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Bioresource Technology 99 (2008) 5556–5560

Supercritical carbon dioxide extraction of astaxanthin from

Haematococcus pluvialis with vegetable oils as co-solvent
Sontaya Krichnavaruk a, Artiwan Shotipruk a, Motonobu Goto b, Prasert Pavasant a,*

a
Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand
b
Department of Applied Chemistry and Biochemistry, Kumamoto University, Kurokami 2-39-1, Kumamoto 860-8555, Japan

Received 3 May 2007; received in revised form 24 October 2007; accepted 29 October 2007
Available online 18 December 2007

Abstract

Soybean oil and olive oil were investigated as continuous co-solvents for supercritical carbon dioxide (SC-CO2) extraction of astaxan-
thin from Haematococcus pluvialis. Without co-solvents, only 25.40 ± 0.79% efficiency was achieved with SC-CO2 extraction at 70 C
and 40 MPa at a continuous flow rate of 3 mL min 1 for 5 h. In the presence of soybean oil or olive oil as a co-solvent, the extraction
efficiency was enhanced, with the most appropriate level of soybean oil in the solvent mixture being 10% by volume. At this concentration
and the above extraction conditions, the highest extraction efficiency of 36.36 ± 0.79% was obtained for soybean oil, a 30% increase in
extraction efficiency compared with SC-CO2 extraction without soybean oil, whereas the 10% olive oil increased the extraction efficiency
further to 51.03 ± 1.08%, which was comparable to that obtained using ethanol as co-solvent.
 2007 Elsevier Ltd. All rights reserved.

Keywords: High pressure extraction; Antioxidants; Carotenoids; Soybean oil; Olive oil

1. Introduction highest amount of astaxanthin with approximately 1.5–

Due to high antioxidant activity, carotenoids are consid-
ered suitable as components of various types of products,
e.g. cancer prevention agents, potential life extenders,
inhibiting agents for heart attack and coronary artery dis-
ease (Li and Chen, 2001; López et al., 2004; Sun and Tem-
elli, 2006). There are several types of carotenoids which can
be obtained from different natural sources such as fruits,
vegetables, and microorganisms. Astaxanthin is one of
the most effective carotenoids whose antioxidant activity
is 10 times stronger than those of any other carotenoids
such as zeaxanthin, lutein, canthaxanthin and b-carotene
and was up to 500 times stronger than vitamin E (Shimidzu
et al., 1996). The compound is generally found in microor-
ganisms such as some yeast and microalgae but of all the
astaxanthin accumulated microorganisms, the green alga,
Haematococcus pluvialis was reported to accumulate the
*
Corresponding author. Tel.: +66 2 2186870; fax: +66 2 2186877.
E-mail address: prasert.p@chula.ac.th (P. Pavasant).
3% by weight (Harker et al., 1996; Gong and Chen, 1997;
Machmudah et al., 2006).
The massive accumulation of astaxanthin in H. pluvialis
was reported to take place due to several inductive condi-
tions, e.g. lag of nutrients, high light intensity and with
the presence of growth inhibiting agents (Kobayashi
et al., 1997; Orasa et al., 2005; Kaewpintong et al., 2007).
However, the use of whole cells of H. pluvialis is generally
not recommended as astaxanthin is produced as an intra-
cellular product, which is surrounded by thick imperme-
able cell walls. Thus, extraction of astaxanthin from the
cell structure is often necessary. Organic solvents are often
introduced for the extraction of astaxanthin from H. pluvi-
alis cells, i.e. dichloromethane, acetone, hexane, etc. How-
ever, some of the organic solvents are potentially toxic and
unacceptable for food and pharmaceutical industries.
Supercritical fluids extraction (SFE) is an efficient alterna-
tive for the extraction of natural substances from foods
(Mendes et al., 2003; Sun and Temelli, 2006). Supercritical
fluids possess excellent extractive properties such as high

