Production of yellow pigments in submerged culture of a mutant of Monascus spp.

JOURNALOF FERMENTATIONAND BIOENGINEERING VOI. 78, NO. 3, 223-228. 1994

Production of Yellow Pigments in Submerged Culture of a Mutant of Monascus spp. B U S A B A Y O N G S M I T H , I * S O M C H A I K R A I R A K , J AND R A P E E P O L B A V A V O D A 2

Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 109001 and Department of Pharmaceutical Botany, Faculty of Pharmaceutical Science, Chulalongkorn University, Bangkok 10400,2 Thailand Received 4 October 1993/Accepted 2 July 1994 This paper communicates our new findings on a mutant of Monascus mold which is capable of producing a high concentration of yellow pigments (2max 370 nm) instead of red pigments (2max 420 and 500 nm) produced by its parent strains. Various factors affecting yellow pigment production have been examined. It was found that the mutant produced the maximal amount of pigment under the optimal conditions for pigment production of its parent strains. The mutation allowed a 10-fold increase in both yellow pigment production and amylolytic enzyme activity over those from a yellow pigment producer isolated previously by us.

There are a number o f reports o f red pigment production ('~max 500nm) a n d / o r yellow pigment production ('~max 400 rim) by Monascus molds (1, 2). Yellow pigments could be further extracted from Monascus red mass. Previously, we investigated p r o d u c t i o n of yellow pigments ('~ma× at 330 nm) by Monascus sp. KB10 (3) which was tentatively identified as Monascus barkari (4). Optimization o f cultivation conditions for yellow pigment p r o d u c t i o n as well as strain improvement had been carried out and reported (3, 5). The m a x i m u m concentration o f yellow pigments (70 U / m l ) was obtained at a low initial p H o f media after investigating its media formulation, though it was hard to improve by mutation. Recently, we have screened a yellow pigment producer by secondary m u t a t i o n of a potent red-pigment-producing m u t a n t o f Monascus sp. KB9, a wild strain which was identified as Monascus kaoliang (4). This mutant also appeared to secrete an intense brownish-yellow pigment into the m e d i u m under a wide variety of conditions and this report is a follow up to observations previously reported. To our knowledge, there has been no report o f such pigmentation in other studies o f cultures of the same species. The present paper describes the screening o f another high yellow-pigment-producing Monascus, the investigation o f culture conditions from a practical point o f view, and its pigment purification.

7H20, 0.2 g o f NaNO3, 0.05 g o f KC1, 0.001 g o f FeSO4. 7H20, 0.3 g of yeast extract, 0.5 g o f casamino acids, and 2.0 g o f agar made up to 1.0 l. The conidia formed were collected and suspended in sterilized water. Ten ml o f spore suspension was exposed to UV light at various times (0, 10, 15, 25 up to 60min). MYS medium was used as a basis for selecting the optimal medium for isolated mutants. The culture was incubated at 28-32°C for 7 d. However, the medium for submerged cultivation was simpler and basically composed o f 30/00 cassava starch and 40/00 soybean flour (SS medium). The 75-ml medium in a 250-ml flask was sterilized at 121°C for 15 min. A b o u t 4 pieces of mycelia of 6 m m diameter from a 10-day-old MYS culture were used as the inocula. Assay of amylolytic enzymes Glucoamylase activity Amylase activity was assayed by the determination o f reducing sugars liberated in the reaction mixture in 3 0 m i n at 65°C. The reaction mixture contained 0.5 ml o f the enzyme solution and 0.5 ml of boiled 1 . 0 ~ soluble starch in acetate buffer o f p H 4.7 at the final concentration o f 0.05 M. The reducing sugar liberated was determined by the method o f Nelson (7) as modified by Somogyi (8) using a spectrophotometer (UV-240 recording d o u b l e - b e a m spectrophotometer; Shimadzu, Kyoto) at 5 2 0 n m absorbance. Glucose was used as the standard. One unit of glucoamylase activity was defined as the amount o f the enzyme that liberated reducing sugars equivalent to 1 microgram o f glucose per ml in one min at 65°C. a-Amylase activity a - A m y l a s e activity was assayed by the determination o f starch digested in the reaction mixture in 2 0 m i n at 40°C. The reaction mixture contained 0.1 ml o f the enzyme solution and 2.0 ml o f 1 . 0 ~ boiled soluble starch in acetate buffer o f p H 5.5 at the final concentration o f 0.05 M. The sugars liberated in the reaction mixture were mixed with iodine solution thoroughly and transmittance was measured at 670nm. One unit of a-amylase activity was defined as the a m o u n t of enzyme that digested 10 mg of starch per ml in one min at 40°C. Detection and extraction of Monascus pigments Estimation of pigment concentration The fermentation broth was extracted with 50%0 w / v ethanol and cen-

