Supercritical carbon dioxide extraction of oil and α-tocopherol from almond seeds

Transcription

1 Journal of the Science of Food and Agriculture J Sci Food Agric 85: (5) DOI: 1.2/jsfa.2244 Supercritical carbon dioxide extraction of oil and α-tocopherol from almond seeds Lucia Leo, 1 Leonardo Rescio, 2 Loredana Ciurlia 2 and Giuseppe Zacheo 1 1 CNR, Istituto di Scienze delle Produzioni Alimentari, Via Lecce-Monteroni, 73 Lecce, Italy 2 PIERRE CHIMICA srl, SS 476 Km 17, 65 Zona Industriale, Galatina (LE), Italy Abstract: The objective of this study was to extract oil and tocopherols from almond seeds using supercritical carbon dioxide and to compare this extraction with a traditional solvent method. Oil and tocopherol extraction rates were determined as functions of the pressure (35 55 bar), temperature (35 5 C) and CO 2 flow rate (1 3 kg h 1 ), using a 1-l vessel. The effects of matrix particle size on extraction yield were also studied and it was demonstrated that extraction yield is greatly influenced by particle size. Maximum recovery was obtained in the first 2 3 h of extraction at a pressure of 42 bar, a temperature of 5 C and a flow rate of 3 kg h 1 CO 2. These results suggest that the elevated initial oil and tochopherol solubility is related to the increased proportion of fatty acids in the initial extract. The results were compared with those obtained when hexane/methanol was used as a solvent. 5 Society of Chemical Industry Keywords: carbon dioxide; extraction; oil; solubility; tocopherol INTRODUCTION Carbon dioxide (CO 2 ) is often used in the development of supercritical fluid extraction (SFE) instead of the organic solvents normally employed in conventional extraction methodologies. The main advantages of using carbon dioxide fluid are: a reduced potential for the oxidation of extracted solutes, higher selectivity, increased sample throughput, shorter extraction time and a low critical temperature. The latter is beneficial in extracting thermally labile compounds, such as natural vegetable products. Supercritical carbon dioxide also has chemical inertness, suitable solvent strength, permits the separation of compounds of widely different polarities and molecular masses, has low cost and, what is more, can be removed from the extracted products without leaving any chemical residue. In addition, carbon dioxide is both non-toxic and non-explosive and its use can reduce the consumption of organic solvents; this is especially useful for the production of natural products used in foods and pharmaceuticals. There are several excellent articles on the use of supercritical carbon dioxide methodologies in various analytical areas, including the extraction of vegetable oil and of fat-soluble vitamins. 1 4 Knowledge of the solubility of vegetable oil and its fat-associated products in supercritical CO 2 along with pressure and temperature are important for the successful application of this technology. The extraction conditions for specific solutes or classes of solutes from the vegetable matrix can also be optimised by changing the flow rate of the supercritical fluid or its pressure or temperature. Recently, an increasing interest in both the detection and the search for new oil and vitamin extraction techniques has been reported, and supercritical CO 2 extraction methods have been successfully applied in the extraction of oils from orange peel, 5 hazelnut, 6 olive, 7 blackcurrant and vineyard grape. 8 Despite the large number of matrices processed, 9 only some models of supercritical CO 2 have been published. Nevertheless, to optimize the extraction conditions, a relationship between the composition of vegetable oils and their solubility in supercritical CO 2 must be taken in account. 