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
1
1
2
1,
Tanya V. Rasheva , Trayana S. Nedeva , Jean-Noel Hallet and Anna V. Kujumdzieva *
1

Biological Faculty, Department of General and Industrial Microbiology, The Soa University St. Kliment
Ohridski, 8 Dragan Tsankov St., 1164 Soa, Bulgaria; 2 Universite de Nantes, Institut Universitaire
Professionnel de Chimie Biologie, 2 rue de la Houssiniere, BP92208, F44322 Nantes Cedex 3, France;
* Author for correspondence (e-mail: kujumdzieva@ biofac.uni-soa.bg; phone: 1359 2 6330 255; fax: 1359
2 668619)
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 12 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 X / C 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 17 , C 20 and C 22
fatty acids and did not produce citrinin.
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

Introduction
Monascus fungi have been used for preparation of
oriental fermented foods such as red rice, red soyabean cheese and red rice wine (Su and Huang 1980).
Recently, these molds have begun to be adopted in the
fermentative pigment production for coloring foodstuffs (Miyake et al. 1984; Kujumdzieva et al. 1997).
The genus Monascus has been studied from the
view points of biochemical, physiological and taxonomical aspects (Carels and Shepherd 1977;
Shepherd 1977; Wong and Bau 1977). Regarding the
pigmentation, up to now there is no clear understand-

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).
Using this rational tool, a non-pigment producing
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
Microorganisms and media. A non-pigment producing mutant Monascus purpureus M 12 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 following the procedure of Hawksworth and Pitt (1983)
on solid CYA, MEA and GNA media. Cultivation was
performed in Petri dishes at 25 8C for 7 d. Additionally, cultivation on CYA medium at 4 8C and 37 8C was
carried out.
Determination of colony radial growth rate: colony
radial growth rate (Kr), dened as an increase in the
colony diameter per hour was determined on solid CD
medium with NH 4 Cl 2.5 g / l or NaNO 3 - 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
ability of both strains was studied in submerged
cultures on CD medium with NaNO 3 as a nitrogen
source and different carbon sources (4 % each). The
cultivation was carried out in 500 ml Erlenmeyer
asks 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
et al. (1971), respectively.
Fermentation of carbon sources. The fermentation
capacity of both strains was studied on a medium
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
Durhams tubes at 30 8C for 28 d were carried out.
Residual sugar concentration was determined as mentioned above, and ethanol formation was analyzed
according to the method of Dawes et al. (1971).
Growth yields coefcients. Y x / c and Y eth / c , dened
as biomass / ethanol produced per carbon source utilized (g / g), were calculated according to Pirt (1975).
All data presented are mean values of at least three
individual measurements.
Cell-free extract preparation. Fresh biomass, harvested by ltration, was washed twice with distilled
water and mixed with silica beads and 0.05 M potassium phosphate buffer, pH 7.8 in 1:3:2 ratio. Liquid

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.
Protein assay. Protein content was determined
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 3 - 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
dened as the amount of protein, which caused release of 1 mmol tyrosine per minute.
Glucoamylase activity. Two cultivation media, CD
with 1 % glucose and CD with 1 % soluble starch as
carbon sources, were used. The submerged cultivation
was carried out as mentioned above. Glucoamylase
activity was determined in the culture ltrate 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.
Fatty acid determination. Lipid extraction as well
as determination of fatty acid composition were carried out according to Rasheva et al. (1997).
Assay for monacolins. Submerged cultivation on
CD medium with 3 % glucose and 7 % glycerol as a
carbon source and NaNO 3 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 quantitative analysis was performed on a Waters Millipore
HPLC column, isocratic mode, mBondapak
C 18 039830 (Waters Assoc. USA), mobile phase 51:49
CH 3 CN / ddH 2 O: 0.05 % TFA, detection at 237 nm.
As a standard for monacolin K, lovastatin extracted
from a commercial preparation (Mevinakor, MSD,
Germany) was used. The standard lovastatin showed
RT 24.8 min.
Assay for citrinin. For investigation of the citrinin
content both strains were cultivated on the culture
medium described by Fabre et al. (1993). Cultivation

