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Monascus purpureus mutant strain" data-type="pdf-document" id="pdf-document">
Antonie van Leeuwenhoek 83 : 333 – 340 , 2003.  2003 Kluwer Academic Publishers

Antonie van Leeuwenhoek 83: 333340, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands.

333

Characterization of a non-pigment producing Monascus purpureus mutant strain

Tanya V. Rasheva , Trayana S. Nedeva , Jean-Noel Hallet

1

1

2

and Anna V. Kujumdzieva

1, *

1 Biological Faculty, Department of General and Industrial Microbiology, The Sofia University ‘‘ St. Kliment

Ohridski’’, 8 ‘‘ Dragan Tsankov’’ St., 1164 Sofia, Bulgaria;

Professionnel de Chimie Biologie, 2 rue de la Houssiniere, BP92208, F44322 Nantes Cedex 3, France; * Author for correspondence (e-mail: kujumdzieva@biofac.uni-sofia.bg; phone: 1359 2 6330 255; fax: 1359

2 668619)

2 Universite de Nantes, Institut Universitaire

Received 4 September 2001; accepted in revised form 10 October 2002

Key words: Albino strain, Citrinin, Glycoamylase, Monacolin, Monascus, Protease

Abstract

A characterization of a non-pigment producing mutant Monascus purpureus M compared with its parental strain Monascus purpureus Went CBS 109.07 has been performed aiming to investigate the relation between pigment biosynthesis and other characteristics of these fungi. A comparison has been made of morphological features, some physiological properties and biochemical activities of both strains. The albino mutant exhibits an anamorph life cycle, high conidia forming capability, slower radial growth rate and temperature sensitivity. The assimilation capacity of both strains for mono-, disaccharides and some alcohols is in the same range (Y 0.2 – 0.35), while the red strain has a higher fermentation capacity. In a selected albino mutant, the growth rate, metabolic activity and capacity for production of typical for Monascus fungi secondary metabolites were reduced considerably. Hydrolytic activity towards natural substrates expressed through glucoamylase and protease was approximately 10 fold lower in the non pigment producing strain (0.05 – 0.08 U/mg protein and 0.01 – 0.07 U/mg protein respectively) compared with the red one. Important qualitative differences between both strains was found in fatty acid composition and in the production of citrinin and monacolin. The mutant strain possessed C , C and C fatty acids and did not produce citrinin.

12

X/C

17

20

22

Abbreviations: CD – Chapek – Dox, CYA – Chapek – Yeast extract Agar, GNA – 25 % Glycerol Nitrate Agar, Kr – colony radial growth rate, MEA – Malt Extract Agar, MEPAG – Malt Extract - Peptone - Agar - Glucose

ing of the mechanism of biosynthesis and the peculiarities of this phenomenon, although numerous Monascus mutant strains with enhanced ability for pigment production have been isolated and studied (Hiroi et al. 1979; Wong and Koehler 1981; Blanc et al. 1995).

fermentative pigment production for coloring food- Using this rational tool, a non-pigment producing

Introduction

Monascus fungi have been used for preparation of oriental fermented foods such as red rice, red soya- bean cheese and red rice wine (Su and Huang 1980). Recently, these molds have begun to be adopted in the

stuffs (Miyake et al. 1984; Kujumdzieva et al. 1997). The genus Monascus has been studied from the view points of biochemical, physiological and tax- onomical aspects (Carels and Shepherd 1977; Shepherd 1977; Wong and Bau 1977). Regarding the pigmentation, up to now there is no clear understand-

mutant strain from Monascus purpureus CBS 109.07 has been selected and studied. In the present paper some characteristic traits of the strain connected with pigmentation are revealed, and some aspects of the functional rationality of pigment biosynthesis are discussed.

334

Materials and methods nitrogen was added and the resultant suspension was ground in a mortar with a pestle for 15 min at 4 8C.

