Antonie van Leeuwenhoek 83: 333340, 2003. 333 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Characterization of a non-pigment producing Monascus purpureusmutant strain

1 1 2 1,*Tanya V. Rasheva , Trayana S. Nedeva , Jean-Noel Hallet and Anna V. Kujumdzieva1Biological Faculty, Department of General and Industrial Microbiology, The Soa University St. Kliment

2Ohridski, 8 Dragan Tsankov St., 1164 Soa, Bulgaria; Universite de Nantes, Institut UniversitaireProfessionnel 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: 13592 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 mutantMonascus purpureus M compared with its parental strain12Monascus purpureus Went CBS 109.07 has been performed aiming to investigate the relation between pigmentbiosynthesis 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 anamorphlife cycle, high conidia forming capability, slower radial growth rate and temperature sensitivity. The assimilationcapacity of both strains for mono-, disaccharides and some alcohols is in the same range (Y 0.2 0.35), whileX /Cthe red strain has a higher fermentation capacity. In a selected albino mutant, the growth rate, metabolic activityand 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 10fold lower in the non pigment producing strain (0.05 0.08 U/mg protein and 0.01 0.07 U/mg proteinrespectively) compared with the red one. Important qualitative differences between both strains was found in fattyacid composition and in the production of citrinin and monacolin. The mutant strain possessed C , C and C17 20 22fatty 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 ing of the mechanism of biosynthesis and thepeculiarities of this phenomenon, although numerous

Monascus fungi have been used for preparation of Monascus mutant strains with enhanced ability fororiental fermented foods such as red rice, red soya- pigment production have been isolated and studiedbean cheese and red rice wine (Su and Huang 1980). (Hiroi et al. 1979; Wong and Koehler 1981; Blanc etRecently, these molds have begun to be adopted in the al. 1995).fermentative pigment production for coloring food- Using this rational tool, a non-pigment producingstuffs (Miyake et al. 1984; Kujumdzieva et al. 1997). mutant strain from Monascus purpureus CBS 109.07The genus Monascus has been studied from the has been selected and studied. In the present paper

view points of biochemical, physiological and tax- some characteristic traits of the strain connected withonomical aspects (Carels and Shepherd 1977; pigmentation are revealed, and some aspects of theShepherd 1977; Wong and Bau 1977). Regarding the functional rationality of pigment biosynthesis arepigmentation, up to now there is no clear understand- discussed.

334

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

Microorganisms and media. A non-pigment produc- This procedure was performed three times and theing mutantMonascus purpureusM and its wild type obtained mixture was centrifuged at 4 500 rpm for 1012parent strain Monascus purpureus Went CBS 109.07 min at 4 8C for separation of the beads and cell debris.were used in this study. Morphological and cultural The supernatant was centrifuged again at 12 500 rpmcharacteristics of both strains were determined fol- 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 determinedon solid CYA, MEA and GNA media. Cultivation was following the method of Lowry et al. (1951). Bovineperformed in Petri dishes at 25 8C for 7 d. Additional- serum albumin was used as a standard.ly, cultivation on CYA medium at 4 8C and 37 8C wascarried out. Enzyme activitiesDetermination of colony radial growth rate: colony

radial growth rate (Kr), dened as an increase in the Protease activity. Submerged cultures were preparedcolony diameter per hour was determined on solid CD on CD medium with 1 % casein, skim milk, gelatin ormedium with NH Cl 2.5 g/ l or NaNO - 3.0 g/ l as albumin as a sole carbon source and NaNO - 0.3 %.4 3 3a nitrogen source. MEPAG medium was used as a Cultivation was performed on a rotor shaker at 220control. The cultivation was performed at 30 8C for 7 rpm, at 28 8C for 7 d. Proteolytic activity of thedays. cell-free extracts was determined according to YasudaGrowth temperature. The growth temperature was and Soeishi (1984). One unit protease activity was

determined on MEPAG medium in the range of 4 50 dened as the amount of protein, which caused re-8C. lease of 1 mmol tyrosine per minute.

