HAYATI Journal of Biosciences, June 2008, p 61-66 Vol. 15, No.

2
ISSN: 1978-3019

Optimizing Angkak Pigments and Lovastatin Production

By Monascus purpureus
HASIM DANURI

Department Biochemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Darmaga Campus,
Bogor 16680, Indonesia; Phone/Fax: +62-251-8423267, E-mail: [email protected]

Received June 4, 2007/Accepted May 7, 2008

Angkak pigments and lovastatin had been reported very useful as natural coloring agents, as an agent to increase
thrombocyte level in Dengue hemorrhagic fever, and also as a compound that was able to control blood cholesterol level.
Three strains of fungus Monascus purpureus AKI, AKII, and 915 were selected to produce angkak pigments and lovastatin
in potato dextrose agar (PDA) medium. The best fungus strain, which is AKII, was then applied in three kinds of rice
media (white rice IR-42, red rice BP-1804-IF-9, and a combination of 1:1 (w/w) white IR-42 and red rice BP-1804-IF-9 for
solid fermentation. The best medium and fermentation times were determined for the production of angkak pigments and
lovastatin separately. Results showed that strains, media, and duration of fermentations gave significant effect on the
amount of pigment produced. Strain AKII produced highest concentration of angkak pigments. The combination of rice
(White IR-42 and red rice BP-1804-IF-9) produced the highest pigment than the individual white and red rie it self. The
optimum duration of fermentation was 16 days for strains AKI and AKII, but only 15 days for strain 915. Therefore the
strain AKII with media combination of rice and a fermentation time of 16 days were used to investigate the additional
effect of various minerals. Addition of the mineral individually gave significant increased on angkak pigment production
by AKII, where as the addition of minerals mixture in the forth tube did not.

Key words: angkak, pigments, lovastatin, monascus purpureus

___________________________________________________________________________

INTRODUCTION Lovastatin is as a competitive inhibitor for 3-hydroxy-3-

methyl-glutaryl Coenzyme-A reductase (HMG-CoA
Nowadays, people are using more synthetic coloring in reductase) that is an important enzyme for cholesterol
foods. However, previous reports have indicated that synthetic biosynthesis. When this enzyme is inhibited, cholesterol
food colorings have a higher risk to human health as they synthesize will blocked (Szakacs et al. 1998). As natural
may be carcinogenic (Blanc et al. 1994). Since this negative secondary metabolite production of microbes is limited, the
information about synthetic food colorings, people have microbes and the media must be modified to obtain sufficient
attempted to develop natural food colorings. amounts of natural secondary metabolites for preparation of
Angkak pigment is an example of natural colorings and commercial products.
has been used in Asian countries e.g. Japan, China, Thailand, Pigments and lovastatin production is influenced by pH,
Philippines, and Indonesia. This pigment is not dangerous temperature, moisture, the medium components (e.g. organic
and does not cause allergic reactions in mice (Fardiaz et al. nutrients), and the fungal strain. Rice strains can be used as
1989). Besides being a natural coloring for food and beverages, the medium to produce angkak and the researches have shown
angkak pigment has also been used as food preservative due that rice gives better results than sticky rice. According to
to its antibacterial properties (Wong & Koehler 1981) and it is Santoso and Satiawiharja (1985), white rice contains high
used for increasing thrombocyte level in Dengue Hemorrhagic amylose and low amylopectin, giving a suitable medium to
Fever (DHF), a viral infection with drops of blood thrombocyte produce angkak, while the sticky rice has high amylopectin.
level (Nurhidayat 2004). Pigment production can also be increased by adding zinc
Angkak is a fermentation product from Monascus (Zn) and magnesium (Mg; Lin & Demain 1991). Chiu and
purpureus (Steinkraus 1983) although the species M. Poon (1993) also noted that Tween 80 [polyoxyethylene (20)
rubropunctatus, M. rubiginosus, M. anka, M. major, and M. sorbitan monoleate] can increase pigment production by
bakeri can also produce the pigment (Carels & Shepherd Monascus sp.
1977). Research objectives were to determine the optimum
Monascus purpureus naturally produces the secondary condition in producing pigments and lovastatin, parameters
metabolite monakalin K (Lovastatin) that can inhibit cholesterol observed include the effects of M. purpureus strain, various
biosynthesis in patients with hypercholesterolemia. Therefore, combination of rice media, duration of fermentation, and
Lovastatin has been used as the first curative for patients also the effect of adding various minerals to fermentation
with a high risk of heart attack due to hypercholesterolemia. mixtures.
62 DANURI HAYATI J Biosci

