J Sci Food Agric 1998, 76, 189È194

Supercritical Carbon Dioxide Extraction of OnionOleoresinAgnes Sass-Kiss,1* Balint Czukor,2* Yanxiang Gao,1” Pal Stefanovits2 andFerenc Boross1

1 Department of Analytical Chemistry, Central Food Research Institute, Budapest, Hungary2 Department of Food Technology, Central Food Research Institute, Budapest, Hungary

(Received 9 July 1996 ; revised version received 25 April 1997 ; accepted 13 June 1997)

Abstract : Supercritical carbon dioxide (SC- extraction on a laboratory-CO2)scale, was applied to produce oleoresin from dried onion (Allium cepa L). Thegoal of this work was to determine the optimal conditions of extraction forproducing oleoresin with the highest yield and the best quality. A polynomialmodel was developed on the data. The square of correlation coefficient wasR2\ 0É9022 (at PO 0É05). The statistical analysis showed that the extractionpressure, temperature, and time have signiÐcant inÑuence on the yield of theoleoresin. Increasing these parameters resulted in an increase in the yield.Maximum yield was obtained when the extraction pressure and temperaturewere above 350 bar and 57¡C, respectively. At the lowest pressure (150 bar)applied the highest concentration of sulphur was produced in the oleoresin. Theconcentration of sulphur increased as a function of temperature when the pres-sure was maintained at 300 and 400 bar. The composition of volatile compoundsof oleoresin di†ered from that of distilled onion oil. 1998 SCI.(

J Sci Food Agric 76, 189È194 (1998)

Key words : supercritical extraction ; onion ; yield ; sulphur ; volatile com-CO2pounds

INTRODUCTION

Owing to the many advantages of carbon dioxide, suchas solvency, extraction properties at moderate tem-peratures (25È60¡C), non-toxicity, low cost, low criticaltemperature and inertness (Hubert and Vitzthum 1980 ;Rizvi et al 1986), supercritical carbon dioxide extraction(SC- has been extensively applied in the foodCO2)industry.

extraction has been used in industry for theSC-CO2deca†eination of co†ee and tea and the extraction ofÑavour and oleoresin from spices (Sharpe and Crabb1980 ; Lee 1988 ; Zosel 1989 ; King and Bott 1993).Removal of fat and cholesterol from animal products

* To whom correspondence should be addressed.” Present address : Department of Food Engineering TianjinUniversity of Light Industry, Tianjin, PeopleÏs Republic ofChina.

(Bradley 1989 ; Froning et al 1990), extraction of naturalpigments from plant materials (Spanos et al 1993 ;Mendes et al 1995) and extraction of Ñavours frommedicinal plants (Vuorela et al 1990 ; Reverchon et al1994) have also been reported as useful applications of

extraction.SC-CO2Onion (Allium cepa L) is an important Ñavouring that

commands universal application as seasonings and Ña-vourings in the food industry with the development ofsnacks and prepared foods. Furthermore, onion is usedin folk therapy worldwide because of its biologicallyactive sulphur compounds (Small et al 1947 ; Adamu etal 1982).

The characteristic Ñavour of onion is formed enzy-matically from odourless amino acid precursors namelyfrom trans-(])-S-1-propenyl-, (])-S-propyl-, (])-S-methyl-L-cysteine sulphoxide by the action of Alliinase(alliin alkyl-sulphenate-lyase), when the cells of theonion bulb are damaged (Whitaker 1976). The primary

1891998 SCI. J Sci Food Agric 0022-5142/98/$17.50. Printed in Great Britain(

190 A Sass-Kiss et al

products of enzyme reaction are sulphenic acids, whichimmediately transform into thiosulphinates (Schwimmerand Friedman 1972). The propanal-S-oxide(lacrymatory factor) which is a more stable form thanpropenyl sulphenic acid is produced from the trans-(])-S-1-propenyl-L-cysteine sulphoxide during enzymaticreaction (Freeman and Whenham 1975). Thiosulphin-ates decompose into disulphides, thiosulphonates, tri-sulphides containing methyl, propyl and 1-propenylgroups. While alkyl thiosulphonates, alkyl thiosulphin-ates are associated with fresh onion Ñavour (Boelens etal 1971 ; Block et al 1992a) the sulphides, di-, tri-, tetra-sulphides are characterised by a cooked onion-likeÑavour (Boelens et al 1971 ; Brodnitz et al 1969).