MATERIALS AND METHODS Microorganisms and cultivation M. kaoliang KB9 was used as the parent strain for stepwise mutagenesis. The m e d i u m used for culture maintenance was MYS (malt extract-yeast extract-cassava starch) agar, which consisted o f 30g o f cassava starch, 3 g of malt extract, 3 g o f yeast extract, 5 g o f p e p t o n e and 15 g o f agar made up to 1 l. UV radiation was used for mutagenesis. F o r isolation o f mutants exhibiting excessive production o f pigments, M. kaoliang KB9 was cultured at 3032°C for 10 d on m e d i u m C (6) which was composed o f 10g o f sucrose, 0 . 1 g o f KHzPO4, 0.05g o f MgSO 4.

* Corresponding author. 223

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trifuged at 7,000 rpm for 15 min. The optical density of the clear broth was measured at 370nm using the uninoculated medium as the blank (9, 10).

Extraction and isolation of fractions containing yellow pigment After a spray-drying treatment, the Monascus pigments were extracted with c h l o r o f o r m , methanol and water, in that order. The brownish-yellow extract obtained by centrifugation was combined and concentrated, and was applied to a silica gel column. The column (3 ~ 5 cm) was eluted with benzene : acetone with increasing polarities (9 : 1, 8 : 2, 7 : 3, 6 : 4 and 2 : 8). Fractions containing yellow pigment from the column were evaporated under reduced pressure to dryness. ~HNMR spectra were obtained in CDC13 or in a mixture of DCI3 and D M S O d6 at 90 M H z on a J E O L FX 90 spectrophotometer. A ~3C N M R spectrum was obtained at 22.5 MHz. Mass spectra were obtained on a DX 300 (JEOL) spectrophotometer operated at 70eV. Infrared a b s o r p t i o n spectra were measured on a PerkinElmer Model 283 spectrophotometer.

RESULTS Screening of hyper-pigment production mutants Our previous experiments (11) showed that treatment with various kinds of mutagens such as UV irradiation, p r a y exposure, ethylmethanesulfonate (EMS) and Nmethyl-N-nitrosoguanidine (NTG) led to improved pigment productivity. All mutants selected from UV treatment showed better stability over those from other treatments. One o f primary mutants (KBIOM16) gave a twofold increase in the concentration o f red pigments. After treatment o f this strain at various times (0-60 min) o f exposure to UV light, it was found that many clones selected on the basis o f the yellow color a r o u n d colonies grown on MYS agar gave good results on yellow pigment production. A b o u t 7 of the 13 isolated colonies from 20-min UV treatment showed a yellow zone different from the others on MYS agar. Then we examined yellow pigment p r o d u c t i o n by them in submerged cultivation. It was shown that the 7 isolates gave good yield of yellow pigments in submerged cultivation using cassava starch and soybean flour. Strain 20M10.2 which gave the densest

yellow color was subjected to spectral determination for comparison with its parent strain, a wild strain and KBIOM16. The maximum absorptions were found at 420 and 500 nm for its parent strain and at 370 nm for this mutant. The absorption spectra of pigments derived from M. kaoliang KB9, KB10M16 and KB20M10.2 are shown in Fig. 1. In the visible region o f the absorption spectrum, two m a x i m a were found at 420 and 500 nm in the cases of M. kaoliang KB9 and KB10M16, whereas that from the culture o f KB20MI0.2 was found only at 370 nm. It meant that the color changed from red to yellow. The UV spectrum showed that these three strains had an absorption maximum at 280nm, and suggested that these three pigments formed a complex of proteins.

