J.

of Supercritical Fluids 30 (2004) 155161

Supercritical uid extraction of red
pepper (Capsicum frutescens L.)
Carla Duarte
a
, Margarida Moldo-Martins
b
, Ana F. Gouveia
b
,
Sara Beiro da Costa
b
, A. Eduardo Leito
c
, M. Gabriela Bernardo-Gil
a,
a
Centro de Engenharia Biolgica e Qumica, DEQ, IST, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
b
Centro de Microbiologia e Indstrias Agrcolas, DAIAT, ISA, Tapada da Ajuda, 1349-017 Lisboa, Portugal
c
Centro de Estudos de Produo e Tecnologia Agrcolas, IICT, Tapada da Ajuda, Apartado 3014, 1301-901 Lisboa, Portugal
Received 10 February 2003; received in revised form 23 June 2003; accepted 18 July 2003
Abstract
The aim of this work was to assess supercritical uid extraction (SFE) of red pepper (Capsicum frutescens L.) oleoresins. The
inuence of pressure, and supercial velocity of supercritical CO
2
at 313 K, on the Capsicum frutescens oleoresins yield and
capsaicinoids content was studied. The central composite, non-factorial design was used to optimise the extraction conditions,
using the Statistica, version 5 software (Statsoft). The results were compared with those obtained when n-hexane was used
for the extraction of red pepper oleoresin in a Soxhlet apparatus. At 10 min of extraction time an optimal value of the yield
was determined (5.2% (w/w)) for pressure of 21.5 MPa and supercial velocity of 0.071 cms
1
. An optimum value for the
yield in capsaicinoids (0.252% (w/w)) occurred at 20.5 MPa and 0.064 cms
1
. The extract of the supercritical uid process
obtained at 21.5 MPa, 0.074 cms
1
of CO
2
supercial velocity during 10 min was chosen, in order to perform a sensorial
analysis. Selected extract was suspended in extra virgin olive oil (0.7% acidity) at three capsaicinoids contents (0.0006%
(w/w), 0.0011% (w/w) and 0.0015% (w/w)) and analysed in terms of pungency, aroma, taste and after taste. Commercial
red pepper avoured olive oil (0.0011% (w/w) capsaicinoids) was used as reference. Panel preferred taste of the olive oil
added with extract that contains 0.0006% (w/w) of capsaicinoids. At this concentration SFE extract did not inuence olive oil
colour.
2003 Published by Elsevier B.V.
Keywords: Capsicum frutescens; CO
2
extraction; Capsaicinoids; Sensorial analysis; Colour
1. Introduction
Capsicum frutescens L., commonly known as red
pepper, is used world-wide as a natural condiment in

Corresponding author. Tel.: +351-21-841-7582;

fax: +351-21-841-9176.
E-mail address: gbernardo.gil@ist.utl.pt (M.G. Bernardo-Gil).
food as well as raw material for pharmaceutical in-
dustries. The fruit is commonly red when ripe, oblong
lanceolate and 1.52.5 cm long.
Pungency (hot avour) is the most important qual-
ity attribute of red pepper. The substances responsible
for pungency are capsaicinoids. The capsaicinoids
are odourless, colourless, avourless and non-nutrient
compounds [1]. More than 20 capsaicinoids were
characterised on the Capsicum genus [1]. The most
0896-8446/$ see front matter 2003 Published by Elsevier B.V.
doi:10.1016/j.supu.2003.07.001
156 C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155161
representative are capsaicin (C) and dihydrocapsaicin
(D) (8090% of the total).
Another attribute of the Capsicum genus fruits is
the red colour due to the natural pigments, carotenoids
that are synthesised massively during fruit ripening.
The mainly responsible carotenoids for the nal colour
of the fruits are capsanthin and capsorubin [2].
Oily Capsicum extracts among pungent compounds
contains considerable amounts of carotenoids. Com-
monly the oily extracts from pungent or non-pungent
red pepper has been prepared by traditional extrac-
tion methods using organic solvents, such as acetone
and cyclohexane [3]. Such extractions, using organic
solvents, have limitations in obtaining solvent-free
extracts. Compressed carbon dioxide has been widely
applied to the extraction of oil and oleoresins from
different plant products [39]. This separation tech-
nique offers extraction yields very similar to those
obtained by conventional extraction processes using
liquid solvents, but does require a certain combina-
tion of operating conditions, depending on pressure,
temperature, particle size or supercial velocity. Its
advantages, compared to organic solvents, are that
CO
2
is non-toxic, non-ammable, and that it is cheap
and readily available in bulk quantity, with a high
degree of purity. In processing terms, CO
2
has a
low critical temperature and pressure (304.1 K and
7.38 MPa, respectively), which makes it the ideal
solvent for natural products, since they do not suffer
thermal degradation during the process.
del Valle et al. [4] had obtained a very attractive
light yellow tinge oleoresin from Capsicum annuum,
at 40

