2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering

EXTRACTION OF GARLIC OIL WITH QUASI-CRITICAL SOLVENTS
A.E. Andreatta, G. Foco, G. Mabe and S.B. Bottini∗
PLAPIQUI, Universidad Nacional del Sur-CONICET Abstract. Garlic oil can be extracted from fresh garlic bulbs using traditional hydro-distillation or steam stripping processes or alternatively by applying new techniques such as high-pressure extraction with nearcritical fluids. In this respect, carbon dioxide is the preferred near-critical solvent, because it has a low critical temperature, it is non-toxic, non-flammable, easily available and relatively cheap. Garlic oil contains sulfur organic compounds such as allyl sulfides, produced by the action of an enzyme (alliinase) on precursor amino acids. The oil is used not only as a flavor agent, but also as a nutraceutical. In this work garlic oil has been extracted from fresh crushed bulbs, applying the traditional techniques of hydro-distillation and steam stripping at atmospheric pressures, and a high-pressure Soxhlet extractor working with near-critical carbon dioxide at a temperature of 288K and a pressure equal to 51 bar. The extracts were analyzed by gas and liquid chromatography (HPLC and GC-MS). The oil extracted with liquid CO2 has the typical flavor of fresh garlic, while the oils obtained by hydro-distillation and steam stripping have the characteristic smell of cooked garlic. The presence of sulfoxides in the extracts obtained with near-critical CO2 give an indication of the difference in quality between the processes applied.

Keywords: Garlic, Extraction, Quasi-Critical CO2.

1. Introduction
The processing of garlic bulbs has considerable advantages for the food industry, since it reduces the problems derived from seasonal variations, handling and transport of the fresh product. The oil of garlic, rich in sulfur organic compounds, is used not only as flavor agent, but also in the prevention and treatment of several illnesses. Cysteine-sulfoxides and γ-glutamyl-cysteines are the main sulfur compounds contained in fresh garlic bulbs and they are present at approximately equal quantities. While the first group of compounds are the precursors of the characteristic garlic flavor, the second group are considered as a reserve for the first ones, since γ-glutamyl-alk(en)cysteines can turn into alk(en)cysteine-sulfoxides during storage and bulb sprouting (Yu et al.,1994). The olfactory and gustatory principles of freshly cut garlic are associated with the presence of alk(en)yl thiosulfinate RS-(O)-SR and related compounds, produced by the action of the enzyme allinase on precursor amino acids such us alk(en)cysteine-sulfoxides, when the cellular tissue is disrupted (Whitaker, 1976). Allicin (diallyl thiosulfinate) is the main thiosulfinate, responsible of many of the bioactive attributes of garlic extracts. Harsh processing conditions such as hydro-distillation or steam stripping convert the thiosulfinates into saturated and unsaturated polysulfurs and sulfur heterocycles (Block et al., 1992). The alkyl thiosulfinates and thiosulfonates have been associated with the flavor of fresh garlic, while the alk(en)yl polysulfurs have the typical flavor of cooked garlic. In this work an alternative extraction process to obtain garlic oil using near-critical carbon dioxide is evaluated and compared to the traditional methods of hydro-distillation and steam stripping.

Address: PLAPIQUI, Camino la Carrindanga Km 7, casilla717, 8000 Bahía Blanca - Argentina. E-mail: sbottini@plapiqui.edu.ar

