Journal of Oleo Science

Copyright 2014 by Japan Oil Chemists Society

doi : 10.5650/jos.ess13184
J. Oleo Sci. 63, (8) 769-777 (2014)

Co-solvent Selection for Supercritical Fluid

Extraction of Astaxanthin and Other Carotenoids
from Penaeus monodon Waste
Shazana Azfar Radzali1, Badlishah Sham Baharin1 , Rashidi Othman2,
Masturah Markom3 and Russly Abdul Rahman1
1

Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang, Malaysia
International Institute for Halal Research and Training (INHART), Herbarium Unit, Department of Landscape Architecture, Kulliyyah of
Architecture and Environment Design, International Islamic University Malaysia, Kuala Lumpur, Malaysia
3
Department of Chemical and Process Engineering and Built Environment, National University of Malaysia, Bangi, Malaysia
2

Abstract: In recent years, astaxanthin is claimed to have a 10 times higher antioxidant activity than that of
other carotenoids such as lutein, zeaxanthin, canthaxanthin, and -carotene; the antioxidant activity of
astaxanthin is 100 times higher than that of a-tocopherol. Penaeus monodon (tiger shrimp) is the largest
commercially available shrimp species and its waste is a rich source of carotenoids such as astaxanthin and
its esters. The efficient and environment-friendly recovery of astaxanthins was accomplished by using a
supercritical fluid extraction (SFE) technique. The effects of different co-solvents and their concentrations
on the yield and composition of the extract were investigated. The following co-solvents were studied prior
to the optimization of the SFE technique: ethanol, water, methanol, 50% (v/v) ethanol in water, 50% (v/v)
methanol in water, 70% (v/v) ethanol in water, and 70% (v/v) methanol in water. The ethanol extract
produced the highest carotenoid yield (84.02 0.8 mg/g) dry weight (DW) with 97.1% recovery. The ethanol
extract also produced the highest amount of the extracted astaxanthin complex (58.03 0.1 g/g DW) and
the free astaxanthin content (12.25 0.9 mg/g DW) in the extract. Lutein and -carotene were the other
carotenoids identified. Therefore, ethanol was chosen for further optimization studies.
Key words: Penaeus monodon, astaxanthin, carotenoids, SFE, HPLC
1 INTRODUCTION
In recent years, the global production of shrimp has been
growing gradually and this trend is predicted to continue1.
In the last 20 years, shrimp has contributed to about 20
of the total value of exported fishery products2. Penaeus
monodon or Tiger shrimp is one of the most significant
commercial species found in Malaysian waters, and in
terms of production, it has continued to be the most important species for the past 5 years at a value of USD 160,
1863. Shrimp processing plants produce a large volume of
waste products. The shrimp body parts that are processed
for human consumption comprises only 70 of the overall
shrimp landing4. Hence, a remarkable tonnage of shrimp
waste is generated, from which astaxanthin, one of the
major carotenoids, can be obtained. Astaxanthin is a potential source of carotenoids for the aquaculture and
poultry industries, which need enormous supplies of this

carotenoid.
Astaxanthin3, 3-dihydroxy-, -carotene-4, 4-dioneis
the most valuable ketocarotenoid, both from a biotechnological and commercial point of view. It has been found in
most crustaceans like shrimps, crabs, and lobsters5. This
carotenoid pigment is also found in birds like flamingo, and
in insects, microorganisms, and micro-green algaHaematococcus pluvialis5. It exhibits a vibrant red color and
higher antioxidant activity compared to other carotenoids
such as -carotene, -carotene, lutein, lycopene, canthaxanthin, and vitamin E 6, 7. Thus, it has been applied as a
food colorant and in pharmaceutical products8, 9. Recently,
some studies proved that astaxanthin inhibits the invasion
of cancer cells10. Astaxanthin has been used as a carotenoid pigment in the aquaculture and poultry industries
and recently, it gained attention in the nutraceutical and
pharmaceutical industries 5. Nowadays, the demand for

Correspondence to: Badlishah Sham Baharin, Department of Food Technology, Faculty of Food Science and Technology, Universiti
Putra Malaysia, Serdang, MALAYSIA
E-mail: badli@food.upm.edu.my
Accepted March 11, 2014 (received for review October 30, 2013)

Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online

http://www.jstage.jst.go.jp/browse/jos/http://mc.manusriptcentral.com/jjocs
769

S. A. Radzali, B. S. Baharin, R. Othman et al.

natural carotenoids is increasing because of the consumer

s
concerns for food safety and strict government regulations.
Normally, organic solvents such as dichloromethane and
acetone are used for the extraction of astaxanthin;
however, the usage of the aforementioned solvents may
cause safety issues. The increasing demand for natural
foods has encouraged research on the extraction of astaxanthin from natural sources. The poor yieldsalmost 50
of the pigments are lostand considerable environmental
concern of the traditional extraction methods have motivated further studies on supercritical fluid extraction
SFEas an effective alternative to the traditional
methods11. SFE is used for the extraction of bioactive
compounds from foods12, 13. This method is more significant when thermo-labile compounds are present. In addition, the use of toxic solvents can be avoided, since carbon
dioxideCO2is inexpensive and generally recognized as a
safeGRASsolvent, which is easy to separate from the
extract1416. Supercritical fluids have outstanding extractive properties such as liquid-like density, high compressibility, high diffusivity, and low viscosity17.
Numerous studies have been conducted on the supercritical carbon dioxideSC-CO2extraction of carotenoids
from crustacean and marine animal waste1820. However,
in those studies, the recovery of astaxanthin was relatively
low compared to that of conventional methods, and the selectivity of the compounds extracted from the shrimp
waste remained uncertain. With the addition of a suitable
modifier or co-solvent to the SC-CO2, the extraction of the
solute could be enhanced by increasing the yield, solubility,
and extraction rate21. The extraction efficiency could be
improved by fine-tuning the solvent selectivity, and the solubility of the carotenoids in the mixture could be increased
by introducing different types and concentrations of cosolventsentrainersto the SC-CO2. In addition, the relatively low solubility of astaxanthin in SC-CO2 requires the
addition of polar modifiers or high pressures
40 MPa
to
improve its dissolution in the mixture with the simultaneous enhancement in extraction yields17, 18, 22, 23.
Astaxanthin is the main carotenoid extracted from the P.
monodon species24, 25. Despite Malaysia being one of the
most important producers of this species, the shrimp processing waste generated in Malaysia is not commercially
exploited for the recovery of astaxanthins1. To the best of
our knowledge, there is no report on the determination of
carotenoid contents from the wild Malaysian P. monodon
waste. Thus, this study will offer new insights into the determination of carotenoids in the P. monodon waste from
Malaysia. From a green technology point of view, the health
food industry will be benefitted if this precious pigment
could be extracted from an inexpensive raw material
instead of it being chemically synthesized. Therefore, this
study was designed to extract astaxanthins from P.
monodon waste owing to the abundance of this species in

Malaysia. The effects of different types and concentrations

of co-solvents on the amounts of the total carotenoids extracted, astaxanthin complex extracted, and free astaxanthin content in the extract were determined. The best cosolvent will be used for further optimization of the SFE
process.

2 EXPERIMENTAL PROCEDURES
2.1 Sample preparation
Samples of fresh P. monodon5060 counts kg1were
obtained from fishermen at the Tanjung Bidara Beach,
Malacca. The shrimps were immediately transported to the
laboratory in an expanded polystyrene box under iced conditions4
. The waste comprising of the head and carapace were freeze-dried for 72 h using a FDU-2100 model
freeze dryer Tokyo Rikakikai Co., Japan. Then, the
sample was grounded into fine powder and kept at 80
until further analysis. Most of the solvents and chemicals
used were of analytical gradeethanol, methanol, hexane
and chloroform
Fisher Scientific, USA. Ethyl acetate
and acetonitrile were of high-performance liquid chromagradeMerck, Germany.
tographyHPLC
2.2 Extractions
2.2.1 Solvent Extraction
The total amount of the carotenoids and astaxanthin
complex present in the sample were determined by a
solvent extraction procedure, which was performed after
slight modification of a previously reported method26. 1.0 g
of the powdered, freeze-dried sample was rehydrated by
adding distilled water and was extracted with a mixture of
acetone and methanol7:3, v/vat room temperature until
the biomass turned colorless. The crude extract was centrifuged for 5 min at 10,000 g in a Thermo Scientific,
Sorvall Biofuge Primo RGermanycentrifuge machine and
the supernatant was stored at 4 in the dark prior to analysis. Then, an equal volume of hexane and distilled water
were added to the combined supernatants in order to
extract the carotenoids. The solution was allowed to separate and the upper layer containing the astaxanthin
complex was collected. The combined upper layer was
then completely dried under a gentle stream of oxygenfree nitrogen and was immediately stored at 80 until
subsequent analysis.
2.2.2 Supercritical fluid extractionSFE
The effects of different modifiers and their concentrations on the carotenoid yield were studied by performing
SFE with CO2 on the sample. The SFE system comprised
of a PU-2080 model CO 2 pumpJASCO Corporation,
Japan, Series III solvent pumpLab Alliance, USA, BP1580-81 model back-pressure regulatorBPR
JASCO Corporation, Japan, extractor vessel enclosed in a FX22

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J. Oleo Sci. 63, (8) 769-777 (2014)

