Food Bioprocess TechnolDOI 10.1007/s11947-008-0072-z
Optimization of Fermentation Parameters for HigherLovastatin Production in Red Mold Rice through Co-cultureof Monascus purpureus and Monascus ruber
Bibhu Prasad Panda & Saleem Javed & Mohammad Ali
Received: 4 January 2008 / Accepted: 19 February 2008
Springer Science + Business Media, LLC 2008
Abstract Monascus, fermented rice (red mold rice), has
3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA)
been found to reduce the serum total cholesterol and
reductase, which catalyzes the reduction of HMG-CoA to
triglyceride due to presence of lovastatin. Lovastatin acts
mevalonate during cholesterol biosynthesis (Alberts et al.
as an inhibitor of 3-hydroxy-3-methyl glutaryl coenzyme A
; Hajjaj et al. This natural statin was the first
reductase. Coculture of Monascus purpureus MTCC 369
fungal secondary metabolite to obtain approval from the US
and Monascus ruber MTCC 1880 was used to produce red
Food and Drug Administration in August 1987 (Tobert
mold rice by solid-state fermentation. Optimization of
; Demain ; Manzoni and Rollini ). Lovastat-
different fermentation process parameters such as temper-
in is produced by Monascus pilosus, Aspergillus terreus,
ature, fermentation time, inoculum volume, and pH of the
Monascus ruber, Monascus purpureus, and Penicillium
solid medium was carried out by Box–Behnken’s factorial
species (Hajjaj et al. Miyake et al. , Chang
design of response surface methodology to maximize
et al. However, some hyperproducing strains of A.
lovastatin concentration in red mold rice. Maximum
terreus produces high amount of lovastatin under sub-
lovastatin production of 2.83 mg/g was predicted at 14th
merged fermentation (Porcel et al. but the liquid
day in solid medium under optimized process condition.
medium containing lovastatin produced by A. terreus is notsuitable for consumption directly by human beings since it
Keywords Coculture . Monascus purpureus . Monascus
is not coming under the “generally regarded as safe”
ruber . Lovastatin . Response surface methodology .
designation. Therefore, complex chromatographic and
solvent extraction procedures were followed to downstreamthe lovastatin from the fermented broth.
M. ruber and M. purpureus are nonpathogenic fungi and
used frequently by Chinese for the production of red moldrice (Kohama et al. Chen and Hu Chiu et al.
Lovastatin (mevinolin and monacolin K), a hypocholestro-
; Lee et al. There are several reports on the
mic agent, competitively inhibit the rate-limiting enzyme
production of lovastatin and red mold rice by usingmonocultures of Monascus species (Lee et al. ; Chiu
et al. ; Miyake et al. ; Su et al. In nature,
B. P. Panda M. AliPharmaceutical Biotechnology Laboratory, Faculty of Pharmacy,
solid substrate fermentation is carried out by mixed cultures
of different fungal species. The coculture of fungi during
fermentation may provide help for better biomass and
secondary metabolite productions; moreover, it helps in
proper utilization of substrate. There are several reports of
Molecular Biology and Biotechnology Laboratory,
coculture of fungal species found to enhance enzyme,
Faculty of Science, Jamia Hamdard (Hamdard University),
organic acid production, and microbial bioconversion
reaction (Banerjee et al. Pandey et al. ; Temudo
New Delhi 110062, Indiae-mail: firstname.lastname@example.org
et al. Unfortunately, no study has been carried out
for production of lovastatin by coculture or mixed culture
9.68 g/l, dextrose 38.90 g/l, MnSO4.H2O 1.96 g/l, and
of Monascus species under solid-state fermentation.