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For that purpose a thermistor is located near the discharge siphon of the Soxhlet. Salt.1 % by weight (Shultz and Randall. the pressure inside the cylinder was equal to the vapor pressure of CO2 at the corresponding experimental temperature. No extra water was added in the extractions with steam stripping process and liquid CO2.25 mm internal diameter and 0. 0. fruit acids and amino acids are insoluble. Analytical tools Four different techniques were applied to characterize the garlic extracts: GC-MS. 2. A metal heating tape that embraces the steel cylinder heats and evaporates the solvent at the bottom. non-flammable. whereas water is soluble in liquid carbon dioxide up to 0. It consists basically of a standard glass Soxhlet placed inside a 5 liters steel cylinder. and ketones. The operating conditions were as follows: injector temperature 250ºC. A cold finger hanging from the lid of the cylinder allows solvent condensation and dripping onto the extraction material. The experiments were carried out at a temperature of 15°C. nutraceuticals. 2000). A capillarity HP fused silica column of 30 m length. UV and FTIR. CO2 is also non-toxic. flavors and aromas is attractive. Further details of the apparatus and experimental procedure are given elsewhere (Marchiaro. prior to the extraction. The garlic oil is finally removed from the Soxhlet flask.1. the absence of oxygen in the extraction system and the facility of solvent recovery. due to the low operation temperatures required.2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering The use of near-critical carbon dioxide in the processing of food. High-pressure Soxhlet extractor Figures 1 and 2 show a scheme of the high-pressure Soxhlet extractor used in the extractions with nearcritical CO2. 1970). The apparatus is based on the design by Jenning (1981) and was built at our workshop. HPLC. 200 g of fresh garlic bulbs were chopped into small pieces using a domestic blender at medium speed during 1 minute. sample volume 5ml. energy of ionization 70 eV. An expansion valve connected to the lid of the cylinder is then opened and CO2 slowly discharges into the atmosphere. sugars.2. After a certain number of cycles the extraction is considered completed and the experiment is stopped. steam stripping and liquid CO2 at nearcritical conditions. 2 . The GC-MS analysis were carried out in a Hewlett Packard HP 6890 gas chromatograph equipped with a selective mass spectrometer detector (Hewlett Packard HP 5972A). initial oven temperature 60ºC (4 minutes) and a heating ramp of 4ºC/min up to 200ºC. Experimental Extractions were carried out using traditional hydro-distillation. aldehydes.25µm film thickness was used. detector temperature 280ºC. 2. 2. gas carrier (He) 1 ml/min. The number of extraction cycles is registered by following the sudden change of temperature produced after completion of every cycle. Liquid carbon dioxide is a selective solvent for the extraction of aromatic compounds such as esters. the garlic paste was homogenized in 200 ml distilled water at room temperature and macerated during 1 hour. For the hydro-distillation experiments. easily available and relatively cheap.

BA REF: thermostatic bath. Organosulfur compounds were detected by UV absorption at 254nm. SU CAL: heating resistance. 2. a Nicolet 520 FTIR Spectroscope was used to analyze the possible group configuration of the different garlic extracts. A constant flow rate of 1.2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering Fig. T: thermistor. High-pressure Soxhlet extractor: CLACL: closing clamps. A SEEN Varian 5500 liquid chromatograph with a photodiode array detector (Waters 996) was used for the HPLC analysis. 3 . DR: rupture disk. CC: cycle counter. SE: extracting material. SICAL: thermistor. TR: condenser.1. The column applied was a Varian MicroPack Sy-10µm. and a hold at 10% for 9 min. SICAL: thermistor. CT: temperature controller. G2: CO2 reservoir. Finally. BALSOX: Soxhet flask. MA: pressure gauge. CCE: extraction chamber. REG: register. SU CAL: heating resistance. SIF: siphon. CIE: closing clamps. before returning to the initial composition over 5 min.8 ml/min was used. a linear increase of 10% 2-propanol during 10 min. Fig. Experimental set-up: V: valves. TP: solvent port. 300mm length x 4mm internal diameter. BA REF: thermostatic bath. A SHIMADZU 160A UV-visible spectrophotometer of simple bundle with a deuterium lamp as source of excitation and a detector of diodes between 200 and 1000 nm was also used to analyze the garlic extracts. with an initial composition of 2% 2-propanol / 98% hexane during 6 min. CCE: extraction chamber. S3VAP: cycle counter.