Co-solvent Selection for SFE of Astaxanthin from Penaeus monodon Waste

model air-circulating ovenSheldon Manufacturing, USA,

682-8 model pressure transmitter Dwyer Instrument,
USAand sample collectorFig. 1. The commercial grade
was purchased from Gas Pantai
liquefied CO99.9
2
Timur, Malaysia. The CO 2 was chilled to 2 using a
chillerProtech Electronic, Malaysiain order to retain its
liquid state before it was pumped to the extractor. The extractor comprised of a high-pressure stainless steel vessel,
which was filled with the freeze-dried shrimp waste. A
back-pressure regulator was used to sustain the system
pressure and the flow of the SFE process was controlled by
the needle valves.
Seven types of co-solvents
water, 50
v/v
methanol in
water, 50v/vethanol in water, 70v/vmethanol in
water, 70v/vethanol in water, pure methanol and
ethanolwere studied. The parameters were set at a temperature of 60 and operating pressure of 20 MPa. The
effect of co-solvent was investigated at a moderate pressure of 20 MPa and at this pressure, the rate of extraction
was assumed to be moderate. A higher extraction rate
might be applicable if the moderate extraction rate was effective. Besides, the experiments were carried out at 20
MPa since the maximum capacity of the co-solvent pump
was 40 MPa. The co-solvent pump required a higher pressure than the CO2 pump. Hence, if the experiment was
performed at 40 MPa, the capacity of co-solvent pump
must be higher than 50 MPa. The flow rate of the entrainer
was set in order to achieve the desired entrainer to CO2
concentration of 15v/v. 5.0 g of the freeze-dried
shrimp waste powder was extracted in the static mode for
15 min, followed by a dynamic extraction for 120 min
2 h
.
The extract fractions were collected every 20 min. All the
carotenoid extracts were centrifuged at 10,000 g in a
Thermo Scientific, Sorvall Biofuge Primo R
Germanycen-

trifuge machine and the supernatants were dried to completion under a gentle stream of oxygen-free nitrogen.
Then, the dried extracts were stored in the dark at 80
until subsequent analysis.
2.3 Determination of total carotenoid concentration
The total carotenoid concentration was determined by
the spectrophotometric method which is similar to that reported previously27. The dried carotenoid was resuspended
in a known volume of ethyl acetate. Then, 50 L of the redissolved sample was diluted with 950 L of chloroform for
spectrophotometric analysis. The carotenoid-containing
solution was measured at three different wavelengths: 480
nm, 648 nm and 666 nm using a Varian Cary 50 UV-Vis
as shown in
spectrophotometer. The Wellburn Equation28
Eq. 1-Eq. 3was used to obtain the total carotenoid
content in chloroform as described below:
Ca10.91A6661.2A648

Eq. 1

Cb16.36A6484.57A666

Eq. 2

Cxc1000A4801.42Ca46.09Cb
/202

Eq. 3

where Caconcentration at 666 nm; Cbconcentration at

648 nm; and Cxctotal carotenoid concentration at 480
nm.
2.4 Determination of the astaxanthin yieldastaxanthin
complex
The astaxanthin yield was calculated according to the
previous method29. The resulting concentrated carotenoids
were diluted in a known volume of petroleum ether and the
amount of astaxanthin was quantified by measuring the absorbance at 468 nm using a Varian Cary 50 UV-Vis spectro-

Fig. 1Design of the experimental system used for the supercritical fluid extraction system with co-solvent21).
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J. Oleo Sci. 63, (8) 769-777 (2014)

S. A. Radzali, B. S. Baharin, R. Othman et al.

photometer. In this study, the astaxanthin yield was calculated as that of theastaxanthin complex. The term
complexcorresponded to the mixture of free and esterified astaxanthin with other related astaxanthin derivatives.
The yield of the astaxanthin complex was calculated using
the following equation
Eq. 4
:
Astaxanthin complex yieldg astaxanthin complex/g
A nmVextractDilution factor
Eq. 4
sample
468
0.2Wsample
where A468 is the absorbance at 468 nm, Vextract is volume of
extract, 0.2 is the A468 of 1 g/mL of standard astaxanthin
and Wsample is the sample weight in gram.
2.5 Determination of free astaxanthin and other carotenoid contents
The astaxanthin content free astaxanthinin P.
monodon waste extracts was determined by using the
astaxanthin standardSigma-Aldrich Corporation, USA.
The astaxanthin standard was dissolved in HPLC grade
acetone and was prepared in a concentration range of
0.21.0 g/mL. The data for the standard curve was obtained with the coefficient of determination, R20.995.
The lutein and -carotene standards
Sigma-Aldrich Corporation, USAwere dissolved in the HPLC grade ethyl
acetate at concentrations of 0.02 0.12 g/L. The linear
regression analysis data of -carotene and lutein also demonstrated a good linear relationship with R2 of 0.995 and
0.996, respectively. The chromatographic peaks were identified by comparing their retention times and spectra
against the known standards.
2.6 HPLC Analysis
The HPLC analysis was performed according to a previous method on an Agilent model 1200 series comprising a
binary pump with autosampler injector, micro vacuum degassers, photodiode arrayPDAdetector and thermostatted column compartment31. The separation column used

