Ind. Eng. Chem. Res. 2001, 40, 3781-3784
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An Improved Fractional Crystallization Method for the Enrichment of γ-Linolenic Acid in Borage Oil Fatty Acid Tor-Chern Chen and Yi-Hsu Ju* Department of Chemical Engineering, National Taiwan University of Science and Technology, 43, Sec.4, Keelung Road, Taipei 106-07, Taiwan
A two-stage, low-temperature solvent fractional crystallization process was developed in this work for the enrichment of γ-linolenic acid (GLA, 18:3 n6) in free fatty acids (FFAs) derived from saponified borage oil. Acetonitrile was the solvent in the first stage. At an acetonitrile to FFA ratio of 62.5 mL/g and after 48 h of storage at -40 °C, the GLA content in the liquid phase can be increased from 23.4% to 66.1% with a corresponding yield of 93.0%. In the second stage, a mixture of 30% acetonitrile and 70% acetone was employed as the solvent. At a solvent to FFA ratio of 90 mL/g and after 24 h of storage at -80 °C, the GLA content in the liquid phase was further raised from 66.1% to 93.9% with a corresponding yield of 92.4% (overall yield 86.0%). More than half of the GLA in the solid can be recovered after the second-stage operation was applied to the solid phase. Introduction γ-Linolenic acid (GLA; all cis-6,9,12-octadecatrienoic acid) is a precursor of arachidonic acid, which in turn is a precursor of the eicosanoids which include prostaglandins, leukotrienes, and thromboxanes.1 GLA also possesses important physiological functions, such as modulating immune and inflammatory response.2 Recently, GLA-rich acylglycerols have been applied for the treatment of certain diseases such as atopic eczema,3 multiple sclerosis,4 and rheumatoid arthritis.5 The obtainment of GLA concentrate or GLA-rich acylglycerol from natural sources is important for pharmaceutical and dietary purposes. Various techniques are available for the enrichment of polyunsaturated fatty acids (PUFAs) from natural sources. Haagsma et al.6 applied a urea inclusion method to prepare an ω3 fatty acid concentrate from cod liver oil. They pointed out that one-third of the fatty acid obtained exists as fatty acid methyl ester. The tendency of forming a complex between PUFA and silver ion was applied for the concentration of PUFA.7,8 Solvent winterization has been employed for the increase of the GLA content of fungal oil.9 GLA ethyl ester was concentrated by using Y zeolite.10 Recently, enzymecatalyzed reactions have become popular methods for obtaining concentrated PUFA. In a two-step process, which involved enzyme-catalyzed hydrolysis of borage oil and the esterification of the resulting free fatty acid (FFA) with lauryl alcohol, a 93.7% GLA content in FFA with a corresponding yield of 67.5% can be obtained.11 Shimada et al.12 used a series of enzyme-catalyzed reactions, distillations, and urea fractionation to obtain a FFA that contains 98.6% GLA with a yield of 49.4%. Low-temperature solvent crystallization was developed for the concentration of PUFA decades ago.13 The basic principle of this method is that the solubility of a fatty acid or its ester in a solvent depends on its chain length as well as the number of double bonds. Uksila and Lehtinen14 used acetonitrile or a mixture of aceto* To whom correspondence should be addressed. Fax: 8862-27376644. E-mail:
[email protected].
nitrile and water as the solvent and obtained FFA that contains 84.7% R-linolenic acid with a corresponding yield of 15% from saponified linseed oil. Borage oil is one of the most important natural sources of GLA. In this work, a two-stage, low-temperature solvent crystallization process was developed for the concentration of GLA in FFA derived from saponified borage oil. The effects of the solvent, storage temperature, and solvent to FFA ratio on the GLA content and yield were systematically investigated. Experimental Section Materials. Borage oil was purchased from Sigma (St. Louis, MO). Except for 20:1, 22:1, and 24:1 that were provided by Nu Chek Prep Inc. (Elysian, MN), all other standards were provided by Sigma. All solvents and reagents used were of HPLC or ACS grade. Preparation of FFA. FFAs obtained by the saponification of borage oil were prepared according to the method described by Haagsma et al.6 Typically, a NaOH solution was prepared by dissolving 48 g of NaOH and 0.5 g of Na2EDTA in 160 mL of water. To this solution was added 160 mL of ethanol. A mixture containing 100 g of oil and 200 mL of a NaOH solution was heated at 60 °C with magnetic stirring at 550 rpm for 1 h. Forty milliliters of water and 400 mL of hexane were then added, and the