MATERIALS AND METHODS Lovastatin Analysis. Standards of soluble lovastatin were

prepared (120, 200, 500, and 1000 parts per million, ppm) then
Potato Dextrose Agar Media (PDA). Ten mg of boiled D- the absorption was measured at 237 nm using a UV
glucose was added to 500 ml potato extract 50% (w/v). 20 g of spectrophotometer. Standard curves were prepared and used
agar was then added and dissolved by heating. The solution to determine the concentration of Lovastatin in samples.
was made up to 1 using distilled water. When it had been cold, Angkak powder (100 mg) was added into 900 ml of ethanol
5 ml PDA was poured into a reaction tube placed in a sloped and then it was vortexed for a few minutes and centrifuged at
position before being sterilized by autoclaving at 121 oC in 9,520 rpm for 15 minutes. The supernatant was removed and
1 atmosphere pressure for 1 hour before being left to cool. retained, while the pellet was extracted with another 900 l of
Rejuvenation and Inoculums (Starter) Preparation. Three 75% ethanol. The supernatant from these extractions was then
strains of M. purpureus were used in this study; M. purpureus mixed before the lovastatin content was analyzed as for the
915, AKI, and AKII are from the collection at the Microbiology standards.
laboratory, LIPI, Cibinong, Bogor, Indonesia. Rejuvenation Statistical Analysis. The data was analyzed using analysis
of the M. purpureus isolates was carried out in laminar cabinet of variance (ANOVA) with confidence limits of 95% and a
using aseptic conditions. Pure isolates of M. purpureus are standard error of 0.005 using SPSS software.
inoculated into PDA, and then those were incubated at 25-30 oC
for 16 days. RESULTS
Starter medium was made from 40% (w/v) rice flour as a
source of carbon (Sutrisno 1987), 0.15% (w/v) NH4NO3 as The Growth of Monascus purpureus in PDA Medium.
source of nitrogen, and minerals (KH2PO4 0.25% (w/v); During the growth of M. purpureus, a process of granular
MgSO47H2O 0,10 % (w/v)). The mixture was then dissolved liquid extrusion occurred through the point of hyphae growth.
in 100 ml distilled water (Jenie & Facda 1991). pH was adjusted During culture growth in PDA medium, this granular liquid is
to 6 before the medium was sterilized. Two scratches of spores initially white but gradually the color develops from yellowish
were used for inoculation and the starter culture was to orange and finally to red, or (Table 1, Figure 1). This coloring
incubated for a week at 25-30 oC. These M. purpureus cultures consists of two pigments, a red pigment (monascorubin) and
were then used as starter cultures for solid medium a yellow pigment (monascoflavin) (Wong & Kohler 1981).
fermentation. The mycelium growth of M. purpureus is also shown in Table 1.
Growth of M. purpureus on Solid Media. The production Media and Strains Effect on the Red and Yellow Pigments
of pigment and lovastatin were initiated by washing the rice Production by M. purpureus. To test the production angkak