Optimization of the yellow pigment production of strain KB20M10.2 Our previous experiments showed that a certain strain, Monascus KB10 (M. barkari) had a tendency to produce yellow pigments ()~...... 370 nm with initial pH of 4.0 or ),....... 3 3 0 n m with initial pH o f 2.5) (3). The optimization of pigment production was markedly affected by medium formulation. The following is the composition o f a recommended medium: 49a (w/v) cassava starch, 0.49/oo (w/v) peptone and 0.1% (w/v) glutamic acid with initial pH of 2.5. The maximal concentration of yellow pigments at 7 0 U / m l , ~...... 330 nm was obtained (3). Improvement of this strain by UV light treatment of cells as well as protoplast has been carried out. Unfortunately, this strain could not been improved to increase yellow pigments production in sub200 MYS-m

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VoL. 78, 1994

YELLOW PIGMENT FORMATION BY A MUTANT OF MONASCUS SPP. 600

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FIG. 5. Effect of incubation temperature on yellow pigment production by M. kaoliang KB20MI0.2. Cultivation was carried out on rotatory shaker (300 rpm) at the indicated temperature for 7 d in the medium composed of 3% cassava starch and 5% soybean flour.

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FIG. 6. Effectof inoculum size on yellow pigment production by M. kaoliang KB20M10.2. Cultivation was carried out in a 75-ml pigment production medium in 250-ml flask on a rotatory shaker at 300 rpm for 7 d at 28°C. Symbols: • , pigment; A, residual starch. flour made up to 75 ml in a 250-ml shaking flask (300rpm). The mold consumed cassava starch quickly up to the 4th day of cultivation and slowed down after that. Yellow pigment production increased along with enzyme activity, both of which reached the m a x i m u m at 6 9 3 U / m l (A37onm) and 12,293U/ml, respectively. These values increased by tenfold over those from a Pigment (U/ml, A 370) 100 ,

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Day FIG. 8. Time course of yellow pigment production by M. kaoliang KB20MI0.2. Cultivation was carried out on a rotatory shaker (300 rpm) at 28°C for days, with 75-ml medium containing 3.0%0 cassava starch and 5.094 soybean flour in 250-ml Erlenmeyer flask. Symbols: A, pigment; o, pH; m, glucoamylase activity; L , residual starch. yellow pigment producer previously reported by us (3). Purification of yellow pigment Purification was carried out to determine the chemical structure of the yellow pigments. Fractions of the yellow pigments were crystallized by evaporation. Then the NMR, IR and mass spectra of the yellow fractions obtained by the methods described before (3) were examined. The results of two potent yellow fractions are as follows. C o m p o u n d yellow 1, bright yellow crystal; M* 358 (C21H2605), m / z 358 (100%), 162 (100%, C I I H I 4 0 ) , 134 (55.4), 119 (27.5), 91 (38.5), and 69 (77.4). 'H NMR (90 MHz, in CDC13); d ppm, from tetramethysilane, 0.87 (t, 3H); 1.44 (S, 3H); 1.87 (d, 3H); 1.83-1.44 (m, -CH2 protons); 2.35-3.85 (m, 5H); 3.67 (d, J = 1 2 H z , 1H); 4.60 (k, J - 1 2 Hz, 1H); 5.0 (d, J = 1 2 Hz, IH); 5.27 (s, 1H); 5.89 (d, J = 1 2 H z , 1H); 6.40 (m, 1H). C o m p o u n d yellow 2, yellow crystal; M* 386 (C23H3005), m / z 386 (15°6) 359 (20), 358 (87.5), 163 (32.5), 162 (100), 134 (31.2), 199 (18.6). ~H NMR (200 MHz. in CDCI3); d 0.87 (t, 3H); 1.20-1.41 (m, 6H); 1.45 (s, 3H); 1.62 (m, 2H); 1.88 (d, 3H); 2.35-2.75 (m, 3H); 2.9-3.35 (m, 2H); 3.60-3.70 (d, 1H); 4.60-4.80 (d, 1H); 5.0-5.15 (d, 1H); 5.27 (s, IH); 5.89 (d, J :12Hz, ld); 5.89 (d, J 12Hz, 1H); 6.50 (m, IH).

