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1 The Korean Journal of Microbiology, Vol. 43, No. 1, March 2007, p Copyright 2007, The Microbiological Society of Korea w y x 1 y 1, * w t w w w y w w» w ³ e mw. x³ w y w 4% rice powder, 0.15% Bacto-peptone, 0.1% glycine, 0.01% FeSO 4 7H 2 O, 0.1% MgSO 4 7H 2 O, 0.25 % KH 2 PO 4, ph 4.5. x³ spores/ml s xk 50 ml w 30 o C 150 rpm 5 w, g/l ³ s y, ƒƒ 3.25 unit, 1.59 unit, 0.88 unit w, sü y, unit, unit, unit w. Key words ý Monascus pigment, Monascus purpureus, optimal conditions, pigment overproducing mutant y ³ ù m l, Monascus purpureus, M. ruber, M. pilosus, M. kaoliang (6). y ³ w, y (15, 16, 22, 23), y xanthomonasin A Bƒ» w (21). t ƒ ƒ Ÿ š. y ³ w ƒ t y monacolin K (lovastatin)ƒ cholesterol w w z HMG-CoA (3-Hydroxy-3-Methyl Glutaryl CoA) reductase y ww x cholesterol w (4, 14, 17, 19). Monascus³ w x w š y (11, 12, 13, 24), w x ùkù w y (7), y ³ w w k (1, 23, 24). ³ w s w y ³ s x w s y ³ w y ³ w y ³ ³ w ³ w. M. purpureus l NTG w w KM 1001 (5) y ³ ³ w ³ mw» w *To whom correspondence should be addressed. Tel: , Fax: tsyu@kmu.ac.kr wš w. x Potato Dextrose Agar (PDA) Difco (Difco, Detroit, USA) t w, NTG (N-methyl-N'-nitro- N-nitrosoguanidine)»k Sigma (Sigma-Aldrich Co. St. Louis, USA) t w. x³ x w x³ M. purpureus KCCM l NTG w w l w x³ w. s xk x³ PDA 30 o C, 7 w plate ³ 0.08% Tween-80 ƒw ³ w s ³ xkw z, cotton filter w s xk ( spores/ml) w. w w» w Lin w (10). w 3% rice powder, 0.25% KH 2 PO 4, 0.15% NaNO 3, 0.1% MgSO 4 7H 2 O, ph 6.0» 50 ml w 121 o C 15 ³ jš, ³ s xk 100 µl ( spores/ml) ³ w 30 o C rpm 59

2 60 Young-Eun Seo et al. Kor. J. Microbiol k w. ³ d ³ d w ³ filter paper (Whatman No. 4) w ³ w, 80 C w w ¾ w o filter paper ³ w. y d s filter paper (Whatman No. 4) w 12,000 rpm 10 w Ÿ d w, sü ƒƒ ³ 0.05 g 80% k 10 ml ƒw z, 150 rpm, 30 o C 1 k g wš 12,000 rpm 10 w z, w ŸŸ (TECHNE Co. Specgene, England) w Ÿ d w. w w z, y 400 nm, 470 nm, 500 nm Ÿ (OD) d w sü ƒƒ ³ w sü y w t w. 1 unit ƒƒ Ÿ Ÿ 1 1 unit w. k w KM 1001 ³ e k w» w Table 1 ùkü ƒƒ k 3% ƒw» w ³ süá Table 1. Effect of carbon sources on the production of pigment from Carbon Dry cell Pigments (unit) sources weight Yellow Orange Red (3%) (g/l) Extra Intra Extra Intra Extra Intra Rice powder Corn starch Potato starch Wheat starch Rice starch Glucose Sucrose Maltose Lactose Fructose Glycerol Dextrin The inoculated media containing various carbon sources were incubated on 150 rpm rotary shaker at 30 o C for 5 days (ph 6.0). d w ƒ yw k w. ³ e k» w k 0.5%, 1-5% ƒw s xk k z, 30 o C rpm k w e k w. w k ƒw» Table 3 ùkü ƒƒ 0.15% ƒw x³ w z, ³ ƒ yw w. k ƒw» ³ ƒ yw ƒƒ 0-1% ƒw s xk w z, 30 o C rpm k w ³ ƒ yw w. ³ e ph w x³ ³ e ph w Table 2. Effect of concentrations of rice powder on the production of pigment from Rice powder Dry cell weight Intracellular pigments (unit) (%) (g/l) Yellow Orange Red The inoculated media containing various concentrations of rice powder were incubated on 150 rpm rotary shaker at 30 o C for 5 days (ph 6.0). Table 3. Effect of nitrogen sources on the production of pigment from Nitrogen sources Dry cell weight Intracellular pigments (unit) (0.15%) (g/l) Yellow Orange Red NaNO (NH 4 ) 2 SO NH 4 NO KNO Yeast extract Malt extract Bacto-peptone Bacto-soytone Bacto-tryptone MSG The inoculated media containing 4% rice powder and various nitrogen sources (0.15%) were incubated on 150 rpm rotary shaker at 30 o C for 5 days (ph 6.0).

