.10 1 1.50 þ1 10.00 þ1 4.5 70.282 1.93
8 3.00 þ1 1.50 þ1 10.00 þ1 0.54 70.0141 1.77
9  0.89 α 0.80 0 6.50 0 1.0 70.212 0.91
10 3.99 þ α 0.80 0 6.50 0 2.09 70.070 4.03
11 1.55 0  0.38 α 6.50 0 3.0 70.071 4.71
12 1.55 0 1.98 þ α 6.50 0 1.0 70.084 1.15
13 1.55 0 0.80 0 0.61 α 0.91 70.212 1.04
14 1.55 0 0.80 0 12.39 þ α 6.0 70.210 7.73
15 1.55 0 0.80 0 6.50 0 9.8 70.141 9.64
16 1.55 0 0.80 0 6.50 0 9.2 70.141 9.64
17 1.55 0 0.80 0 6.50 0 8.2 70.070 9.64
18 1.55 0 0.80 0 6.50 0 12.0 70.141 9.64
19 1.55 0 0.80 0 6.50 0 8.0 70.070 9.64
20 1.55 0 0.80 0 6.50 0 11.0 70.070 9.64
 V.S. Prajapati et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 140–145 143

soyabean meal, and peptone or chitin powder in the fermentation As reported, media pH plays an important role in activating key
medium lead to the production of water soluble pigment. enzymes involved in pigment production and excretion by M.
The addition of amino acid in the fermentation medium lead to pupureus CCT3802 (Orozco and Kilikian, 2008). Besides pigment
the production of more red pigment than the yellow pigment excretion by the cultures, alkaline pH promotes a higher stability
(Yongsmith et al., 1993; Juzlova et al., 1996). A comparison of the of this colorant relative to acidic values (Fabre et al., 1993;
relative amounts and color quality of the pigments produced using Carvalho et al., 2005). From the present study using Plackett–
several amino acid show that yellow and orange pigment are Burman design, glucose, tryptone and pH were shortlisted and
unaffected by the nitrogen source, whereas red pigment differ in studied further for optimization of their concentration require-
tone and solubility (Jung et al., 2003). In addition to that supple- ment and to check their interaction effect. Methodology of Plack-
mentation of organic nitrogen results in higher growth and hence ett–Burman was thus found to be very useful for determination of
gives more red pigment production compared to the inorganic relevant variables for further optimization. Ahmad et al. (2009)
nitrogen source. used the Plackett–Burman design for screening the nutrient
A statistical approach has been used to screen the most parameter for red pigment production under submerged fermen-
effective medium components and select their concentration to tation and also reported that the dextrose is one of the best carbon
achieve highest possible red pigment production by M. purpureus source for the maximum pigment production. Plackett–Burman
MTCC 410 under submerged fermentation. Usually, for the fer- experiment used only to screen the significant components but
mentation processes, initial screening of the ingredients is carried did not give the optimum level of the significant components. In
out to understand the significance of their effect on the product the present study, significant components were screened using
formation. Subsequently better ingredients are selected for further Plackett–Burman design followed by their level optimization
optimization. Plackett–Burman design was used to screen eight employing response surface central composite design.
different medium components including carbon and nitrogen
sources along with standard salt solution and a physical process
3.2. Response surface methodology (RSM)
parameter (pH) in a 12 run experiment with 2 level concentration
of each variable. Studies were carried out under submerged
The central composite design was employed to evaluate the
fermentation at 120 rpm, 30 1C for 8 days. The independent
interaction among the significant factors and also to determine
variables and their respective high and low concentrations used
their optimal levels. In the present work, experiments were
in optimization study are represented in Table 1, whereas the
planned to obtain a quadratic model consisting of 23 trials. The
Plackett–Burman experimental design for 12 trials with two level
plan included twenty experiments and two levels of concentration
concentrations of each variable followed for the screening of
for each factor. In order to study the combined effect of these
medium components for red pigment production is given in
variables, experiments were performed at different combinations.
Table 2. The variable X1–X9 represents the medium constituents
The central composite experimental plan along with the predicted
and D1–D2 represents the dummy/unassigned variables. Results of
and observed response for each individual experiment is summar-