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.10.049
 S. Krichnavaruk et al. / Bioresource Technology 99 (2008) 5556–5560
compressibility, liquid-like density, low viscosity, high diffu-
sivity (Lim et al., 2002). They have been widely used in many
industrial applications, i.e. the decaffeination of coffee, the
extraction of hops, the synthesis of polymers, the purification
and the formation of nano particles (Lim et al., 2002; Kopcak
and Mohamed, 2005; Machmudah et al., 2006). For extrac-
tion of natural substances, supercritical carbon dioxide
(SC-CO2) is generally used. It has a greater ability to diffuse
through the ultra fine complex matrix than conventional
organic solvents and can be easily separated from the prod-
ucts by depressurizing process. Furthermore, low critical
temperature of carbon dioxide means that the SC-CO2 sys-
tem could be operated at moderate temperature, preventing
the degradation of the substance due to heat induction
(López et al., 2004; Vasapollo et al., 2004; Machmudah
et al., 2006). As a result, the obtained product is pure and
of great quality, and thus safe for use as nutritional additives
and for pharmaceutical applications (Mendes et al., 2003).
Since CO2 is nonpolar, pure SC-CO2 is not suitable for
extraction of slightly polar solute due to low solute solubil-
ity. In such case, polar co-solvents such as ethanol is often
used to enhance the solute solubility in SC-CO2 by interact-
ing with the solute, and thus improving the extraction effi-
ciency. Moreover, the addition of ethanol causes the
swelling of the matrix and increasing the contacting surface
area of the cell with SC-CO2. Although astaxanthin mole-
cule is considered containing no strong polar moieties, its
large molecule (MW = 596.8) inhibited its solubility in SC-
CO2 due to its low volatility (Dandge et al., 1985). Ethanol
can also be used as a co-solvent for extraction of other high
MW carotenoids (Vega et al., 1996; Baysal et al., 2000),
however, it is important that ethanol be separated from
the product, which requires the use of heat. Vegetable oils
have also been proposed instead of ethanol to enhance
SC-CO2 extraction. The advantage of using vegetable oils
is that there are no needs for subsequent separation of such
edible oil from the product. Evidences on the attempts to use
vegetable oil as co-solvent for the SC-CO2 extraction include
the extraction of lycopene from tomatoes (Vasapollo et al.,
2004) using hazelnut oil and extraction of carotenoids from
carrot using canola oil (Sun and Temelli, 2006).
This work investigated the potential use of soybean oil
and olive oil as co-solvents for the SC-CO2 extraction of
astaxanthin from H. pluvialis. The objective of this study
was to evaluate the use of soybean oil as co-solvent by
determining the effect of various extraction parameters,
i.e. temperature, pressure, SC-CO2 flow rate, and co-sol-
vent concentration on the percent of astaxanthin extracted.
The results were then compared with the extractions with
other co-solvents such as olive oil and ethanol.
2. Methods
2.1. Chemicals
Dried samples of feed grade H. pluvialis were purchased
from Cyanotech Corporation, Hawaii Ocean, Science and
5557
Technology Park, USA. The samples were kept in a tight
aluminium pack at 5 C to prevent degradation. CO2
(99.95%) was purchased from Uchimura Co., Japan Stan-
dard astaxanthin at 94% purity, ethanol 99.5%, dichloro-
methane 99.0%, and 99.8% HPLC grade acetonitrile used
for the analysis were purchased from Wako Pure Chemical
Industries Ltd., Japan.
2.2. Extraction experiment
CO2 was supplied to the CO2 pump (Jasco PU-2080-
100MPa, Japan) where a chiller (Shibata, Coolman C-
560, Japan) was connected to decrease CO2 temperature
down to 5 C. The pressure was adjusted in the range
from 30 to 50 MPa by a back pressure regulator (AKICO
Co., Japan). The resulting liquid CO2 was mixed in the sol-
vent mixer with the co-solvent which was delivered by
another pump (Jasco PU-980, Japan). The flow rate of
the co-solvent was adjusted to achieve the desired oil con-
centration of 0–12% v/v oil to CO2. The mixture was then
fed into the extractor where the total flow rate was varied
between 2 and 4 mL min 1. In the 50 mL extraction col-
umn (Thar Tech, Inc., USA), 6 g of dried H. pluvialis
was loaded in the middle of the column where the remain-
ing top and bottom sections were trapped with glass beads.
The extractor was placed in the heating chamber (ESPEC,
ST-110, Japan) to maintain the operating temperature
between 50 and 80 C. Extraction was carried out for up
to 5 h. The extracted stream from the extractor was then
depressurized through a pressure restrictor and the sample
was collected at a 1 h interval in brown test tubes to pre-
vent light-degradation. The samples were kept at 20 C
until analyzed. All extractions are performed in duplicate.
2.3. Analysis
For the determination of astaxanthin, the extract was
analyzed using high performance liquid chromatography
(HPLC, Jasco, Japan). The astaxanthin standard and the
sample extract were diluted with a mixture of dichloro-
methane:ethanol at 1:4 by volume before being injected
with a 10 lL loop. The separation column was STR
ODS-II 250 · 4.6 mm, 5 lm s-type (Shinwa Chemical
Industries) equipped with UV/VIS detector (Jasco UV-
970). The isocractic elution was provided 1 mL min 1 with
a mobile phase consisting of acetonitrile, dichloromethane
and ethanol at the volume ratio of 5:10:85. Astaxanthin
was detected at the wavelength of 470 nm. The amount
of astaxanthin in the extract was analyzed on the basis of
peak area compared to the standard astaxanthin. As the
basis for the determination of extraction efficiency, the
total amount of astaxanthin in H. pluvialis was determined
using the conventional Soxhlet apparatus where 1 g of