3 Vol. 43, No. 1 y y 61 Table 4. Effect of ph on the production of pigment from Monascus sp. KM 1001 ph Dry cell weight Intracellular pigments (unit) (g/l) Yellow Orange Red were incubated on 150 rpm rotary shaker at 30 o C for 5 days. Table 5. Effect of metal salts on the production of pigment from Metal salts Dry cell weight Intracellular pigments (unit) (0.01%) (g/l) Yellow Orange Red CaCl CuSO 4 5H 2 O FeCl 3 6H 2 O FeSO 4 7H 2 O MnCl 2 4H 2 O MnSO 4 H 2 O MgCl ZnCl ZnSO 4 7H 2 O were incubated on 150 rpm rotary shaker at 30 o C for 5 days (ph 4.5).» w, k ƒw» ph w s xk w 30 o C 150 rpm 5 k w z, ³ ƒ yw ph w. w» w 50 ml spores/ml s xk 0.1 ml w 150 rpm o C w 5 w ³ d w. k w x³ e k w» w s xk w 30 o C k rpm w 5 w z, ³ d w ³ ƒ yw z rpm w. y w x³ e y w» w, y Tween 20, Tween 80 Triton-X 100 Table 6 t» ƒw x³ w w z, w. 0.01% ƒƒ ƒw 30 o C 150 rpm 5 w z ³ e w w. ³ e w ƒw s xk 0.1 ml w 30 o C 150 rpm 5 w ³ e w w w. š k w ³ e k w Table 1 ùkù, w k rice powderƒ ƒ yw. p, rice powder 7.33 g/l ƒ ³ ùkü, KM 1001 ƒ w s y, 3.96 unit, 0.70 unit, 0.44 unit w, sü y unit, unit, š unit w. ù lactose k x w, ³ w. Tseng (20) Monascus purpureus k sucroseù lactose w w š ùk ü. ³ yw k rice powder Table 2 ùkù, 5% rice powder x ³ ³ š w, 9.78 g/l ³ ƒ. ù 4% rice powder 8.78 g/l ³ w ùkü ù, 4% rice powder ƒ ùkû. Rice powder 4% ƒw» x³ ƒ w sü y unit, unit, š unit w. x³ k 4% rice powderƒ ³ ƒ yw k ùkû. w ³ e w Table 3 ùkù,» Bacto-peptone ƒw ƒ g/l ³ ƒ,» KNO 3 ƒ û 5.00 g/l ³ ƒ. Bacto-

4 62 Young-Eun Seo et al. Kor. J. Microbiol peptone 0.15% w x³ ƒ w sü y, ƒƒ unit, unit, unit w. ³ e Bacto-peptone 0.15%, 0.50% Bacto-peptone ³ ( ). Kim (7) y yeast extract peptone w ³» NaNO 3 ƒ ƒ ùk ü, Carels (1) Monascus ammonium nitrate w š yeast extractù w ƒ ƒ š. w x³ ùkû»» ³ w w. ph w x³ ³ e ph w m w Table 4 ùkù, ³ ph g/l ƒ ³ ùkü ù, ph 4.5 ùkü. ph 4.5 x³ sü y, unit, unit, š unit w. x³ ph 6.0 ³, ph 4.5. Chang (2) Monascus sp. CS-2³ w y ph ph 4.5, Park (18) Monascus sp. KS2³ w ph 6.0 ùkü. w Chang (2) w, x w ³ ³ ph y ph 6.0 Park (18) w. w w mw Fig. 1 ùkü. Fig. 1 ùkù, x³ ³ 30 o C ƒ yw ùkü. x³ 30 o C g/l ƒ ³ ƒ, sü y, unit, unit unit w. Juzlova (6) Monascus ³ ƒ 25 o C-37 o C, ƒ ww ƒ 30æ w. k w k w mw Fig. 2 ùkü. x³ Fig. 1. Effect of temperature on the production of pigment from. The inoculated media containing 4% rice powder and 0.15% Bacto-peptone were incubated on 150 rpm rotary shaker at 20, 24, 30, 37, 40 o C for 5 days (ph 4.5). Symbols ø, yellow; ù, orange; ÿ, red;, dry cell weight. Fig. 2. Effect of shaking speed on the production of pigment from. The inoculated media containing 4% rice powder and 0.15% Bacto-peptone were incubated on 0, 50, 100, 150, 180 rpm rotary shaker at 30 o C for 5 days (ph 4.5). Symbols ø, yellow; ù, orange; ÿ, red;, dry cell weight. ³ 100 rpm w k ³ w ù, 150 rpm ƒ yw ùkü g/l ƒ ³ w, sü y, ƒƒ unit, unit unit w. ù 150 rpm k ³ w.