According to the publications alkyl sulphides, di-, tri-,tetrasulphides appear mainly in distilled onion oil(Carson and Wong 1961 ; Brodnitz et al 1969 ; Bandyo-padhyay et al 1970 ; Boelens et al 1971 ; Sass-Kiss et al1989).

The compounds characterised by a fresh onion-likeÑavour appear Ðrst in the solvent (Bayer et al 1989 ;Block et al 1992a,b) and extracts (Sinha et alSC-CO21992 ; Calvey et al 1994).

Information on the e†ects of extraction parameterson the yield and quality of oleoresins is not found in thecase of extraction of dried onion.SC-CO2

The aim of the present work was to investigate thefeasibility of extraction for producing of oleo-SC-CO2resin from dried onion and to study the e†ect of extrac-tion parameters on the yield and the quality of onionoleoresin.

EXPERIMENTAL

Fresh onion was purchased from a local retail market.The outer skins and sprout of bulbs were removed,washed and cut into 0É20 cm thick slices. They weredried in the oven at 60¡C for 48 h until the watercontent was below 10% and ground before extraction.

extractionSC-CO2

extraction was performed by using theSC-CO2laboratory-scale SFX 2-10 type supercritical Ñuidextraction instrument (Isco Inc, USA) with the series Dpump controller and the model 260 D syringe pump.The used for the extraction was of 99É99% purity.CO2

The Ñow diagram of extraction is presentedSC-CO2in Fig 1.

Five grams of onion powder were packed into 10 mlextraction chamber, the liquid was compressedCO2through the extraction chamber at the pressure andtemperature adjusted. In all cases extracts were trappedin 15 ml hexane, evaporated to dryness at 40¡C, and theweight of extracts was measured.

Fig 1. Diagram of laboratory-scale extraction apparatus.

Distillation of onion volatile oil

Seventy grams of onion powders were mixed with1200 ml distilled water and incubated at room tem-perature for 1 h. The distillation was carried out for 4 hin a special steam distillation apparatus (Sass-Kiss et al1989). The distillate was circulated through a V-shapedtrap with a screw-cap on top of it. Onion oil was col-lected in the trap. A portion of onion oil appeared inthe bottom of the trap the other one could be found onthe surface of distillate (emulsion). Thirty microlitres ofonion oil were collected and dissolved in diethyl etherafter removing a large portion of emulsion. The trapwas washed with diethyl ether several times. Solution ofdiethyl ether collected was Ðltered through an anhy-drous bed and evaporated at 38¡C.Na2SO4

Determination of sulphur concentration

Scho� nigerÏs method (1956) modiÐed (Bata Iunpublished) was used for the determination of sulphurconcentration in oleoresin and distilled oil. The prin-ciple of this method is that burning the samples in anoxygen atmosphere the oxygen makes organic sulphurtransform to sulphate that can be titrated. The onion oilwas packed in Ðltration paper (ash free) was taken in aplatinum mesh. After setting light to the sample, it wasplaced in an Erlenmeyer Ñask containing 5 ml waterand 5 drops hydrogen peroxide and Ðlled with oxygen.The time of burning was 30 min. The ash was washedwith 20 ml ethanol and the solution was titrated bybarium perclorate (0É01 mol dm~3) with 5 drops ofthorin indicator (0É01 mol dm~3). The factor of thetitrant was calculated by titration of organic com-pounds of known sulphur content.

