Lycopene and β-Carotene Extraction from Tomato Processing Waste

Ind. Eng. Chem. Res. 2003, 42, 6641-6646

6641

Lycopene and β-Carotene Extraction from Tomato Processing Waste Using Supercritical CO2 E. Sabio,† M. Lozano,‡ V. Montero de Espinosa,‡ R. L. Mendes,§ A. P. Pereira,| A. F. Palavra,| and J. A. Coelho*,|,⊥ Escuela Ingenierı´as Industriales, Universidad de Extremadura, Avenida Elvas s/n, 06071 Badajoz, Spain, Instituto Tecnolo´ gico Agroalimentario, Junta de Extremadura, Apartado 20107, 06071 Badajoz, Spain, Complexo I, CQE, Instituto Superior Te´ cnico, Avenida Rovisco Pais, 1096 Lisboa Codex, Portugal, Departamento de Engenharia Quı´mica, ISEL, Rua Conselheiro Emı´dio Navarro, 1949-014 Lisboa, Portugal, and Departamento de Energias Renova´ veis, INETI, Estrada do Pac¸ o do Lumiar, 1649-038 Lisboa, Portugal

Tomato skins and their mixtures with seeds were submitted to supercritical CO2 extraction using a flow apparatus at pressures of 250 and 300 bar and temperatures of 60 and 80 °C. Two different mean particle sizes (80 and 345 µm) were used at two solvent flow rates (0.792 and 1.35 kg/h). The yields of lipids, lycopene, and β-carotene obtained by supercritical fluid extraction were compared with those obtained by conventional organic solvent extraction. Supercritical fluid extraction from tomato skins at 300 bar and 80 °C allowed the recovery of 80% of the lycopene and 88% of the β-carotene, using about 130 g of CO2 per gram of matrix at the lower flow rate of CO2. Introduction Carotenoids are compounds widely used as colorants that are added directly to many food products such as butter, popcorn, salad dressings, and beverages. Lycopene and β-carotene are authorized food ingredients that cover a broad range of colors (from yellow to red), although lycopene has a color intensity 6-8 times higher than that of β-carotene.1 The importance of both of these carotenoids has increased because of the more extensive use of natural compounds in the food, cosmetic, and pharmaceutical industries, following EU directives in favor of natural rather than synthetic compounds.2 Lycopene can be classified chemically as a hydrocarbon, being an oligomer of isoprenoid type consisting of eight isoprene units joined together into a symmetrical chain containing 11 conjugated double bonds and another 2 bonds that are nonconjugated, a total of 13 double bonds.3 This high number of double bonds is greater than the number in any of the other carotenoids, including β-carotene, which has the same molecular weight as lycopene and also has a chain with 11 double conjugated bonds, although the β-carotene molecule contains a six-membered ring at each end (C40H56). Currently, lycopene is also of continuously increasing interest because of its unique antioxidant properties.4,5 Epidemiological studies have reported an important inverse correlation between the intake of lycopene in the diet and the development of some types of chronic illnesses, such as coronary heart disease and cancer.6 This protective effect seems to be related to lycopene’s ability to act as an antioxidant7 and/or singlet oxygen quencher.8 On the other hand, β-carotene plays a * To whom correspondence should be addressed. Tel.: 351218317066. Fax: 351-218317267. E-mail: [email protected]. † Universidad de Extremadura. ‡ Instituto Tecnolo´gico Agroalimentario. § INETI. | Instituto Superior Te´cnico. ⊥ ISEL.