5 Vol. 43, No. 1 y y 63 w z w z w z» w ù,,, y w n w (3). w mw Table 5 ùkü. Table 5 ùkù, x³ ³ ferrous sulfate(feso 4 7H 2 O) ƒw ƒ yw, g/l ³ w sü y, ƒƒ unit, unit unit w. Monascus anka, MnSO 4 ƒw ƒ ùkù Kim (9) š ew ù, Monascus purpureus ATCC Fe w Lee (10) š ùkü. z y y w v w (9). y w y e t j, k y j (8). y ƒƒ x³ ³ sü, š sü s n e w mw Table 6 ùkü. Table 6 ùkù, x³ ³ 0.1% Tween 80 ƒ ³ g/l ƒ ùkû, x³ ƒ w s y, 4.60 unit, 2.60 unit, 1.40 unit ƒƒ w, sü y, unit, unit unit ƒ w. ù Tween 20 Triton-X 100 Tween 80 w ³ sü w ùkü. y Triton-X 100 ƒ x³ s y, 5.35 unit, 3.24 unit, 2.04 unit ùkü ùkù s ùkü y w sü ƒ s n w š dw. w w» w ƒw x³ s xk 0.1 ml w 30 o C 150 rpm 5 w Table 7 ùkü. Table 7 ùkù, L-lysine ƒw ³ g/l ƒ ùkû ù, glycine Table 6. Effect of surfactants on the production of pigment from Pigments (unit) Dry cell Surfactant weight Yellow Orange Red (%) (g/l) Extra Intra Extra Intra Extra Intra None Tween 20 (0.10) (0.30) Tween 80 (0.10) (0.30) Triton-X 100 (0.10) (0.30) were incubated on 150 rpm rotary shaker at 30 o C for 5 days (ph 4.5). Table 7. Effect of amino acids on the production of pigment from Amino acids Dry cell weight Intracellular pigments (unit) (0.1%) (g/l) Yellow Orange Red L-Arginine L-Asparagine L-Aspartic acid L-Alanine Cystine L-Glutamic acid Glycine L-Leucine L-Lysine DL-Methionine Nicotinic acid DL-Tryptophan L-Tyrosine DL-Valine were incubated on 150 rpm rotary shaker at 30 o C for 5 days (ph 4.5). L-glutamic acid ƒw ùkû. x³ ƒ glycine ƒw w sü y, ƒƒ unit, unit, š unit w. x³ KM 1001 w ³ lysine yw glycine glutamic acid ƒw yw. Kim (7) glutamic acid arginine ƒw ƒ y z š