was a ZORBAX Eclipse XDB-C18 end capped5 m, 4.6

150 mmreverse phase column Agilent Technologies,
USA. The solvents used wereA
acetonitrile:water9:1 v/
vandBethyl acetate. The solvent gradient was developed as follows: 040 solvent B020 min, 4060
solvent B2025 min, 60100 solvent B2525.1 min,
100 solvent B25.135 minand 1000 solvent B35
35.1 min
at a flow rate of 1.0 mL/min. The injection volume
was 10 L. The column temperature was maintained at
20. The individual carotenoids were detected at the
maximum absorption wavelength of the carotenoids in the
mobile phase: -carotene454 nm, lutein447 nm, and
astaxanthin480 nm. Compounds were identified by cochromatography with standards and by elucidation of their
spectral characteristics using a PDA detector. The carotenoid peaks were detected in the range of 350550 nm.
The individual carotenoid concentrations were calculated
by comparing their relative proportions, as reflected by the
integrated HPLC peak areas to the total carotenoid content
determined by spectrophotometry. The total and individual
carotenoid concentrations were expressed in terms of microgram per 1.0 g dry weight of freeze-dried matterg/g
DW
.

3 RESULTS AND DISCUSSION

3.1 Carotenoid recovery
The recoverymass of extracted compounds/mass of extracted compounds by acetone and methanol7:3, v/vX
100and yields mass of extracted compounds/mass of
biomassof the carotenoids for the performed extractions
are shown in Table 1. The maximum recovery100was
obtained with acetone and methanol7:3, v/vat room temperature. The highest total carotenoid recovery97.1of
SFE was obtained when CO2-ethanol was used as the cosolvent. The total carotenoid content in the extract was
84.020.8 g/g under the aforementioned extraction conditions. This value was very similar to that obtained

Table 1Yield and recovery of total carotenoids from Penaeus monodon waste for all extractions.
Extraction methods and solvents

Total carotenoid (g/g DWB) (S.D.)

Recovery (%)

(Solvent extraction method) Acetone and methanol (7:3, v/v)

86.520.1

SFE, CO2 and methanol

82.510.3

95.4

SFE, CO2 and ethanol

84.020.8

97.1

5.000.2

5.8

SFE, CO2 and 50% methanol

17.490.9

20.2

SFE, CO2 and 50% ethanol

15.531.7

17.9

SFE, CO2 and 70% methanol

30.740.4

43.2

SFE, CO2 and 70% ethanol

51.790.1

59.9

SFE, CO2 and water

*DWB-dry weight basis; Average of triplicate analyses standard deviation.

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J. Oleo Sci. 63, (8) 769-777 (2014)

100

Co-solvent Selection for SFE of Astaxanthin from Penaeus monodon Waste

through the solvent extraction method. Hence, the beneficial use of SC-CO2 for the recovery of the bioactive compounds was demonstrated. The least carotenoid content
was recovered when water was used as the co-solvent
5.8
for the SFE.
Pure SC-CO2 is nonpolar. Usually, a slightly polar solute
like astaxanthin exhibits low solubilities in pure SC-CO2.
However, the addition of polar entrainers could increase
the solubility of a less soluble solute in the solvent mixture,
which in turn improves the extraction efficiency. The enhancement in solubility is possibly due to the increase of
solvent density, polarity as well as the specific chemical interactions like hydrogen bonding 32. In this study, the
ethanol helped to swell the pores of the biomass, which in
turn improved the interaction of the SC-CO2 with the cell.
The high molecular weight of astaxanthinMW596.8
slowed down its release in SC-CO2 due to its low volatility33. Ethyl alcohol is also suitable to be applied as an entrainer for the extraction of the other high MW carotenoids34, 35. Since astaxanthin was more soluble in organic
solvents, which are less polar than water, the use of methanol and ethanol as the co-solvents gave better results than
that obtained using water. Although the results obtained
using methanol were similar to that using ethanol, the use
of methanol could cause potential health issues and is intolerable for pharmaceutical and food industries.
The presence of water in the sample could influence the
SFE process and there had been applications of aqueous
samples for SFE36. One recent study37 showed that the
70 methanol entrainer increased the extraction yield of
essential oils and bioactive compounds from Polygonum
minus
kesum
compared to that obtained using only water
or alcohol. However, in our study, the greatest yield and selectivity of the extracted carotenoids were obtained from
pure ethanol. This was explained by the differences in the