Longer periods did not give higher yields. (1892) established the importance of diallyl disulfide and diallyl trisulfide in the flavor of these garlic extracts. 1997. (1971) suggests that the absence of thiosulfinates in the garlic oils obtained by hydro-distillation and steam stripping is due to the 4 . the main components of these oils are methyl and methyl-allyl polysulfides. A maceration period of 45 minutes prior to extraction of the garlic paste was sufficient. The yields and physical properties of garlic oils obtained by hydro-distillation.. while the oils obtained by hydrodistillation and steam stripping have the characteristic smell of cooked garlic. The thermoliability of thiosulfinates and thiosulfonates precludes the identification of these compounds by GC-MS.18 5 3. these compounds decomposes at the high temperatures of the GC injector and detector (Block et al. 1993). Table 1. The results are compared with previous work from the literature on onion oil (Dron et al.08 0. extraction periods of 2h 30’ were sufficient to exhaust the raw material. Boelens et al. For high-pressure extractions with quasi-critical CO2.01 Liquid CO2 Dark yellow 1. Table 2 shows the garlic oil compositions determined by GC-MS. 8 cycles were adequate. These compounds are missing in the oils obtained by hydro-distillation and steam stripping due to the high operating temperatures applied in these processes.1. Quality of the extracts obtained by the different extraction processes The oil extracted with liquid CO2 has the typical flavor of fresh garlic..21 0. Simandi et al. di. 2000).. This organoleptic difference is attributed to the presence of saturated and unsaturated thiosulfinates and thiosulfonates in the extracts obtained with near-critical CO2. While GC-MS operating conditions will not alter the composition of the garlic oils obtained by hydro-distillation and steam stripping because they have been already subjected to high temperatures.01 Steam Stripping Light yellow 1. 1989).. Mono-. cryogenic GC and supercritical chromatography have been suggested as appropriate techniques to avoid thermal decomposition.2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering 3..08 0.22 0. An alternative method should then be explored to determine the presence of thiosulfinates and thiosulfonates in these extracts. In hydrodistillation and steam stripping. steam stripping and high-pressure extractions with CO2 are given in Table 1. HPLC. the situation is different for the extracts obtained with liquid CO2. The chemical composition of the extracts was analyzed using the analytical tools described above. Also the addition of extra water to the garlic paste did not affect the yields. Experimental Results A series of extractions were carried out to evaluate the effect of process variables on yield and quality of extracts. Block et al. Semmler et al. The remaining aqueous phase in these processes is white and it contains some volatile water-soluble compounds (Yu et al.1992. Physical Properties and Yields of Garlic Extracts Colour Density (g/ml) Yield (weight %) Hydro-distillation Light yellow 1.and trisulfides were the main components determined by GC-MS in the garlic oils obtained by hydro-distillation and steam stripping.

1-thio-.164 0.trithiepane 2.248 0.3H)-Pyrimidinedithione 1.420 3.5-dimethyl. Hydro-distillation 2.212 0.-dimethylethoxy) 2.Furanmethanol 2.2.992 0.2.4.887 4.570 2.231 2.165 0.458 0. 5-(1.thiouracil Thiopheno Dihydro-3 methyl 2.635 0.346 0.5 Furandione Propane.146 0.220 8.774 25.5 dimethyl Di 1-propenyl sulfide 5 methyl 1. Results of the GC-MS analysis of garlic extracts Common compound Diallyl sulfide Diallyl trisulfide Allyl methyl sulfide Allyl methyl disulfide Dimethyl trisulfide Diallyl disulfide Diallyl tetrasulfide Dimethyl disulfide Dimethyl tetrasulfide Allyl methyl trisulfide 1-propenyl methyl disulfide 1 oxa-4-6-diazocyclooctane-5thione Toluene 3.166 0.326 0.932 1.304 34.5 diethyl 1.783 0.463 0.398 0.893 7. mol.117 0.3.489 5 .318 0.276 1.4 dimethyl thiopheno 4 methyl thiocyclohexanone 1.487 0.2.260 0.108 7.2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering Table2.051 27.443 0.147 0.1-dimethylethyl).236 0. 5 methyl5-acetoxymethyl-2-furaldehyde 4-mercaptophenol 5-methyl-2-thiophenecarboxaldehyde 3-(1.336 3.147 0.370 2.054 0.104 0. thiadiazole.127 0.(methylthio).4 (1H. 1 isothiocyanate 2 methyl .897 1.5 heptadiene Propenyl ethynyl sulfoxide 2.2.285 0.019 0.3 Butadiene Ethyl Thiourea 3.509 0.176 15.4 trithiolane Allyl isothiocyanate 1-3 Dithiane 3H-1.3thidiazole 1-propenyl methyl sulfide (Z) 1.5.S-allyl ester 2 Furancarboxaldehyde 2.289 0. cis Oxirane Acetic acid Acetoacetic acid.183 1.1.174 0.838 1.2-dithiol-3-one.738 0.207 24.366 0.099 1.436 7.689 0.5.3-dimethyl-.thiophene Sulfur.538 1.4 dimethyl 1.105 0.341 % peak area Steam Stripping Liquid CO2 2.253 0.988 2.961 1.Pyrazine Furyl hydroxymethyl ketone Methyl 2-furoate 5-isopropyltetrahydrothiophen-3-one 2-thiophenecarboxilic acid.253 0.661 72.606 0.050 0.Furan 3 ethyl-2.718 0.366 2.192 2.138 13.122 0.