This procedure was performed three times and the

obtained mixture was centrifuged at 4 500 rpm for 10 min at 4 8C for separation of the beads and cell debris. The supernatant was centrifuged again at 12 500 rpm for 15 min at 4 8C to obtain a clear cell-free extract.

lowing the procedure of Hawksworth and Pitt (1983) Protein assay. Protein content was determined

on solid CYA, MEA and GNA media. Cultivation was performed in Petri dishes at 25 8C for 7 d. Additional- ly, cultivation on CYA medium at 4 8C and 37 8C was carried out. Determination of colony radial growth rate: colony radial growth rate (Kr), defined as an increase in the colony diameter per hour was determined on solid CD medium with NH Cl – 2.5 g/l or NaNO - 3.0 g/l as a nitrogen source. MEPAG medium was used as a control. The cultivation was performed at 30 8C for 7 days. Growth temperature. The growth temperature was determined on MEPAG medium in the range of 4 – 50

8C.

Assimilation of carbon sources. The assimilation Glucoamylase activity. Two cultivation media, CD

with 1 % glucose and CD with 1 % soluble starch as

Microorganisms and media. A non-pigment produc- ing mutant Monascus purpureus M and its wild type parent strain Monascus purpureus Went CBS 109.07 were used in this study. Morphological and cultural characteristics of both strains were determined fol-

12

following the method of Lowry et al. (1951). Bovine serum albumin was used as a standard.

Enzyme activities

Protease activity. Submerged cultures were prepared on CD medium with 1 % casein, skim milk, gelatin or albumin as a sole carbon source and NaNO - 0.3 %. Cultivation was performed on a rotor shaker at 220 rpm, at 28 8C for 7 d. Proteolytic activity of the cell-free extracts was determined according to Yasuda and Soeishi (1984). One unit protease activity was defined as the amount of protein, which caused re- lease of 1 mmol tyrosine per minute.

3

4

3

ability of both strains was studied in submerged cultures on CD medium with NaNO as a nitrogen source and different carbon sources (4 % each). The cultivation was carried out in 500 ml Erlenmeyer flasks with 100 ml medium on a rotor shaker at 220 rpm at 30 8C for 7 d. Dry weight of the biomass was determined as described before (Rasheva et al. 1997). Residual sugar and polyol content was determined

following the methods of Somogyi (1952) and Dawes Fatty acid determination. Lipid extraction as well et al. (1971), respectively. as determination of fatty acid composition were car-

carbon sources, were used. The submerged cultivation was carried out as mentioned above. Glucoamylase activity was determined in the culture filtrate and in the cell-free extract, following the procedure of Yasuda and Kuwae (1989). One unit glucoamylase activity was expressed as the amount of protein causing release of 1 mmol glucose per min.

3

Fermentation of carbon sources. The fermentation

ried out according to Rasheva et al. (1997).

capacity of both strains was studied on a medium Assay for monacolins. Submerged cultivation on

containing (g/l): YNB (Difco) - 6.7, sugar – 10.0, pH 6.0. The anaerobic cultivation in bottles with a stopper and a needle as well as a static cultivation with Durham’s tubes at 30 8C for 28 d were carried out. Residual sugar concentration was determined as men- tioned above, and ethanol formation was analyzed

according to the method of Dawes et al. (1971). HPLC column, isocratic mode, mBondapak

C 039830 (Waters Assoc. USA), mobile phase 51:49 CH CN/ ddH O: 0.05 % TFA, detection at 237 nm. As a standard for monacolin K, lovastatin extracted from a commercial preparation (Mevinakor, MSD,

individual measurements. Germany) was used. The standard lovastatin showed

CD medium with 3 % glucose and 7 % glycerol as a carbon source and NaNO as a nitrogen one was performed on a rotor shaker at 220 rpm, at 30 8C for 7 d. Extraction of monacolins from the biomass was done as described by Endo et al. (1985). The quantita- tive analysis was performed on a Waters Millipore

3

18

3

2

Growth yields coefficients. Y and Y , defined as biomass/ ethanol produced per carbon source uti- lized (g/ g), were calculated according to Pirt (1975). All data presented are mean values of at least three

x/ c

eth/ c

Cell-free extract preparation. Fresh biomass, har-

RT ¯ 24.8 min.

vested by filtration, was washed twice with distilled Assay for citrinin. For investigation of the citrinin

content both strains were cultivated on the culture medium described by Fabre et al. (1993). Cultivation

water and mixed with silica beads and 0.05 M potas- sium phosphate buffer, pH 7.8 in 1:3:2 ratio. Liquid