8 Almond is one of the major nut tree crops of the Mediterranean region. Almond kernel, analysed by using a conventional solvent method for the isolation of oil and fat-soluble vitamins, is shown to consist mainly of (g kg 1 ): lipids (45 6), proteins (), and carbohydrates (), with other elements, such as tocopherols, present in much smaller quantities (tocopherols.4.8) The major lipid species were found to be (g kg 1 total fatty acids): oleic acid, linoleic acid ( 17), and palmitic acid (55 7). The objective of our study was to develop a supercritical CO 2 extraction method for almond seed oil and subsequently to characterise the oil and tocopherols present in seed extracts. The supercritical CO 2 method for extraction of the lipid components and tocopherols used a 1-l pilot plant. The effect of pressure, temperature, solvent flow and particle size on the extraction rate were analysed. The results of Correspondence to: Giuseppe Zacheo, CNR, Istituto di Scienze delle Produzioni Alimentari, Via Lecce-Monteroni, 73 Lecce, Italy giuseppe.zacheo@ispa.cnr.it (Received 1 September 4; revised version received 27 January 5; accepted 16 March 5) Published online 2 June 5 5 Society of Chemical Industry. J Sci Food Agric /5/$

2 LLeoet al the SFE extraction were also compared with those obtained by using the conventional hexane/methanol method. EXPERIMENTAL Materials and sample pre-treatment Almond seeds collected in 2 were acquired from producers and were preserved in a dry chamber at 2 C. The almond seeds were ground in a blender, using different degrees of grinding in order to obtain different particle sizes; samples were separated on the basis of their size by sieving. Particle size diameters were determined by microscopic analysis. Grinding was performed just before the extraction and within a 5-s time interval in order to prevent the heating of the almond matrix and the related degradation of oil components. Liquid carbon dioxide with a purity of 99 g kg 1 was supplied by Air Liquide srl. (Milan, Italy). Solvents (HPLC grade) were purchased from JT Baker (Milan, Italy). Standard compounds for tocopherol and lipid characterizations were obtained from Sigma- Aldrich (Milan, Italy). Oil extraction by solvent Lipid fraction was extracted from 2 g of powdered almond seeds by a modified Bligh and Dyer method. 13 Whole and ground almonds were stirred for 1 h with 2 ml of hexane/methanol (2:1) mixture and centrifuged for 1 min at g. The upper layer was separated off, the residual solvent was evaporated with a rotavapor, and the oil content was determined by weighing the lipid fraction present in the extract samples. Aliquots were immediately used for further analyses or stored in the dark at 2 C. Oil content wasexpressedasmgg 1 of almond fresh weight. Oil and tocopherols extraction by supercritical CO 2 The schematic pilot apparatus used for CO 2 supercritical extraction of almond seeds is shown in Fig 1. The system was composed of an extractor (1 l); three separators: S1 (1.5 l), S2 (1.5 l) and S3 (.3 l); a pump; three heat exchangers; a tank for CO 2 ;a filter and the instruments and all the devices necessary for the management of the automatic control of the process, including emergency jettisoning of the fluid CO 2. The matrix (3 4 kg of almond) was arranged in a small cylindrical basket (6.8 l) which was inserted inside the extractor. Pure CO 2 was condensed and/or cooled to approximately 3 C (condenser E1), collected in a 1-l tank (buffer tank) and compressed to a desired pressure by means of a pump. CO 2 was then heated to the predefined temperature in order to reach the supercritical state before it was passed into the extractor. From the extractor, the solution (CO 2 + extracted compounds) moved into the first separator (S1) which was equipped with an internal coil that further lowers the CO 2 temperature to the predefined value. The decreased temperature reduced the solubility of the less soluble components, in particular waxes and water, which then collected on the bottom of the separator. From S1 the solution passed to the S2 separator, where there was a drastic Figure 1. Diagram of SFE pilot plant J Sci Food Agric 85: (5)

3 Supercritical extraction of oil and α-tocopherol from almond seeds reduction of pressure and, therefore, of the solubility of the majority of the oil components. The reduction of solubility in S2 could be controlled by the valve PV1 situated at the entrance to the separator. The solution then left S2 and flowed into S3 where, by means of the pressure-reducing valve PV2, there was a further fall in pressure and the separation of the solvent and the solute. The CO 2 from S3 flowed through a filter, was newly cooled and recycled in the service tank. The extracts were manually recovered by opening the valves at the bottom of S1, S2 and S3 at certain time intervals. The extracted samples were collected, weighed and then stored at 2 C for the further analyses. Oil characterisation The almond oil obtained by conventional and CO 2 extraction was separated into polar and neutral fractions by using the standardised IUPAC method. 