6 64 Young-Eun Seo et al. Kor. J. Microbiol w ùkü. w w (M ) ww, w w w m y l ww x w. š x 1. Carels, M. and D. Shepherd The effect of different nitrogen sources on pigment production and sporulation of Monascus species in submerged. Can. J. Microbiol. 23, Chang, U., H.S. Kim., C.H. Son., J.C. Bae, and J.C. Yu Studies on the yellow pigment produced by Monascus sp. CS-2. Korean J. Microbiol. Bioeng. 8, Chung, M.S. and M.S. Lee Stability of naphthoquinone pigments isolated from the roots of Lithospermum erythrorhizon by various temperatures and metal ions. Korean J. Food. Sci. Technol. 27, Friedrich, J., M. Zuzek, M. Bencina, A. Cimerman, A. Strancar, and I. Radez High-performance liquid chromatographic analysis of mevinolin as mevinolic acid in fermentation broths. J. Chromatogr. A. 704, Jung, H.J. and T.S. Yu Production of mycelium and Monacolin K from Monascus sp. on rice solid culture. J. Microbiol. 40, Juzlova, P., L. Martinkova, and V. Krent Secondary metabolites of the fungus Monascus. J. Ind. Microbiol. 16, Kim, H.S., D.H. Kim., H.S. Yang., Y.R. Pyun, and J.H. Yu Studies on the red pigment produced by Monascus sp. in submerged culture. Korean J. Microbiol. Bioeng. 7, Kim, J.S., H.S. Song., N.H. Chung, and W.G. Bang Optimization of production conditions of biosurfactant from Bacillus sp. and its purification. J. Korean Soc. Appl. Biol. Chem. 48, Kim, M.H., T.K. Lee, and H.C. Yang Red pigment production from Monascus anka albidus. Korean J. Food. Sci. Technol. 24, Lee, B.K., N.H. Park, H.Y. Pyao, and W.J. Chung Production of red pigments by Monascus purpureus in submerged culture. Biotechnol. Bioprocess Eng. 6, Lee, Y.K., D.C. Chen, S. Chauvatcharin, T. Seki, and T. Yoshida Production of Monascus pigments by a solid-liquid state culture method. J. Ferment. Bioeng. 79, Li, C., Y. Zhu, Y. Wang, J.-S. Zhu, J. Chang, and D. Kritchevsky Monascus purpureus-fermented rice (Red Yeast Rice): A natural food product that lowers blood cholesterol in animal models of hypercholesterolemia. Nutri. Res. 18, Lotong, N. and P. Suwanarit Fermentation of ang-kak in plastic bags and regulation of pigmentation by initial moisture content. J. Appl. Bacteriol. 68, Manzoni, M. and M. Rollini Biosynthesis and biotechnological production of statins by filamentous fungi and application of these cholesterol-lowering drugs. Appl. Microbiol. Biotechnol. 58, Martinkova, L., P. Juzlova, and D. Vesely Biological activity of polyketide pigments produced by the fungus Monascus. J. Appl. Bacteriol. 79, Martinkova, L., P. Juzlova, V. Krent, Z. Kucerova, V. Havlicek, P. Olsovsky, O. Hovorka, and B. Rihova Biological activities of oligoketide pigments of Monascus purpureus. Food Addit. Contam. 16, Morovjan, G., G. Szakacs, and J. Fekete Monitoring of selected metabolites and biotransformation products from fermentation broths by high-performance liquid chromatography. J. Chromatogr. A. 763, Park, H.E., C.H. Kim, and K.H. Min Isolation of pigmentproducing mutants from Monascus sp. KS2 and optimization of cultural conditions. Kor. J. Mycol. 19, Strode, J.T.B., L.T. Taylor, A.L. Howard, and D. Ip Feasibility of lovastatin analysis by packed column supercritical fluid chromatography with ultraviolet detection. J. Pharm. Biomed. Anal. 20, Tseng, Y.Y., M.T. Chen, and C.F. Lin Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrate, polyphosphate and various sugars. J. Appl. Microbiol. 88, Watanabe, T., A. Yamamoto, S. Nagai, and S. Terabe Separation and determination of Monascus yellow pigments for food by micellar electrokinetic chromatography. Anal. Sci. 13, Wild, D., G. Toth, and H. Humpf New Monascus metabolite isolated from red yeast rice. J. Agric. Food Chem. 50, Wu, W.T., P.M. Wang, Y.Y. Chang, T.K. Huang, and Y.H. Chien Suspended rice particles for cultivation of Monascus purpureus in a tower-type bioreactor. Appl. Microbiol. Biotechnol. 53, Yongsmith, B., S. Krairak, and R. Bavavoda Production of yellow pigments in submerged culture of a mutant of Monascus spp. J. Ferment. Bioeng. 78, (Received November 10, 2006/Accepted March 27, 2007)

7 Vol. 43, No. 1 y y 65 ABSTRACT : Optimization of Production of Pigment from Monascus sp. in Liquid Culture Young-Eun Seo, Hyuck-Jun Jung 1, Soon-Myung Hong, and Tae Shick Yu 1, * (Department of Food and Nutrition, Ulsan University, Ulsan , Korea, 1 Department of Microbiology, Keimyung University, Daegu , Korea) The optimal conditions for Monascus pigments production of, pigment overproducing mutant, in submerged culture was investigated. The optimal medium for the production of pigment from KM 1001 mutant is determined to be composed of 4% rice powder, 0.15% Bacto-peptone, 0.1% glycine, 0.01% FeSO 4 7H 2 O, 0.1% MgSO 4 7H 2 O, 0.25% KH 2 PO 4, ph 4.5. On optimal conditions, 10.0 g/l of the cell mass was obtained at 30 o C for 5 days. Yellow, orange and red pigment of were produced 3.25 units, 1.59 units and 0.88 units in extracellular part, and units, units and units in intracellular part, respectively.

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