Gas chromatographic analysis

The volatile compounds of distilled onion oil and oleo-resin were separated by Carlo Elba 2900 gas chromato-graph equipped with FID. A 30 m fused silica columnof 0É25 mm id, Supelcowax 10 with a Ðlm thickness of

SC-CO2 extraction of onion oleoresin 191

TABLE 1Independent variables and variation levels used in the small-scale experiments

V ariables V ariation levels Number of experiments

[1 0 ]1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Extraction pressure (bar) 150 300 450 [1 ]1 [1 ]1 [1 ]1 [1 ]1 0 0 0 0 0 0 0Extraction temperature (¡C) 35 50 65 [1 [1 ]1 ]1 0 0 0 0 [1 ]1 [1 ]1 0 0 0Extraction time (min) 30 60 90 0 0 0 0 [1 [1 ]1 ]1 [1 [1 ]1 ]1 0 0 0

0É25 lm was used. The operating conditions were asfollows : injector temperature 220¡C, detector tem-perature 250¡C, the head pressure of hydrogen carriergas 0É7 bar, oven temperature 50È190¡C with7¡C min~1 linear heating, the split ratio 1/20. The con-centration of samples was 10 g dm~3 in diethyl ether.One microlitre of the sample solution was injected.

Experimental design

Response surface methodology (RSM) was used todesign the small-scale extraction of onion oleo-SC-CO2resin according to the literature (Henika 1972). A three-variable, three-level fractional factorial design with tworeplicates at the centre point was used. The independentvariables, variation levels and the centre point of thedesign were chosen based on our preliminary experi-mental work. The outline of experimental design is pre-sented in Table 1.

Data analysis

All related experimental data were submitted to theanalysis of response surface by using the Statgraphiccomputer program (Statistical graphic system by Sta-tistical Graphic Corporation, Version 5, 1991).

RESULT AND DISCUSSION

extraction of onion oleoresinSC-CO2

The yield (Y ) of onion and oleoresin obtained under thedi†erent extraction variables can be found in Table 2and the regression analysis and the analysis of variance(ANOVA) are present in Table 3 and Table 4.

The data of Table 2 were used to develop the poly-nomial model shown in eqn (1) according to Table 3.

Y \4É56]3É18]10~2A[9É61]10~2B]5É28]10~2C

[1É93]10~4AB]1É70]10~4AC

[3É94]10~4BC[3É46]10~5A2

]2É17]10~3B2[3É02]10~4C2 (1)

The analysis of variance (Table 4) of the model devel-oped for this process indicated that the model Ðtted to

TABLE 2The e†ect of extraction variables on the yield of onion oleo-

resin (means, n \ 2)

Run Pressure T emperature T ime Y ield(bar) (¡C) (min) (g kg~1)

1 300 50 60 3É532 300 35 90 4É933 150 50 90 1É424 300 35 30 1É115 150 35 60 0É326 300 65 90 6É027 150 50 30 0É688 300 50 60 3É209 450 35 60 5É25

10 300 65 30 2É9111 150 65 60 2É0912 450 65 60 5É2813 450 50 30 2É0014 450 50 90 5É8015 300 50 60 3É85

the experimental data well. The correlation coefficient ofdetermination for the Ðt was R2\ 0É9022. The modelfailed to show any signiÐcant interaction e†ects betweenthe variables (P[ 0É05) and indicated that the contribu-tion of each variable to the yield could be ranked in thefollowing order : extraction pressure[ extractiontime[ extraction temperature.

Three dimensional response surface for the extractionprocess (time 60 min) is shown in Fig 2.