the Plackett–Burman experiment with respect to red pigment
ized in Table 4. It also shows the production of red pigment (OD U/
production, the effect, standard error, t(xi), p and confidence level
ml) corresponding to combined effect of all three components in
of each component are represented in Tables 2 and 5. The
the specified ranges.
components were screened at the confidence level of 95% on the
The optimum levels of selected variables were obtained by
basis of their effects. When components show significance at or
solving the regression equation and by analyzing the response
above 95% confidence level and its effect is negative, it is
surface contour and surface plots. The larger the magnitude of the
considered effective for production but the amount required may
t-value and smaller the p-value, more significant is the corre-
be lower than the indicated (as low,  1) concentration in Plack-
sponding coefficient (Myers and Montgomery, 2002). The regres-
ett–Burman experiment. If the effect is found positive, the amount
sion equation obtained after the analysis of variance (ANOVA)
required may be higher than indicated (as high, þ1) concentra-
provides an estimate of the level of red pigment production as a
tion. In our experiment, glucose and tryptone gave confidence
function of pH, glucose and tryptone concentration.
level4 95% and could be considered significant. Remaining com-
The production of red pigment may be best predicted by the
ponents, i.e., malt extract, yeast extract, peptone, sucrose, lactose
following model:
and salt solution showed confidence level o95% and were
considered insignificant in the study. pH also showed o95% Y ¼ 0:96 þ ð0:093nAÞ–ð0:11nBÞ þ ð0:20nCÞ
significance but our initial studies on effect of pH on the red –ð0:30nAnBÞ þ ð0:20nAnCÞð0:20nBnCÞ
pigment production, and many research reports have demon-
ð0:25nA2 Þð0:24nB2 Þð0:19nC 2 Þ; ð5Þ
strated its importance in red pigment production and hence we
have selected this component for further level optimization. where Y¼ red pigment production (OD U/ml), A¼glucose (g%),
B ¼Tryptone (g%) and C ¼pH.
The statistical significance of the second-order model equation
Table 5 was evaluated by F-test analysis of variance as shown in Table 6
Statistical analysis of components for red pigment production by M. purpureus
which revealed that this regression is statistically highly signifi-
MTCC 410.
cant for red pigment production. The model F-value 6.18 implies
Components Effect Standard error t-Value P Confidence (%) that the model is significant. There is only a 0.44% chance that a
large model F-value could occur due to noise. P-value less than
Malt extract 0.07 0.052 1.413 0.293 70.68 0.050 indicate that the model terms are significant. Thus, in this
Sucrose 0.145 0.052 2.913 0.100 89.96
study C, AB, A2, B2 and C2 are significant model terms (Table 6). The
Lactose  0.008 0.052  0.16 0.887 11.24
Glucose 0.301 0.052 6.033 0.026 97.36 Lack of fit F-value of 4.82 implies the lack of fit is not significant
Tryptone 0.452 0.052 9.046 0.012 98.80 relative to the pure error. Non-significant lack of fit is good for the
Yeast extract 0.058 0.052 1.173 0.361 63.85 model to fit. The R2 value (multiple correlation coefficient) closer
Peptone 0.065 0.052 1.306 0.321 67.86 to 1 denotes better correlation between observed and predicted
Salt solution 0.015 0.052 0.313 0.783 21.63
pH 0.107 0.052 2.146 0.164 83.50
values. The coefficient of variation (CV) indicates the degree of
precision with which the experiment is compared. The lower
144 V.S. Prajapati et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 140–145

Table 6
Analysis of variance (ANOVA) for the fitted quadratic polynomical model for level optimization of red pigment production using M. purpureus MTCC 410.

Source Sum of Squares Df Mean square F-value P-value prob4F

Model 4.011796 9 0.445755 6.181912 0.0044 Significant

A-Glucose 0.117603 1 0.117603 1.630968 0.2304
B-Tryptone 0.152756 1 0.152756 2.118488 0.1762
C-pH 0.540156 1 0.540156 7.491104 0.0209 Significant
AB 0.709241 1 0.709241 9.836035 0.0106 Significant
AC 0.309685 1 0.309685 4.29483 0.0650
BC 0.306545 1 0.306545 4.251284 0.0662
A2 0.925971 1 0.925971 12.84174 0.0050 Significant
B2 0.812171 1 0.812171 11.26352 0.0073 Significant
C2 0.498323 1 0.498323 6.910948 0.0252 Significant
Residual 0.721063 10 0.072106
Lack of fit 0.597263 5 0.119453 4.824422 0.0546 Not significant
Pure error 0.1238 5 0.02476
Cor total 4.732859 19

R2 ¼0.84; Adeq Pre¼ 8.41.