14 Total lipids (1 g) were separated on a silica gel column by eluting sequentially the neutral lipids (NL) with chloroform and then the polar lipids (PL) with a methanol/chloroform (6:1 v/v) mixture. The solvents were evaporated and the remaining lipid fractions were weighed and used for further analysis. NL were further separated into their subclasses by TLC on.25 mm silica gel plates (Merck, Darmstadt, Germany) using a hexane/diethyl ether/formic acid (8:2:1 v/v) mixture. The plates were air dried and analytes detected by exposure to iodine vapour. Individual bands were identified with the aid of the standard Sigma (Milan, Italy) procedure and fractions corresponding to each lipid type were extracted from the plates with ml l 1 methanol in diethyl ether and weighed to calculate chemical yelds. 8 Fatty acid methyl esters (FAMEs) were prepared by adding 5 ml of sodium hydroxide (.5 M) in methanol at C to.5 g of oil in a water bath for 1 min. The solution was cooled to room temperature, added to 5 ml of 15 mg g 1 boron trifluoride in methanol and heated at C for a further 1 min. After cooling, 5 ml of hexane and 1 ml of aqueous saturated solution of sodium chloride were added and the mixture was vigorously shaken. The organic layer was separated and concentrated under vacuum. FAMEs were analysed by a Shimadzu (Kyoto, Japan) GC-17A using a gas chromatograph equipped with a flame ionisation detector (FID) and a 6 m Stabilwax (Restek Corp, Bellafonte, PA, USA) capillary column (.25 mm ID,.25 µm film thickness). The oven temperature was programmed as follows: C for 1 min, then to 24 Cat8 Cmin 1 and maintained at this temperature for 3 min. The injector and detector temperatures were 25 and 28 C, respectively. Helium was used as carrier gas at a flow rate of 1 ml min 1. Tocopherol analysis Collected oil samples were analysed by HPLC using a Beckman-Coulter (Milan, Italy) chromatograph equipped with a diode array (System Gold 168; 19 6 nm) detector, a Shimadzu spectrofluorimetric detector (RF-1AXL) and a Marathon autosampler. Tocopherols were separated by isocratic elution in a normal phase column (Ultrasphere Silica, 4.6mm 2.5 cm, Beckman-Coulter, Fullerton, CA, USA.). The mobile phase was hexane/2-propanol (99.5:.5 v/v) flowing at.5ml min 1. The elute was monitored with fluorescence detector at an excitation wavelength of 289 nm and an emission wavelength of 33 nm and with UV detector at 294 nm. Quantification was performed by comparing the sample peak areas to those of known amounts of standard compounds. Tocopherol content was expressed as µgg 1 of almond dry weight. RESULTS AND DISCUSSION Oil extraction The almond oil obtained from the various fractions, extracted with either hexane/methanol or supercritical CO 2, was intensely yellow in colour having a variable content of tocopherols. After oil extraction, the residual defatted almond was a white flour with variable grain size, more or less fine, depending on the degree of grinding of the fresh matrix and on the effect of the operating conditions (pressure, flow rate, temperature) used to remove the oil. Influence of particle size on conventional oil extraction Mature dry almond seeds with water content estimated at 5 6 g kg 1 dry weight were homogenized and sieved to obtain four representative sub-samples: whole, broken (4 8 mm), milled (.5 3 mm) and powered samples. The influence of particle size on the conventional extraction process of almond seeds is reported in Table 1. The oil value obtained from the powdered almond seeds was assumed to be the maximum possible extractable oil and was used as an absolute value (%) to calculate the proportion of extracted oil. Influence of particle size on CO 2 supercritical oil extraction Preliminary tests allowed us to determine suitable operating conditions (temperature, pressure and flow rate). At the same time the ability of the process to concentrate the almond oil components selectively in the separator vessel was also established. From these tests it was evident that the yield of extraction not only depended on the utilised pressure, temperature and flow rate of the solvent through the extraction bed, but also on the chemical and physical characteristics of the matrix such as oil composition, moisture content and particle size. The almond matrix was blended at various particle sizes in order to obtain data on the extraction rate and the total yield of oil. The yield, ie the proportion of oil extracted by our pilot apparatus in relation J Sci Food Agric 85: (5) 2169