Assimilation of carbon sources. The assimilation Glucoamylase activity. Two cultivation media, CDability of both strains was studied in submerged with 1 % glucose and CD with 1 % soluble starch ascultures on CD medium with NaNO as a nitrogen carbon sources, were used. The submerged cultivation3source and different carbon sources (4 % each). The was carried out as mentioned above. Glucoamylasecultivation was carried out in 500 ml Erlenmeyer activity was determined in the culture ltrate and inasks with 100 ml medium on a rotor shaker at 220 the cell-free extract, following the procedure ofrpm at 30 8C for 7 d. Dry weight of the biomass was Yasuda and Kuwae (1989). One unit glucoamylasedetermined as described before (Rasheva et al. 1997). activity was expressed as the amount of proteinResidual sugar and polyol content was determined causing release of 1 mmol glucose per min.following the methods of Somogyi (1952) and Dawes Fatty acid determination. Lipid extraction as wellet al. (1971), respectively. as determination of fatty acid composition were car-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 oncontaining (g / l): YNB (Difco) - 6.7, sugar 10.0, pH CD medium with 3 % glucose and 7 % glycerol as a6.0. The anaerobic cultivation in bottles with a stopper carbon source and NaNO as a nitrogen one was3and a needle as well as a static cultivation with performed on a rotor shaker at 220 rpm, at 30 8C for 7Durhams tubes at 30 8C for 28 d were carried out. d. Extraction of monacolins from the biomass wasResidual sugar concentration was determined as men- done as described by Endo et al. (1985). The quantita-tioned above, and ethanol formation was analyzed tive analysis was performed on a Waters Milliporeaccording to the method of Dawes et al. (1971). HPLC column, isocratic mode, mBondapakGrowth yields coefcients. Y and Y , dened C 039830 (Waters Assoc. USA), mobile phase 51:49x / c eth / c 18

as biomass /ethanol produced per carbon source uti- CH CN/ddH O: 0.05 % TFA, detection at 237 nm.3 2lized (g /g), were calculated according to Pirt (1975). As a standard for monacolin K, lovastatin extractedAll data presented are mean values of at least three from a commercial preparation (Mevinakor, MSD,individual measurements. Germany) was used. The standard lovastatin showedCell-free extract preparation. Fresh biomass, har- RT 24.8 min.

vested by ltration, was washed twice with distilled Assay for citrinin. For investigation of the citrininwater and mixed with silica beads and 0.05 M potas- content both strains were cultivated on the culturesium phosphate buffer, pH 7.8 in 1:3:2 ratio. Liquid medium described by Fabre et al. (1993). Cultivation

335

Table1.