Fig. 1. Response surface graph showing the interaction effect of glucose and tryptone on red pigment production.

Fig. 2. Response surface graph showing the interaction effect of glucose and pH on red pigment production.

reliability of the experiment is usually indicated by high value of
CV. In the present case low CV (53.43) denotes that the experiment
performed is reliable. Adequate precision measures the signal to
noise ratio. A ratio greater than 4 is desirable. In present study, the
ratio is 8.417 which indicate an adequate signal.
The effect of interaction of variables on red pigment yield was
studied by changing levels of any two independent variables while
keeping the third independent variable at its constant level. The
response surface plots or contour plots can be used to predict the
optimal values for different test variables. Therefore, three
response surface plots were obtained by considering all the
possible combinations. Fig. 1 shows the effect of interaction
between glucose and tryptone on red pigment production, which
revealed that both the components at their lower level have no
significant effect on the red pigment production but increase in
glucose concentration leads to gradual increase in the pigment
production while increase in tryptone concentration leads to
decrease in the pigment production. Glucose and tryptone con-
centration at higher level showed negative effect on pigment
production but glucose at higher level and tryptone at lower level
showed positive effect on the red pigment production. As shown
in Fig. 2 higher glucose concentration and alkaline pH (above 7)
lead to maximum pigment production while higher glucose
concentration and acidic pH showed negative effect on red pig-
ment production. Thus, both the parameters at their maximum
level were found to be significant for red pigment production. The
 V.S. Prajapati et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 140–145
Fig. 3. Response surface graph showing the interaction effect of tryptone and pH on red pigment production.
interaction between tryptone and pH also play an important role
in red pigment production; higher pH (alkaline pH) at lower level
of tryptone was found to be significant for red pigment produc-
tion. It was noticed that tryptone concentration up to certain level
supports red pigment production but at higher concentration it
negatively affects the red pigment production (Fig. 3). According to
the response surface point prediction analysis, glucose concentra-
tion of 28 g/l, tryptone (1 g/l) with 8.5 pH of medium could give
maximum red pigment yield up to 12.47 OD U/ml.
3.3. Validation of the quadratic model
In order to confirm the above mentioned optimized medium
constitution and condition an experiment for pigment production
was performed in duplicate. Under these suggested condition the
mean value of the pigment yield was found to be 14.50 OD U/ml,
which was higher than the predicted value of 12.47 OD/U ml. Thus,
the model developed was accurate and reliable for predicting the
production of red pigment by M. purpureus MTCC 410.
4. Conclusions
To the best of our knowledge, majority of the experiment on the
red pigment production by M. purpureus have been carried out using
one factor at a time methodology under solid state fermentation. Only
few reports are available on the statistical optimization of red pigment
production using submerged culture of M. purpureus. Present study
has allowed rapid screening of a number of nutrients influencing red
pigment production. Interestingly tryptone as nitrogen source has
been found to support good pigment production where majority of
the investigators have reported monosodium glutamate as effective
for the same. The pigment yield and the production were found to be
significantly influenced by initial pH of medium, glucose and tryptone
concentration. The data obtained after optimization has resulted in
14.50 OD U/ml red pigment production.
References
Ahmad, M., Nomani, S., Panda, B., 2009. Screening of nutrient parameters for red
pigment production by Monascus purpureus MTCC 369 under submerged fermenta-
tion using Plackett–Burman design. Chiang Mai Journal of Science 36, 104–109.