4 LLeoet al Table 1. Influence of particle size on conventional almond oil and tocopherol extractions Oil Tocopherol Almond As mg g 1 of fresh weight a As mg g 1 of extractable oil As µgg 1 of dry weight a As mg g 1 of extractable tocopherol Powdered 545 ± 23 ± ± 25 ± 6 Milled (.5 3 mm) 298 ± ± 4 16 ± ± 1 Broken (4 8 mm) 151 ± ± 8 44 ± 1 11 ± 2 Whole 55 ± 4 ± 7 3±.2 a Mean of four values ± standard deviations Milled almond 8 Oil yield (as g kg -1 extractable oil) Broken almond Whole almond CO 2 kg kg -1 matrix Figure 2. Comparison of oil extraction curves for various particle sizes of almond seeds. Experiments carried out at 5 ± 2 C, 42 ± 2 bar, 2 ± 5kgh 1 (mean ± SD, n = 4). to the amount extracted by solvent in powdered seeds, is given as a function of the ratio of the CO 2 mass used and the initial seed mass loaded into the extractor (Fig 2). As expected, it was observed that extraction yield strongly increased in broken and milled almonds. During the first part of the extraction process the curves were characterised by a linear increase, subsequently they approached a constant value. The latter was caused by a reduction in oil concentration in the matrix as well being more transfer-controlled in the latter stages of extraction. Nevertheless, under identical operating conditions and using the same quantity of CO 2 ( g kg 1 of CO 2 per kg of matrix) broken almonds yielded g kg 1 oil as whole almonds and milled almonds as much as 7 g kg 1 (Fig 2). This means that, at predetermined operating conditions, the reduction of particle size resulted in an increase in oil yield. This increase was explained by the increase of contact surface in the milled matrix compared with that available in the whole almonds. Oil yield (as g kg -1 extractable oil) P=55 bar P=45 bar P=35 bar CO 2 kg kg -1 matrix Figure 3. Effect of pressure on SFE oil extraction yield at a temperature of 5 ± 2 C and a flow rate of 2 ± 5kgh 1 (mean ± SD, n = 4). Influence of pressure, temperature and flow rate on CO 2 supercritical oil extraction The functional relationships between the various physical parameters such as pressure, temperature and flow rate were investigated. The influence of pressure on the CO 2 supercritical oil extraction from almond seeds was determined at pressures of 35, 45 and 55 bar, respectively, and at a constant temperature (5 ± 2 C) and a constant flow rate (2 ± 5kgh 1 ). These results are reported in Fig 3 where the oil yield is plotted versus kg of CO 2 per kg of matrix. The extraction curves obtained at 45 and 55 bar were characterised by an initial period in which the oil yield rose more steeply as the pressure was increased and a second period characterised by a higher value of the plateau as the pressure increased. The extraction curve showed a linear period without a flex point at low pressure (35 bar). In view of these results it can be stated that at the beginning of the extraction process, when the physical phenomenon 217 J Sci Food Agric 85: (5)

5 Supercritical extraction of oil and α-tocopherol from almond seeds of quantitative extraction mass depends mainly on the available amount of oil extracted into the CO 2,an increment of the extraction pressure determines a yield increment of oil. Figure 3 shows that at a flow rate of 3 kg CO 2 kg 1 an increase in pressure of 28% from 35 to 45 bar resulted in a % increase in yield, while an increase of 57% from 35 to 55 bar in the extraction pressure led to a further increase in yield. As expected, using the same quantity (4 kg of CO 2 per kg of matrix), as pressure increased the solubility of the almond oil in CO 2 increased, as found in other studies on oil extraction from seeds The results of the pressure effect on the oil yield suggest that the increased yield resulted from the increasing density of the solvent and consequently