TABLE 3Regression coefficients for yieldÈ3-factor study

Constant [4É56A: pressure 3É18 ] 10~2B: temperature [9É61 ] 10~2C: time 5É28 ] 10~2AB [1É93 ] 10~4AC 1É7 ] 10~4BC [3É94 ] 10~4AA [3É46 ] 10~5BB 2É17 ] 10~3CC [3É02 ] 10~4

192 A Sass-Kiss et al

TABLE 4Analysis of variance for yieldÈ3-factor studya

E†ect Sum of squares df Mean squares F-Ratio P-value

A: pressure 23É8741 1 23É8741 66É10 0É0005B: temperature 2É7495 1 2É7495 7É61 0É0392C: time 16É4451 1 16É4451 45É53 0É0011AB 0É7569 1 0É7569 2É10 0É2074AC 2É3409 1 2É3409 6É48 0É0515BC 0É1260 1 0É1260 0É35 0É5864AA 2É2440 1 2É2440 6É21 0É0550BB 0É8790 1 0É8790 2É43 0É1795CC 0É2733 1 0É2733 0É76 0É4330Total error 1É8058 5 0É3612

Total (corr) 51É4946 14

a R-squared \ 0É9651. R-squared (adjusted for df) \ 0É9022.

The best way of Ðnding the optimum conditionwithin the experimental space under investigation fromthese response surfaces was to generate a contour plotof the response surface. A contour plot showing thee†ect of the extraction pressure and temperature on theyield of onion oleoresin is presented in Fig 3.

When the extraction time was Ðxed, the yield ofonion oleoresin increased with the increase of extractionpressure and temperature. At an extraction pressurebetween 350 and 450 bar and at an extraction tem-perature between 57 and 65¡C the maximum yield ofonion oleoresin was reached.

The Ñavour and biologically active compounds ofonion are attributed mainly to sulphur-containing com-pounds therefore the concentration of sulphur in oleo-resin was studied. The concentration of sulphur inoleoresin and distilled onion oil and the yield of

Fig 2. Three-dimensional response surface of the e†ect ofextraction pressure and temperature on yield (time 60 min).

extracted sulphur referred to raw material are shown inTable 5.

The concentration of sulphur in extracts wasSC-CO29É3È22É3 g kg~1 depending on the condition of theextraction. When the temperature and time were kept atconstant values (50¡C, 90 min or 35¡C, 60 min), theconcentration of sulphur in onion oleoresin was higherby 25% and 58% at 150 bar than at pressure 300 and450 bar, respectively.

The concentration of sulphur in oleoresin at 300 barand 400 bar increased by rising the temperature from 35to 65¡C. However, at 150 bar its concentrationdecreased. At all pressure levels applied the recovery ofsulphur increased in parallel with the yield when thetemperature was arised from 35 to 65¡C. The change ofthe quantity of extracted sulphur and the yield as afunction of rising temperature is shown in Table 6.

Fig 3. Counter plot showing the e†ect of extraction pressureand temperature on the yield of oleoresin (time 60 min).

SC-CO2 extraction of onion oleoresin 193

TABLE 5The change of sulphur by extraction parameters (means, n \ 2)

Extraction conditions Concentration Y ieldof of

Pressure T emperature T ime sulphur sulphur(bar) (¡C) (min) (g É kg~1 (g É kg~1 raw

oleoresin) material)

150 35 60 22É3 0É007150 50 90 20É1 0É028150 65 60 13É4 0É028300 35 90 10É9 0É054300 50 60 10É5 0É037300 65 90 14É9 0É090450 35 60 9É3 0É049450 50 90 16É2 0É093

Distilled onion oil 418

The change of yield in Table 6 is nearly proportionalto the change of non-sulphur-containing compounds.According to this it can be established, that at 150 barthe increase of the quantity of the non-sulphur com-pounds from 35 to 65¡C was larger (4É4È6É5 times) thanthat of sulphur compounds (4É0 times). Consequently,the concentration of sulphur decreased in oleoresin. At300 and 450 bar the recovery of sulphur-containingcompounds increased (1É7È1É9 times) as a result of risingtemperature while that of the others showed much lessincrease (1É2È1É1 times). As a consequence sulphur con-centration of oleoresin substantially increased.

Summarising, it can be established that at pressure300 and 400 bar the onion Ñavour can be strengthenedin the oleoresin by arising the temperature.

From the data of Table 5 it can be seen that the dis-tilled onion oil contains 19È45 times more sulphur thanthe extracts obtained by Distilled onion oil isSC-CO2 .