dried sample was extracted with 200 mL of 99% dichloro-
methane for 2 h or until the observed color of the condense
solvent in the apparatus became clear, and the extract was
then analyzed with HPLC. The percentage of astaxanthin
5558 S. Krichnavaruk et al. / Bioresource Technology 99 (2008) 5556–5560
extracted or extraction efficiency was defined as the ratio
between the amount of astaxanthin in the extract and the
total amount of astaxanthin in the sample.
3. Results and discussion
3.1. Extraction with pure SC-CO2
The effect of temperature on the cumulative amount of
astaxanthin extracted from H. pluvialis with SC-CO2 with-
out co-solvent was examined at 40 MPa and flow rate of
3 mL min 1. It was observed that the amount of astaxan-
thin extracted increased steadily with temperature from
19.57 ± 0.47% at 50 C to 25.4 ± 0.79% at 70 C. An
increase in temperature exerted multiple effects on the
extraction. Firstly, an increase in temperature resulted in a
reduction in SC-CO2 density, leading to a decrease in solute
solubility. However, the solute vapor pressure rose with an
increase in operating temperature and this enhanced the sol-
ubility of astaxanthin in the solvent (Fuente et al., 2006). In
other words, the solubility of organic compounds in SC-CO2
depended largely on the balance between fluid density and
solute vapor pressure, which were both controlled by fluid
temperature. In addition, the increase in temperature
increased the diffusivity of the solvent, which thus increased
the mass transfer and enhanced the interaction between SC-
CO2 and the solute in the complex cellular matrix. However,
the maximum astaxanthin in the extract occurred at 70 C
whereby a further increase in temperature to 80 C did not
have significant effect on the amount of astaxanthin in the
extract. In fact, the extraction at this temperature seemed
to deteriorate astaxanthin where the extracted percentage
slightly went down to 24.68 ± 0.69%. Visual observation
revealed that there was a significant quantity of sample being
excessively burnt after leaving the extraction chamber. It
was suggested therefore that the extraction should not be
performed at temperature higher than 70 C.
The results on the amount of astaxanthin being
extracted with pure CO2 at 70 C with a solvent flow rate
of 3 mL min 1 suggested that the extraction increased with
extraction pressure. An increase in operating pressure
resulted in an increase in the density of SC-CO2 (the extrac-
tion went up from 17.14 ± 0.76% at 30 MPa to 34.71 ±
0.97% at 50 MPa), and although the increased pressure
led to a reduction in the solvent diffusivity which dimin-
ished the extraction rate, an increase in the SC-CO2 density
was reported to increase the solvating power which
enhanced the overall extraction efficiency (Macı́as-Sánchez
et al., 2007). The resulting extraction rate was therefore
controlled by the trade-off between these two opposing
mechanisms.
3.2. SC-CO2 extraction with the presence of soybean oil
Fig. 1 illustrates the performance of the extraction with
the mixture of SC-CO2 and soybean oil at various volume
ratio compared with the extraction with pure SC-CO2.
Although the pressure that gave the highest astaxanthin
yield was found to be 50 MPa, the pressure employed in
this and subsequent experiment was 40 MPa. This was
because the purpose of the use of co-solvents was to facil-
itate the extraction without the need for high pressure oper-
ation. The results in Fig. 1 shows that similar time profiles
of the extract percentage were obtained from the various
mixtures where the extraction occurred more rapidly at
the first 2 h and after which continued at a slightly lower,
constant rate. During the early stage, astaxanthin at the
surface was extracted and the process took place mostly
due to convection. After a certain period of time, astaxan-
thin at the surface became exhausted, and the astaxan-
thin within in the cell matrix started to be extracted. This
stage of extraction was controlled by a slower, diffusion
mechanism.
Fig. 1 also reveals that the diffusion rate of astaxanthin
in the cell matrix did not vary significantly with the mixture
composition. The difference seemed to occur at the early
stage where convection prevailed. It was therefore sug-
gested that the diffusivities of astaxanthin in various sol-
vent mixtures were within the same range but the
external mass transfer rate depended more significantly
on the solvent composition. It should also be noted that
the extraction was continued for 5 h, and still the rate of
extraction, or the slope of the curve in Fig. 1, was still quite
linear. This demonstrated that the amount of astaxanthin
was still not completely extracted. All the comparisons in
this work were based on the results of the extraction at 5 h.
The results here indicated that the presence of soybean
oil as co-solvent was found to be beneficial for the extrac-
tion based on the results on the extraction efficiency. This
could be due to two reasons: (i) higher solubility of asta-
xanthin in the mixture of soybean oil and SC-CO2 than
that in pure SC-CO2 and (ii) a faster mass transfer rate
of the astaxanthin through the swollen algal matrix caused
by the solvent mixture. These results agreed with those of
Bamberger et al. (1988) which reported that the solubility
of a less volatile lipid component was significantly
enhanced by the presence of a more volatile triglyceride
species in the system. Furthermore, soybean was a good
solvent for astaxanthin in the liquid state, thus considered
50
Astaxanthin extracted (%)
6% oil 10% oil
40 12% oil pure CO2
30
20
10
0
0 60 120 180 240 300 360
Extraction time (min)
Fig. 1. Extraction of astaxanthin with soybean oil at 70 C, 40 MPa, and
solvent flow rate of 3 mL min 1.
 S. Krichnavaruk et al. / Bioresource Technology 99 (2008) 5556–5560 5559