C, 120 bar, during 4 h of extraction.

The aim of this work was to study the inuence
of pressure, and supercial velocity of supercritical
CO
2
on the extraction of Capsicum frutescens oleo-
resins. The extracts were characterised and the results
were compared with those obtained when n-hexane
was used for the extraction of red pepper oleoresin in
Soxhlet.
2. Materials and methods
2.1. Raw material
The red peppers were acquired from a Portuguese
producer and were preserved at 18

C until they
were ground using a refrigerated knife mill (IKA
Universal muhle M20, 20000 min
1
, Jank & Kunkel
GmBH & Co KG, Labortechnik Staufen, Germany).
A sieving machine Retsch KS 1000 (Retsch, Haan,
Germany) was used to adjust the medium diameter of
the particles (0.23 mm) without the use of any solvent.
The total oleoresin content was obtained by Soxh-
let extraction using n-hexane as solvent. The value
determined was 10.1% (w/w) at 8 h of extraction.
2.2. Solvents
The used carbon dioxide had a purity of 99.995%
(N45) and was supplied by AR LIQUIDO-Portugal.
All other solvents and reagents used in analytical de-
terminations were purchased at Merck, as pro anal-
ysis type, except the external standards for the cap-
saicinoids analysis, which were from Sigma of HPLC
grade.
2.3. Supercritical uid extraction
2.3.1. Equipment
Extraction measurements were carried out in a
semi-batch ow extraction apparatus built in Instituto
Superior Tcnico (Lisbon). Supercritical carbon diox-
ide (SCCO
2
) was used as solvent. The supercritical
apparatus is identical to that described by Esquvel
and Bernardo-Gil [7]. The extraction experiments
were performed with a tubular extractor of 0.2 dm
3
capacity (section area = 16.395 cm
2
), assembled as
described [7].
Liquid carbon dioxide from the supply cylinder
passes through a cold bath (about 275 K) and then
is pumped with an air driven liquid pump; model
MCP-71 (Haskel, Inc., USA) and heated in a tubular
heat exchanger to the extraction temperature. Pressure
is controlled with a backpressure regulator (Tescom
Co., Minnesota, USA, model number 26-1722-024-
043).
The extractor, containing the raw material to be
extracted, was placed in a controlled temperature
water bath, the temperature inside the extractor be-
ing controlled by a digital controller (Ero Electronic,
Italy) within an accuracy of 0.05 K. The pressure
at the exit of the extractor was measured by using a
manometer (Skalenwert 2, Germany) with an accuracy
of 0.2 MPa. After leaving the extractor, the stream of
C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155161 157
carbon dioxide loaded with extract owed through
an on/off valve and a sequence of pressure expan-
sion valves (Hoke needle valve (Hoke, Inc., Cresskill,
USA)). The stream pressure was in this way reduced
down to atmospheric pressure, and the oily extract was
recovered in a glass collector. Water and volatile com-
ponents were deposited in a second collector that was
immersed in a dry ice-acetone bath at approximately
203 K.
A dry test meter (American Meter Company,
Philadelphia, USA, DTM-200A) with accuracy of
0.005 l was used to measure the delivered volume
of carbon dioxide, being measured the pressure and
temperature conditions. The estimated accuracy of
the pressure measurement was 0.01 MPa and tem-
perature was measured with a mercury thermometer
to within 0.1 K.
Using this equipment, the mass of oleoresin extract
(and hence the yield of extract) was determined as
a function of extraction time and CO
2
mass passed
through the ground red pepper bed, at each of the
conditions studied.
2.3.2. Extraction procedure
Before each set of yield determinations at given
extraction conditions, the extractor was manually lled
with a weighed quantity of ground red pepper (about
0.050 kg) and CO
2
was pumped into the extractor until
the desired extraction pressure value. After ensuring
that there was no leak in the equipment, the expansion
valves were opened and a steady stream of the solvent
was allowed to pass upward through the bed of ground
particles, at predetermined pressure and temperature
values. After a given extraction period these valves
were closed, and the weight of the extract deposited
in the collector was determined.
The expansion valves and pipes leading from the
extractor to the collector were washed with n-hexane
to remove any extract trapped in this region. To wash
the pipes, the n-hexane was fed through a cap tting
placed just after the rst expansion valve (which was
closed during the washing operation). The n-hexane
was left in contact with the inside surfaces of the
pipe for about 10 min and then was sucked out with
a vacuum pump. The washings were collected and
the oleoresin was subsequently separated from the
solvent using a rotary evaporator VV2000 (Heidolph,
Germany), and weighed.
Table 1
Coded and uncoded matrix of the experiments (T = 313 K)
Run no. P V
s
P (MPa) V
s
(cms
1
)
CO
2
density
(kg m
3
)
1 1 1 16.2 0.046 759.99
2 1 +1 16.2 0.075 759.99
3 +1 1 21.8 0.047 844.13
4 +1 +1 21.8 0.074 844.13
5 0 0 19 0.065 799.71
6 0 0 19 0.059 799.71
7 0 0 19 0.059 799.71
8 0 0 19 0.060 799.71
9 +

