Production of the secondary metabolites γ-aminobutyric acid and monacolin K by Monascus

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O R I G I N A L P A P E R

Yuan-Chi Su Æ Jyh-Jye Wang Æ Tzu-Tsen Lin

Tzu-Ming Pan

Production of the secondary metabolites c-aminobutyric acid

and monacolin K by

Monascus

Received: 14 May 2002 / Accepted: 11 September 2002 Society for Industrial Microbiology 2003

Abstract c-Aminobutyric acid (GABA), a hypotensive

agent, and monacolin K, a cholesterol-lowering drug, can

be produced by Monascus spp. Under optimal culture

conditions, the products of fermentation using Monascus

spp. may serve as a multi-functional dietary supplement

and can prevent heart disease. In this study, Monascus

purpureus

CCRC 31615, the strain with the highest

amount of monacolin K, was identiﬁed from 16 strains

using solid fermentation. Its GABA productivity was

particularly high. Addition of sodium nitrate during

solid-state fermentation of M. purpureus CCRC 31615

improved the productivity of monacolin K and GABA

to 378 mg/kg and 1,267.6 mg/kg, respectively. GABA

productivity increased further to 1,493.6 mg/kg when

dipotassium hydrophosphate was added to the medium.

Keywords Monascus sp. Æ c-Aminobutyric acid Æ

Monacolin K

Introduction

Red mold rice has been used as a part of Chinese food for

thousands of years and has also been considered an

essential part of wine making and other fermented food

products. There are many reports on the medical eﬀects

of red mold rice both as a folk remedy and in scientiﬁc

publications [7, 14, 25]. Red mold rice fermented using

Monascus

spp. is eﬀective in decreasing blood pressure

[20], lowering plasma cholesterol levels [3, 4], and

has antibacterial activity [24]. c-Aminobutyric acid

(GABA) has several physiological functions, such as

neurotransmitting, hypotensive, and diuretic eﬀects [8,

21]. GABA is produced by the decarboxylation of

glu-tamic acid by a glutamate decarboxylase. In the process

of making red mold rice, glutamic acid is produced from

steamed rice by an acid protease and an acid

carbox-ypeptidase that are secreted upon growth of koji mold

[17]. Red mold rice contains a large amount of GABA

and possesses anti-hypertensive eﬀects for humans [10].

GABA productivity, which can be measured, is

con-sidered to have a major eﬀect in lowering blood pressure.

Monacolin K is a secondary metabolite of Monascus

strains. Endo [4] discovered that M. ruber produces an

active methylated form of compactin known as

mon-acolin K (lovastatin; mevinolin) in liquid fermentation.

Monacolin K functions as an inhibitor of

3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is the

regulatory and rate-limiting enzyme of cholesterol

biosynthesis [1]. The fact that red mold rice can suppress

the synthesis of cholesterol has also been conﬁrmed [16].

High blood cholesterol leads to atherosclerosis and is a

causal factor in many types of coronary heart disease, a

leading cause of human death. These ﬁndings have been

widely noted in medical circles [12]. Further research

has established that substances with similar molecular

structures have the same eﬀect [15].

This study focused on screening the pharmacologically

active compounds from a series of Monascus strains with

the aim of identifying a strain with maximum productivity

of both chemicals.

Materials and methods

Microorganism and growth conditions

Screening for GABA and monacolin K production was carried out on 16 species and strains of the genus Monascus, purchased from the Culture Collection and Research Center (CCRC) and kept in our laboratory (M. ruber CCRC 31538, M. purpureus CCRC 31497, 31498, 31499, 31501, 31504, 31530, 31540, 31542, 31615, 32966, Monascussp. CCRC 32807, 32808, 32809 and other M. anka M-13, Monascussp. S2). The cultures were maintained on potato dextrose agar (PDA) slants at 10C, and transferred monthly.

DOI 10.1007/s10295-002-0001-5

Y.-C. Su Æ J.-J. Wang Æ T.-T. Lin Æ T.-M. Pan (&) Department of Agricultural Chemistry,

National Taiwan University, 1, Sec. 4, Roosevelt Road, Taipei, Taiwan, ROC E-mail: [email protected]

Tel.: +886-2-23630231 ext 3813; Fax: +886-2-23627044 J.-J. Wang

Department of Industrial Safety and Hygiene, Tajen Institute of Technology, Pingdon, ROC

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Chemicals

GABA, monacolin K and citrinin were purchased from Sigma (St. Louis, Mo.). LC grade acetonitride was purchased from Merck (Darmstadt, Germany). Tryptone, yeast extract, peptone, malt extract, PDA broth and Bacto-agar were purchased from Difco (Detroit, Mich.). Reagent grade ethyl acetate was purchased from ALPS (Taiwan).