in distilled water, draining it before placing 25 g rice and pigments by 3 strains of M. purpureus (AKII, 915, and AKI),
12.5 ml distilled water into Erlenmeyer flask and adjusting the we had inoculated cultures on white rice (IR-42) contained
pH to 5 by adding of 0.1 M HCl or 0.25 M KOH. The rice media high amylase (25-33%), red rice (BP-1924-IE-52), and a 1:1
was then sterilized by autoclaving at 121 oC for one hour. combination of red and white rice medium and then tested
After cooling, the medium was inoculated with 2 ml starter pigment levels between from 0 to 16 days (Figures 2, 3, and 4).
culture, and then incubated at room temperature (28-33 oC) for Maximum production of red and yellow pigment by the AKII
9-16 days. Fermentation process was ended in 16 days; the strain occurred on 16th day of fermentation (Figure 2). The
substrate was dried in an oven at 56 oC for 3 days. maximum amount of red pigment was obtained from
Growing at M. purpureus Solid Media with Minerals combination rice medium, while the maximum amount of yellow
Variations. When medium supplemented with minerals was pigment was obtained from white rice. The maximum amount
required, mineral salt mixtures were added before autoclaving of both red and yellow pigment produced by strain 915 occurred
the rice medium as follows. There were five different
treatments: 1st treatment medium was added with 12.5 ml distilled Table 1. The development of M. purpueus in PDA media
water, 2nd treatment was added 12.5 ml mixture of minerals of Duration of Change in colony Mycelium number
KH2PO4 0.25% (w/v), NaNO3 0.75% (w/v), MgSO47H2O 0.5% growth (days) color AKI AKII 915
(w/v), CaCl 22H2O 0.005% (w/v), 3rd treatment was added 1 White + + +
12.5 ml ZnSO 4 7H 2 O 0.03 mM, 4 th treatment was added 2 white ++ ++ ++
3 white ++ +++ ++
12.5 mg MgSO47H2O 12 mM, last treatment was added with 4 White-yellowish +++ +++
++++
12.5 ml Tween 80 0.4%. All treatments were placed in 5 White-yellowish +++ ++++ +++
Erlenmeyer glass. Fermentation process was ended in 16 days; 6 White-yellowish ++++ ++++ +++
the substrate was dried in an oven and refined in mortar. 7 White-yellowish ++++ +++++ ++++
8 Orange ++++ +++++ +++++
Analysis of Angkak Pigment. Angkak powder (0.05 g) 9 Orange +++++ ++++++ +++++
from the fermentation above was added to 10 ml methanol in 10 Dark Orange +++++ ++++++ +++++
glass tube in order to extract the pigment. Extraction was carried 11 Dark Orange ++++++ ++++++ +++++
out for 24 hours in shaking incubator, and then it was vortexed 12 Orange-red ++++ +++++ +++
13 orange-red ++++ +++++ +++
and drained to obtain the filtrate. The pigment absorption 14 dark-red +++ ++
++++
was measured using a spectrophotometer. Yellow and red 15 dark-red +++ ++++ ++
pigments were measured at 390 and 500 nm respectively with 16 dark-red ++ +++ +
methanol as a blank. +: few, ++: medium, +++: many, ++++: numerous.
Vol. 15, 2008 ANGKAK PIGMENTS AND LOVASTATIN PRODUCTION 63