Exudates

CONIDIA
Shape
Colour
Type of conidia chain
Number of conidia
Size
Ability to form conidia
PERITHECIA
Shape
Colour
Formation capability
Diameter
ASCOSPORE
Shape
Colour
Formation capability
Diameter
COLONY
Diameter
Colour
Shape
Aerial mycelium

characters

and morphological

Cultural

Short, white, rare

ColoUrless

Long, abundant,white
Red

2.5 mm
White
Flat

No growth

WENT CBS 109.07

31.0 mm
Red
Flat sparse

23.0 mm
White
Regular raised
Short, abundant, white

No growth

M 12

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

Globose
No
1111
3035 mm

Globose
No
Straight
1
1011 mm
6

WENT CBS 109.07

4 8C

Oval
No
1111
45 mm

No

Oval
No
Straight
12
67 mm
1111
No

M 12

37 8C

No growth

No growth

M 12

Culture media, 7 d of cultivation

Oval
No
111
45 mm

Globose
No
111
3035 mm

Globose
No
Straight
1
1011 mm
1

WENT CBS 109.07

25 8C

Chapec Yeast Extract Agar

Short, red-orange
Orange

18.0 mm
Orange
lava

Oval
No
1111
45 mm

Globose
No
11111
3035 mm

Globose
No
Straight
1
1011 mm
1

20.0 mm
White
Regular raised

No

Oval
No
Straight
13
68 mm
1111
No

M 12

Short, abundant, white

Colorless

25 8C

Malt Extract Agar

WENT CBS 109.07

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

Short, white, rare

5.5 mm
White
Flat sparse

Oval
No
1
45 mm

Globose
No
1
3035 mm

Oval
No
Straight
12
67 mm
11

WENT CBS 109.07

25 8C

Short, white, rare

6.0 mm
White
Flat folded

No

Oval
No
Straight
12
67 mm
11
No

M 12

Glycerol 25 % Nitrate Agar

335

336
was performed on a rotor shaker at 220 rpm, 30 8C for
7 d. The culture broth (200 300 ml) was freezedried and the resultant powder extracted three times
for 3 h using a magnetic stirrer with 50 ml chloroform
- methanol 1:1. The combined extracts were evaporated to dryness and dissolved in a minimum amount
of chloroform - methanol 1:1. Analysis of citrinin was
performed by TLC under the following conditions:
one dimensional vertical TLC in a chamber without
saturation, stationary phase Kieselgel 60 F 254 HPTLC
plates 10 3 20 cm (Merck), mobile phase: chloroform
- 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 3 reagent (1 % solution of AlCl 3 in 95 % ethanol). The uorescence of citrinin changed from yellow 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.

Results and discussion

Biosynthesis of Monascus pigments is a complex
biochemical process depending upon the physiological 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

of pigment synthesis with other typical features of

Monascus purpureus fungi.
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 morphological characteristics of both strains are given in
Table 1. The white strain differed from the ascosporogenous 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 constant conditions for both strains it could be concluded
indirectly that the specic 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). Determining 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.

Figure 1. Radial growth rate (Kr) of Monascus purpureus Went CBS 109.07 (-d-) and Monascus purpureus M 12 (-q-) on MEPAG medium at
different temperatures.

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
(g / l)

Yx / c

Dry Weight
(g / l)

Yx / c

11.65
6.85
10.2
2.12
12.55
9.05
0.03
2.24
2.36
0.36
0.03
0.01
2.64
1.38
0.95
0.35

0.292
0.179
0.256
0.385
0.318
0.236
0.020
0.326
0.345
0.179
0.043
0
0.261
0.310
0.312
0.265

9.15
7.89
13.1
3.83
13.99
10.77
0.03
0.01
2.42
2.31
0.76
1.81
1.64
2.27
0.36
0.41

0.229
0.206
0.328
0.355
0.354
0.280
0.020
0
0.300
0.242
0.200
0.190
0.184
0.231
0.193
0.222

Glucose
Galactose
Maltose
Rafnose
Sucrose
Xylose
Lactose
Ethanol
Mannitol
Ribitol
Sorbitol
Erythritol
Glycerol
Xylitol
Inositol
Galactitol

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 coefcient (Y x / c ), are presented in
Table 2. The assimilation characteristics did not differ
signicantly in the parent and mutant strains. Both
strains utilize different mono- and disaccharides as
well as some alcohols with similar efciency (Y x / c 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
Monascus fungi utilize very efciently (Blanc et al.
1999). Instead, an increased possibility for assimilation of sorbitol and erytritol appeared.
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-

ciency of the red parent strain was twice as high as

that of the albino one. The assimilation and fermentation 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 explained by the psychrotolerance of the white strain.
The production of exoenzymes, - glucoamylase and
protease (Nishikawa et al. 1988) is also a characteristic trait of Monascus fungi, the latter are used for the
production of these enzymes through cultivation on
different natural substrates (Hesseltine 1983).
The glucoamylase enzyme activity of cell-free
extracts as well as that the extracellular one were
studied. Glucoamylase activity was detected in both
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
Sugar
Glucose
Galactose
Maltose
Sucrose
Lactose