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Yellow pigment production through a series of mutagenesis processes was carried out with M. kaoliang KB9 for the improvement of pigment yield. Two potent mutants, designated KB10MI6 and KB20MI0.2 were obtained as good red and yellow pigment producers in submerged culture, respectively. Strain KB20M10.2 was found to produce yellow pigments showing a maximum absorption at 3 7 0 n m whereas its parent strain KB10MI6 and a wild-type strain KB9 produced red pigments showing m a x i m u m absorptions at 420 and 5 0 0 n m . Yellow pigments having a single absorption m a x i m u m at lower wavelengths or having an absorption maximum which

VOL. 78, 1994

YELLOW PIGMENT FORMATION BY A MUTANT OF MONASCUS SPP.

227

TABLE 2. List of Monascus pigments Yielda Strain

Culture conditions

Yellow 330 nm 370 nm

400 nm

Red 420 nm

500 nm

Reference

Submerged culture M. kaoliang

M. anka V-250

M. barkari KBI0

M. barkari KBI0

M. kaoliang 20M10.2

Polished rice powder 3°Jo MgSO4.7H20, 0.1°~o; KNO3, 0.15°~o; KHzPO4, 0.25%0; initial neutral pH; 72 h at 35°C Polished rice powder, monosodium glutamate, 0.15°/ooMgSO4.7H20, 0.1~o; KH2PO4, 0.25~; initial neutral pH; 144 h at 30°C Cassava starch, 4%o; peptone 0.4%o; glutamic acid, 0.1~; initial pH 2.5; 168 h at 28°C Cassava starch, 3~; malt extract, 0.3%o; yeast extract, 0.3%o; peptone, 0.5~; initial neutral pH; 158 h at 28°C Cassava starch, 3.00/oo;soybean flour, 5.09/00; initial neutral pH; 168 h at 30°C

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(3) This study

Solid culture M. kaoliang R-10847 M. barkari KB 10 Monascus NP

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a For submerged culture, data are presented as units of absorbance per milliliter. For solid culture, data are presented as units of absorbance per gram of dried medium. coincides with that of red pigments at higher wavelengths have been reported in submerged and solid culture by m a n y investigators (Table 2). Yellow, orange and red pigments are normally present as a mixture in M o n a s c u s culture (1, 15, 16; Moll, H . R . and Farr, D . R . , US patent, 3993789, 1976). A report showed that most of the yellow pigments could be extracted chemically from a mixture of pigments from a M o n a s c u s culture (17). Our previous study found that under stressed conditions of low initial pH (pH 2.5) and a certain medium formulation, the production of orange-red pigments having absorption maxima at 370, 420 and 500 n m by M . barkari could be changed to that of yellow pigment with a single absorption maximum, with a decrease in orange and red pigments. In our present study, the wild-type strain and its firstgeneration m u t a n t , KB10MI6, have red pigmentation with absorption maxima at 420 n m and 500 nm in the optimal medium containing 3.0% (w/v) of cassava starch and 4.09/00 (w/v) of soybean flour at various initial pH (2.5-7.0). In contrast, yellow pigments of strain KB20MI0.2 (second-generation mutant) with the predominating single absorption m a x i m u m at 3 7 0 n m was produced in the similar optimal medium at various pH ranging from 2.5-7.0, resulting in the m a x i m u m concentration of pigments in the broth of KB20M10.2 (693 U / m l , A370nm). The two red pigments, r u b r o p u n c t a m i n e and m o n a s c o r u b r a m i n e , are nitrogen analogues of the orange pigments, r u b r o p u n c t a t i n and m o n a s c o r u b r i n (17). It seemed that the wild-type strain and its firstgeneration m u t a n t produced a high a m o u n t of such red pigments in the nitrogen-rich medium, whereas that the second-generation m u t a n t (KB20M10.2) could exclusively produce the yellow pigments in the same nitrogen-rich medium at various pH (2.5-7.0). Therefore, this phen o m e n o n on yellow pigment production of strain KB20MI0.2 is not affected by environmental factors such as medium formulation or pH but rather, it is