Seed cultures

Seed cultures were prepared by transferring a loopful of spores from a PDA agar slant into a 500 ml Hinton ﬂask containing 100 ml basal medium (100 g dextrose, 10 g peptone, 2 g KNO3, 2 g NH4H2PO4, 0.5 g MgSO4Æ7H2O, 0.1 g CaCl2in 1,000 ml distilled water; pH adjusted to 6.0 [9, 19]). Cultures were incubated at 30C for 48 h at 110 rpm. A 5% inoculum was then transferred to submerged or solid-state fermentation medium.

Submerged culture for GABA and monacolin K production Submerged culture GABA production was carried out in 500 ml Hinton flasks containing 100 ml medium (20 g rice powder, 40 g glucose, 10 g monosodium L-glutamate (MSG), 10 g peptone in 1,000 ml distilled water; pH 5.0) [22]. The flasks were incubated at different temperatures (25C, 30C, 37C) on a rotary shaker at 110 rpm for 14 days. The GABA concentration was measured at intervals. Submerged culture monacolin K production was carried out in 500 ml Hinton flasks containing 120 ml medium (60 g glu-cose, 25 g peptone, 5 g corn steep liquor, 5 g ammonium chloride in 1,000 ml distilled water) [3]. The flasks were incubated at 30C for 14 days on a rotary shaker at a speed of 110 rpm. The mon-acolin K concentration was measured at intervals.

Solid-state culture for GABA and monacolin K production Long-grain rice was purchased from a local supermarket and was used as the substrate for monacolin K and GABA production under solid-state cultivation. Except for the parameter under investigation, rice substrate was prepared as follows: 500 g rice was soaked in distilled water for 8 h. Water was then removed using a sieve. The soaked rice was autoclaved for 20 min at 121C in a ‘‘koji-dish’’ (the koji-dish is made of wood, dimensions 30·20·5 cm). After being cooled, the rice substrate was inoculated with a 5% spore suspension of a Monascus sp. and the inoculated substrate was cultivated at 30C for 14 days. After cultivation, monacolin K and GABA concentrations were measured [19]. Eﬀect of medium components on GABA

and monacolin K production

The eﬀect of the nitrogen source (peptone, ammonium sulfate, sodium nitrate, MSG, or yeast extract) and inorganic salts (po-tassium dihydrogen phosphate, magnesium sulfate, calcium chlor-ide, manganese sulfate) on GABA and monacolin K production in solid-state culture was analyzed. After the rice was sterilized and cooled, various nitrogen sources (1%) and inorganic salts (0.1%) were added individually to determine their eﬀect on GABA and monacolin K production.

Determination of the monacolin K concentration

Methods for determination of the monacolin K concentration under diﬀerent conditions were according to Wang et al. [23]. Submerged culture

Fermented broth (5 ml) from the ﬂasks was homogenized and adjusted to pH 3.0 with 2 N H3PO4and then extracted with 5 ml

ethyl acetate. The extract was centrifuged at 3,000 g for 8 min. The supernatant (1 ml) was mixed with triﬂuoroacetic acid (10 ml, 1%) for lactonization of the monacolin K. The resulting mixture was then concentrated to dryness under reduced pressure. The residue was resuspended in 1 ml acetonitrile for HPLC analysis.

Solid-state culture

Mature red mold rice (1 g) was suspended in 5 ml ethyl acetate at 70C for 1.5 h. The suspension was then filtered through filter paper. The filtrate was evaporated under vacuum. After lactonization, the resulting mixture was added to 1 ml acetonitrile and subjected to preparatory HPLC analysis.

Chromatographic conditions

Chromatographic separation was achieved on a Beckman Ultrasphere ODS column (150·4.6 mm ID). Acetonitrile:water (72:28, v/v) was used as the mobile phase. The eluent was pumped at a ﬂow-rate of 0.5 ml/min. The UV detector was at 238 nm.

Determination of the GABA concentration

GABA concentrations were determined as in [11]. Mature red mold rice (5 g) was soaked in 40 ml 60% ethanol solution at 25C for 24 h. After ﬁltration (0.45 lm), the ﬁltrate was analyzed using a Beckman 6300 amino acid analyzer (column: spherical cation-ex-change resin 10 cm, Lithium column PN 338051).

Qualitative analysis of citrinin using thin-layer chromatography Thin layer chromatography (TLC) of citrinin was performed according to [2]. Red mold rice was extracted with acetonitrile. The ﬁltered extract was twice defatted using isooctane. After adding an equal amount of water and acidifying to pH 4.5 with H2SO4(50:50, v/v), the extract was partitioned with CHCl3. The lower phase was evaporated to dryness, and then dissolved in methanol before analyzing by TLC. TLC was performed on 10·20 cm aluminum sheets pre-coated with silica gel 60. Prior to analysis, the plates were impregnated with oxalic acid (10% in methanol). Citrinin and samples were spotted on silica gel plates. Chloroform-acetone (90:10, v/v) followed by toluene-ethyl acetate-formic acid (60:30:10, v/v/v) were used to conduct two-dimensional analysis.

