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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 Solid-state fermentation 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 Jamia Hamdard (Hamdard University), of different fungal species. The coculture of fungi during fermentation may provide help for better biomass and New Delhi 110062, India secondary metabolite productions; moreover, it helps in S. Javed (*) 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: [email protected] et al. Unfortunately, no study has been carried out Food Bioprocess Technol 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 response surface methodology (RSM).
different levels (Table Extraction of Lovastatin Materials and Methods 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. ).
al. Chang et al. ).
Quantitative Analysis of Lovastatin Preparation of Mixed Seed Cultures 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 Results and Discussion 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 ratio of 1:1.
the solid medium are selected for lovastatin productionunder coculture of M. purpureus MTCC 369 and M. ruber Solid-state Fermentation 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 Food Bioprocess Technol Table 1 Box–Behnken design for process parameters with lovastatin concentration (actual and predicted) under the coculture system Temperature (°C) Lovastatin (mg/g) 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 Regression analysis of model 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.
Correlation coefficient (R2) Coefficient of variation (CV%) Multiple Nonlinear Regression Model: Test for lack of fit 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- Adequate precision tation time −0.17×temperature×inoculum volume+ Food Bioprocess Technol 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 Fermentation time pH of the solid medium Temperature×pH of the solid medium Fermentation time×Inoculum volume Fermentation time×pH of the solid Inoculum volume×pH of the solid Fig. 1 Response surface plots showing relative effects of different process parameters on lovastatin production during solid-statefermentation Fermentation time2 pH of the solid medium2 Food Bioprocess Technol 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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