strain-specific. Sweeny et al. (17) reported that yellow pigments of M o n a s c u s , monascoflavin and ankaflavin are the reduced forms of the orange pigments (rubropunctatin and monascorubrin). Therefore, based on their report, our yellow-pigment producing m u t a n t is thought to lack the capability of catalyzing the Schiff-base reaction involved in the conversion of orange pigments to red ones, but to possess the ability of catalyzing the reduction of orange pigments to yellow ones in a nitrogen-rich medium. Using these three M o n a s c u s strains as models, future studies on the pathway or regulation of enzymes involved in the production of pigments must be undertaken. Moreover, we found that not only did the yellow pigment production increase, but the amylolytic enzyme activity of strain KB20MI0.2 also increased under a neutral pH medium. It should be noted that glucoamylase was produced at a higher concentration than ~-amylase. (~-Amylase from M o n a s c u s p u r p u r e u s had been reported (18) as well as glucoamylase (10, 19, 20). Most of the experimenters conducted the assay for glucoamylase activity by incubating the reaction mixture at 40°C, which is lower than the temperature required to achieve optimal enzyme activity (19, 20). We found that the temperature requred to attain optimal activity of crude or partially purified glucoamylase from strain KB20M10.2 is 65°C at p H 4 . 7 . This thermostable enzyme is capable of digesting cassava starch. On the basis of the results obtained in this paper, the yellow-pigment-producing m u t a n t appeared to have some commercial potential in the utilization of low-cost substrates to produce yellow food colors at high concentration. This m u t a n t is notable in that it is species-specific in the production of yellow pigments exclusively at neutral pH medium and its activity is stable. It seems that c o m p o u n d yellow 1 and 2, the most a b u n d a n t yellow pigments, are monascin and ankaflavin,

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YONGSMITH ET AL.

respectively. H o w e v e r , c h e m i c a l a n d s t r u c t u r a l elucidication as well as i d e n t i f i c a t i o n o f t h e r e m a i n i n g m i n o r f r a c t i o n s are r e q u i r e d to s t u d y . In c o n c l u s i o n , s t r a i n K B 2 0 M 1 0 . 2 was s h o w n to be a p o t e n t yellow p i g m e n t p r o d u c e r in s u b m e r g e d c u l t i v a t i o n using locally a v a i l a b l e s u b s t r a t e s , c a s s a v a s t a r c h a n d soyb e a n flour, w h i c h m i g h t be u s e d in t h e f u t u r e f o r the p r o d u c t i o n o f yellow p i g m e n t s f o r i n d u s t r i a l use. ACKNOWLEDGMENT

J. FERMEN'I. B1OENG., method. J. Biol. Chem., 195, 19 (1952). 9. Yongsmith, B. and Tabloka, W.: Food colors fermentation from cassava by Monascus sp. The Kasetsarl J.: Natural Sci., 19, 45-50 (1985).

10. Yongsmith, B., Tabloka, W., Yongmanitchai, W., Kampee, T., and Suwanna-adth, M.: Cassava as a potential substrate for yellow and red pigments produced by Monascus sp. KBII304. Microbial Utiliz. Renewable Resource, 5, 260267 (1986).

11. Yongsmith, B., Chansiripotha, S., Limtong, S., 'Fantiyapurn, S., and Bavavoda, R.: Mutagenesis of Monascus kaoliang for

This paper is a part of project supported by National Center of Genetic Engineering and Biotechnology.

t2.

REFERENCES

13.

1. Lin, C.F. and Suen, S. J. T.: Isolation of hyperpigment productive mutants of Monascus sp. F-2. J. Ferment. Technol., 51, 757-759 (1973). 2. Lin, C. F. and Iizuka, H.: Production of extracellular pigment by a mutant of Monascus kaoliang sp. nov. Appl. Environ. Microbiol., 43, 671-675 (1982).

14.

15.

3. Yongsmith, B., Tabloka, W., Yongmanitchai, W., and Bavavoda, R.: Culture conditions for yellow pigment formation by Monascus sp. KB10 grown on cassava medium. World J. Microbiol. Biotechnol., 9, 85-90 (1993).

4. Yongsmith, B., Tabloka, W., Bavavoda, R., Vaisiriroj, V., Yoshida, T., and Tanaka, A.: Characterization of cassavautilizing Monascus spp. and their pigments. Microbial Utiliz. Renewable Resources, 6, 340-348 (1988).

5. Yongsmith, B., Bavavoda, R., Tabioka, W., and Vaisiriroj, V.: Production and purification of yellow pigment from Monascus sp. KBI0 grown on cassava medium. Princess Congress. I,

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