polarity and solubility of the targeted compounds in

SC-CO2 and co-solvent as well as the matrix characteristics.
This might also be due to the fact that the solvent concentration range from 5070v/vin the mixture was not
in accordance with the polarity of the analyte. Therefore, it
was most likely that astaxanthin was less soluble in the
mixture.
Normally, water can diminish or enhance the extraction
process depending on whether it can swell the matrix,
enlarge the pores, aid the fluid in accessing the analytes,
and improve the flow through the matrix. Water usually improves the fluid polarity and increases the recoveries of the
polar species. Nevertheless, the SFE recovery would be
low when the leftover water remained in the extractor
vessel, as the highly water-soluble analytes would be partitioned in the aqueous phase. In addition, carotenoids are
considered lipids and therefore are insoluble in water. The
water-insoluble materials would precipitate from the
mixture and even if the analytes were soluble in the
mixture, the leftover water would disturb the flow of
analyte to fluid mixture 36.
3.2 Carotenoids extract composition
The composition of the extract yield and the total and
individual carotenoid contentsg/g DWin P. monodon
waste for all extractions are presented in Table 2. The best
results of total carotenoids TC
84.020.8 g/g DW,
amount of astaxanthin complex58.030.1 g/g DW, free
astaxanthin content12.250.9 g/g DWand other carotenoids were obtained when ethanol was used as the co-solvent. The yield of astaxanthin complex obtained in this
study58.030.1 g/gwas 32.2 higher than the astaxanthin yield of P. indicus waste harvested from Indian
waters43.90.7 g/greported previously38. The existing
literature showed that the range of carotenoid content in

Table 2Composition of yield of extract, total and individual carotenoid content (g/g DW) in Penaeus monodon
waste for all extractions.
(Free
astaxanthin)
(g/g DWB)
(S.D.)

Extraction methods and solvents

Total
carotenoids
(g/g DWB)
(S.D.)

Amount of
astaxanthin
complex (g/g
DWB)(S.D.)

Acetone and methanol (7:3, v/v)

86.520.1

69.740.3

ND

SFE, CO2 and methanol

82.510.3

53.171.1

10.871.5

SFE, CO2 and ethanol

84.020.8

58.030.1

12.250.9

5.000.2

2.700.3

0.381.2

SFE, CO2 and 50% methanol

17.490.9

12.240.7

2.330.3

SFE, CO2 and 50% ethanol

15.531.7

10.870.4

1.810.6

SFE, CO2 and 70% methanol

30.740.4

21.521.6

4.270.1

0.110.3

0.430.1

8.680.2

SFE, CO2 and 70% ethanol

51.790.1

36.260.2

8.160.2

0.060.5

0.690.6

14.781.9

SFE, CO2 and water

-carotene
(g/g DWB)
(S.D.)

Unidentied
(g/g DWB)
(S.D.)

ND

ND

16.790.7

0.390.4

1.130.3

27.821.2

0.470.9

2.530.1

22.991.5

0.090.2

0.051.1

2.160.3

0.100.6

0.160.4

4.990.9

0.091.5

0.190.2

4.380.1

Lutein
(g/g DWB)
(S.D.)

*ND- Not Detected. DWB-dry weight basis; Average of triplicate analysesstandard deviation.
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J. Oleo Sci. 63, (8) 769-777 (2014)

S. A. Radzali, B. S. Baharin, R. Othman et al.

the exoskeleton of P. monodon from waters of the IndoPacific region were between 23 to 331 g/g39. The highest
carotenoid contents were obtained from the shrimps
having a dark grey body color and the lowest carotenoid
contents were obtained from those having a pale blue body
color 39.
The discrepancy in the appearance of the shrimp and
the composition of the carotenoid extract was assumed to
depend on the species, seasons, area of catch and other
physical and environmental conditions. The amount of esterified astaxanthin was determined by the background
color of the shrimp during the time of harvesting and the
changed chemical form that affected the pigment
s location
in the chromophores40. In addition, the physiological and
morphological color changes in crustaceans were related to
the rapid and slow transformations resulting from the hormonal and environmental factors 41, 42. Astaxanthin is
mainly found as carotenoproteins in the exoskeleton. While
cooking, the color of the pigment changes from blue-green
to red because of the separation of the astaxanthin from
the protein43. The crustaceans and other animals obtain
astaxanthin from their diet because the animals cannot
synthesize astaxanthin and in wild shrimps, this carotenoid
is derived from the conversion of other consumed carotenoids such as -carotene and zeaxanthin24. Therefore,
the presence of astaxanthin in shrimps was dependent on
its availability from the diet, ability to be digested, delivered and deposited in specific location as well as other
physical and environmental conditions.
The pigmentation of the shrimps is determined by the
presence of carotenoid pigmentsmostly astaxanthinthat
plays a key role in influencing a consumer
s choice to buy
shrimps and thus would impact their market value40. The
results also proved that the TC has a positive correlation