2 0 15 0.061 742.97
10

2 0 23 0.060 854.51
11 0 +

2 19 0.081 799.71
12 0

2 19 0.041 799.71
After washing, the valves leading from the extrac-
tor were reopened and extraction was resumed for a
further period as above. This procedure was repeated
until sufcient oleoresin had been extracted to dene
the form of the extraction curve at the extraction con-
ditions in study. The extractor was then unloaded and
cleaned.
An experimental design matrix (Table 1) was used
to study the inuence of pressure (P) and supercial
velocity (V
s
) on yields. The temperature was main-
tained constant at 313 K, based on studies of capsaicin
solubilities at different temperatures [10].
2.4. HPLC analysis
Identication and quantication of capsaicin and
dihydrocapsaicin were performed on a HPLC ap-
paratus (Beckman System Gold Chromatograph,
USA) composed of a 126 gradient pump and a 168
diode-array detector connected to a Gold software
data module. The eluates were detected at 280 nm,
on a LiChrospher 100 RP-18, 5 m, 250 mm 4 mm
column with acetonitrile:water (70:30 (v/v)) as mo-
bile phase and 1 ml min
1
ow rate. Capsaicin and
dihydrocapsaicin were identied and quantied using
external standards.
Before injection (20 l) supercritical extracts were
diluted in acetone (50 l:900 l).
2.5. Sensorial analysis
According to the total yield and the capsaicinoids
contents a SFE extract was chosen, in order to perform
158 C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155161
a sensorial analysis. Selected extract (obtained at
21.5 MPa, 0.074 cms
1
of CO
2
supercial velocity
during 10 min) was suspended in extra virgin olive
oil (0.7% acidity) supplied by a local producer in the
Southwest of Portugal. As reference a commercial
avoured olive oil with Capsicum (0.0011% (w/w)
of capsaicinoids) (CFO) was used. Three concentra-
tions of capsaicinoids were tested: (FOO1) 0.0006%
(w/w), (FOO2) 0.0011% (w/w) and (FOO3) 0.0015%
(w/w). Samples were ranked in the following way:
pungency, aroma, taste and after taste.
Sensory evaluation was conducted in the sensory
laboratory, Department of Food Science and Technol-
ogy, ISA/UTL. Sensorial analysis was carried out by
20 panel test persons (12 females and 8 males), stu-
dents and staff of the food department and they can
be considered as experienced panel test persons. All
of them were non-smokers and their age ranged from
2056 years old. The room was equipped with seven
isolated sensory booths.
The samples were presented to the panellists in
blue glasses, generally used for olive oil sensory
analysis, with a shape that allows an evaluation of
aroma and prevents interference of light. The sam-
ples for each assessor were prepared simultaneously
about 1 h prior to the sessions to allow the headspace
to develop. Samples were three-digit coded and

0
20
40
60
80
100
0 20 40 60 80 100 120 140 160 180
g CO
2
/ g ground red pepper
O
l
e
o
r
e
s
i
n
s

r
e
c
o
v
e
r
e
d

b
y

S
F
E

(
w
/
w

%
)
run 12
run 9
run 5
run 7
run 10
run 11
run 4
run 3
run 1
run 2
Fig. 1. Red pepper oleoresins recovered by SFE in function of mass of CO
2
per unity of initial mass of sample, at various values of
pressure and supercial velocity.
the order of serving was determined by random
permutation.
A rank preference test [11] was performed. Results
were statistically interpreted by Friedman test, which
gives the maximum opportunities for demonstrating
recognition by the assessors of differences between
samples.
2.6. Colour analysis
The colour of avoured olive oils with red pep-
per SFE extracts was evaluated with a colorimeter
(CR 300 Minolta, Osaka, Japan) by measuring L

,
a

and b

parameters. A white tile (L

= 97.10;
a

= 0.08; b

= 1.80) was used as reference. The

L

value indicates lightness, a

value indicates chro-

maticity on a green () to red (+) axis, and b

value
represents chromaticity on a blue () to yellow (+)
axis.
3. Results and discussion
Fig. 1 shows the quantity of oleoresins from red
pepper recovered by SFE as a function of mass of
carbon dioxide per unit of initial mass of red pepper
for several values of pressure and supercial velocity.
C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155161 159
0
10
20
30
40
50
60
70
80
90
100
720 740 760 780 800 820 840 860 880
CO
2
density (kg m
-3
)
O
l
e
o
r
e
s
i
n
s

r
e
c
o
v
e
r
e
d

b
y

S
F
E

(
w
/
w

%
)
Vs=0.061 cm/s
Vs=0.046 cm/s
Vs=0.074 cm/s
Vs=0.041 cm/s
Vs=0.081 cm/s
Fig. 2. Red pepper oleoresins recovered by SFE in function of CO
2
density, at 50 min of extraction time for different values of supercial
velocity (V
s
).
As it can be seen, the higher the pressure, the higher
are the extraction yields.
In Fig. 2, the percentages of red pepper oleoresins
recovered by SFE at 50 min of extraction time are
presented as a function of carbon dioxide density at
different supercial velocity conditions.
The percentage of oleoresins recovered by SFE in-
creases with CO
2
density and also with the supercial
velocity.
Studies using the Statistica, version 5, software
(Statsoft, Tulsa, OK, USA) were developed to ob-
tain functional relations between the yield of red
pepper oleoresins after 10 min of extraction (Y10)
and after 50 min (Y50) as functions of pressure (P)
and supercial velocity (V
s
). The yield of capsaici-
noids (capsaicin + dihydrocapsaicin) after 10 min of
extraction was also investigated.
The response surface of the yields of oleoresins af-
ter 10 min of extraction is shown in Fig. 3 and it is
described as:
Y10 = 44.56 +3.777P 0.1027P
2
+258.1 V
s
3157 V
2
s
+8.901P V
s
,
R
2
= 0.89, R
2
Adj
= 0.79 (P < 0.05) (1)
Fig. 3. Response surface showing the relation between the red
pepper oleoresins recovered by SFE at 10 min of extraction time
as a function of pressure (P) and supercial velocity (V
s
).
160 C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155161
Fig. 4. Response surface showing the relation between the red
pepper oleoresins recovered by SFE at 50 min of extraction time
as a function of pressure (P) and supercial velocity (V
s
).
where P represents the P-level, which is a measure
of the statistical signicance. R
2
can be interpreted
as the proportion of the variability in the data, and
R
2
Adj
is the adjusted R
2
, which depends on the R
2
and
the number of degrees of freedom [12,13]. When R
2
and R
2
Adj
differ dramatically there is a good chance
that non-signicant terms have been included in the
model [13].
At 10 min of extraction time an optimal value of
the yield was determined for pressure of 21.5 MPa and
supercial velocity of 7.1 10
4
ms
1
.
The response surface of the yields of oleoresins af-
ter 50 min of extraction is shown in Fig. 4 and it is
described as
Y50 = 39.29 +4.106P 0.08964P
2
+139.8V
s
5.912PV
s
,
R
2
= 0.88, R
2
Adj
= 0.78 (P < 0.05) (2)
At 50 min of extraction, an optimum value of yield
of red pepper oleoresin occurred for the pressure of
18.6 MPa.
The yields in capsaicinoids (capsaicin + dihydro-
capsaicin) after 10 min of extraction, in function of
pressure and supercial velocity, are represented by
Fig. 5. Response surface showing the relation between the cap-
saicin (C) + dihydrocapsaicin (D) content of red pepper oleoresins
recovered by SFE at 10 min of extraction time as a function of
pressure (P) and supercial velocity (V
s
).
the response surface of Fig. 5, which is described
as
C +D 10 = 2.638 +0.2309P 0.995631P
2
+16.61V
s
129.1V
s
2
,
R
2
= 0.86, R
2
Adj
= 0.77 (P < 0.05) (3)
The surface showed that an optimum value for the
yield in capsaicinoids occurred within the experimen-
tal pressure and supercial velocity ranges, being the
optimal values of pressure and supercial velocity, re-
spectively, 20.5 MPa and 6.4 10
4
ms
1
.
In what respects sensorial analysis, the attribute
aroma did not show to be signicantly different for the
four samples (F < critical value from the Friedman
test at 0.05 level). Taste responses were signicantly
different (P < 0.001). Panel preferred taste of the
olive oil added with extract which conferrers 0.0006%
(w/w) of capsaicinoids (FFO1). Commercial one was
bad scored as residual taste.
The results demonstrate that the avouring with
supercritical uid red pepper extracts did not signi-
cantly (P > 0.05%) affect the colour parameters (L

,
a

and b

) of the original virgin olive oil (Table 2).

These results are in accordance to those reported by
C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155161 161
Table 2
Colour parameters
L

OO 84.8 1.7 15.0 0.6 60.9 6.7

FOO1 84.5 1.0 15.5 0.1 69.7 3.6
FOO2 86.7 2.6 13.6 2.5 56.3 8.7
FOO3 85.8 3.3 15.1 0.2 61.5 5.7
CFO 79.1 2.5 8.4 1.3 92.4 3.6
Duarte et al. [14], referring that capsaicinoids are
easily extracted by supercritical uid extraction, in
opposition to the pigments that are only extracted at
higher extraction pressures.
Colour parameters of commercial avoured olive
oil (CFO) are signicantly different (P < 0.01) from
those of the olive oils avoured with SFE extracts.
4. Conclusions
The supercritical carbon dioxide can be used as sol-
vent to obtain extracts fromCapsicumfrutescens. High
extraction yields in oleoresins and capsaicinoids were
obtained at pressures around 2022 MPa and about
0.060.07 cms
1
.
Extracts obtained at these conditions give pungen-
cies to the virgin olive oil similar to that found in com-
mercial avoured olive oils, without modication of
original colour.
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