fundamental role in human nutrition as pro-vitamin A, which is also an antioxidant agent.9 Mammals cannot synthesize carotenoids, as these pigments are obtained from vegetables and fruits. For instance, lycopene is found in moderate concentrations in watermelon and red grapefruit and is especially abundant in tomatoes, in which it can reach a concentration between 20 and 200 mg/kg on a dry weight basis. The concentration of this carotenoid is quite variable and depends on both the type of tomato and the maturation temperature.10 Lycopene and β-carotene are soluble in organic solvents, which are usually toxic, expensive, and hazardous to handle. Consumer concerns about health and environment have increased the interest in clean technologies and alternative and reliable extraction methods for these carotenoids.11,12 Supercritical fluid extraction is an environmentally benign alternative to conventional industrial solvent extraction, with the important advantage of giving products that are completely free from toxical residues.13 One of the most frequently used supercritical fluids is carbon dioxide. In addition to the advantage of being neither toxic nor flammable, carbon dioxide is also available at low cost and high purity. Because of its moderate critical temperature, CO2 can be used to extract thermally labile compounds.2 Taking into account these characteristics, carbon dioxide is an ideal solvent in the food, dye, pharmaceutical, and cosmetic industries. Seeds and skins constitute the main waste of the tomato processing industry. The skins can contain about 5 times more lycopene than tomato pulp.14 Actually, the utilization of waste tomato skins is essentially limited to animal feed, making this byproduct a suitable raw material for lycopene production. Supercritical CO2 extraction of lycopene and β-carotene from ripe tomatoes,2 from tomato processing waste,15-18 has been studied. The aim of the present work is to assess the influence of pressure, temperature, solvent flow rate, particle size, and vegetable matrix on the supercritical carbon dioxide

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Figure 1. Diagram of the supercritical fluid extraction apparatus: S1, ice cooler; F, filter; CP, circulating pump; BP, back-pressure regulator; M1-M4, manometers; S2, heat exchanger; V, extraction vessel; SP1 and SP2, separators; V1 and V2, valves; FL, rotameter; DTM, dry test meter.

extraction of lipids, lycopene, and β-carotene from tomato processing waste (skins and seeds), as well as from only skins. Material and Methods Raw Material and Extraction. For the supercritical fluid extraction experiments, a flow apparatus was used (Figure 1). This equipment allows studies to be performed at temperatures ranging from 25 to 120 °C and at pressures up to 300 bar.19,20 The liquid CO2 flowing from the cylinder is compressed with a diaphragm pump, CP (Dosapro Milton Roy, model Milroyal B), into the 1-L extraction vessel,V (HIP, model TOC-27-40), after passing a heat exchanger (S2), where it reaches supercritical conditions. The preset extraction temperature is achieved with the aid of a water jacket enveloping the extraction vessel and controlled through temperature measurements at four different points ((0.5 °C). The pressure, measured with a Bourdon-type manometer, M2 (Omega Engineering, model Q-8638, (0.7 bar), is controlled by a backpressure regulator, BP (Tescom Corporation, model 261722-44-043). The supercritical CO2 flowing upward through the fixed vegetable bed in the extraction vessel is expanded in two 0.27-L SS-316 separators (SP1 and SP2) whose pressures were measured by two oil manometers (Jumo) at 2.5 and 1.0 bar, respectively. The total volume of CO2 is determined with a dry test meter, DTM (American Company, model DTM-200A, (0.01 L). The CO2 (99.995% purity) was supplied by Air Liquide (Alges, Portugal). The tomato wastes used in this study (skins and seeds) were supplied by CONESA (a tomato processing company in Extremadura, Spain). The raw material had a moisture content of around 80% and was dried in a Vaciotem vacuum drier (Selecta) at 40 °C for 24 h. Prior to extraction, the raw material was ground using a laboratory grinder (Retsch Grindomix GM-100, Christison), sieved, packed under vacuum with a FC-360 vacuum packer (Friulmed), and stored at -20 °C. Finally, the moisture content of the samples (skins + seeds, 2.0 wt %; skins, 2.3 wt %) was measured following the standard methods described elsewhere.21 Supercritical fluid extraction was carried out using 40-50 g of biomass. The total volume of the vessel was filled with tomato waste in the middle and glass beads and filters at the top and bottom, respectively. About 130 g of CO2 per gram of tomato waste was used in all extractions. The extraction conditions were as follows: CO2 flow rates of 0.792 and 1.35 kg/h, pressures of 250 and 300 bar, and temperatures of 60 and 80 °C. Two particle sizes were tested: 80 and 345 µm.