MgSO4.7H2O 0.730 g/l) obtained by a Plackett–Burman
Therefore, the objective of this research was to produce
design and RSM were added, and the pH of the medium
the finest-quality rice-based nutraceutical-containing maxi-
was adjusted as per the experimental design with 0.1 M
mum amount of a hypocholestromic agent (lovastatin). Two
HCl or NaOH and autoclaved for 20 min at 121 °C. After
filamentous fungi, M. purpureus MTCC 369 and M. ruber
being cooled, the rice-based medium was inoculated with
MTCC 1880, were used together as inocula for the
mixed seed cultures of M. purpureus and M. ruber. Box–
production of the nutraceutical under solid-state fermenta-
Behnken response surface design (Sayyad et al. was
tion. As the fermentation process is highly regulated by
followed to design fermentation process conditions such as
different fermentation process conditions, interactions of
temperature, fermentation time, inoculum volume, and pH
parameters and their optimum levels were determined by
of the solid medium for different experimental runs at
Fermented rice (1 g) was suspended in 5 ml ethyl acetate
and kept in a shaker incubator at 180 rpm and 70 °C for1.5 h. The mixtures were centrifuged at 3,000×g for 8 min,
Fungal cultures of M. purpureus MTCC 369 and M. ruber
supernatant (1 ml) was collected, and 1% trifluoroacetic
MTCC 1880 were obtained from the Institute of Microbial
acid (10 ml) was added for lactonization of the lovastatin.
Technology, Chandigarh, India. Fungal cultures were
The resultant was concentrated at 80 °C (without applying
maintained routinely on a potato dextrose agar medium
vacuum), diluted to 1 ml with acetonitrile and filtered
containing agar (1.5%), diced potatoes (30%), and glucose
through a 0.45-μm filter for high-performance liquid
(2%) and subcultured in every 30-day interval (Sayyad et
chromatography (HPLC) analysis (Su et al. ).
Procedure given by Samiee et al. for HPLC analysis
Spore suspensions of M. purpureus and M. ruber was
was slightly modified. Lovastatin was estimated by
prepared separately from actively growing slants in sterile
HPLC (SHIMADZU, Japan) using 250 × 4.6 mm ID
water and diluted to a concentration 5.7×103 spores per
Lichrosper® 100 C18 column of 5 μm particle size, 20 μl
milliliter. Spore counting was carried out using a hemocy-
loop injector, and Shimadzu CLASS-VP version 5.032
tometer. Spore suspension (7.5 ml) of M. purpureus was
software. Acetonitrile/water (65:35 v/v), acidified with
inoculated to conical flasks containing 50 ml basal medium
ortho-phosphoric acid to the concentration 0.1%, was used
(100 g dextrose, 10 g peptone, 2 g KNO3, 2 g NH4H2PO4,
as mobile phase with a flow rate of 1.5 ml/min, and
0.5 g MgSO4.7H2O, 0.1 g CaCl2 in 1,000 ml distilled
detection was carried out by UV detector (SPD10A VP) at
water; adjusted to pH 6.0) and incubated at 30 °C for 48 h
235 nm (Samiee et al. Sayyad et al. ).
in a shaker incubator at 110 rpm (Sayyad et al. ; Su etal. ). Spore suspension (7.5 ml) of M. ruber wasinoculated to conical flasks containing 50 ml of potato
dextrose broth, incubated at 30 °C for 4 days with shakingat 150 rpm (Chang et al. ). Finally both the seed
Fermentation process parameters such as temperature,
cultures of M. purpureus and M. ruber were mixed at a
fermentation time periods, inoculum volume, and pH of
the solid medium are selected for lovastatin productionunder coculture of M. purpureus MTCC 369 and M. ruber
MTCC1880 during solid-state fermentation, and RSM forprocess optimization was followed.