with the amounts of astaxanthin complex, free astaxanthin,

and other carotenoids present in the extractTable 2.
This relationship showed that astaxanthin and its derivatives were the most predominant carotenoids, which accumulated in the P. monodon waste. Previous studies
showed that the main carotenoid pigments that affect the
color of P. monodon were free astaxanthin, esterified
astaxanthin, and minor amounts of other pigments such as
-carotene39, 44. Small quantities of other carotenoids such
as zeaxanthin and lutein had been discovered in P.
monodon30, 45.
Another interesting aspect of this study was the comparison between the carotenoid profiles obtained from the
acetone and methanol7:3, v/vand SFE extracts, using
ethanol as co-solvent. The carotenoid profiles of these extracts were not similar. The acetone and methanol
7:3, v/v
extract was found to have only the astaxanthin estersFig.
2. As can be seen in Fig. 3, the free astaxanthin, astaxanthin esters, lutein and -carotene were detected in the SFE
extract using ethanol as the entrainer. The difference in
the carotenoid profiles might be due to the difference in
the solubility and polarity of the individual carotenoid
content in the extracts. The final carotenoid extract was
collected from the hexane fraction in the solvent extraction
method. Slightly polar carotenoids such as lutein and free
astaxanthin were difficult to dissolve in the nonpolar
hexane. The polarity of carotenes-carotenewas slightly
below those of the xanthophyllslutein, astaxanthin. In
addition, the free carotenoids were relatively polar, while
the monoesters and diesters were relatively non-polar46.
Therefore, the variation in the procedure and the different
types of solvents used for extraction also affected the determination of astaxanthin.
Both the HPLC profiles of the carotenoid extracts indi-

Fig. 2HPLC analysis of carotenoids extracted using solvent from Penaeus monodon waste, astaxanthin esters (AE).
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J. Oleo Sci. 63, (8) 769-777 (2014)

Co-solvent Selection for SFE of Astaxanthin from Penaeus monodon Waste

Fig. 3HPLC analysis of carotenoids extracted using SFE with ethanol as a co-solvent, at 60 and 20 MPa from
Penaeus monodon waste (1) Free astaxanthin, (2) Lutein, (3) -carotene and astaxanthin esters (AE).
cated that the free astaxanthin and its derivatives were the
main pigments present in the P. monodon species. The
results obtained in this study were in good agreement with
those of the previous study30 which stated that about
6492 of the carotenoid content in P. monodon,
Metapenaeus dobsonii, Parapenaeopsis stylifera and P.
indicus obtained from shallow waters off the Indian coast
was contributed by astaxanthin and its esters. The carotenoid composition and accumulation level vary with the
extraction method and environmental factors47, 48. The
astaxanthin content in the SFE extract of the Haematococcus pluvialis microalgae was significantly influenced
by the properties of the solvent, which was used as the
modifier49.

4 CONCLUSIONS
In this study, a rapid, selective and environment-friendly
SFE technique was proposed for the extraction of carotenoids from shrimp waste. The presence of ethanol as the
entrainer at 60 and 20 MPa gave the highest yield of the
total carotenoids84.020.8 g/g DWwith 97.1 recovery. The ethanol extract produced the highest value for the
amount of astaxanthin complex extracted
58.030.1 g/g
DWand the free astaxanthin content in the extract
12.25
. Hence, ethanol was preferred to methanol,
0.9 g/g DW
which is also highly toxic to humans. Therefore, the recovery of astaxanthin from P. monodon waste using SFE with
the addition of ethanol as the entrainer has great potential
to be developed for the pilot scale production of astaxanthins.

ACKNOWLEDGMENT
The authors would like to thank Universiti Putra
Malaysia UPMfor awarding the Research University
GrantsGrant No. 02-01-11-1138RUthat made this study
possible. We wish to acknowledge the Department of
Chemical and Process Engineering and Built Environment,
National University of MalaysiaUKMfor the SFE equipment. Thanks also to Herbarium Laboratory, KAED, International Islamic University MalaysiaIIUM
for the facilities
provided for the HPLC analysis.

References
1Kongkeo, H. Cultured aquatic species information programme: Penaeus monodon. FAO Fisheries and
Aquaculture Department, Rome. Available online from
www.fao.org/fishery/culturedspecies/Penaeus_monodon/en
2005
Accessed on March 16, 2012.
2CAC. Discussion Paper on Risk Management Strategies for Vibrio spp. in Seafood. Food and Agriculture
Organization / World Health Organization, Rome, Italy
2002.
3Mazuki, H. National Aquaculture Sector Overview. Malaysia. National Aquaculture Sector Overview Fact
Sheets. FAO Fisheries and Aquaculture Department.
Available online from www.fao.org/fishery/countrysector/naso_malaysia/en2008
Accessed on August 31,
2013.
4Sachindra, N. M.; Bhaskar N.; Siddegowda G. S.; Sathisha D.,Suresh P. V. Recovery of carotenoids from ensiled shrimp waste. Bioresource Technol. 98, 164216462007.
775