In each test, the extracts were collected in glass tubes (previously weighed) inside the first separator at atmospheric pressure and at the temperature controlled with an ice bath, after predetermined amounts of CO2 had passed through the bed material, so that a sufficient number of points to build extraction curves could be obtained. The amount of extract (lipids) obtained was assessed gravimetrically by weighing each glass tube on a Mettler M180 balance ((0.0001 g). Each extract was stored under N2 at -20 °C for further determination of the lycopene and β-carotene contents and the fatty acid composition. The process continued until no significant amount of extract could be collected, with about 130 g of CO2 per gram of sample being used in each experiment. Finally, the CO2extracted sample was subjected to a Soxhlet extraction to determine the residual lipids, lycopene, and β-carotene. In all cases, the differences between the remaining residue calculated from the mass balance based on CO2 extraction and the actual residue measured by Soxhlet extraction were lower than 8%. The total lipids were determined gravimetrically after extraction with hexane (Merck, pa) using a Soxhlet apparatus for 4 h. The total amounts of lycopene and β-carotene were determined by HPLC with an HP 1050 chromatograph equipped with a 10-µm Lichrosorb RP18 column (200 × 4.6 mm) and using acetone (solvent A, HPLC grade from Merck) and water (solvent B) as eluents. An isocratic elution was used: first, 10 min with a solvent composition of 75% A/25% B and, then, 20 min with 95% of solvent A/5% of solvent B. By comparing the retention times of the two pigments in the extract mixture with those of their respective standard compounds (Sigma), lycopene and β-carotene were identified. Qualitative analysis of lycopene was also carried out by comparing the UV-visible spectrum of the obtained sample with that of the standard compound. Fatty acids were analyzed by gas chromatography in a gas chromatograph (HP 6890) equipped with an automatic injector, an FID detector, and a capillary column BPX-70 (50 m × 0.25 mm i.d.). The initial temperature of the column was 200 °C, and after 10 min, the temperature was raised to 240 °C at a rate of 10 °C/min. Results and Discussion As in most vegetables, the major lipid components present in tomato seeds and skin are essentially triglycerides.22 Table 1 shows the fatty acid composition of the extracted lipids (mainly triglycerides). The fatty acid compositions of both skins and skins + seeds are very similar and are typical of a vegetable oil, compris-

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Figure 2. Yields of lipids, lycopene, and β-carotene obtained from the mixture of tomato skins and seeds at the temperature of 60 °C and different pressures for the particle size of 0.345 mm and the CO2 flow rate 1.35 kg/h. Table 1. Profile of Fatty Acids in Lipids from Tomato Waste (wt %) fatty acids

skins and seeds

skins

C16:0 C18:0 C18:1 C18:2 C18:3

14.4 5.7 18.9 57.9 3.1

16.5 5.6 16.9 55.6 5.4

Figure 3. Yields of lipids, lycopene, and carotenes at 300 bar and 60 °C from a mixture of tomato skins and seeds with a particle size of 0.345 mm.