TABLE 6The change of the quantity of extractable sulphur and the

yield by arise of the temperature comparing to 35¡C

Pressure T emperature T ime Quantity (t¡C)a(bar) (¡C) (min)

Quantity (35¡C)

Sulphur Y ield

150 35 60 1 1150 50 90 4É0 4É4150 65 60 4É0 6É5

300 35 90 1 1300 50 60 0É7 0É7300 65 90 1É7 1É2

450 35 60 1 1450 50 90 1É9 1É1

a t¡C: 50¡C or 65¡C.

composed of almost exclusively of volatile sulphur com-pounds (onion Ñavour compounds). Onion oleoresincontains low level of sulphur compounds, it is due tothe high concentration of non-sulphur compoundsextracted. The other thing that may cause this is thatpolar sulphur compounds of onion (eg aroma precur-sors, cycloallin) occurring in large quantity in rawmaterial can not be extracted by non-polar SC-CO2 .The kind of non-sulphur-containing compounds presentin high concentration in onion oleoresin has beenhardly reported yet.

The composition of volatile compounds of oleoresinwas analysed by gas chromatography. Figure 4 showsthe gas chromatogram of oleoresin and distilled onionoil prepared from dried onion also.

The compounds with the same retention time wereconnected with dotted lines in Fig 4. In the oleoresin

Fig 4. Gas chromatogram of distilled oil and extractSC-CO2of dried onion. Column (0É25 mm id, 30 m) coated with Supel-cowax 10. Temperature programme: 50¡CÈ190¡C at

6¡C min~1 linear. A, distilled oil ; B, extract.SC-CO2

194 A Sass-Kiss et al

prepared by extraction several compoundsSC-CO2appeared that did not occur in the distilled oil. Thevolatile compounds representing the distilled oil thatconsists almost of sulphides di-, tri-, tetrasulphides(Brodnitz et al 1969 ; Boelens et al 1971 ; Sass-Kiss et al1986) were found at low concentration in the volatilepart of extracts. It was in compliance with theSC-CO2results of the concentration of sulphur.

CONCLUSION

The results indicated that the extraction can beSC-CO2successfully applied for producing oleoresin from driedonion. The extraction conditions have a†ected the yieldand the composition of oleoresin. The yield of onionoleoresin can be increased by the proper selection of theextraction variables, namely by arising the pressure,temperature and time. In point of view of onion Ñavourthe 300 or 450 bar pressure and the 65¡C temperatureare the most suitable conditions for producing of oleo-resin with stronger onion characteristics.

REFERENCES

Adamu I, Joseph P K, Augusti K T 1982 Hypolipidemicaction of onion and garlic unsaturated oils in sucrose fedrats over a two month period. Experimantia 38 899È900.

Bandyopadhyay C, Srirangarajana A N, Sreenivasan A 1970Studies on Ñavor compounds of onion (Allium cepa) I. Thin-layer chromatographic investigation of onion oil. J Chro-matogr 47 400È407.

Bayer T, Breu W, Seligman O, Wray V, Wagner H 1989 Bio-logically active thiosulphinates and alfa-sulphinyl disulÐdesfrom Allium cepa. Phytochemistry 28 2373È2377.

Block E, Naganathan S, Putman D, Zhao S H 1992a Alliumchemistry : HPLC analysis of tiosulÐnates from onion,garlic, wild garlic (Ramsoms), leek, scallion, shallot, ele-phant (great headed) garlic, chive, and Chinese chive.Uniquely high allyl methyl ratios in same garlic samples. JAgric Food Chem 40 2418-2430.

Block E, Putman D, Zhao S H 1992b Allium chemistry :GC-MS analysis of thiosulÐnates and related compoundsfrom onion leek, scallion, shallot, chive, and Chinese chive.J Agric Food Chem 40 2431È2438.

Boelens M, De Valois P J, Wobben H J, Van Der Gen A 1971Volatile Ñavour compounds from onion. J Agric FoodChem 19 984È991.