from increased solubility of almond oil in the CO Temperature is also a critical parameter which affects the oil yield in CO 2 extraction. In Fig 4 the effect of temperatures of 35, 4 and 5 C on the extraction yield is reported. The results obtained clearly indicate that the extraction yield was promoted by an increase in temperature. At fixed conditions of pressure and flow rates, an increase in temperature of 15 C produced an almost fourfold yield increment (Fig 4). An explanation of this phenomenon is that the oil solubility is enhanced in the CO 2. However, the temperature effect on CO 2 extraction is more difficult to assess than the other parameters, pressure and solvent flow, because it influences both the solvent diffusion in the matrix and the oil dissolution in the solvent. 19 As the temperature rises there is a lower solvent strength of the fluid due to the decrease in fluid density. In contrast, an increase in temperature can improve the extraction efficiency despite the decrease in fluid density, since the vapour pressure of the oil is increased. 9,19 To determine the effect of flow rate during almond oil extraction a constant pressure of 42 bar and temperature of 5 C were used. In our pilot plant, the optimum extraction yield was obtained at a flow rate between 2 and 3 kg h 1 of CO 2. From the results reported in Fig 5 it is evident that oil yield, at the initial stage of extraction, increased with increasing CO 2 flow rate from 1 to 3 kg h 1. It seemed that flow rate of supercritical fluid directly affects the extraction rate. Flow rates below 1 kg h 1 resulted in a relatively flat yield curve and, consequently, in an increase in the extraction time, while flow rates beyond 3 kg h 1 resulted in only a slight increase of oil yield (data not shown). The latter observation indicated that for higher flow rates the carbon dioxide leaving the extractor was less saturated and the effect of increasing flow rate on extraction process was very small. In conclusion, these experiments demonstrated that when extracting a biological matrix at the described conditions (see Materials and Methods), the oil yield increased with increasing pressure, temperature and flow rate. Tocopherol extraction and lipid characterisation Lipids from oil extracted by hexane/methanol and CO 2 fraction extracted oil were analysed in order to find the relationship between changes in oil solubility and Oil yield (as g kg -1 extractable oil) T=5 C T=4 C T=35 C Oil yield (as g kg -1 extractable oil) kg h -1 CO 2 2 kg h -1 CO 2 1 kg h -1 CO CO 2 kg kg -1 matrix CO 2 kg kg -1 matrix Figure 4. Effect of temperature on SFE oil extraction yield at a pressure of 42 ± 2 bar and a flow rate of 2 ± 5kgh 1 (mean ± SD, n = 4). Figure 5. Effect of CO 2 flow rate on SFE oil extraction yield at a pressure of 42 ± 2 bar and a temperature of 5 ± 2 C (mean ± SD, n = 4). J Sci Food Agric 85: (5) 2171

6 LLeoet al its components. Tocopherol extraction from almond seeds by the conventional solvent method increased as particle size decreased (Table 1). In powdered almond a mean of.4mgg 1 dry weight (dw) was obtained and this recovery was comparable with results obtained by Zacheo et al. 12 The value was assumed to be the maximum possible extractable almond tocopherols and it was assumed as the reference value for calculating the proportion of tocopherol extracted by the SFE process (Table 1). In all the analyses of solvent and CO 2 extracts, α-tochopherol was the main isomer; β, γ, δ- tochopherols were not detected. In order to assess the ability of the process to obtain oil fractions rich in tocopherol various supercritical extraction conditions were employed. The optimum co-extraction conditions for oil and tocopherols were a temperature of 5 ± 2 C, a pressure of 42 ± 2 bar and a flow rate of 25 ± 5kgh 1 using powdered almond seeds. The oil fractions obtained at the above conditions during the first 2 h of the extraction process contained the highest tocopherol concentration, which decreased in the remaining extraction time. This tendency can be observed in Fig 6, where the proportion of tochopherol obtained is plotted against extraction time. Experiments conducted by varying extraction pressure led to changes of the oil tocopherol concentration during the first 2 h. It can be seen