J. Oleo Sci. 63, (8) 769-777 (2014)

S. A. Radzali, B. S. Baharin, R. Othman et al.

5Guerin, M.; Huntley M. E.; Olaizola, M. Haematococcus

astaxanthin: applications for human health and nutrition. Trends Biotech. 21, 210-216
2003
.
6Naguib, Y. M. A. Antioxidant activities of astaxanthin
and related carotenoids. J. Agric. Food Chem. 48,
1150-1154
2000
.
7Rengel, D.; Dez-Navajas.; A.Serna-Rico, A. Exogenously incorporated ketocarotenoids in large unilamellar vesicles. Protective activity against peroxidation.
Biochim. Biophys. Acta - Biomembranes 1463, 179187
2000
.
8Johnson, E. A.; An, G. H. Astaxanthin from microbial
sources. Crit. Rev. Biotech. 11, 297-326
1991
.
9Lorenz, R. T.; Cysewski, G. R. Commercial potential
for Haematococcus microalgae as a natural source of
astaxanthin. Trends Biotech. 18, 160-167
2000
.
10Kozuki, Y.; Miura, Y.; Yagasaki, K. Inhibitory effects of
carotenoids on the invasion of rat ascites hepatoma
cells in culture. Cancer Lett. 151, 111-115
2000.
11Delgado-Vargas, F.; Paredes-Lopez, O. Carotenoids, in:
F. Delgado-Vargas, O. Paredes-LopezEds., Natural
Colorants for Food and Nutraceutical Uses, CRC
Press, LLC, NW,
Chapter 7
2003
.
12Mendes, R. L.; Nobre, B. P.; Cardoso, M. T.; Pereire, A.
P.; Palavre, A. F. Supercritical carbon dioxide extraction of compounds with pharmaceutical importance
from microalgae. Inorg. Chim. Acta 356, 328-334
2003
.
13Sun, M.; Temelli, F. Supercritical carbon dioxide extractions of carotenoids from carrot using canola oil as
a continuous co-solvent. J. Supercrit. Fluids 37, 397408
2006
.
14Sahena, F.; Zaidul, I. S. M.; Jinap, S.; Saari, N.; Jahurul,
H. A.; Abbas, K. A.; Norulaini, N. A. PUFAS in fish: extraction, fractionation, importance in health. Comp.
Rev. Food Sci. Food Safety 8, 59-74
2009
.
15Mercadante, A. Z. Analysis of carotenoids, in: C. SocaciuEd., Food Colorants: Chemical and Functional Properties, CRC Press Taylor & Francis Group,
LLC, USA, pp. 447-472
2008
.
16Reverchon, E.; De Marco, I. Supercritical fluid extraction and fractionation of natural matter. J. Supercrit.
Fluids 38, 146-166
2006
.
17Lim, G. B.; Lee, S. Y.; Lee, E. K.; Haam, J. S.; Kim, W. S.
Separation of astaxanthin from red yeast Phafa rhodozyma by supercritical carbon dioxide extraction.
Biochem. Eng. J. 11, 181-187
2002
.
18Charest, D. J.; Balaban, M. O.; Marshall, M. R.; Cornell,
J. A. Astaxanthin extraction from crawfish shells by
supercritical CO2 with ethanol as cosolvent. J. Aqua.
Food Pro. Technol. 10, 79-93
2001
.
19Yamaguchi, K.; Murakami, M.; Nakano, H.; Konosu, S.;
Kokura, T.; Yamamoto, H.; Kosaka, M.; Hata, K. Supercritical carbon dioxide extraction of oils from Antarc-

tic krill. J. Agric. Food Chem. 34, 904-907

1986.
20Hardardottir, I.; Kinsella, J. E. Extraction of lipid and
cholesterol from fish muscle with supercritical fluids.
J. Food Sci. 53, 1656-16611988.
21Markom, M. High Pressure Extraction and Fractionation of Tanins from Phyllanthus niruri Linn.
Phd thesis, Kuala Lumpur, Malaysia, University of Malaya2007.
22Lopez, M.; Arce, L.; Garrido, J.; Rios, A.; Valcarcel, M.
Selective extraction of astaxanthin from crustaceans
by use of supercritical carbon dioxide. Talanta 64,
726-7312004.
23Machmudah, S.; Shotipruk, A.; Goto, M.; Sasaki, M.;
Hirose, T. Extraction of astaxanthin from Haematococcus pluvialis using supercritical CO2 and ethanol
as entrainer. Ind. Eng. Chem. Res. 45, 3652-3657
2006.
24Katayama, T.; Hirata, K.; Chichester, C. O. The biosynthesis of astaxanthin-IV. The carotenoids in the prawn,
Penaeus japonicus Bate
Part I. Bull. Jap. Soc. Sci.
Fish. 37, 614-6201971.
25Katayama T.; Kitama, T.; Chichester, C. O. The biosynthesis of astaxanthin in the prawn, Penaeus japonicus BatePart II
. Int. J. Biochem. 3, 363-3681972.
26Othman, R. Biochemistry and genetics of carotenoid
composition in potato tubers. Phd thesis, Christchurch, New Zealand, Lincoln University2009.
27Lewis, D. H.; Bloor, S. J.; Schwinn, K. E. Flavonoid and
carotenoid pigments in flower tissue of Sandersonia
auranticaHook.. Scientia Hort. 72, 179-192
1998.
28Wellburn, A. R. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Phys. 144, 301-313
1994.
29Simpson, B. K.; Haard, N. F. The use of enzymes to extract carotenoprotein from shrimp waste. J. Appl.
Biochem. 7, 212-2221985.
30Sachindra, N. M.; Bhaskar, N.; Mahendrakar, N. S. Carotenoids in different body components of Indian
shrimps. J. Sci. Food Agric. 85, 167-1722005.
31Morris, W. L.; Ducreux, L.; Griffiths, D. W.; Stewart, D.;
Davies, H. V.; Taylor, M. A. Carotenogenesis during Tuber Development and Storage in Potato. J. Exper.
Botany 55, 975-9822004.
32Wang, H.; Chen, C.; Chang, C. J. Carbon dioxide extraction of ginseng root hair oil and ginsenosides.
Food Chem. 72, 505-5092001.
33Dandge, D. K.; Heller, J. P.; Wilson, K. V. Structure solubility correlations: organic compounds and dense
carbon dioxide binary systems. Ind. Eng. Chem. Pro.
Res. Develop. 24, 162-1661985.
34Vega, P. J.; Balaban, M. O.; Sims, C. A.; O
Keefe, S. F.;
Cornell, J. A. Supercritical carbon dioxide extraction