Table 2. Carotenoids in Tomato Dry Waste (mg/kg) carotenoid

skins and seeds

skins

lycopene β-carotene

294 69.7

310 68.9

ing linolenic acid (C18:2) as the main fatty acid, followed by oleic (C18:2) and palmitic (C16:0) acids. However, the lipids content of the waste from the tomato industry (skins + seeds) is much higher than that of skin alone: 8.37 vs 3.37% (dry weight basis). To explain this difference, the tomato seeds were analyzed, and their lipids content was found to be 21%. The obtained extract was an orange-yellowish liquid at ambient temperature. Further analysis showed that this oil contained a small amount of β-carotene and traces of lycopene. This means that seeds do not contribute much to the carotenoids content of the skins + seeds sample and exert a dilution effect on the carotenoids content of the extracts. This can also explain why the lycopene content found in skin is higher than that in skins + seeds (Table 2). Effect of the Pressure. In Figure 2 are represented the yields of lipids, lycopene, and β-carotene obtained from the supercritical extraction of dried tomato waste at the temperature of 60 °C, pressures of 250 and 300 bar, and a CO2 flow rate of 1.35 kg/h for the particle size of 0.345 mm. The increase in density of supercritical carbon dioxide with pressure from 250 to 300 bar leads to a higher lycopene content at the end of the extraction. On the other hand, the influence of pressure on the lipids and β-carotene contents was significant at the beginning of the process. However, beyond 60 kg of CO2/ kg of tomato, almost the same yields of lipids and β-carotene were obtained. Effect of the Solvent Flow Rate. In Figures 3 and 4 are presented the yields of lipids, lycopene, and β-carotene obtained from the dried tomato waste at 300 bar and two different flow rates (0.792 and 1.35 kg/h)

Figure 4. Yields of lipids, lycopene, and carotenes at 300 bar and 80 °C from a mixture of tomato skins and seeds with a particle size of 0.345 mm.

and temperatures of 60 and 80 °C, when about 130 kg of CO2 per kilogram of tomato waste was used. For both temperatures, the higher yield, for any of the three fractions analyzed, was obtained for the lower flow rate. Given that the total amount of CO2 was the same in all the experiments, this result suggests that, for the lower flow rate, the yields are closer of those obtained at equilibrium conditions. In fact, the fluid contact times at 60 °C were 8.9 and 4.7 min, and those at 80 °C were 8.2 and 4.8 min for the flow rates of 0.792 and 1.35 kg/h, respectively. On the other hand, at 60 °C (Figure 5), the concentration of lipids in CO2 corresponding to the initial part of the curves was higher for the lower CO2 flow rate (4.66 and 3.94 g/kg, respectively), and this difference was higher at 80 °C (4.35 and 2.97 g/kg, respectively). These values are lower than the solubility of the vegetable oil in CO2 predicted by the equation of Del Valle and Aguilera.23 A similar behavior was found for β-carotene (Figure 6), where the initial slope in all experiments gave concentration values lower than the corresponding solubility,24,25 which indicates that the

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Figure 5. Lipids extraction curves obtained from a mixture of tomato skins and seeds at the pressure of 300 bar and temperatures of 60 and 80 °C at two different flow rates and the particle size of 0.345 mm.

Figure 6. β-Carotene extraction curves obtained from a mixture of tomato skins and seeds at the pressure of 300 bar and temperatures of 60 and 80 °C at two different flow rates and the particle size of 0.345 mm.

low solubility of this carotenoid was not a limiting parameter of the extraction. Subra26 reached the same conclusion studying the extraction of β-carotene from dried carrots. The same behavior probably occurs for lycopene. However, for lycopene, no experimental solubility data are available for comparison (Figure 7). The effect of the solvent flow rate is higher at 80 °C (Figure 4), especially for lycopene and β-carotene, where there is an important drop in the extraction yield at the higher mass flow rate (1.35 kg/h). Effect of the Temperature. The solubilities of most vegetable oils are similar and can be estimated from a common correlation proposed by Del Valle and Aguilera.23 The application of this equation to the conditions of our experiments predicted almost the same solubilities for 60 and 80 °C (8.4 and 8.3 g/kg, respectively). This could explain the results obtained for lipids at the two temperatures (Figure 8). For the lower flow rate tested, which leads to the higher extraction yield of carotenoids, the increment in temperature seems to have no effect on the extraction yield of β-carotene. In contrast, the percentage of lycopene obtained increases to 73% (Figure 8). This different behavior can be explained in terms of the different localization of these two compounds in the matrix.