that increasing the pressure from 37 to and 42 bar, resulted in an increase in the tocopherol extraction rate (expressed as mg min 1 kg 1 CO 2 ) in the fraction collected during the first 2 h of extracting time, but a further increase in pressure induced a decrease in the extraction rate (Table 2). This result can be explained by considering that the mass transfer of tocopherol is related to its solubility in the oil, and both its initial amount present in the matrix and the subsequent depletion of the solid phase (Fig 6). The tendency of extraction curves can be seen in Fig 7 where the oil and tocopherol SFE extraction rate (expressed as g min 1 kg 1 CO 2 and mg min 1 kg 1 CO 2 respectively) is reported as function of time. The figure clearly indicates that the oil extraction rate during the first stage was very high and remained almost constant at a mean value of.25.3 g min 1 kg 1 CO 2 for a 3 h period Table 2. Tocopherol and oil extraction rate at different pressure values during 2 h of extraction time Pressure (bar) Tocopherol extraction rate (mg min 1 kg 1 CO 2 ) a Oil extraction rate (g min 1 kg 1 CO 2 ) a ±.1 ±. ±.21 ± ±.22 ± ±.22 ± ±.27 ± ±.26 ± ±.27 ±.2 a Mean of four values ± standard deviations. and decreased slowly in the remaining extraction time. The extraction rate of tocopherol was highest (.13 mg min 1 kg 1 CO 2 ) in the initial phase of the process and was characterised by a constant decrease over the remaining time. The high tocopherol concentration during the initial stage could be due to a high proportion of more soluble lipids such as free fatty acids, in the oil extracted (Fig 8). This, in a way, is Tocopherol concentration (as mg g -1 oil) Time (min) Figure 6. Concentration of tocopherols in SFE extracted oil as function of time at a temperature of 5 ± 2 C, a pressure of 42 ± 2 bar and a flow rate of 2 ± 5kgh 1 (mean ± SD, n = 4). Tocopherol extraction rate (mg min -1 kg -1 CO 2 ) Tocopherol Time (min) Figure 7. Comparison of oil and tocopherol extraction rate as function of time at a temperature of 5 ± 2 C, a pressure of 42 ± 2 bar and a flow rate of 2 ± 5kgh 1 (mean ± SD, n = 4). Oil Oil extraction rate (g min -1 kg -1 CO 2 ) 2172 J Sci Food Agric 85: (5)

7 Supercritical extraction of oil and α-tocopherol from almond seeds Table 3. Lipid composition (as mg g 1 weight a ) of almond oil extracted by conventional and CO 2 methods SFE extraction c Hexane/methanol Lipid classes b extraction Fract I Fract II Fract III Fract IV Fract V Fract VI TAGs 75 ± 2 63 ± 3 64 ± 2 71 ± 4 79 ± 2 86 ± 3 84 ± 3 FFA 15 ± 1 31 ± 2 29 ± 2 ± 1 12 ± 4 4± 3 12± 1 DAGs 34 ± 1 29± 2 25± 1 23± 6 32± 1 38± 2 38± 2 MAGs 21 ± 1 25± 2 22± 2 3± 2 32± 2 33± 1 35± 3 Other 15 ± 3 11± 1 11± 5 12± 1 9± 5± 2± a Mean of four values ± standard deviations. b Lipid classes: TAGs (triglycerides), FFA (free fatty acids), DAGs (diglycerides), MAGs (monoglycerides). c The fractions (Fract I VI) were collected at intervals of 1h. Table 4. Fatty acid composition (as mg g 1 of total FAME a ) in hexane and SFE extracted almond oil SFE extraction b Hexane Fatty acid extraction Fract I Fract II Fract III Fract IV Fract V Fract VI Palmitic (C16:) 7 ± 6 68± 4 66± 2 67± 4 67± 4 69± 2 71± 3 Palmitoleic (C16:1) 4 ± 6± 5± 5±.5 ± 5±.5 ± Stearic (C18:) 22 ± 22± 21± 1 17± 2 19± 22± 1 17± Oleic (C18:1) 72 ± 2 71 ± 2 73 ± 2 73 ± 3 71 ± 3 69 ± 2 68 ± 2 Linoleic (C18:2) 177 ± ± 9 18 ± 1 18 ± 1 ± 1 21 ± 1 22 ± 2 Other 7 ± 4± 1± 1± 1± 8± 2± MUFA 72 ± 2 72± 2 73± 2 74± 3 72± 3 69± 2 69± 2 PUFA 17.7 ± ±.9 18± 1 18± 1 2± 1 21± 1 22± 2 a Mean of four values ± standard deviations. b The SFE samples (Fract I VI) were collected at intervals of 1 h. Tocopherol (mg g -1 of oil) Tocopherol Free Fatty Acids Time (min) Figure 8. Comparison of tocopherol and free fatty acid content in the extracted oil as function of time at a temperature of 5 ± 2 C, a pressure of 42 ± 2 bar and a flow rate of 2 ± 5kgh 1 (mean ± SD, n = 4). supported from the data reported in Table 3 where in fraction I (1 h after starting the CO 2 extraction process) 31 mg free fatty acids (FFA) g 1 were observed. During the rest of the extraction process the free fatty acid component gradually decreased from 31 5 Free Fatty Acids (mg g -1 of oil) to 1 mg g 1 while an increase in triglycerides (TAG) fractions was observed. Similar