Short, white, rare –

Flat folded

6–7 mm

6.0 mm

Straight

White

Glycerol 25 % Nitrate AgarChapec

Oval

M 1212121212

1–2

11

No

NoNoASCOSPORE

NoNoPERITHECIA

25 8C

WENT CBS 109.07

Short, white, rare –

30–35 mm

Flat sparse

4–5 mmDiameter

6–7 mm

Globose

5.5 mm

Straight

White

Oval

Oval

1–2

11

No

No

No

1

1

Short, abundant, white Colorless

Regular raised

20.0 mm

6–8 mm

Straight

1111

White

Oval

1–3

No

No

No

M

Malt Extract Agar

25 8C

WENT CBS 109.07

Short, red-orange

30–35 mm

10–11 mm

11111

18.0 mm

4–5 mm

Globose

Globose

Straight

1111

Orange

Orange

Table 1. Morphological and cultural characterization of Monascus purpureus Went CBS 109.07 and Monascus purpureus M 12

Oval

lava

No

No

No

1

1

Culture media, 7 d of cultivation

No growthCOLONY

No growth No growthCONIDIA –

M

WENT CBS 109.07

4 8C

Short, white, rare –

2.5 mm

White

Flat

No growth

M

Yeast Extract AgarCultural

Long, abundant,white

37 8C

WENT CBS 109.07

30–35 mm

10–11 mm

Flat sparse

31.0 mm

4–5 mm

Globose

Globose

Straight

1111

1111

Oval

Red

Red

No

No

No

6

1

23.0 mm White Regular raised Short, abundant, white

ColoUrless

6–7 mm

Straight

1111

Oval

1–2

No

M

25 8C

18.0 mm White, red center Flat Short, abundant, white

WENT CBS 109.07

30–35 mm

10–11 mm

4–5 mm

Globose

Globose

Straight

111

111

Oval

No

No

No

1

1

Shape Colour Type of conidia chain Number of conidia Size Ability to form conidia

Formation capability

Formation capability

Aerial mycelium

and morphological

Diameter

Diameter

Exudates

characters

Colour

Colour

Colour

Shape

Shape

Shape

335

336

was performed on a rotor shaker at 220 rpm, 30 8C for 7 d. The culture broth (200 – 300 ml) was freeze-

dried and the resultant powder extracted three times An albino mutant strain, accumulating high

amounts of lipids, has been selected from the parent, red pigment producing Monascus purpureus Went CBS 109.07 (Rasheva et al. 1997). The main mor- phological characteristics of both strains are given in Table 1. The white strain differed from the ascos- porogenous parent one. It possessed an anamorph life cycle, high conidia forming capability and a very thin aerial mycelim. The radial growth rate (Kr) of the albino mutant during cultivation on CD and MEPAG media was nearly twice lower in comparison with the parent one (0.109 6 0.01 mm/ h and 0.183 6 0.02 mm/ h vs. 0.203 6 0.02 mm/ h, and 0.326 6 0.03 mm/ h). As the results for Kr were obtained in con- stant conditions for both strains it could be concluded indirectly that the specific growth rate (m) of the mutant strain decreased. A similar type of growth rate reduction was found in Neurospora crassa poky and Saccharomyces cerevisiae petit mutants. It could be due to mitochondrial mutation (Ratledge 1982). De- termining the Kr parameter, a growth temperature investigation of both strains was also performed. The obtained Kr values indicated that the albino strain became temperature sensitive; As shown in Figure 1 its minimal growth temperature is 18 8C and the maximal one is 34 8C. Thus the albino mutant, derived from a thermotolerant parent strain (maximal growth temperature 46 8C), became a psychrotolerant one. Since the growth temperature affects m, its reduction in the white strain correlates with the requirement for lower growth temperature.

for 3 h using a magnetic stirrer with 50 ml chloroform

- methanol 1:1. The combined extracts were evapo-

rated to dryness and dissolved in a minimum amount

of chloroform - methanol 1:1. Analysis of citrinin was performed by TLC under the following conditions:

of pigment synthesis with other typical features of Monascus purpureus fungi.

one dimensional vertical TLC in a chamber without saturation, stationary phase Kieselgel 60 F HPTLC plates 10 3 20 cm (Merck), mobile phase: chloroform

254

- ethyl acetate - formic acid 4.5:5.5:1, migration,

distance 8 cm. The plate was separated into 2 halves and equal samples were applied on each side. After development, the mobile phase was removed from the plate in cool air and one half of it was dipped for 3 s in AlCl reagent (1 % solution of AlCl in 95 % etha- nol). The fluorescence of citrinin changed from yel- low to blue after treatment. Commercial pure citrinin obtained from Sigma Chemical Co. was used as reference. The detection limit of the method is 2 ng/ chromatogram zone.