Results

Screening of strains

By using submerged culture or solid culture on the sixteen

strains it was possible to examine initially the productivity

of GABA and monacolin K (Figs. 1, 2). More GABA was

produced in solid culture than in submerged culture

(Fig. 1), with a maximum production of 1,031.13 mg/kg

(M. purpureus CCRC 31540). Figure 2 shows that, except

for strains of M. purpureus CCRC 31499, M. purpureus

CCRC 31615, and M. anka M-13, the productivity of

monacolin K was not very high (<100 mg/kg) for the

remaining 13 strains, regardless of whether they were

cultivated in solid state or submerged culture. The highest

productivity of monacolin K in the 16 strains was M.

purpureus

CCRC 31615 strain, with a yield of 237 mg/kg.

Monacolin K productivity was not as high as that of

GABA when these 16 Monascus strains were used

(Figs. 1, 2). GABA was produced in both solid state and

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submerged cultures. However, diﬀerent strains resulted in

varying monacolin K productivity (Fig. 2). Solid state

cultivation always produced more monacolin K and

GABA than submerged cultivation, perhaps because

monacolin K and GABA were more stable and were easily

released from rice grains under conditions of solid state

cultivation, while submerged cultivation resulted in

accumulation of both monacolin K and GABA on the

mycelium. The yield of Monascus pigment from solid

cultivation was higher than submerged cultivation as

pointed out by Johns and Stuart [6]. Although both

Monascus

pigment and monacolin K are polyketides, it

has yet to be determined whether monacolin K will have

the same eﬀect as Monascus pigment.

Since M. purpureus CCRC 31615 produced the highest

amount of monacolin K and a signiﬁcant amount of

GABA, M. purpureus CCRC 31615 was selected as the

target strain for further study. Furthermore, M. purpureus

CCRC 31615 accumulated ten amino acids in solid-state

cultures (Table 1). The relative amounts of amino acids

produced were in the order: Ala, Glu, GABA, Gln, Asp,

Ser, Cys, Aln, Orn, Lys.

The solid state medium used in this study was rice.

Rice contains suﬃcient carbon sources, therefore further

optimization eﬀorts focused on various nitrogen sources

and inorganic salts.

Eﬀect of inoculum size on the productivity of

GABA and monacolin K

Inocula (20, 25, and 30 ml) were administered to the

prepared rice (500 g). After 10 days at 30C, the

pro-ductivity of GABA and monacolin K were measured

and are shown in Fig. 3. Inoculum size had little eﬀect

Fig. 1 c-Aminobutyric acid (GABA) concentration in solid (closed squares) and submerged (open squares) cultures. The values represent the mean ± SD (n=3)

Fig. 2 Monacolin K concentration in solid (closed squares) and submerged (open squares) cultures. The values represent the mean ± SD (n=3)

Table 1 Production of amino acids by Monascus purpureus CCRC 31615. GABA c-Aminobutyric acid

Amino acid Composition (%) Production (lmol/g)

Asp 6.81 4.45 Ser 5.00 2.31 Glu 15.50 10.53 Gln 13.01 11.03 Aln 3.59 1.29 Ala 16.12 7.57 Cys 3.93 2.26 GABA 14.36 4.70 Orn 3.53 1.23 Lys 2.45 1.13 Other 15.70 –

Fig. 3 Eﬀect of inoculum size on the production of GABA and monacolin K by Monascus purpureus CCRC 31615. Medium: rice, culture conditions: 30C, inoculum size: 20 ml (closed circles); 25 ml (open circles); 30 ml (closed triangles). The values represent the mean ± SD (n=3)

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on GABA productivity. When a 25 ml (5%) inoculum

was used, the yield of GABA reached 965.71 mg/kg. In

contrast, inoculum size had a signiﬁcant eﬀect on the

productivity of monacolin K. Inoculation with 25 ml

resulted in a signiﬁcant increase in monacolin K

pro-ductivity with more than 280 mg/kg produced.

Eﬀect of temperature on GABA

and monacolin K productivity

Because M. purpureus CCRC 31615 performed poorly at

25C (Fig. 4), GABA production was low at that

tem-perature. However, when the temperature reached 30C,

the yield of GABA jumped to 961.35 mg/kg. In contrast,

at 37C for 150 h, the yield of GABA decreased, perhaps

because GABA decomposed quickly above 30C.