Morphological

andcultu

ralc

haracterizationofMonascuspurpureusWentCB

S109.07

andMonascuspurpureusM

12

Cultu

remedia,

7dof

cultivatio

n

Cultu

ralCh

apec

YeastE

xtract

Agar

Malt

Extra

ctAg

arGlycero

l25%

Nitra

teAg

ar

andmorpholo

gical

258C

378C

48C

258C

258C

chara

cters

WEN

TCB

S109.0

7M

WEN

TCB

S109.0

7M

WEN

TCB

S109.07

MWEN

TCB

S109.07

MWEN

TCB

S109.07

M12

1212

1212

CONI

DIA

Nogrow

thNo

grow

thSh

ape

Globose

Oval

Globose

Globose

Oval

Oval

Oval

Colou

rNo

NoNo

No

NoNo

NoTy

peof

conid

iachain

Strai

ght

Strai

ght

Strai

ght

Strai

ght

Strai

ght

Strai

ght

Strai

ght

Numb

erof

conid

ia1

12

1

11

31

21

2Size

1011mm

67mm

101

1mm

10

11mm

68mm

67mm

67mm

Abilitytoform

conid

ia1

1111

6

11111

11

11

PERITH

ECIA

NoNo

NoSh

ape

Globose

Globose

Globose

Globose

Colou

rNo

No

NoNo

Form

ation

capabil

ity111

1111

11111

1

Diam

eter

3035mm

303

5mm

30

35mm

303

5mm

ASCO

SPOR

ENo

NoNo

Shape

Oval

Oval

Ov

alOv

alCo

lour

NoNo

No

NoFo

rmation

capabil

ity111

1111

1111

1

Diam

eter

45mm

45mm

4

5mm

45mm

COLO

NYNo

grow

thNo

grow

thDiam

eter

18.0

mm

23.0

mm

31.0

mm

2.5mm

18.0

mm

20.0

mm

5.5mm

6.0mm

Colou

rWhit

e,redcenter

Whit

eRe

dWhit

eOr

ange

Whit

eWhit

eWhit

eSh

ape

Flat

Regular

raise

dFlat

spars

eFlat

lava

Regular

raise

dFlat

spars

eFlat

folde

dAe

rialm

ycelium

Short,a

bundant,w

hite

Short,a

bundant,w

hite

Long,abundant,w

hite

Short,whit

e,rare

Short,red-orange

Short,a

bundant,w

hite

Short,whit

e,rare

Short,w

hite,rare

Exudate

s

ColoU

rless

Red

Or

ange

Color

less

336

was performed on a rotor shaker at 220 rpm, 30 8C for of pigment synthesis with other typical features of7 d. The culture broth (200 300 ml) was freeze- Monascus purpureus fungi.dried and the resultant powder extracted three times An albino mutant strain, accumulating highfor 3 h using a magnetic stirrer with 50 ml chloroform amounts of lipids, has been selected from the parent,- methanol 1:1. The combined extracts were evapo- red pigment producing Monascus purpureus Wentrated to dryness and dissolved in a minimum amount CBS 109.07 (Rasheva et al. 1997). The main mor-of chloroform - methanol 1:1. Analysis of citrinin was phological characteristics of both strains are given inperformed by TLC under the following conditions: Table 1. The white strain differed from the ascos-one dimensional vertical TLC in a chamber without porogenous parent one. It possessed an anamorph lifesaturation, stationary phase Kieselgel 60 F HPTLC cycle, high conidia forming capability and a very thin254plates 103 20 cm (Merck), mobile phase: chloroform aerial mycelim. The radial growth rate (Kr) of the- ethyl acetate - formic acid 4.5:5.5:1, migration, albino mutant during cultivation on CD and MEPAGdistance 8 cm. The plate was separated into 2 halves media was nearly twice lower in comparison with theand equal samples were applied on each side. After parent one (0.109 6 0.01 mm/h and 0.183 6 0.02development, the mobile phase was removed from the mm/h vs. 0.203 6 0.02 mm/h, and 0.326 6 0.03plate in cool air and one half of it was dipped for 3 s in mm/h). As the results for Kr were obtained in con-AlCl reagent (1 % solution of AlCl in 95 % etha- stant conditions for both strains it could be concluded3 3nol). The uorescence of citrinin changed from yel- indirectly that the specic growth rate (m) of thelow to blue after treatment. Commercial pure citrinin mutant strain decreased. A similar type of growth rateobtained from Sigma Chemical Co. was used as reduction was found in Neurospora crassa poky andreference. The detection limit of the method is 2 Saccharomyces cerevisiae petit mutants. It could beng/chromatogram zone. due to mitochondrial mutation (Ratledge 1982). De-

termining the Kr parameter, a growth temperatureinvestigation of both strains was also performed. Theobtained Kr values indicated that the albino strain

Results and discussion became temperature sensitive; As shown in Figure 1its minimal growth temperature is 18 8C and the

Biosynthesis of Monascus pigments is a complex maximal one is 34 8C. Thus the albino mutant, derivedbiochemical process depending upon the physiologi- from a thermotolerant parent strain (maximal growthcal conditions (Su and Huang 1980). Selection of temperature 46 8C), became a psychrotolerant one.albino strains and comparative morphological and Since the growth temperature affects m, its reductionbiochemical investigations with pigment parent ones in the white strain correlates with the requirement forcould give valuable information about the relationship lower growth temperature.

Figure 1. Radial growth rate (Kr) of Monascus purpureusWent CBS 109.07 (-d-) and Monascus purpureus M (-q-) on MEPAG medium at12different temperatures.