Babitha, S., Soccol, C.R., Pandey, A., 2006. Jackfruit seed—a novel substrate for the
production of Monascus pigments through solid state fermentation. Food
Technology and Biotechnology 44, 465–471.
Carels, M., Shepherd, D., 1977. The effect of different nitrogen sources on pigment
production and sporulation of Monascus sp. in submerged shaken culture.
Canadian Journal of Microbiology 23, 1360–1372.
145
Carvalho, C., Oishi, B.O., Pandey, A., Soccol, C.R., 2005. Biopigments from Monascus:
strain selection, citrinin production and color stability. Brazilian Archives of
Biology and Technology 48, 885–894.
Carvalho, J.C., Pandey, A., Babitha, S., Soccol, C.R., 2003. Production of Monascus
biopigmentation: an overview. Agro Food Industry Hi Tech 14, 619–624.
Chiu, S.W., Poon, K., 1993. Submerged production of Monascus pigments. Mycologia
85, 214–218.
Cladera-Olivera, F., Caron, G.R., Brandelli, A., 2004. Bacteriocin production by
Bacillus licheniformis strain P40 in cheese whey using response surface
methodology. Biochemical Engineering Journal 21, 53–58.
Fabre, C.E., Santerre, A.L., Loret, M.D., Baberian, R., Parailleux, A., Goma, G., et al.,
1993. Production and food application of the red pigments of Monascus ruber.
Journal of Food Science 58, 1099–1102.
Hamdi, M., Blanc, P.J., Goma, G., 1996. Effect of aeration conditions on the
production of red pigments by Monascus purpureus growth on prickly pear
juice. Process Biochemistry 31, 543–547.
Jung, H., Kim, C., Kim, K., Shin, C.S., 2003. Color characteristic of Monascus pigments
derived by fermentation with various amino acids. Journal of Agricultural and
Food Chemistry 51, 1302–1306.
Juzlova, P., Martinkova, L., Kren, V., 1996. Secondary metabolites of the fungus
Monascus: a review. Journal of Industrial Microbiology 16, 163–170.
Kalil, S.J., Maugeri, F., Rodrigues, M.I., 2000. Response surface analysis and
simulation as a tool for bioprocess design and optimization. Process Biochem-
istry 35, 539–550.
Lin, C.F., 1973. Isolation and cultural conditions of Monascus sp. for production of
pigment in a submerged culture. Journal of Fermentation Technology 51, 407–414.
Loret, M.O., Morel, S., 2010. Isolation and structural characterization of two new
metabolites from Monascus. Journal of Agricultural and Food Chemistry 58,
1800–1803.
Miyake, T., Isato, K., Nobuyuki, N., Sammoto, H., 2008. Analysis of pigment
composition in various Monascus cultures. Food Science and Technology
Research 14, 194–197.
Mukherjee, G., Singh, S.K., 2011. Purification and characterization of a new red
pigment from Monascus purpureus in submerged fermentation. Process Bio-
chemistry 46, 188–192.
Myers, R.H., Montgomery, R.C., 2002. Response Surface Methodology: Process and
Product Optimization Using Design Experiments. Wiley, New York.
Orozco, S.F.B., Kilikian, B.V., 2008. Effect of pH on citrinin and red pigment
production by Monascus purpureus CCT3802. World Journal of Microbiology
and Biotechnology 24, 263–268.
Pastrana, L., Blanc, P.J., Santterre, A.L., Lorret, M.O., Goma, G., 1994. Production of red
pigments by Monascus rubber in synthetic media with strictly controlled
nitrogen source. Process Biochemistry 30, 1200–1203.
Plackett, R.L., Burman, J.P., 1946. The design of optimum multifactorial experiments.
Biometrika 33, 305–325.
Silveira, S.T., Daroit, D.J., Brandelli, A., 2008. Pigment production by Monascus
purpureus in grape waste using factorial design. LWT—Food Science and
Technology 41, 170–174.
Srinivas, M.R.S., Naginchand, Lonsane, B.K., 1994. Use of Plackett–Burman design
for rapid screening of several nitrogen sources, growth/product promoters,
minerals and enzyme inducers for the production of alpha-galactosidase by
Aspergillus niger MRSS 234 in solid state fermentation. Bioprocess Engineering
10, 139–144.
Thys, R.C.S., Guzzon, S.O., Cladera-Olivera, F., Brandelli, A., 2006. Optimization of
protease production by Microbacterium sp. in feather meal using response
surface methodology. Process Biochemistry 41, 67–73.
Yongsmith, B., Tabloka, W., Yongmanitchai, W., Bavavoda, R., 1993. Culture condi-
tions for yellow pigments formation by Monascus sp. KB10 grown on cassava
medium. World Journal of Microbiology and Biotechnology 9, 85–90.