data were reported by Sovová et al 8 during the extraction of vegetable oils where a close relationship between the oil solubility and the amount of free fatty acids was observed. The methyl ester fatty acid analysis in both the hexane/methanol and the CO 2 extracted oil is presented in Table 4. No differences in the fatty acid compositions of the various extracts are apparent. From the analysis, the main components of almond oil were as follows (g kg 1 ): oleic acid (7), linoleic acid () and palmitic acid (7). Mono- and polyunsaturated fatty acids accounted for about 7 and g kg 1 respectively of the total fatty acid composition. The use of SFE, provides the advantage of fractioning the components of almond oil and obtaining oil enriched in tocopherol oil. Similar results for the extraction and enriched tocopherol fractions have been obtained using supercritical CO 2 by several authors, as reviewed by Turner et al. 9 CONCLUSION In conclusion, the use of supercritical CO 2 in almond oil extraction, under the specific conditions reported above, resulted in tocopherol-rich oil fractions in the first part of the extraction process. The results clearly demonstrate that the oil extraction rate differed for large and small almond pieces, indicating that particle size is a limiting parameter to the extraction rate. J Sci Food Agric 85: (5) 2173

8 LLeoet al REFERENCES 1 Chester TL, Pinkston JD and Raynie DE, Supercritical fluid chromatography and extraction. Anal Chem 66:16 13 (1994). 2 Chester TL, Pinkston JD and Raynie DE, Supercritical fluid chromatography and extraction. Anal Chem 68: (1996). 3 Chester TL, Pinkston JD and Raynie DE, Supercritical fluid chromatography and extraction. Anal Chem 7:31 32 (1998). 4 Smith RM, Supercritical fluids in separation science-the dreams, the realty and the future. J Chromatogr A 856: (1999). 5 King JW, Favati F and Taylor SL, Production of tocopherol by supercritical fluid extraction and chromatography. Sep Sci Tech 31: (1996). 6 Bernardo-Gil MG, Grenha J, Santos J and Cardoso P, Supercritical fluid extraction and characterisation of oil from hazelnut. Eur J Lipid Sci Technol 14:42 49 (2). 7 De Lucas A, Martinez de la Ossa E, Rincon J, Blanco MA and Gracia I, Supercritical fluid extraction of tocopherol concentrates from olive tree leaves. J Supercrit Fluids 22: (2). 8 Sovová H, Zarevúcka M, Vacek M and Stránský K, Solubility of two vegetable oils in supercritical CO 2. J Supercrit Fluids 2:15 28 (1). 9 Turner C, King JW and Mathiasson L, Supercritical fluid extraction and chromatography for fat-soluble vitamin analysis. J Chromatogr A 936: (1). 1 Kader AA, In-plant storage, in Almond production Manual. Publication N 3364, Micke CW, University of California, Division of Agriculture and Natural Resources, 274 pp (1996). 11 Zacheo G, Cappello AR, Perrone LM and Gnoni GV, Analysis of factors influencing lipid oxidation of almond seeds during accelerated ageing. Lebensm Wiss Technol 31:6 9 (1998). 12 Zacheo G, Cappello MS, Gallo A, Santino A and Cappello AR, Changes associated with post-harvest ageing in almond seeds. Lebensm Wiss Technol 33: (). 13 Bligh EG and Dyer WS, A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: (1959). 14 Ruiz-Gutierreez V and Perez-Camino MC, Update on solidphase extraction for the analysis of lipid classes and related compounds, J Chromatogr A 885: (). 15 Reverchon E, Supercritical fluid extraction and fractionation of essential oils and related products, J Supercrit Fluids 1:1 37 (1997). 16 Eggers R, Supercritical fluid extraction of oil seeds, in Supercritical Fluid Technology in Oil and Lipid Chemistry, ed by King JW and List GR, AOCS Press, Champaign, IL, pp (1996). 17 King JW, Sub- and supercritical fluid processing of agrimaterials: extraction, fractionation and reaction models, in Supercritical Fluids Fundamentals and Applications, edbykirane, Debenedetti PG and Peters CJ, NATO Science Series, Series E, Applied Sciences, Vol 366, Kluwer Academic, Dordrecht, pp (). 18 Del Valle JM and Aguilera JM, An improved equation for predicting the solubility of vegetable oils in supercritical CO 2. Ind Eng Chem Res 27: (1988). 19 Luque de Castro MD, Valcarcel M and Tena MT, Analytical supercritical fluid extraction, Springer-Verlag, New York, 319 pp (1994) J Sci Food Agric 85: (5)