776

J. Oleo Sci. 63, (8) 769-777 (2014)

Co-solvent Selection for SFE of Astaxanthin from Penaeus monodon Waste

efficiency for carotenes from carrots by RSM. J. Food

Sci. 61, 757-765
1996
.
35Baysal, T.; Ersus. S.; Starmans, D. A. J. Supercritical
CO2 extraction of -carotene and lycopene from tomato paste waste. J. Agric. Food Chem. 48, 5507-5511
2000
.
36Lehotay, S. J. Supercritical fluid extraction of pesticides in foods. J. Chromat. A 785, 289-312
1997
.
37Markom, M.; Hassim, N.; Anuar, N.; Baharum, S. N. Cosolvent Selection for Supercritical Fluid Extraction of
Essential Oil and Bioactive Compounds from Polygonum minus. ASEAN J. Chem. Eng. 12, 19-26
2013.
38Sachindra, N. M.; Bhaskar, N.; Mahendrakar, N. S. Recovery of carotenoids from shrimp waste in organic
solvents. Waste Manag. 26, 1092-1098
2006
.
39Okada, S; Nur-E-Borhan, S. A.; Yamaguchi, K. Carotenoid composition in the exoskeleton of commercial
black tiger prawns. Fish. Sci. 60, 213-215
1994
.
40Tume, R. K.; Sikes, A. L.; Tabrett, S.; Smith, D. M. Effect of background colour on the distribution of astaxanthin in black tiger prawn Penaeus monodon: Effective method for improvement of cooked colour.
Aquaculture 296, 129-135
2009
.
41Rao, K. R. Pigmentary effectors, in integuments, pigments and hormonal processes. In: Bliss, D. E., ManEds.
, The Biology of Crustacea, Vol. 9. Actel, L. H.
ademic Press, New York, pp. 395-462
1985
.
42Melville-Smith, R.; Cheng, Y. W.; Thompson, A. W. Factors affecting colour change inwhitewestern rock
lobsters, Panulirus cygnus. J. Exper. Marine Biol.
Ecolog. 291, 111-129
2003
.

43Britton, G.; Armitt, G. M.; Lau, S. Y. M.; Petal, A. K.;

Shone, C. C. Carotenoproteins. In: Britton, G., Goodwin, T. W.
Eds., Carotenoid Chemistry & Biochemistry. Pergamon Press, Oxford, pp. 237-2511981.
44Boonyaratpalin, M.; Thongrod, S.; Supamattaya, K.;
Britton, G.; Schlipalius, L. E. Effects of -carotene
source, Dunaliella salina, and astaxanthin on pigmentation, growth, survival and health of Penaeus
monodon. Aquaculture Res. 32, 182-1902001.
45Pan, C. H.; Chien, Y. H.; Cheng, J. H. Effects of light
regime algae in the water, and dietary astaxanthin on
pigmentation, growth and survival of black tiger prawn
Penaeus monodon post-larvae. Zoolog. Stu. 40, 3713822001.
46Thomason, M. HPLC analysis of astaxanthin from fish
and shrimp feeds containing NatuRoseHaematococcus algae meal. Cyanotech Corporation Technical
Bulletin #012, Cyanotech Corporation2001.
47Norshazila, S.; Irwandi, J.; Othman, R.; Yumi Zuhanis,
H. H. Scheme of obtaining -carotene standard from
pumpkinCucurbita moschataflesh. Int. Food Res.
J. 19, 531-5352012.
48Sachindra, N. M.; Bhaskar N.; Mahendrakar, N. S. Carotenoids in Solonocera indica and Aristeus alcocki,
deep-sea shrimps from Indian waters. J. Aqua. Food
Pro. Technol. 15, 5-16
2006.
49Nobre, B.; Marcelo, F.; Passos, R.; Beiro, L.; Palavra,
A.; Gouveia, L.; Mendes, R. Supercritical carbon dioxide extraction of astaxanthin and other carotenoids
from the microalga Haematococcus pluvialis. Eur.
Food Res. Technol. 223, 787-7902006.

777

J. Oleo Sci. 63, (8) 769-777 (2014)