Figure 7. Total lycopene extraction curves obtained from a mixture of tomato skins and seeds at the pressure of 300 bar and temperatures of 60 and 80 °C at two different flow rates and the particle size of 0.345 mm

Figure 8. Yields of lipids, lycopene, and carotenes obtained at the pressure of 300 bar and the flow rate of 0.792 kg/h from a mixture of tomato skins and seeds with a particle size of 0.345 mm.

These results are basically in agreement with those of other workers. For instance, it was found that the yield of lycopene2 obtained by supercritical CO2 extraction at 276 bar increased with temperature in the range of 60-80 °C, whereas the concentration of β-carotene remained practically constant. Although the two pigments are very similar in structure, they possibly present different solubilities in the fluid and different solubility dependences of temperature. Other authors have found that increasing the temperature increases the solubility of the carotenoids27 and that the temperature dependence of the lycopene extraction yield at a constant pressure (300 bar) was higher than that of β-carotene extraction yield.16 The reason for this behavior could be both the fact that the concentrations of the two pigments in the various parts of the vegetable tissues are different and the fact that lycopene crystallizes as long needles.2,14 In tomatoes, lycopene crystallizes as long needles inside tiny particles called chromoplats. This fact hinders lycopene extraction because the chromoplast cell wall acts as a barrier and the dissolution of a crystallized substance is slower than that of amorphous one because the area/volume ratio is lower and the intermolecular forces in the crystal are stronger. An increase

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Figure 9. Yields of lipids, lycopene, and carotenes obtained at 300 bar, 60 °C, and the CO2 flow rate of 0.792 kg/h from mixtures of tomato skins and seeds with two different particle sizes.

in temperature promotes lycopene extraction by increasing both the CO2 diffusivity (and the ability to penetrate into the solid matrix) and the vapor pressure of lycopene. Effect of the Particle Size. In Figure 9 are shown the results of the experimental study carried out at the pressure of 300 bar, the temperature of 60 °C, and the flow rate of 0.792 kg/h for two different particle sizes. From this figure, it is evident that the lower particle size reduces, to a large extent, the yield obtained in the extractions. This result might seem surprising, because it is known that a reduction of the particle size increases the surface area and the rupture of the cell walls. This process accelerates the extraction and increases the yield, given that diffusion is hindered by the cell walls.28 Therefore, the total amount of solute available for extraction depends on the amount of surface area. Small particles have a high surface area/volume ratio; therefore, more solute is located on the surface than inside the particles. Because more solute is exposed to the solvent, the total yield is higher when small particles are used. Because of their small specific surface area, large particles lead to a distinct, diffusion-dominated extraction and long processing times. For example, decreasing the particle size of ground peanuts from a range of 3.35-4.75 mm to a range of 0.86-1.19 mm was found to increase the total oil recovery from 36 to 82%.28 However, particle sizes that are too small can result in inhomogeneous extractions, because of channeling effects in the fixed bed.29 In our case, an inhomogeneous color distribution in the 80-µm-particle-size solid after the extraction was verified, which indicates an uneven extraction due to an excessively small particle size. Effect of the Solid Matrix. To study the influence of the solid matrix on the extraction process of the three fractions studies, two experiments were carried out differing only in the preparation of the matrix. In Figure 10 are represented the percentagse of lipids, lycopene, and β-carotene extracted at the pressure of 300 bar, the temperature of 80 °C, and the flow rate of CO2 of 0.792 kg/h for a mixture of tomato skins and seeds and for skins only. The yields in lipids were almost the same for the two types of matrix, but the yield of lycopene increased and

Figure 10. Yields of lipids, lycopene, and carotenes obtained at 300 bar, 80 °C, and the flow rate of 0.792 kg/h from a mixture of tomato skins and seeds and from skins only, using 130 g of CO2 per gram of tomato waste at the particle size of 0.345 mm.