58 mg of allicine/g in the extract. The peak at 2 minutes could correspond to decomposition products of thiosulfinates compounds. According to these authors they are present in the aqueous layer and cannot be transferred to the organic phase during the process. internal diameter and particle-size of the chromatographic columns used: a 10µm 300mm x 4mm column was used in our work. The compounds were identified as methyl (Me). (1992). (1992). 12345- AllSS(O)Pro(E) AllS(O)SPro-(Z) AllS(O)SAll AllS(O)SMe MeS(O)SMe minutes Fig. To confirm the presence of allicin (AllS(O)SAll) in this extract. No allicin was detected in this oil. AllS(O)SPro(Z) (2).2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering solubility of these compounds in water. The results of GC-MS for the garlic extracts produced by liquid CO2 show many compounds that are not detected in the oils obtained by hydro-distillation and steam stripping (see Table 2). by comparison with a synthesized standard.6mm column. This discrepancy can be attributed to the difference in length. AllS(O)SAll (3). Though the shape of the chromatogram shown in Figure 3 is very similar to that reported by Block et al. Figure 3 shows the results of HPLC analyses carried out on extracts obtained by high-pressure liquid CO2 to determine the presence of thiosulfinates RS(O)SR’. 3. a sample was sent to the Laboratory of Toxicological Analysis of Residues from Facultad de Ciencias Agrarias (Universidad Nacional de Cuyo) for analysis. while Block and co-workers used a 5 µm 250mm x 4. there is a difference of about 3 minutes in the retention times. allyl (All) and propenyl (Pro) thiosulfinates: AllSS(O)Pro-(E) (1). This laboratory confirmed the present of 3. These compounds were characterized by direct comparison with results reported in the literature by Block et al. Figure 4 shows the results of an HPLC analysis on garlic oil obtained by hydrodistillation. Si-HPLC separation of thiosulfinates in garlic extract obtained by high-pressure liquid CO2 extraction 6 . AllS(O)SMe (4) and MeS(O)SMe (5).

8 0. 4. 5. Figures 6 and 7 show the results of the FTIR spectroscopic measurements.2 0 200 220 240 260 280 300 Absorbance A Hydro-dist. UV Spectra for extracts obtained by hydro-distillation and quasi-critical CO2 extraction These spectra confirm the different nature of the garlic extracts.4 0. The spectrum of the extract obtained with liquid CO2 coincides with the spectrum of allicin (allyl thiosulfinate). 1994). Ext. 7 . Si-HPLC of garlic oil obtained by hydro-distillation Figure 5 shows the UV spectrum of the samples obtained for hydro-distillation and high-pressure liquid CO2 extraction.6 1.2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering minutes Fig.8 1.2 1 0..6 0.4 1. with CO2 wavelength (nm) Fig. 2 1. which presents a maximum of absorption at 250 nm (Calvey et al.