Long-grain, nonglutinous rice was purchased from the
To identify the optimum levels of different process
local market of New Delhi, India, and was used as a base
parameters influencing lovastatin production, solid-state
solid substrate for red mold rice production under solid-
fermentation was carried out in conical flasks containing
state culture. Initially, 20 g of presoaked rice was taken in a
optimized nutrients. Four process parameters (temperature,
250-ml conical flask to which 40 ml of distilled water
fermentation time, inoculum volume, and pH of the solid
containing different optimized nutrients (malt extract
medium) were chosen for study by borrowing methodology
Table 1 Box–Behnken design for process parameters with lovastatin concentration (actual and predicted) under the coculture system
as these process parameters mostly influence the growth ofdifferent fungal strains and secondary metabolite produc-
Table 2 The analysis of variance of the calculated model of process
tion during solid-state fermentation. An experimental
parameters for lovastatin production in the coculture system
design of 29 runs containing five central points was made
according to Box–Behnken’s response surface design forfour selected parameters. The individual and interactive
effects of these (process parameters) variables were studied
by conducting the fermentation run at different levels of all
factors. The response was measured in milligram of
lovastatin per gram of fermented rice. The results of
experimental and simulated values are listed in Table .
Lovastatin production in each experimental run was
analyzed using the software Design Expert 7.1 (Statease,
USA) and fitted into a multiple nonlinear regression model.
The model proposes the following equation.
Lovastatin (mg/g) = 2.81−0.26×temperature−0.023×
fermentation time −0.13×inoculum volume+0.035×
pH of the solid medium −0.12×temperature×fermen-
tation time −0.17×temperature×inoculum volume+
0.12×temperature×pH of the solid medium −0.24×fermentation time×inoculum volume+0.092×fermen-tation time ×pH of the solid medium +0.058×inocu-lum volume × pH of the solid medium − 1.02 ×temperature2−0.49×fermentation time2−0.29×inocu-lum volume2−0.65×pH of the solid medium2
This multiple nonlinear quadratic model resulted in six
response surface graphs. A few representative responsesurface plots of the calculated model for lovastatinproduction are shown in Fig. and c. The analysis ofvariance of the model for lovastatin production is repre-sented in Table
Point prediction of the design expert software was used
to determine the optimum values of the factors formaximum lovastatin production. Finally, the optimumvalues of temperature at 29.46 °C, fermentation time for13.89 days, inoculum volume of 4.95 ml, and at a mediumpH of 6.03 were determined. These values predict2.83 mg/g of lovastatin production by coculture of M.
purpureus and M. ruber under solid-state fermentation.
These optimized values of process parameters werevalidated by solid-state fermentation of rice containingpreviously optimized medium parameters (malt extract9.68 g/l, dextrose 38.90 g/l, MnSO4.H2O 1.96 g/l, andMgSO4.7H2O 0.730 g/l), and an average 2.80 mg/g oflovastatin production in solid substrate was obtained. Thisshows 98.93% validity of the predicted model.
Solid-state fermentation runs were designed according to
Box–Behnken design of RSM at randomly selecteddifferent levels. The process parameters temperature,fermentation time periods, and inoculum volume wasnegatively significant factors, and the pH of the fermenta-
Table 3 Analysis of variance of model parameters
Fig. 1 Response surface plots showing relative effects of different
process parameters on lovastatin production during solid-statefermentation
tion medium was a positively significant factor. From the
quadratic model, it was conformed that the pH of thefermentation medium interacts positively to all the process
Lovastatin concentration in red mold rice (Chinese func-
parameters. Fermentation temperature interacts negatively
tional food) can be increased by mixed-culture fermentation
with fermentation time and inoculum volume, but fermen-
of rice with two different Monascus species (M. purpureus
tation time and inoculum volume interact negatively with
and M. ruber). Solid-state fermentation of rice with pH 6.03
each other. Out of the total model parameters, 30% of the
at 29.46 °C for 13.89 d, predict 2.83 mg/g and yielded
parameters significantly influenced the lovastatin produc-
2.80 mg of lovastatin/gram of fermented rice with 98.93%
tion (Table ). The “lack-of-fit F value” of 2,887.56 was
validity. Moreover, it can be eaten directly to gain better
obtained. A high lack-of-fit value could occur due to noise.
However, adequate precision (measures the signal-to-noiseratio) of the model was found to be at 7.633, and the valueis larger then the desirable value of 4 (Table A highadequate precision ratio indicates an adequate signal in the
quadratic model. Therfore, the model can be used tonavigate the design space.
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