Figure 1. Production of red pigment by M. purpureusisolates AKI,

AKII, and 915 in Potato Dextrose Agar (PDA) medium.

Figure 3. a: Red pigemnt production by M. Purpureus 915 in rice medium

(measured in 500 nm); b: Yellow pigemnt production by M.
Purpureus 915 in rice medium (measured in 390 nm).
b

produce pigment are similar, while AKII strains is differ to

AKI and 915, in which pigment production of AKII is the
highest. Concerning to the media in producing red and yellow
pigment both white rice and combination rice is similar (Figure
2, 3, and 4).
Linear regression analysis indicates that red and yellow
pigments have positive correlation (r2 = 0.933), it means that
when the red pigment increase, the yellow pigment produced
will also increase (Table 2).
Effects of Minerals Addition on the Production of Red
and Yellow Pigments by M. purpureus AKII Using White
Rice and Combination Rice Media (White and Red 1:1). Table
Figure 2. a: Red pigemnt production by M. Purpureus AKII in rice 1 indicates that M. purpureus AKII is the fungus strain which
medium (measured in 500 nm); b: Yellow pigemnt production produce the highest pigment compared to the other strain.
by M. Purpureus AKII in rice medium (measured in 390 nm).
The variance analysis shows that red and yellow pigments
produced by AKII with addition of minerals mixture,
on 15th day of fermentation on combination rice media (Figure ZnSO 4 7H 2 O, MgSO 4 7H 2 O and Tween 80 0.4% are
3). The AKI strain produced maximum pigments after 16 days significantly difference. The effects of different treatments
fermentation with maximum red pigment produced using white on pigment production are shown in Figure 5 and 6. Using
rice medium and maximum yellow pigment using combination white rice medium, the red and yellow pigment production are
of rice medium. Analysis of variance (ANOVA) shows that decreased by 32.12 and 30.23% respectively, in comparison
the strains, rice media, and duration of fermentation with the control treatment (distilled water) when mineral mixture
significantly influence production of red and yellow pigments (0.25% (w/v) KH2PO4, 0,75% (w/v) NaNO3, 0.5% (w/v)
by M. purpureus. MgSO47H2O, 0.005% (w/v) CaCl22H2O) was added to the
The duration of fermentation influences pigment medium. For combination rice medium, red and yellow pigment
production. Table 1 shows that the potency of AKI and 915 production are also decreased to 70.41 and 64.94%
64 DANURI HAYATI J Biosci

respectively, when this mineral mixture is added. Statistical significantly different to the control distilled water treatment
analysis showed these values are significantly difference to (P > 0.05).
the control (P < 0.05). Minerals Addition Effects to Lovastatin Production by
Tween 80 is able to increase pigment production by M. M. purpureus AKII Grown in White Rice Medium. Next,
purpureus (Figure 5, 6, and 7) Using white rice medium, tween we investigated the lovastatin content that has been
80 increases the production of red and yellow pigments by produced by angkak using white rice medium supplemented
15.05 and 9.39% respectively, while on combination rice the with mineral mixture and Tween 80 during fermentation by
increase is 18.09 and 7.61% respectively. However, it is not M. purpureus AKII. The results show that the content

a b

Figure 4. a: Red pigment production by M. Purpureus AKI in rice medium (measured in 500 nm); b: Yellow pigemnt production by M. Purpureus AKI
in rice medium (measured in 390 nm).

Table 2. Correlation test result between red pigment, yellow pigment, lovastatin level, and fermentation length
Correlation (r) Sig probabilities (1-tailed)
Fixed variables Red Yellow Lovastatin Fermentation Red Yellow Lovastatin Fermentation
pigment pigment length pigment pigment length
Red pigment 1.000 0.964 0.520 0.633 - 0.000 0.403 0.000
Yellow pigment 0.964 1.000 0.088 0.665 0.000 - 0.338 0.000
Lovastatin 0.520 0.088 1.000 0.135 0.403 0.338 - 0.261
Fermentation Length 0.633 0.665 0.135 1.000 0.000 0.000 0.261
R 0.971
Determination Coefficient 0.944
R2 0.933
Red pigment absorbance give positive correlation with yellow pigment absorbance (r = 0.964) and red pigment absorbance give no correlation
with lovastatin production (r = 0.052), neither with the yellow pigment absorbance. Fermentation length also make a positive correlation with
red and yellow pigment absorbance (r = 0.633), but lack of correlation with lovastatin production (r = 0.135).