Furthermore, a higher yield of monacolin K was achieved

at 30C (Fig. 4). Consequently, 30C is the optimal

temperature for M. purpureus CCRC 31615 strain to

produce GABA or monacolin K.

Eﬀect of nitrogen source on GABA

and monacolin K productivity

The eﬀects on GABA and monacolin K productivity of

adding various nitrogen sources (to 1%) are shown in

Fig. 5. Ammonium sulfate did not contribute to GABA

production. Rather, the pH value was lower than 3.0

throughout the entire process, and only yellow and

orange pigments were produced.

Other nitrogen sources, like peptone, sodium nitrate,

MSG, and yeast extract, contributed to the production of

GABA, which reached about 1,000 mg/kg with a yield of

1,267.6 mg/kg when sodium nitrate was added. On the

other hand, adding MSG reduced the yield of monacolin

K to zero, whereas adding ammonium sulfate had no

eﬀect on monacolin K yield. Sodium nitrate was the

optimum nitrogen source for M. purpureus CCRC 31615

as it resulted in the highest productivity for both

monacolin K and GABA.

Eﬀect of inorganic salts on GABA and monacolin K

productivity

The addition of 0.1% of various inorganic salts to the

basic medium positively aﬀected GABA and monacolin

K productivity (Fig. 6).

To produce GABA only, the addition of the

in-organic salts K

2

HPO

4

, CaCl

2

Æ2H

2

O and MnSO

4

Æ4H

2

O

could be helpful. When NaNO

3

and MnSO

4

Æ4H

2

O were

Fig. 4 Eﬀect of temperature on the production of GABA and monacolin K by M. purpureus CCRC 31615 Medium: rice, inoculum size: 25 ml/500 g, 25C (closed circles); 30C (open circles); 37C (closed triangles); control (open triangles). The values represent the mean ± SD (n=3)

Fig. 5 Eﬀect of various nitrogen sources on the production of GABA and monacolin K by M. purpureus CCRC 31615 Medium: rice, culture conditions: 30C, nitrogen source concentration: 1% peptone (closed circles); (NH4)2SO4(open circles); NaNO3(closed triangles); yeast extract (open triangles); monosodiumL-glutamate (MSG) (closed squares); control (open squares). The values represent the mean ± SD (n=3)

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both added to the basic medium (Fig. 7), GABA

pro-ductivity reached 1,396.04 mg/kg, while monacolin K

productivity was still very low.

Detection of citrinin during solid

and submerged culture

TLC analysis showed that no citrinin was detected in

either solid or submerged culture (data not shown).

Discussion

Analysis of the 16 strains in submerged culture and solid

culture for both GABA and monacolin K productivity

resulted in the choice of solid culture over submerged

culture. M. purpureus CCRC 31615 was selected as the

best strain for further study because it produced the

highest amount of both GABA and monacolin K in solid

culture. GABA production reached 1,396.04 mg/kg and

monacolin K production reached 26.77 mg/kg when the

basic medium was supplemented with both NaNO

3

and

MnSO

4

Æ4H

2

O. Citrinin, a nephrotoxic agent, is produced by

M. purpureus

or M. ruber in both submerged and

solid-state cultures. According to Blanc et al. [2, 5], there is

some risk of citrinin contamination in the fermentation

process. However, it could be avoided either by

detox-iﬁcation of the red mold rice, use of a species that does

not produce citrinin or by adjusting the fermentation

conditions for citrinin-free production. Nevertheless, no

citrinin was detected in this study, perhaps because the

citrinin concentration was below the level of detection

by TLC. Further analysis using HPLC with

UV/ﬂuor-escence detection or enzyme immunoassays would

clar-ify the existence of citrinin [18].

In this study, a maximum amount of 378 mg/kg of

monacolin K was produced. Although this is less than

reported in other studies [13], this study employed a

diﬀerent approach in that M. purpureus combined with

solid state cultivation to seek optimum conditions for

producing maximum amounts of both monacolin K and

GABA. Although the proposed method did not yield

maximum productivity of monacolin K, it resulted in a

signiﬁcant amount of GABA. Furthermore, the purpose

of this study was to provide a means to produce food

products by direct fermentation on a nutraceutical

containing both active ingredients rather than the

production of the individual medicinal components. In

short, this study lays an emphasis on a dietary product

that could be directly consumed.

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Fig. 6 Eﬀect of inorganic salts on the production of GABA and monacolin K by M. purpureus CCRC 31615. Medium: rice, culture conditions: 30C, mineral concentration: 0.1% KH2PO4 (closed circles); MgSO4Æ7H2O (open circles); CaCl2Æ7H2O (closed triangles); MnSO4Æ4H2O (open triangles; control (closed squares). The values represent the mean ± SD (n=3)

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