as an appropriate co-solvent for SC-CO2 extraction (Haw- secondly, it reduced the contact time. Therefore the final
thorne, 1990). Illustrated also in Fig. 1 is the effect of soy- extraction efficiency depended on the compensation
bean content in the solvent mixture which demonstrated between the two consequences. In other words, the resi-
that the highest extraction efficiency of around 36.4% was dence time of solvent at 2 mL min 1 was sufficient to allow
obtained at the optimal level of soybean oil of 10%. the diffusion of astaxanthin to solvent stream but with a
It is interesting to note here that the presence of soybean lower initial extraction rate. An increase in the flowrate
oil significantly enhanced the effect of temperature on to 4 mL min 1 implied a shorter contacting time which
SC-CO2/soybean oil extraction. Experimental results dem- resulted in a more incomplete extraction, and hence, a
onstrated that, at 70 C, the extraction with SC-CO2 with diluted astaxanthin extracted was obtained.
soybean co-solvent was as high as 36.36 ± 0.79% which
was almost 30% higher than the efficiency at 60 C 3.4. Extraction with the presence of olive oil and ethanol as
(26 ± 1.04%) where an increase in the extraction efficiency co-solvents
was only about 12% greater than that using pure SC-CO2.
These results agree with previous literature which stated This section intended to demonstrate the different
that the addition of co-solvent might accentuate the tem- extraction power of the different solvents. It should be
perature dependence of solute solubility (Güçlü-Üstüngdağ noted that, in general, SC-CO2/co-solvent mixtures should
and Temelli, 2005). be in the supercritical state (completely miscible) at the
operating conditions to achieve the desired co-solvent
3.3. Effect of solvent flow rate effect. In this study, two other solvents other than soybean
oil were investigated including: ethanol and olive oil. Eth-
For the discussion in this section, the results from exper- anol is among one of the most frequently used solvents
iments with the solvent mixture of SC-CO2 with 10% by for the extraction of components from agricultural prod-
volume soybean oil, at 40 MPa and 70 C, were employed. ucts. On the other hand, olive oil can directly be consumed
Fig. 2 illustrates the effect of solvent flow rate on the as high quality oil with high content of unsaturated fatty
amount of extract and the concentration of the astaxanthin acids. The results of the SC-CO2 extraction of astaxanthin
extract with respect to extraction time. The extraction at a in the presence of olive oil and ethanol as co-solvents were
flow rate of 3 mL min 1 resulted in the maximum amount therefore of great interest. The extraction was conducted
of astaxanthin being extracted. The rapid increase of asta- with 10% co-solvent at 70 C, 40 MPa and with the flow
xanthin in the extracted stream was only observed at the rate of 3 mL min 1. With the same extraction conditions,
early stage of extraction. This was because during this soybean oil gave inferior efficiency to that with ethanol,
stage, the extraction of the surface attached astaxanthin indicating a possible limited solubility of soybean oil in
occurred, and increasing the flow rate reduced the external SC-CO2, compared with ethanol. On the other hand, SC-
mass transfer resistance leading to a faster initial rate of CO2 extraction with olive oil gave higher extraction effi-
extraction. However, the extraction at later stage was not ciency than soybean oil and gave comparable results to that
significantly affected by the flow rate. This was because this with ethanol, in which the efficiency of as high as
extraction was controlled mainly by diffusion of astaxan- 51.03 ± 1.08% was obtained. It could therefore be drawn
thin from the cell matrix, and increasing the flow rate could from these results that the solubility of olive oil in SC-
not manipulate the rate of diffusion and therefore similar CO2 was presumably higher than that in soybean oil. This
rate of extraction at later stage became apparent. is consistent with the existing literatures which reported
In fact, the effect of flow rate was twofold. Firstly, an lower temperature and pressure required for extraction of
increase in the flow rate increased the mass transfer, but olive oil (12–18 MPa) than soybean oil (30–60 MPa)
(Simões and Brunner, 1996; Hurtado-Benavides et al.,
2004).
Astaxanthin concentration (mg L-1)