3

3

Results and discussion

Biosynthesis of Monascus pigments is a complex biochemical process depending upon the physiologi- cal conditions (Su and Huang 1980). Selection of albino strains and comparative morphological and biochemical investigations with pigment parent ones could give valuable information about the relationship

ones could give valuable information about the relationship Figure 1. Radial growth rate (Kr) of Monascus

Figure 1. Radial growth rate (Kr) of Monascus purpureus Went CBS 109.07 (-d-) and Monascus purpureus M different temperatures.

12 (-q-) on MEPAG medium at

337

Table 2. Assimilation of carbon sources by Monascus purpureus Went CBS 109.07 and Monascus purpureus M 12

Carbon source

Monascus purpureus WENT CBS 109.07

 

Monascus purpureus

M 12

Dry Weight

Y

x/c

Dry Weight

Y

x/c

(g/l)

(g/l)

Glucose

11.65

0.292

9.15

0.229

Galactose

6.85

0.179

7.89

0.206

Maltose

10.2

0.256

13.1

0.328

Raffinose

2.12

0.385

3.83

0.355

Sucrose

12.55

0.318

13.99

0.354

Xylose

9.05

0.236

10.77

0.280

Lactose

0.03

0.020

0.03

0.020

Ethanol

2.24

0.326

0.01

0

Mannitol

2.36

0.345

2.42

0.300

Ribitol

0.36

0.179

2.31

0.242

Sorbitol

0.03

0.043

0.76

0.200

Erythritol

0.01

0

1.81

0.190

Glycerol

2.64

0.261

1.64

0.184

Xylitol

1.38

0.310

2.27

0.231

Inositol

0.95

0.312

0.36

0.193

Galactitol

0.35

0.265

0.41

0.222

Both strains were compared in their ability to utilize different carbon substrates. The data for the assimilation capacity of the strains, evaluated through the growth yield coefficient (Y ), are presented in Table 2. The assimilation characteristics did not differ significantly in the parent and mutant strains. Both

strains utilize different mono- and disaccharides as The production of exoenzymes, - glucoamylase and

protease (Nishikawa et al. 1988) is also a characteris- tic trait of Monascus fungi, the latter are used for the production of these enzymes through cultivation on different natural substrates (Hesseltine 1983).

Monascus fungi utilize very efficiently (Blanc et al. The glucoamylase enzyme activity of cell-free

1999). Instead, an increased possibility for assimila-

tion of sorbitol and erytritol appeared. studied. Glucoamylase activity was detected in both

Comparative investigation of fermentation showed that both strains could ferment glucose, galactose and maltose in strictly anaerobic conditions, but not in static cultivation where a limited amount of oxygen is available (Table 3). This indicates that the effect of Pasteur takes place. Besides this, the fermentation effi-

extracts as well as that the extracellular one were

well as some alcohols with similar efficiency (Y is about 0.2 – 0.35) and differ only in the assimilation of ethanol, sorbitol and erythritol. The mutant strain has lost the ability to assimilate ethanol, a substrate which

ciency of the red parent strain was twice as high as that of the albino one. The assimilation and fermen- tation characteristics of the red parent strain and the mutant one showed that the metabolic activity of the white strain is lower. This observation can be ex- plained by the psychrotolerance of the white strain.

x/c

x/c

strains cultivated on CD medium with glucose, and it was predominantly endocellular (Table 4). These results show that the glucoamylase enzyme in the studied strains is of a constitutive nature, as it is the case with many other fungi (Yokotsuka 1992); the parent strain possessed higher values. The dynamics

Table 3. Fermentation of carbon sources by Monascus purpureus Went CBS 109.07 and Monascus purpureus M 12

Y

eth/c

at anaerobic conditions M. purpureus

Y M. purpureus WENT CBS 109.07

eth/c

at static cultivation M. purpureus M

Sugar

M. purpureus

WENT CBS 109.07

M

12

12

Glucose

0.319

0.143

0.019

0.070

Galactose

0.256

0.158

0.019

0.036

Maltose

0.373

0.073

0.0006

0.035

Sucrose

0.030

0.048

0.000

0.010

Lactose

0.107

0.078

0.019

0.011

338

Table 4. Endo- and exocellular glucoamylase activity of Monascus purpureus Went CBS 109.07 and Monascus purpureus M , cultivated on CD 1 glucose (2 d) and CD 1 soluble starch (7 d) media.