that of β-carotene decreased slightly when only skins was submitted to supercritical CO2 extraction. The seeds contain a higher percentage of triglycerides, which are preferentially extracted over lycopene and other carotenes not only because of the difference in solubility, but also because of the different localization of the compounds in the seeds and skins.2,14 On the other hand, it seems that a higher concentration of triglycerides helps the extraction of β-carotene, by working as an entrainer against the extraction of lycopene. Conclusions It was shown that the contents in lipids and carotenoids and the type of the matrix, particle size, and flow rate of the solvent, as well as the pressure and temperature, play an important role in the extraction process. For instance, increases in the temperature and pressure and ain the flow rate of solvent favor the extraction of lycopene, as does the use of only tomato skins as the matrix. At the lower flow rate studied and extraction conditions of up to 300 bar and 80 °C, it was possible to extract 80% of the lycopene and 88% of the β-carotene from the raw tomato waste material (only skins). Literature Cited (1) Nir, Z.; Hartal, D.; Raveh, Y. Lycopene from Tomatoes. Int. Food Ingredients 1993, 6, 45. (2) Cadoni, E.; De Giorgi, R.; Medda, E.; Poma, G. Supercritical CO2 Extraction of Lycopene and β-Carotene from Tomatoes. Dyes Pigm. 2000, 44, 27. (3) Ray, K.; Misra, T. N. Photophysical Properties of Lycopene Organized in Langmuir-Blodgett Films: Formation of Aggregates. J. Photochem. Photobiol. A: Chem. 1997, 107, 201. (4) Burton, G. W. Antioxidant Action of Carotenoids. J. Nutr. 1989, 119, 109. (5) Diplock, A. T. Antioxidant Nutrients and Disease Prevention: An Overview. Am. J. Clin. Nutr. 1991, 53, 189. (6) Giovannucci, E. Tomatoes, Tomato-Based Products, Lycopene, and Cancer: Review of the Epidemiologic Literature. J. Natl. Cancer Inst. 1999, 91 (4), 317. (7) Bohm, V.; Puspitasari-Nienaber, N. L.; Ferruzzi, M. G.; Schwartz, S. J., Trolox Equivalent Antioxidant Capacity of Dif-

6646 Ind. Eng. Chem. Res., Vol. 42, No. 25, 2003 ferent Geometrical Isomers of Alpha-Carotene, Beta-Carotene, Lycopene, and Zeaxanthin. J. Agric. Food Chem. 2002, 50 (1), 221. (8) Di Mascio, P.; Kaiser, S.; Sies, H. Lycopene as the Most Efficient Biological Carotenoid Singlet Oxygen Quencher. Arch. Biochem. Biophys. 1989, 274, 532. (9) Britton, G. Struture and Properties of Carotenoids in Relation to Function. FASEB J. 1995, 9 (15), 1551. (10) Porreta, S. Il Controllo della Qualita´ dei Derivati del Pomodoro; Stazione Sperimentale per l’Industria delle Conserve Alimentari: Parma, Italy, 1993; p 7. (11) Bruno, T. J.; Castro, C. A. N.; Hamel, J. F. P.; Palavra, A. M. F. In Recovery Process for Biological Materials; Kennedy, J. F., Cabral, J. M. S., Eds.; Wiley: New York, 1993; Chapter 11. (12) Phelps, C. L.; Smart, N. G.; Wai, C. M. Past, Present, and Possible Future Applications of Supercritical Fluid Extraction Technology. J. Chem. Educ. 1996, 73(12), 1163. (13) Hauthal, W. H. Advances with Supercritical Fluids (Review). Chemosphere 2001, 43, 123. (14) Sharma, S. K.; Le Maguer, M. Lycopene in Tomatoes and Tomato Pulp Fractions. Ital. Int. Food Sci. 1996, 48, 107. (15) Favati, F.; Galgano, N.; Lanzarini, G. Supercritical CO2 extraction of lycopene and β-carotene from by-products of the tomato industry. In Proceedings of the Fourth Italian Conference on Supercritical Fluids and their Applications; Reverchon, E., Ed.; Capri: Napoli, Italy, 1997; p 121. (16) Baysal, T.; Ersus, S. Y.; Starmans, D. A. J., Supercritical CO2 Extraction of β-Carotene and Lycopene from Tomato Paste Waste. J. Agric. Food Chem. 2000, 48, 5507. (17) Rozzi, N. L.; Singh, R. K.; Vierling, R. A.; Watkins, B. A. Supercritical Fluid Extraction of Lycopene from Tomato Processing Byproducts. J. Agric. Food Chem. 2002, 50, 2638. (18) Ollanketo, M.; Hartonen, K.; Riekkola, M. L.; Holm, Y.; Hiltunen, R. Supercritical Carbon Dioxide Extraction of Lycopene in Tomato Skins. Eur. Food Res. Technol. 2001, 212, 561. (19) Reis-Vasco, E. M. C.; Coelho, J. A. P.; Palavra, A. M. F. Extraction of pennyroyal oil (Mentha pulegium L.) with supercritical CO2. In Proceedings of the Fourth Italian Conference on Supercritical Fluids and their Applications; Reverchon, E., Ed.; Capri: Napoli, Italy, 1997; p 163.