50 1000
2 mL min-1 3 mL min-1 -1
4 mL min-1
4. Conclusions
Astaxanthin extracted (%)

-1 -1 -1
2 mL min-1 3 mL min-1 4 mL min-1
40 800
Soybean oil and olive oil used as co-solvents were inves-
30 600
tigated for SC-CO2 extraction of astaxanthin from H. plu-
20 400
vialis. The initial experiment was carried out to determine
the appropriate level of soybean oil in the solvent mixture
10 200 and the results indicated that the optimal level of soybean
oil was 10% by volume, in which the highest extraction effi-
0 0 ciency was 36.36 ± 0.79% at 70 C, 40 MPa, and at the sol-
0 60 120 180 240 300 360 vent flow rate of 3 mL min 1. In addition, the result of the
Extraction time (min) SC-CO2 extraction of astaxanthin with the presence of 10%
Fig. 2. Effect of solvent flow rate on SC-CO2 extraction of astaxanthin by volume olive oil showed the higher increase in the
from H. pluvialis with 10% soybean oil as co-solvent at 70 C and 40 MPa. amount of astaxanthin extracted. This was comparable to
5560 S. Krichnavaruk et al. / Bioresource Technology 99 (2008) 5556–5560
that obtained with 10% ethanol co-solvent. The results in
this study demonstrated clearly the superior extraction
power of having co-solvents such as soybean oil and olive
oil for the high pressure extraction of astaxanthin from
H. pluvialis.
Acknowledgements
The author would like to acknowledge the Thailand Re-
search Fund (TRF) and Kumamoto University 21st COE
‘‘Pulse Power Science’’ Program for their financial support.
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