12

Glucoamylase activity (U/mg protein)

CD 1 glucose

CD 1 soluble starch

Strain

Endocellular

Exocellular

Endocellular

Exocellular

 

2d

2d

2d

5d

7d

2d

5d

7d

M. purpureus WENT CBS 109.07 M. purpureus M

12

0.032

0.022

0.050

0.117

0.127

0.100

1.330

1.620

0.015

0.011

0.007

0.007

0.007

0.05

0.070

0.080

of endocellular glucoamylase activity of the red In order to estimate the level of divergence between

strain, cultivated on CD medium with soluble starch, showed values of about 0.05 – 0.13 U/mg protein, while the M strain exhibited only trace activity under the same conditions. Regarding the exocellular enzyme, it was more than 10 fold higher for M.

purpureus Went CBS 109.07 as compared with M colored one (Rasheva et al. 1997). An interesting

both strains, an investigation of their fatty acid com- position, when cultivated on MEPAG medium, was performed. The results are presented in Table 5. It is known that the amount of saturated fatty acids is considerably higher in the albino-strain than in the

12

12

and increased gradually with cultivation. These data

indicate that the inducer (soluble starch) stimulated an increase of glucoamylase activity in the pigment producing strain. Alternatively, the albino strain showed a strongly restricted ability for exocellular secretion of glucoamylase. Thus, there is a correlation between pigment production and biosynthesis and

excretion of glucoamylase. selection procedure.

Results for protease activity of both strains indi- It is known that pigments synthesized by Monascus

fungi are polyketide derivatives and that their bio- synthesis is generally coupled with production of mixtures of additional compounds – monacolins (K, J, M, L and X) (Endo 1979; Endo et al. 1985) and citrinin (Blanc et al. 1995). These secondary metabo- lites possess structures close to those of pigments and probably are functionally connected to the relation- ships of Monascus fungi with other microflora in ecosystems.

selective pressure of nutritional limitation in natural Monacolin production by both strains was studied ecosystems. during cultivation on CD medium with ammonium or

difference is the presence of three saturated fatty acids - heptadecasanoic (C17), arachidonic (C20) and behenic (C22) in the mutant strain. Nishikawa et al. (1989) reported that these fatty acids are entirely absent or occur rarely in Monascus fungi. The appear- ance of these fatty acids in the mutant strain is unclear and could be due to genetic changes during the

cated higher activity towards globular (albumin) and insoluble (gelatin) proteins expressed definitely by the parent strain (0.03 and 0.08 U/mg protein, respective- ly), while both strains utilized casein and skim milk with similar efficiency (0.06 – 0.09 U/mg protein). Therefore, the considerable reduction of amylase and protease activity in the white mutant indicates that the mutation has changed important properties of the Monascus purpureus fungus selected by the high

Table 5. Fatty acid composition of a colony of Monascus purpureus Went CBS 109.07 and Monascus purpureus M , cultivated on MEPAG at 25 8C for 7 d. The amount of the individual fatty acids is shown as a percentage the total one.

12

Strain

Saturated fatty acids fatty acid

Unsaturated fatty acids fatty acid

 

%

%

M.

purpureus

C16

22.40

C16:1

6.12

 

M

12

C17

1.25

 

C18

10.62

C18:1

46.80

C20

9.61

C22

3.20

M.

purpureus WENT CBS 109.07

C16

17.50

C16:1

2.42

C18

7.10

C18:1

37.40

 

C18:2

27.20

Table 6. Citrinin and monacolin synthesis by Monascus purpureus Went CBS 109.07 and Monascus purpureus M

12 .

339

Monascus purpureus WENT CBS 109.07

Monascus purpureus

M 12

citrinin, mg/ g freeze dried culture broth monacolin (lovastatin), mg/ g dry weight

65

88

6 0.05

6 0.07

not detected* 16.4 6 0–002

* No citrinin was detected in the investigated sample. If a recalculation in accordance with the method’s resolution is made, the amount of citrinin in the albino mutant, if available could be less than 0.1mg/ g freeze dried culture broth.

nitrate as a source of nitrogen. It is shown that

strains produce one and the same monacolin com-

pounds - monacolin K (lovastatin) and probably other substances from the same group (Table 6). These results indicate that biosynthesis of pigments and

monacolins is quite independent and the white

preserved the ability for monacolin biosynthesis al-

though it is nearly twice reduced. This could be due to

the low growth rate of the mutant strain. 1360–1372.

The other important citrinin, coupled with a pig- ment biosynthesis metabolite, is identified as a yel- low-pigmented substance first isolated from Penicil- lium citrinum and Aspergillus terreus (Endo et al. 1976). Thus, experiments for citrinin production of both strains were performed in accordance with Blanc et al. (1995). Our results indicate that the albino mutant strain lacks the ability to produce citrinin (Table 6). Regardless of the prototrophy and monacolin bio- synthesis, the albino mutant indicates some important biochemical differences, which allow some specula- tions on the nature of the mutation that has taken place. Obviously, a change in inheritance has occurred which would be due to mutation in the

gene regulation mechanism or in extrachromosomal heredi- ty. It is possible to speculate that the mutation, which took place in the albino Monascus mutant, is a pleot- ropic one. The change in one property (lack of pig- mentation) results in the alteration of several other important characteristics of Monascus fungi and causes a reduction of growth rate, metabolic activity

both

Pareilleux A. et al. 1995. Characterization of monascidin A from Monascus as citrinin. Int. J. Food Microbiol. 27: 201–213. Blanc P.J., Loret M.O. and Goma G. 1999. Pigments and citrinin production during cultures of Monascus in liquid and solid media. Advance in Solid State Fermentation. Chapter 32 Sec- ondary Metabolites, Aroma, Pigments and Biopesticides, pp.

393–406.

Carels M. and Shepherd D. 1977. The effect of different nitrogen sources on pigment production and sporulation of Monascus species in submerged shaken culture. Can. J. Microbiol. 23:

Dawes E.A., McGill D.J. and Midgley M. 1971. Analysis of fermentation products. In: Norris J.R. and Ribbons D.W. (eds), Methods in Microbiology Vol. 6A. Academic Press, London, New York, pp. 99103. Endo A., Kuroda M. and Tsujita Y. 1976. ML-236A, ML-236B, and ML-236 C, new inhibitors produced by Penicillium citrinum. J. Antibiot. 29: 1346–1348. Endo A. 1979. Monacolin K, a new hypocholesterolemic agent produced by Monascus species. J. Antibiot. 32: 852–854. Endo A., Hasumi K., Nakamura T., Kunishima M. and Masuda M.

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strain

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Nishikawa J., Sato Y., Kashimura J. and Iizuka H. 1989. Cellular fatty acids composition of the genus Monascus. J. Basic Mi- crobiol. 29: 369–374. Nishikawa J., Watanabe Y., Kashimura J., Aso K. and Iizuka H.

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of pigment production with the discussed properties of Monascus fungi and emphasize the possibility of using white strains for revealing the complexity of the process at a genetic level.

These results throw some light on the

relationship

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Wong H.-C. and Bau Y.-S. 1977. Pigmentation and antibacterial activity of fast-neutron and X-ray induced strains of Monascus purpureus Went. Plant Physiol. 60: 578–581. Yasuda M. and Kuwae M. 1989. Purification and properties of two forms of glucoamylase from Monascus purpureus sp. No. 3403 - isolation and characterization. Agric. Biol. Chem. 53: 247–149. Yasuda M. and Soeishi K. 1984. Purification and properties of acid protease from Monascus purpureus no. 3403, isolated from beni koji. Agric. Biol. Chem. 48: 1637–1639. Yokotsuka T. 1992. Non-proteinaceous fermented foods and bever- age, produced with koji molds. In: Arora D.K., Mukerji K.G. and Marth E.H. (eds), Handbook of Applied Mycology Vol. 3. Marcel Dekker Inc., New York, pp. 293–373.