Chiang Mai J. Sci. 2009; 36(1) : Contributed Paper

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1 104 Chiang Mai J. Sci. 2009; 36(1) Chiang Mai J. Sci. 2009; 36(1) : Contributed Paper Screening of Nutrient Parameters for Red Pigment Production by Monascus purpureus MTCC 369 Under Submerged Fermentation Using Plackett- Burman 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 India. *Author for correspondence; bibhu_panda31@rediffmail.com Received: 7 August 2008 Accepted: 11 January 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, Cl, (, NaCl, KH 2, O, CaCl 2. O, O were screened along with two dummy factors, in twelve experimental runs as per the design. From standard Plackett- Burman data analysis it was conformed that, Cl, NaCl, KH 2, O and O had contributed to a large extent, dextrose, CaCl 2. O had little impact, while, ( 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

2 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 purpureus MTCC 369 under submerged fermentation. 2. MATERIALS AND METHODS 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 Potato- Dextrose Agar (PDA) medium at 4 o C and subcultured every 30 days. 2.2 Preparation of Seed Culture Ascospore suspension of Monascus purpureus MTCC 369 was prepared from actively growing slants in sterile water and diluted to a concentration spores per ml. Fifteen percent spore suspension was inoculated to conical flasks containing the basal medium (100g dextrose, 10g peptone, 2g KNO 3, 2g H 2, 0.5g O, 0.1g CaCl 2 in 1000 ml distilled water; adjusted to ph 6.0). These cultures were incubated at 30 o C for 48 hrs in a shaker incubator at 110 rpm [6,14]. 2.3 Plackett-Burman Experimental Design Dextrose, Cl, (, NaCl, KH 2, O, CaCl 2. O, O 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 Plackett- Burman experimental design [12] for eleven variables: nine nutritional components 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 X 1 Dextrose X 2 Cl X 3 ( X 4 NaCl X 5 KH X 6 O X 7 Dummy X 8 CaCl 2. O X 9 O X 10 FeSO 4 O X 11 Dummy 2 - -

3 106 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 (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 o C for 15 min. Finally each flask were inoculated with 10 % seed culture and incubated at 30 o C 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 (Stat Ease Inc USA). The effect of each variable was determined with the following equation. E xi = 2 (ΣH xi - ΣL xi ) / N Where, E xi is the concentration effect of the tested variable, H xi and L xi 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. Red Trial X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 pigment (mg l -1 )

4 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 V xi = (ΣH xi - ΣL xi / N The experimental error was calculated by averaging the mean squares of the dummy variables. R = ΣV xd / n Factor showing larger effects were identified using F-test. F = V xi / R Where, R is experimental error (mean square for error), V xd is mean square of dummy variable; V xi 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 Among the nine nutrient components used in study, Cl, NaCl, KH 2, O and O had contributed to a large extent for biopigment production. Dextrose, CaCl 2. O had little impact, while, ( contributes moderately in production of red pigment. Between O, contribution of O for pigment synthesis found to be more with F value of than the FeSO 4 O with F value Table 3. Influence of medium variables on red pigment production. Designation Variable Σ H Σ L Mean Experimental Effect F-value Square error X 1 Dextrose X 2 Cl X 3 ( X 4 NaCl X 5 KH X 6 O X 7 Dummy X 8 CaCl 2. O X 9 O X 10 FeSO 4 O X 11 Dummy

5 108 Chiang Mai J. Sci. 2009; 36(1) (Table 3). Dextrose, Cl, NaCl, KH 2, O were influences the production in their higher concentration, whereas (, CaCl 2. O and O 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 Cl was found to be better than ( 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, Cl 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 Cl 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, Cl, KH 2, O and O for red pigment production by M. purpureus MTCC 369 is necessary. 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: [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: [3] Juzlova P., Martinkova L., and Kren, V. Secondary metabolites of the fungus Monascus: A review, J. Ind. Microbiol., 1996; 16: [4] Pattanagul P., Pinthong R., Phianmongkhol A., and Leksawasdi N., Review of Angkak Production (Monascus purpureus), Chiang Mai J. Sci., 2007; 34: [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: [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: [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: [8] Endo A., Monacolin K, a new hypocholesterolemic agent produced by Monascus species, J. Antibiot., 1979; 32: [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:

6 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: [11] Panda B.P., Javed S., and Ali M., Fermentation process optimization, Res. J. Microbiol., 2007; 2: [12] Plackett R.L., and Burman J.P, The design of optimum multifactorial experiments, Biometrika., 1946; 33: [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: [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: [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: [16] Johns M.R., and Stuart D.M, Production of pigments by Monascus purpureus in solid culture, J. Ind. Microbiol., 1991; 8: [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: [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: [19] Yu X., Hallett S.G., Sheppard J., and Watson A.K. Application of Plackett- Burman experimental design to evaluate nutritional requirements for the production of Collectotrichum coccodes spores, Appl Microbiol Biotechnol., 1997; 47: