J. of Supercritical Fluids 30 (2004) 155–161

Supercritical fluid extraction of redpepper (Capsicum frutescensL.)

Carla Duartea, Margarida Moldão-Martinsb, Ana F. Gouveiab,Sara Beirão da Costab, A. Eduardo Leitãoc, M. Gabriela Bernardo-Gila,∗

a Centro de Engenharia Biológica e Qu´ımica, DEQ, IST, Av. Rovisco Pais, 1049-001 Lisboa, Portugalb Centro de Microbiologia e Indústrias Agr´ıcolas, DAIAT, ISA, Tapada da Ajuda, 1349-017 Lisboa, Portugal

c Centro de Estudos de Produção e Tecnologia Agr´ıcolas, 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 fluid extraction (SFE) of red pepper (Capsicum frutescensL.) oleoresins. Theinfluence of pressure, and superficial velocity of supercritical CO2 at 313 K, on theCapsicum frutescensoleoresins yield andcapsaicinoids 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 whenn-hexane was usedfor the extraction of red pepper oleoresin in a Soxhlet apparatus. At 10 min of extraction time an optimal value of the yieldwas determined (5.2% (w/w)) for pressure of 21.5 MPa and superficial velocity of 0.071 cm s−1. An optimum value for theyield in capsaicinoids (0.252% (w/w)) occurred at 20.5 MPa and 0.064 cm s−1. The extract of the supercritical fluid processobtained at 21.5 MPa, 0.074 cm s−1 of CO2 superficial velocity during 10 min was chosen, in order to perform a sensorialanalysis. 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. Commercialred pepper flavoured olive oil (0.0011% (w/w) capsaicinoids) was used as reference. Panel preferred taste of the olive oiladded with extract that contains 0.0006% (w/w) of capsaicinoids. At this concentration SFE extract did not influence olive oilcolour.© 2003 Published by Elsevier B.V.

Keywords: Capsicum frutescens; CO2 extraction; Capsaicinoids; Sensorial analysis; Colour

1. Introduction

Capsicum frutescensL., commonly known as redpepper, 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, oblonglanceolate and 1.5–2.5 cm long.

Pungency (hot flavour) is the most important qual-ity attribute of red pepper. The substances responsiblefor pungency are capsaicinoids. The capsaicinoidsare odourless, colourless, flavourless and non-nutrientcompounds[1]. More than 20 capsaicinoids werecharacterised on theCapsicumgenus[1]. The most

0896-8446/$ – see front matter © 2003 Published by Elsevier B.V.doi:10.1016/j.supflu.2003.07.001

156 C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155–161

representative are capsaicin (C) and dihydrocapsaicin(D) (80–90% of the total).

Another attribute of theCapsicumgenus fruits isthe red colour due to the natural pigments, carotenoidsthat are synthesised massively during fruit ripening.The mainly responsible carotenoids for the final colourof the fruits are capsanthin and capsorubin[2].

Oily Capsicumextracts among pungent compoundscontains considerable amounts of carotenoids. Com-monly the oily extracts from pungent or non-pungentred pepper has been prepared by traditional extrac-tion methods using organic solvents, such as acetoneand cyclohexane[3]. Such extractions, using organicsolvents, have limitations in obtaining solvent-freeextracts. Compressed carbon dioxide has been widelyapplied to the extraction of oil and oleoresins fromdifferent plant products[3–9]. This separation tech-nique offers extraction yields very similar to thoseobtained by conventional extraction processes usingliquid solvents, but does require a certain combina-tion of operating conditions, depending on pressure,temperature, particle size or superficial velocity. Itsadvantages, compared to organic solvents, are thatCO2 is non-toxic, non-flammable, and that it is cheapand readily available in bulk quantity, with a highdegree of purity. In processing terms, CO2 has alow critical temperature and pressure (304.1 K and7.38 MPa, respectively), which makes it the idealsolvent for natural products, since they do not sufferthermal degradation during the process.

del Valle et al.[4] had obtained a very attractivelight yellow tinge oleoresin fromCapsicum annuum,at 40◦C, 120 bar, during 4 h of extraction.

The aim of this work was to study the influenceof pressure, and superficial velocity of supercriticalCO2 on the extraction ofCapsicum frutescensoleo-resins. The extracts were characterised and the resultswere compared with those obtained whenn-hexanewas used for the extraction of red pepper oleoresin inSoxhlet.

2. Materials and methods

2.1. Raw material

The red peppers were acquired from a Portugueseproducer and were preserved at−18◦C until they

were ground using a refrigerated knife mill (IKA—Universal muhle M20, 20000 min−1, Jank & KunkelGmBH & Co KG, Labortechnik Staufen, Germany).A sieving machine Retsch KS 1000 (Retsch, Haan,Germany) was used to adjust the medium diameter ofthe particles (0.23 mm) without the use of any solvent.The total oleoresin content was obtained by Soxh-let extraction usingn-hexane as solvent. The valuedetermined 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, aspro anal-ysis type, except the external standards for the cap-saicinoids analysis, which were from Sigma of HPLCgrade.

2.3. Supercritical fluid extraction

2.3.1. EquipmentExtraction measurements were carried out in a

semi-batch flow extraction apparatus built in InstitutoSuperior Técnico (Lisbon). Supercritical carbon diox-ide (SCCO2) was used as solvent. The supercriticalapparatus is identical to that described by Esquı́veland Bernardo-Gil[7]. The extraction experimentswere performed with a tubular extractor of 0.2 dm3

capacity (section area= 16.395 cm2), assembled asdescribed[7].

Liquid carbon dioxide from the supply cylinderpasses through a cold bath (about 275 K) and thenis pumped with an air driven liquid pump; modelMCP-71 (Haskel, Inc., USA) and heated in a tubularheat exchanger to the extraction temperature. Pressureis controlled with a backpressure regulator (TescomCo., Minnesota, USA, model number 26-1722-024-043).

The extractor, containing the raw material to beextracted, was placed in a controlled temperaturewater bath, the temperature inside the extractor be-ing controlled by a digital controller (Ero Electronic,Italy) within an accuracy of±0.05 K. The pressureat the exit of the extractor was measured by using amanometer (Skalenwert 2, Germany) with an accuracyof 0.2 MPa. After leaving the extractor, the stream of

C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155–161 157

carbon dioxide loaded with extract flowed throughan 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 reduceddown to atmospheric pressure, and the oily extract wasrecovered in a glass collector. Water and volatile com-ponents were deposited in a second collector that wasimmersed in a dry ice-acetone bath at approximately203 K.

A dry test meter (American Meter Company,Philadelphia, USA, DTM-200A) with accuracy of±0.005 l was used to measure the delivered volumeof carbon dioxide, being measured the pressure andtemperature conditions. The estimated accuracy ofthe pressure measurement was±0.01 MPa and tem-perature was measured with a mercury thermometerto within ±0.1 K.

Using this equipment, the mass of oleoresin extract(and hence the yield of extract) was determined asa function of extraction time and CO2 mass passedthrough the ground red pepper bed, at each of theconditions studied.

2.3.2. Extraction procedureBefore each set of yield determinations at given

extraction conditions, the extractor was manually filledwith a weighed quantity of ground red pepper (about0.050 kg) and CO2 was pumped into the extractor untilthe desired extraction pressure value. After ensuringthat there was no leak in the equipment, the expansionvalves were opened and a steady stream of the solventwas allowed to pass upward through the bed of groundparticles, at predetermined pressure and temperaturevalues. After a given extraction period these valveswere closed, and the weight of the extract depositedin the collector was determined.

The expansion valves and pipes leading from theextractor to the collector were washed withn-hexaneto remove any extract trapped in this region. To washthe pipes, then-hexane was fed through a cap fittingplaced just after the first expansion valve (which wasclosed during the washing operation). Then-hexanewas left in contact with the inside surfaces of thepipe for about 10 min and then was sucked out witha vacuum pump. The washings were collected andthe oleoresin was subsequently separated from thesolvent using a rotary evaporator VV2000 (Heidolph,Germany), and weighed.

Table 1Coded and uncoded matrix of the experiments (T = 313 K)

Run no. P Vs P (MPa) Vs

(cm s−1)CO2 density(kg m−3)

1 −1 −1 16.2 0.046 759.992 −1 +1 16.2 0.075 759.993 +1 −1 21.8 0.047 844.134 +1 +1 21.8 0.074 844.135 0 0 19 0.065 799.716 0 0 19 0.059 799.717 0 0 19 0.059 799.718 0 0 19 0.060 799.719 +√

2 0 15 0.061 742.9710 −√

2 0 23 0.060 854.5111 0 +√

2 19 0.081 799.7112 0 −√

2 19 0.041 799.71

After washing, the valves leading from the extrac-tor were reopened and extraction was resumed for afurther period as above. This procedure was repeateduntil sufficient oleoresin had been extracted to definethe form of the extraction curve at the extraction con-ditions in study. The extractor was then unloaded andcleaned.

An experimental design matrix (Table 1) was usedto study the influence of pressure (P) and superficialvelocity (Vs) on yields. The temperature was main-tained constant at 313 K, based on studies of capsaicinsolubilities at different temperatures[10].

2.4. HPLC analysis

Identification and quantification of capsaicin anddihydrocapsaicin were performed on a HPLC ap-paratus (Beckman System Gold Chromatograph,USA) composed of a 126 gradient pump and a 168diode-array detector connected to a Gold softwaredata module. The eluates were detected at 280 nm,on a LiChrospher 100 RP-18, 5�m, 250 mm× 4 mmcolumn with acetonitrile:water (70:30 (v/v)) as mo-bile phase and 1 ml min−1 flow rate. Capsaicin anddihydrocapsaicin were identified and quantified usingexternal standards.

Before injection (20�l) supercritical extracts werediluted in acetone (50�l:900�l).

2.5. Sensorial analysis

According to the total yield and the capsaicinoidscontents a SFE extract was chosen, in order to perform

158 C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155–161

a sensorial analysis. Selected extract (obtained at21.5 MPa, 0.074 cm s−1 of CO2 superficial velocityduring 10 min) was suspended in extra virgin oliveoil (0.7% acidity) supplied by a local producer in theSouthwest of Portugal. As reference a commercialflavoured olive oil withCapsicum(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 sensorylaboratory, Department of Food Science and Technol-ogy, ISA/UTL. Sensorial analysis was carried out by20 panel test persons (12 females and 8 males), stu-dents and staff of the food department and they canbe considered as experienced panel test persons. Allof them were non-smokers and their age ranged from20–56 years old. The room was equipped with sevenisolated sensory booths.

The samples were presented to the panellists inblue glasses, generally used for olive oil sensoryanalysis, with a shape that allows an evaluation ofaroma and prevents interference of light. The sam-ples for each assessor were prepared simultaneouslyabout 1 h prior to the sessions to allow the headspaceto develop. Samples were three-digit coded and

0

20

40

60

80

100

0 20 40 60 80 100 120 140 160 180g CO2 / g ground red pepper

Ole

ores

ins

reco

vere

d by

SF

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 CO2 per unity of initial mass of sample, at various values ofpressure and superficial velocity.

the order of serving was determined by randompermutation.

A rank preference test [11] was performed. Resultswere statistically interpreted by Friedman test, whichgives the maximum opportunities for demonstratingrecognition by the assessors of differences betweensamples.

2.6. Colour analysis

The colour of flavoured 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. TheL∗ value indicates lightness, a∗ value indicates chro-maticity on a green (−) to red (+) axis, and b∗ valuerepresents chromaticity on a blue (−) to yellow (+)axis.

3. Results and discussion

Fig. 1 shows the quantity of oleoresins from redpepper recovered by SFE as a function of mass ofcarbon dioxide per unit of initial mass of red pepperfor several values of pressure and superficial velocity.

C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155–161 159

0

10

20

30

40

50

60

70

80

90

100

720 740 760 780 800 820 840 860 880

CO2 density (kg m-3)

Ole

ores

ins

reco

vere

d by

SF

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 CO2 density, at 50 min of extraction time for different values of superficialvelocity (Vs).

As it can be seen, the higher the pressure, the higherare the extraction yields.

In Fig. 2, the percentages of red pepper oleoresinsrecovered by SFE at 50 min of extraction time arepresented as a function of carbon dioxide density atdifferent superficial velocity conditions.

The percentage of oleoresins recovered by SFE in-creases with CO2 density and also with the superficialvelocity.

Studies using the Statistica, version 5, software(Statsoft, Tulsa, OK, USA) were developed to ob-tain functional relations between the yield of redpepper oleoresins after 10 min of extraction (Y10)and after 50 min (Y50) as functions of pressure (P)and superficial velocity (Vs). The yield of capsaici-noids (capsaicin + dihydrocapsaicin) after 10 min ofextraction was also investigated.

The response surface of the yields of oleoresins af-ter 10 min of extraction is shown in Fig. 3 and it isdescribed as:

Y10 = 44.56 + 3.777P − 0.1027P2 + 258.1 Vs

− 3157 V 2s + 8.901P Vs,

R2 = 0.89, R2Adj = 0.79 (P < 0.05) (1)

Fig. 3. Response surface showing the relation between the redpepper oleoresins recovered by SFE at 10 min of extraction timeas a function of pressure (P) and superficial velocity (Vs).

160 C. Duarte et al. / J. of Supercritical Fluids 30 (2004) 155–161

Fig. 4. Response surface showing the relation between the redpepper oleoresins recovered by SFE at 50 min of extraction timeas a function of pressure (P) and superficial velocity (Vs).

where P represents the P-level, which is a measureof the statistical significance. R2 can be interpretedas the proportion of the variability in the data, andR2

Adj is the adjusted R2, which depends on the R2 andthe number of degrees of freedom [12,13]. When R2

and R2Adj differ dramatically there is a good chance

that non-significant terms have been included in themodel [13].

At 10 min of extraction time an optimal value ofthe yield was determined for pressure of 21.5 MPa andsuperficial velocity of 7.1 × 10−4 m s−1.

The response surface of the yields of oleoresins af-ter 50 min of extraction is shown in Fig. 4 and it isdescribed as

Y50 = 39.29 + 4.106P − 0.08964P2

+ 139.8Vs − 5.912PVs,

R2 = 0.88, R2Adj = 0.78 (P < 0.05) (2)

At 50 min of extraction, an optimum value of yieldof red pepper oleoresin occurred for the pressure of18.6 MPa.

The yields in capsaicinoids (capsaicin + dihydro-capsaicin) after 10 min of extraction, in function ofpressure and superficial velocity, are represented by

Fig. 5. Response surface showing the relation between the cap-saicin (C) + dihydrocapsaicin (D) content of red pepper oleoresinsrecovered by SFE at 10 min of extraction time as a function ofpressure (P) and superficial velocity (Vs).

the response surface of Fig. 5, which is describedas

C + D 10 = −2.638 + 0.2309P − 0.995631P2

+ 16.61Vs − 129.1Vs2,

R2 = 0.86, R2Adj = 0.77 (P < 0.05) (3)

The surface showed that an optimum value for theyield in capsaicinoids occurred within the experimen-tal pressure and superficial velocity ranges, being theoptimal values of pressure and superficial velocity, re-spectively, 20.5 MPa and 6.4 × 10−4 m s−1.

In what respects sensorial analysis, the attributearoma did not show to be significantly different for thefour samples (F < critical value from the Friedmantest at 0.05 level). Taste responses were significantlydifferent (P < 0.001). Panel preferred taste of theolive oil added with extract which conferrers 0.0006%(w/w) of capsaicinoids (FFO1). Commercial one wasbad scored as residual taste.

The results demonstrate that the flavouring withsupercritical fluid red pepper extracts did not signifi-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) 155–161 161

Table 2Colour parameters

L∗ a∗ b∗

OO 84.8 ± 1.7 15.0 ± 0.6 60.9 ± 6.7FOO1 84.5 ± 1.0 15.5 ± 0.1 69.7 ± 3.6FOO2 86.7 ± 2.6 13.6 ± 2.5 56.3 ± 8.7FOO3 85.8 ± 3.3 15.1 ± 0.2 61.5 ± 5.7CFO 79.1 ± 2.5 8.4 ± 1.3 92.4 ± 3.6

Duarte et al. [14], referring that capsaicinoids areeasily extracted by supercritical fluid extraction, inopposition to the pigments that are only extracted athigher extraction pressures.

Colour parameters of commercial flavoured oliveoil (CFO) are significantly different (P < 0.01) fromthose of the olive oils flavoured with SFE extracts.

4. Conclusions

The supercritical carbon dioxide can be used as sol-vent to obtain extracts from Capsicum frutescens. Highextraction yields in oleoresins and capsaicinoids wereobtained at pressures around 20–22 MPa and about0.06–0.07 cm s−1.

Extracts obtained at these conditions give pungen-cies to the virgin olive oil similar to that found in com-mercial flavoured olive oils, without modification oforiginal colour.

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