b
a

ZnSO47H2O
ZnSO47H2O
MgSO47H2O
MgSO47H2O

Figure 5. The effect of adding minerals to production of a: Red pigment in white rice medium by M. Purpureus AKII (measured in 500 nm); b:
Yellow pigment in white rice medium by M. Purpureus AKII (measured in 390 nm).
Vol. 15, 2008 ANGKAK PIGMENTS AND LOVASTATIN PRODUCTION 65

of lovastatin is varying, depends on the treatment highest concentration of lovastatin is obtained from angkak
(Figure 7). when Tween 80 is added in its production. The average value
Variance analysis indicates that treatments gave for 5 replicates is 254.934 + 9.656 ppm or an increase of 40.78%
significantly effect on lovastatin produced (Figure 7). The compared to the control. This result was supported by t-Test
which shows a significant difference from the control. In
a contrast, the average lovastatin content from angkak
produced with added mineral mixture was increased by 8.81%
but this is not significantly different to control using the
ZnSO47H 2O Duncan multiple range tests. The addition of ZnSO47H2O
MgSO47H2O increased lovastatin content to 22.53% respectively (not
significant compared to control group), whereas adding
MgSO47H2O will decrease the pigment content by 4.11%
(significant compared to control group). ANOVA test indicated
that red pigment production correlated positively to the yellow
pigment production (r = 0.96) and that there is no positive
correlation between red pigment and lovastatin (r = 0.052) or
yellow pigment and lovastatin (r = 0.088).

DISCUSSION
b
The results show that AKII and AKI strains produce
maximum pigment on day 16 of fermentation, while 915 strain
produced maximum pigment on the 15th day of fermentation.
According to Jenie and Facda (1991), fermentation to produce
pigment needs 16 days. The decrease of pigment during
fermentation is caused by decomposition of pigment and
change in pigment structure. The chromophor group of
pigment is degraded, which results in decreased pigment
absorption.
Wong and Koehler (1981) indicated that the growth of
M. purpureus and pigment production are influenced by the
amount of carbon and nitrogen in the media, and that the
amount of carbon and nitrogen will determine the amount and
the type of pigments.
Figure 6. The effect of adding minerals on production of a: Red
pigment in combination rice (red:white 1:1) by M. Purpureus Steinkraus (1983) said that pigment can be produced from
AKII (measured in 500 nm); b: Yellow pigment in several rice strains with high amylose (25-33%), more pigment
combination rice (red:white 1:1) by M. Purpureus AKII production will be obtained using rice with low amylopectin
(measured in 390 nm). (Santoso & Satiawiharja 1985). The use of sticky rice will inhibit
the growth of M. purpureus.
M. purpureus produces protease and amylase enzymes
which break down proteins and amylose respectively. Every
strain of M. purpureus has the capability to produce enzyme
amylase. The more amylase which the strain produces, the
more amylose can be hydrolyzed into glucose that will produce
more pigment. Glucose is needed for source of energy during
the development of secondary metabolic. Amylose in white
rice is higher than in red rice; therefore, white rice will produce
more red and yellow pigment (Santoso & Satiawihardja 1985).
Rice consists of carbohydrate, protein, vitamin B1,
phosphate, potassium, and zinc. Vitamin B1 in red rice is higher
than in white rice (Santoso & Satiawihardja 1985). Vitamin B1
(Lin 1973), amino acids, and zinc (Lin & Demain 1991) can
influence pigment production. Vitamin B1 (Thiamin
ab a ab c bc Pyrophosphate) is as coenzyme or prosthetic group in
Treatments, n = 5 for each group complex of a pyruphate dehydrogenase enzyme that catalyses
Figure 7. The effect of adding minerals on lovastatin levels (ppm) the conversion of pyruvate into acetyl-CoA in glucose
in angkak production by M. Purpureus AKII. Key: Same metabolism. According to Chen and Johns (1994), M.
letter under each treatment indicates that the treatments
give no significant results.
purpureus pigment is produced through a polyketide pathway
66 DANURI HAYATI J Biosci