(20) Reis-Vasco, E. M. C.; Coelho, J. A. P.; Palabra; A. M. F. Comparison of Pennyroyal Oils Obtained by Supercritical CO2 Extraction and Hydrodistillation. Flavour Fragrance J. 1999, 14, 156. (21) Official Methods of Analysis of AOAC International, 17th ed.; Horwitz, W., Ed.; AOAC International: Gaithersburg, MD, 2000, Chapter 42, Method No. 984.25, p 14. (22) Lazos, E. S.; Kalathenos, P. Composition of Tomato Processing Wastes. Int. J. Food Sci. Technol. 1988, 42, 212. (23) Del Valle, J. M.; Aguilera, J. M. An Improved Equation for Predicting the Solubility of Vegetable Oils in Supercritical CO2. Ind. Eng. Chem. Res. 1998, 27, 1551. (24) Johannsen, M.; Brunner, G., J. Solubilities of the FatSoluble Vitamins A, D, E, K in Supercritical Carbon Dioxide. Chem. Eng. Data 1997, 42, 106. (25) Mendes, R. L.; Nobre, B. P.; Coelho, J. P.; Palavra, A. F.; Solubility of β-Carotene in Supercritical Carbon Dioxide and Ethane. J. Supercrit. Fluids 1999, 16, 99. (26) Subra, P.; Castellani, S.; Jestin, P.; Aoufi, A. Extaction of β-Carotene with Supercritical Fluids. Experiments and Modelling. J. Supercrit. Fluids 1998, 12, 261. (27) Vega, P. J.; Balaban, M. O.; O’Keefe, S. F.; Cornell, J. A. Supercritical Carbon Dioxide Extraction Efficiency for Carotenes from Carrots by RSM. J. Food Sci. 1996, 61, 757. (28) Goodrum, I. W.; Kilgo, M. K.; Santerre, C. R. Oilseed Solubility and Extraction Modeling. In Supercritical Fluid Technology in Oil and Lipid Chemistry; King, I. W., List, G. R., Eds.; AOCS: Champaign, IL, 1996; Chapter 5. (29) Eggers, R. Supercritical Fluid Extraction (SFE) of Oilseeds/ Lipids in Natural Products. In Supercritical Fluid Technology in Oil and Lipid Chemistry; King, I. W., List, G. R., Eds.; AOCS: Champaign, IL, 1996; Chapter 3.

Received for review February 10, 2003 Revised manuscript received August 11, 2003 Accepted September 24, 2003 IE0301233