Bradley R L Jr 1989 Removal of cholesterol from milk fatusing supercritical carbon dioxid. J Dairy Sci 72 2834È2843.

Brodnitz M H, Pollock C L, Vallon P P 1969 Flavour com-ponents of onion oil. J Agric Food Chem 17 760È763.

Calvey E M, Matusik J E, White K D, Betz J M, Block E,Littlejohn M H, Naganathan S, Putman D 1994 O†-line

supercritical Ñuid extraction of thiosulÐnates from garlicand onion. J Agric Food Chem 42 1335È1341.

Carson J F, Wong F F 1961 The volatile Ñavor compounds ofonion. J Agric Food Chem 9 140È143.

Freeman G G, Whenham R J 1975 A rapid spectro-photometric method of determination of thiopropanalS-oxid (lachrymator) in onion (Allium cepa L) and its signiÐ-cance in Ñavour study. J Sci Food Agric 26 1529È1543.

Froning G W, Wehling R L, Cuppett S L, Pierce M M,Niemann L, Siekman D K 1990 Extraction of cholesteroland other lipids from dried egg yolk using supercriticalcarbon dioxide. J Food Sci 55 95È98.

Henika R G 1972 Simple and e†ective system for use withresponse surface methodology. Cereal Sci T oday 17 309È312, 314, 334.

Hubert P, Vitzthum O 1980 Fluid extraction of hops, spicesand tobacco with supercritical gases. In : Extraction withSupercritical Gases, eds Schneider G M, Stahl E & Wilke G.Verlag Chemie, DeerÐeld Beach, FL, USA, pp 25È43.

King M B, Bott T R 1993 Extraction of Natural Productsusing Near-Critical Solvents. Blackie Academic & Pro-fessional, Glasgow, UK, p 325.

Lee S 1988 Supercritical deca†eination. T ea Co†ee T rade J160 3È5.

Mendes R L, Fernandes H L, Coelho J P, Reis E C, CabralJ M S, Novais J M, Palavra A F 1995 Supercritical CO2extraction of carotenoids and other lipids from Chlorellavulgaris. Food Chem 53 99È103.

Revechon E, Ambruosi A, Senatore F 1994 Isolation of pep-permint oil using supercritical extraction. Flavour &CO2Fragrance J 9 19È23.

Rizvi S S H, Benado A L, Zollweg J A, Daniels J A 1986Supercritical Ñuid extraction : fundamental principles andmodeling methods. Food T echnol 40 55È65.

Sass-Kiss A, Petro� -Turza M, Szarfo� ldi-Szalma I, Pino J,Rosado A 1989 Investigation on the volatile oil of Mako�onions. Die Nahrung 33 413È421.

Schwimmer S, Friedman M 1972 Genesis of volatile sulphur-containing food Ñavours. Flavour Ind 3 137È145.

Sharpe F R, Crabb D 1980 Pilot plant extraction of hops withliquid carbon dioxide and the use of these extracts in pilotand production scale brewing. J Inst Brew 86 60È64.

Sinha N K, Guyer D E, Gage D A, Lira C T 1992 Super-critical carbon dioxide extraction of onion Ñavors and theiranalysis by gas chromatographyÈmass spectrometry. JAgric Food Chem 40 842È845.

Small L D V, Bailey J H, Cavallito C J 1947 Alkyl-thiosulÐnates. J Am Chem Soc 69 1710È1713.

Spanos G A, Hao-Chen, Schwartz S J 1993 Supercritical CO2extraction of beta-carotene from sweet potatoes. J Food Sci58 817È820.

Vuorela H, Holm Y, Hiltunen R, Harvala T, Laitinen A 1990Extraction of the volatile oil in chamomile Ñowerheadsusing supercritical carbon dioxide. Flavour Fragrance J 581È84.

Whitaker J R 1976 Development of Ñavour, odour and pun-gency in onion and garlic. Adv Food Res 22 73È133.

Zosel K 1989 Process for deca†einationmof of co†ee. GermanFederal Patent 2905078C2.