Y eth / c at anaerobic conditions

M. purpureus
M. purpureus
WENT CBS 109.07
M 12
0.319
0.256
0.373
0.030
0.107

0.143
0.158
0.073
0.048
0.078

Y eth / c at static cultivation

M. purpureus
M. purpureus
WENT CBS 109.07
M 12
0.019
0.019
0.0006
0.000
0.019

0.070
0.036
0.035
0.010
0.011

338
Table 4. Endo- and exocellular glucoamylase activity of Monascus purpureus Went CBS 109.07 and Monascus purpureus M 12 , cultivated on
CD 1 glucose (2 d) and CD 1 soluble starch (7 d) media.
Glucoamylase activity (U / mg protein)
CD 1 glucose
Strain

M. purpureus WENT CBS 109.07

M. purpureus M 12

CD 1 soluble starch

Endocellular

Exocellular

2d

2d

2d

5d

7d

2d

5d

7d

0.032
0.015

0.022
0.011

0.050
0.007

0.117
0.007

0.127
0.007

0.100
0.05

1.330
0.070

1.620
0.080

of endocellular glucoamylase activity of the red

strain, cultivated on CD medium with soluble starch,
showed values of about 0.05 0.13 U / mg protein,
while the M 12 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 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.
Results for protease activity of both strains indicated higher activity towards globular (albumin) and
insoluble (gelatin) proteins expressed denitely by the
parent strain (0.03 and 0.08 U / mg protein, respectively), while both strains utilized casein and skim milk
with similar efciency (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
selective pressure of nutritional limitation in natural
ecosystems.

Endocellular

Exocellular

In order to estimate the level of divergence between

both strains, an investigation of their fatty acid composition, 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
colored one (Rasheva et al. 1997). An interesting
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 appearance of these fatty acids in the mutant strain is unclear
and could be due to genetic changes during the
selection procedure.
It is known that pigments synthesized by Monascus
fungi are polyketide derivatives and that their biosynthesis 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 metabolites possess structures close to those of pigments and
probably are functionally connected to the relationships of Monascus fungi with other microora in
ecosystems.
Monacolin production by both strains was studied
during cultivation on CD medium with ammonium or

Table 5. Fatty acid composition of a colony of Monascus purpureus Went CBS 109.07 and Monascus purpureus M 12 , cultivated on MEPAG at
25 8C for 7 d. The amount of the individual fatty acids is shown as a percentage the total one.
Strain
M. purpureus
M 12

M. purpureus
WENT CBS 109.07

Saturated fatty acids

fatty acid
C16
C17
C18
C20
C22
C16
C18

%
22.40
1.25
10.62
9.61
3.20
17.50
7.10

Unsaturated fatty acids

fatty acid

C16:1

6.12

C18:1

46.80

C16:1
C18:1
C18:2

2.42
37.40
27.20

339
Table 6. Citrinin and monacolin synthesis by Monascus purpureus Went CBS 109.07 and Monascus purpureus M 12 .

citrinin, mg / g freeze dried culture broth

monacolin (lovastatin), mg / g dry weight

Monascus purpureus
WENT CBS 109.07

Monascus purpureus
M 12

65 6 0.05
88 6 0.07

not detected*
16.4 6 0002

* No citrinin was detected in the investigated sample. If a recalculation in accordance with the methods 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 both

strains produce one and the same monacolin compounds - 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 strain
preserved the ability for monacolin biosynthesis although it is nearly twice reduced. This could be due to
the low growth rate of the mutant strain.
The other important citrinin, coupled with a pigment biosynthesis metabolite, is identied as a yellow-pigmented substance rst isolated from Penicillium 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 biosynthesis, the albino mutant indicates some important
biochemical differences, which allow some speculations 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 heredity. It is possible to speculate that the mutation, which
took place in the albino Monascus mutant, is a pleotropic one. The change in one property (lack of pigmentation) results in the alteration of several other
important characteristics of Monascus fungi and
causes a reduction of growth rate, metabolic activity
and production of typical secondary metabolites.
These results throw some light on the relationship
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.

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