and needs acetyl-CoA in which is produced from glucose by ACKNOWLEDGEMENT

pyruvate acid. Therefore indirectly, vitamin B1 is involved in
the production of pyruphate acid from glucose during the The author would like acknowledge and thank Dudi S.
process of pigment biosynthesis. The study shows that Sastraatmadja from LIPI for his kindness in allowing the use
pigment level produced using red rice medium is lower than of Monascus purpureus; and Lisna Vitria Manjasari, Mega
that using white rice medium. This might be vitamin B1 in red Safitri, and Dimas Andrianto for their help in this study.
rice is higher than in white rice.
The study shows that red and yellow pigments levels are REFERENCES
lowered compared to the control (distilled water group) when
a mineral mixture is added. Wong and Koehler (1981) stated Blanc PJ et al. 1994. Pigments of Monascus. J Food Sci 59:862-865.
that the growth of M. purpureus and pigment production are Carels M, Shepherd D. 1977. The effect of different nitrogen sources
on pigment production and sporulation of Monascus spesies in
influenced by the proportion of carbon and nitrogen. Organic submerged shaken culture. Can J Mikrobiol 23:1360-1372.
nutrients and minerals are important for the development of Chen MH, Johns MR. 1994. Effect of pH and nitrogen source on
the angkak pigment, and mineral are supporting elements pigment produced by Monascus purpureus. Appl Microbiol
during metabolism of the microorganism during fermentation Biotechnol 40:132-138.
Chiu SW, Poon YK. 1993. Submerged production of Monascus pigment.
(Sutrisno 1987). When the amount of carbon is increased, Mycologia 85:214-218.
more nitrogen and minerals are needed to reach the optimum Fardiaz D, Apriyantono A, Puspitasari NL, Sedarwati, Budiyantono S.
growth. Decreasing pigment level in this study maybe caused 1989. Analisis Pangan. Bogor: PAU Pangan dan Gizi IPB.
by the proportion and composition of minerals added is Jenie BSL, Facda F. 1991. Pemanfaatan onggok dan dedak padi
untuk produksi pigmen angkak oleh Monascus purpureus.
optimal (Figures 5 & 6). Lind and Demain (1991) indicated that Pertemuan Ilmiah Tahunan. Bogor: Perhimpunan Mikrobiologi
high phosphate level (> 70 mM) can hinder the growth of M. Indonesia.
purpureus and pigment production. In contrast, high MgSO4 Lin CF. 1973. Isolation and cultural conditions of Monascus sp. for
(> 16 mM) will improve the growth of M. purpureus but hinder the. production of pigment in a submerged culture. J Ferment
Technol 51:407-414.
pigment production. Lin CF, Demain A. 1991. Effect of nutrition of Monascus sp. on the
Figure 7 shows that Tween 80 can increase lovastatin level formation of red pigments. Appl Microbiol Biotechnol 36:70-75.
by M. purpureus. Tween 80 apparently an emulsifier that Nurhidayat N. 2004. Angkak meningkatkan jumlah trombosit. Bogor:
stimulates cell development and increases the permeability of http://www.pikiran-rakyat.com.
Santoso G, Satiawiharja B. 1985. Produksi pewarna alami angkak dengan
the cell membrane, so that nutrient transport from both outside media fermentasi beras Sosoh. Media Teknol Pangan 1:34-35.
and inside the cell are increased and consequently cell Steinkraus KH. 1983. Handbook of Indigenous Fermented Foods.
metabolism will increase too. New York: Marcel Dekker Inc.
Finally, pigment production will achieve best result using Sutrisno AD. 1987. Pembuatan dan peningkatan kualitas zat warna
alami yang dihasilkan oleh M purpureus. In: Fardiaz S, Dewanti R,
fermentation by M. purpureus AKII strains in combination of Budijanto S (eds). Risalah Seminar Bahan Tambahan Kimiawi.
white and red rice for 16 day fermentation, addition of Bogor: PAU Pangan dan Gizi IPB.
MgSO47H2O 12 mM will also increase pigment production. Szakacs G, Morovjan G, Tengerdy RP. 1998. Production of lovastatin
Whereas lovastatin production an be generated by adding by wide strain of Aspergillus terreus. Bacteriol Let 20:411-415.
Wong HC, Koehler PE. 1981. Mutant of Monascus pigmen production.
Tween 80 in the fermentation medium. These results provide J Food Sci 46:956-957.
more information in producing pigment and lovastatin for
commercial purpose described above.