337

Table 2. Assimilation of carbon sources by Monascus purpureus Went CBS 109.07 and Monascus purpureus M12Carbon source Monascus purpureus Monascus purpureus

WENT CBS 109.07 M12DryWeight Y DryWeight Yx / c x / c

(g / l) (g / l)Glucose 11.65 0.292 9.15 0.229Galactose 6.85 0.179 7.89 0.206Maltose 10.2 0.256 13.1 0.328Rafnose 2.12 0.385 3.83 0.355Sucrose 12.55 0.318 13.99 0.354Xylose 9.05 0.236 10.77 0.280Lactose 0.03 0.020 0.03 0.020Ethanol 2.24 0.326 0.01 0Mannitol 2.36 0.345 2.42 0.300Ribitol 0.36 0.179 2.31 0.242Sorbitol 0.03 0.043 0.76 0.200Erythritol 0.01 0 1.81 0.190Glycerol 2.64 0.261 1.64 0.184Xylitol 1.38 0.310 2.27 0.231Inositol 0.95 0.312 0.36 0.193Galactitol 0.35 0.265 0.41 0.222

Both strains were compared in their ability to ciency of the red parent strain was twice as high asutilize different carbon substrates. The data for the that of the albino one. The assimilation and fermen-assimilation capacity of the strains, evaluated through tation characteristics of the red parent strain and thethe growth yield coefcient (Y ), are presented in mutant one showed that the metabolic activity of thex / cTable 2. The assimilation characteristics did not differ white strain is lower. This observation can be ex-signicantly in the parent and mutant strains. Both plained by the psychrotolerance of the white strain.strains utilize different mono- and disaccharides as The production of exoenzymes, - glucoamylase andwell as some alcohols with similar efciency (Y is protease (Nishikawa et al. 1988) is also a characteris-x / cabout 0.2 0.35) and differ only in the assimilation of tic trait of Monascus fungi, the latter are used for theethanol, sorbitol and erythritol. The mutant strain has production of these enzymes through cultivation onlost the ability to assimilate ethanol, a substrate which different natural substrates (Hesseltine 1983).Monascus fungi utilize very efciently (Blanc et al. The glucoamylase enzyme activity of cell-free1999). Instead, an increased possibility for assimila- extracts as well as that the extracellular one weretion of sorbitol and erytritol appeared. studied. Glucoamylase activity was detected in bothComparative investigation of fermentation showed strains cultivated on CD medium with glucose, and it

that both strains could ferment glucose, galactose and was predominantly endocellular (Table 4). Thesemaltose in strictly anaerobic conditions, but not in results show that the glucoamylase enzyme in thestatic cultivation where a limited amount of oxygen is studied strains is of a constitutive nature, as it is theavailable (Table 3). This indicates that the effect of case with many other fungi (Yokotsuka 1992); thePasteur takes place. Besides this, the fermentation effi- parent strain possessed higher values. The dynamics

Table 3. Fermentation of carbon sources by Monascus purpureus Went CBS 109.07 and Monascus purpureus M12Y at anaerobic conditions Y at static cultivationeth / c eth / c

Sugar M. purpureus M. purpureus M. purpureus M. purpureusWENT CBS 109.07 M WENT CBS 109.07 M12 12

Glucose 0.319 0.143 0.019 0.070Galactose 0.256 0.158 0.019 0.036Maltose 0.373 0.073 0.0006 0.035Sucrose 0.030 0.048 0.000 0.010Lactose 0.107 0.078 0.019 0.011

338

Table 4. Endo- and exocellular glucoamylase activity of Monascus purpureusWent CBS 109.07 and Monascus purpureus M , cultivated on12CD 1 glucose (2 d) and CD 1 soluble starch (7 d) media.