S-C absorption between 800700 cm-1 and S-S absorption between 500-400 cm-1. IR Spectrum of garlic extract obtained with near-critical CO2 100 90 80 70 60 50 40 30 20 10 0 Transmitance Hydro-dist. thiosulfinate C-S(O)SC absorption between 1095-1080 cm-1 (Block et al. S-C absorption between 800-700 cm-1 and S-S absorption located between 500-400 cm-1. The presence of the biologically active thiosulfinates in the extracts obtained with near-critical CO2 gives an indication of the difference in quality between the processes applied.absorption between 1350-1300 cm-1 and 1160-1120 cm-1. since it produces higher yields and extracts of better quality than the traditional hydro-distillation and steam stripping processes. CH3 absorption between 3100-2900cm-1 and 1470-1430cm-1. 7. C=C absorption between 1680-1625 cm-1. while the oils obtained by hydro-distillation and steam stripping have the characteristic smell of cooked garlic. CH absorption between 1400-1200cm-1. sulfone -(SO2). 8 .2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering 100 90 80 70 60 50 40 30 20 10 0 Transmitance Ext. IR Spectrum of garlic extract obtained by hydro-distillation The IR spectrum of the sample extracted with near-critical CO2 displayed sulfoxide C-S(O)-C stretch between 1070-1035 cm-1. 4. Conclusions The use of liquid carbon dioxide at near-critical conditions represents an attractive option for the extraction of garlic oil. in addition to strong C=C absorption between 1680-1625 cm-1. with liquid CO2 3900 3700 3500 3300 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900 700 wavenumbers (cm-1) Fig. 1986). 6.The IR spectrum of the sample extracted by hydrodistillation displayed CH3 absorption between 3100-2900cm-1 and 1470-1430cm-1. 3900 3700 3500 3300 3100 2900 2700 2500 2300 2100 1900 1700 1500 1300 1100 900 700 wavenumbers (cm-1) Fig. The extracts obtained with liquid CO2 have the typical flavor of fresh garlic.

R. Extracción de Aceite de Rosa Mosqueta con gases densos. J. components of distilled oils and extracts of Allium species.T. J. Volatile flavor compounds from onion. US. Hartman. Ho. Liou. Volatile Compounds Generated from Thermal Degradation of Alliin and Deoxyalliin in Aqueous solution. M. R.. Apitz-Castro.G. 20.. Liquid carbon dioxide for selective aroma extraction. Lira C.(1971)... Arch Pharm. E. And Sawinsky J. Food Technol. (1997). 46. 73.. Adv. S. 2418. 984.... Calvey... Sass-Kiss A. Eng. C. (2000). Acknowledgments The authors are grateful to CONICET and Universidad Nacional del Sur for financial support... 22. Yu. (1986). A. Van der Gen. Yield and quality of onion flavor obtained by supercritical fluid extraction and other methods. Pilot-scale extraction and fractional separation of onion oleoresin using supercritical carbon dioxide. R. T. (2000). odor and pungency in onion and garlic. 2235. Csordas A...2nd Mercosur Congress on Chemical Engineering 4th Mercosur Congress on Process Systems Engineering References Block. Prechl A. Ahmad. mechanistic. Yu. Dron A. Zhao. 230.. (1970). Boelens. 9 . J. Y. Wu.. Block. Patent 4. UNS. Food Chem. of Food Engineering. Food Chem. F.D. Uniquely high allyl to methyl ratios in some garlic samples. J. shallot. Agric. M.. P. Block E.. garlic. Simándi B. Marchiaro. Czukor B.M.4-dimethyl-2-thienyl disulfides... Food Chem.... (1976). Wu. Antithrombotic organosulfur compounds from garlic: structural. Block.40. leek. E. 183. 37. and Chinese chive. Food Chem. D. M. Off-Line Supercritical Fluid Extraction of Thiosulfinates from Garlic and Onion. Shultz. Naganathan. Jain. J... W. Volatile Compounds from garlic.. 1282. Littlejohn M. 41. Guyer D. 107. Agric. (1993)... wild garlic (ramsoms). de Valois. 24. H.Development of flavor.42. (1989).. S. scallion. J.M.J.. C. White K. 108(22). Rosen. Food Res.. Putman.. Naganathan S... 42(6). A.R. Matusik J. Whitaker J. Agric. 1335.. J. Randall. J. Deak A. Catalfamo.W. Semmler. Food Chem.. elephant (great-headed) garlic. Chem. Agric. Master Thesis in Chemical Engineering. Thiruvazhi. Allium chemistry: Synthesis of alk(en)yl 3. Betz J. Rosen. Am...(1994). E. C. High Presure Soxlet Extractor design. Soc. 725. Food Process.. 19. 146. (1992). J. Agric.. 7045. (1994). and Putman D. E. T. J..265. Agric. Gage D.J. Wobben. Allium chemistry: HPLC analysis of thiosulfinates from onion. H. E.A. Jenning (1981). Food Chem. S. chive.E. and synthetic studies.. (1892). T.