Glucoamylase activity (U/mg protein)CD 1 glucose CD 1 soluble starch

Strain Endocellular Exocellular Endocellular Exocellular

2 d 2 d 2 d 5 d 7 d 2 d 5 d 7 d

M. purpureusWENT CBS 109.07 0.032 0.022 0.050 0.117 0.127 0.100 1.330 1.620M. purpureus M 0.015 0.011 0.007 0.007 0.007 0.05 0.070 0.08012

of endocellular glucoamylase activity of the red In order to estimate the level of divergence betweenstrain, cultivated on CD medium with soluble starch, both strains, an investigation of their fatty acid com-showed values of about 0.05 0.13 U/mg protein, position, when cultivated on MEPAG medium, waswhile the M strain exhibited only trace activity performed. The results are presented in Table 5. It is12under the same conditions. Regarding the exocellular known that the amount of saturated fatty acids isenzyme, it was more than 10 fold higher for M. considerably higher in the albino-strain than in thepurpureus Went CBS 109.07 as compared with M colored one (Rasheva et al. 1997). An interesting12and increased gradually with cultivation. These data difference is the presence of three saturated fatty acidsindicate that the inducer (soluble starch) stimulated an - heptadecasanoic (C17), arachidonic (C20) andincrease of glucoamylase activity in the pigment behenic (C22) in the mutant strain. Nishikawa et al.producing strain. Alternatively, the albino strain (1989) reported that these fatty acids are entirelyshowed a strongly restricted ability for exocellular absent or occur rarely inMonascus fungi. The appear-secretion of glucoamylase. Thus, there is a correlation ance of these fatty acids in the mutant strain is unclearbetween pigment production and biosynthesis and and could be due to genetic changes during theexcretion of glucoamylase. selection procedure.Results for protease activity of both strains indi- It is known that pigments synthesized byMonascus

cated higher activity towards globular (albumin) and fungi are polyketide derivatives and that their bio-insoluble (gelatin) proteins expressed denitely by the synthesis is generally coupled with production ofparent strain (0.03 and 0.08 U/mg protein, respective- mixtures of additional compounds monacolins (K,ly), while both strains utilized casein and skim milk J, M, L and X) (Endo 1979; Endo et al. 1985) andwith similar efciency (0.06 0.09 U/mg protein). citrinin (Blanc et al. 1995). These secondary metabo-Therefore, the considerable reduction of amylase and lites possess structures close to those of pigments andprotease activity in the white mutant indicates that the probably are functionally connected to the relation-mutation has changed important properties of the ships of Monascus fungi with other microora inMonascus purpureus fungus selected by the high ecosystems.selective pressure of nutritional limitation in natural Monacolin production by both strains was studiedecosystems. during cultivation on CD medium with ammonium or

Table 5. Fatty acid composition of a colony ofMonascus purpureusWent CBS 109.07 andMonascus purpureus M , cultivated on MEPAG at1225 8C for 7 d. The amount of the individual fatty acids is shown as a percentage the total one.

Strain Saturated fatty acids Unsaturated fatty acidsfatty acid % fatty acid %

M. purpureus C16 22.40 C16:1 6.12M C17 1.2512

C18 10.62 C18:1 46.80C20 9.61C22 3.20

M. purpureus C16 17.50 C16:1 2.42WENT CBS 109.07 C18 7.10 C18:1 37.40

C18:2 27.20

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Table 6. Citrinin and monacolin synthesis by Monascus purpureus Went CBS 109.07 and Monascus purpureus M .12Monascus purpureus Monascus purpureusWENT CBS 109.07 M12

citrinin, mg/g freeze dried culture broth 65 6 0.05 not detected*monacolin (lovastatin), mg/g dry weight 88 6 0.07 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 ofcitrinin in the albino mutant, if available could be less than 0.1mg/g freeze dried culture broth.

Pareilleux A. et al. 1995. Characterization of monascidin A fromnitrate as a source of nitrogen. It is shown that bothMonascus as citrinin. Int. J. Food Microbiol. 27: 201213.strains produce one and the same monacolin com-

Blanc P.J., Loret M.O. and Goma G. 1999. Pigments and citrininpounds - monacolin K (lovastatin) and probably other production during cultures of Monascus in liquid and solidsubstances from the same group (Table 6). These media. Advance in Solid State Fermentation. Chapter 32 Sec-

ondary Metabolites, Aroma, Pigments and Biopesticides, pp.results indicate that biosynthesis of pigments and393406.monacolins is quite independent and the white strain

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