solution was stirred for 1 h. The upper layer containing unsaponifiable matter was removed and discarded. One hundred and sixty milliliters of water was added to the lower layer, and 12 N hydrochloric acid was then added until it had pH 1. The resulting lower layer was removed by using a separating funnel and was discarded. The FFA-containing upper layer was dried with anhydrous magnesium sulfate, and the solvent was evaporated in a vacuum rotary evaporator at 35 °C. First-Stage Solvent Crystallization. A fixed amount of acetonitrile was added to a vial containing 5 g of FFA derived from saponified borage oil. This mixture was stirred with a magnetic stirrer at 30-35 °C under a nitrogen atmosphere until all FFAs were dissolved. The solution was cooled to room temperature and then
10.1021/ie010024u CCC: $20.00 © 2001 American Chemical Society Published on Web 07/27/2001
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Table 1. Compositions of FFA before and after the Solvent Crystallization content (wt %) FFA
before
16:0 18:0 18:1 18:2 GLA 20:1 22:1 24:1 yield of GLA (%) overall yield of GLA (%)
10.34 ( 0.14 3.61 ( 0.03 15.89 ( 0.06 37.28 ( 0.08 23.35 ( 0.07 4.25 ( 0.05 3.16 ( 0.16 2.12 ( 0.09
after the 1st stagea
after the 2nd stageb
after the 1st stagec
after the 2nd staged
0.12 ( 0.00 NDe 4.34 ( 0.07 28.63 ( 0.10 66.09 ( 0.07 0.70 ( 0.14 0.12 ( 0.02 ND 93.04 ( 0.10 93.04
0.05 ( 0.01 ND 0.69 ( 0.05 5.31 ( 0.13 93.91 ( 0.18 0.04 ( 0.00 ND ND 92.41 ( 0.41 85.98
0.43 ( 0.04 0.08 ( 0.03 12.54 ( 0.05 50.98 ( 0.04 32.73 ( 0.10 2.61 ( 0.08 0.55 ( 0.02 0.10 ( 0.02 93.44 ( 0.28 93.44
0.07 ( 0.01 ND 0.66 ( 0.01 5.53 ( 0.05 93.7 ( 0.04 0.05 ( 0.00 ND ND 87.79 ( 0.41 82.03
a Solvent ) acetonitrile, solvent/FFA ) 62.5 mL/g, storage temperature ) -40 °C. b Solvent ) 30% acetonitrile + 70% acetone, solvent/ FFA ) 90 mL/g, storage temperature ) -80 °C. c Solvent ) acetonitrile, solvent/FFA ) 50 mL/g, storage temperature ) -20 °C. d Solvent ) 30% acetonitrile + 70% acetone, solvent/FFA ) 50 mL/g, storage temperature ) -80 °C. e Not detectable.
stored in an ultralow-temperature freezer (Sanyo MDF192, Japan) chamber for 48 h at -20 or -40 °C. The solid formed was removed by using a Buchner funnel operated at room temperature. GLA-rich FFA was obtained after the solvent in the remaining liquid was removed in a vacuum rotary evaporator operated at 35 °C. Second-Stage Solvent Crystallization. All procedures were the same as those described in the first-stage solvent crystallization except the following: the solvent was a mixture of 30% acetonitrile and 70% acetone, the storage temperature was -80 °C, and the storage time was 24 h. Recovery of GLA from the Solid Phase. The second-stage solvent crystallization process as described previously was applied to the solid collected from the two-stage operation. The resulting liquid phase was collected and its composition analyzed. Determination of FFA Composition. The composition of FFA was determined by gas chromatography. FFAs were transformed into their corresponding fatty acid methyl esters (FAME) using trimethylsulfonium hydroxide.15 The composition of the FAME mixture was analyzed by a China Chromatography model 8700F (Taipei, Taiwan) gas-liquid chromatograph, equipped with a flame ionization detector. The column used was SP-2330 (30 × 0.25 mm i.d., Supelco, Bellefonte, PA). The temperatures of the injector and the detector were set at 250 and 270 °C, respectively. The column was held at 180 °C for 10 min, then increased to 235 °C at a constant rate of 15 °C/min, and kept at 235 °C for 3 min. The GLA yield is defined as the amount of GLA recovered in the product divided by the amount of GLA that exists in the original FFA. All data presented were the average of three independent experiments. Results and Discussion First-Stage Solvent Crystallization. As shown in Table 1, FFA derived from saponified borage oil contains 23.4% GLA. Other components that exist in a significant amount are palmitic acid (16:0, 10.3%), oleic acid (18:1, 15.9%), and linoleic acid (18:2, 37.3%). PUFAs have higher solubility in a hydrophilic solvent, such as acetone and acetonitrile, than saturated and monounsaturated fatty acids. Aqueous acetonitrile is a useful solvent in the separation of saturated from unsaturated fatty acids.16 Acetonitrile was used as the solvent in the first stage for the separation of PUFA from saturated and monounsaturated fatty acids. As can be seen from Table 1, most of the saturated and monounsaturated
fatty acids were removed by the first-stage operation. In the concentration of PUFA by low-temperature solvent crystallization, both the PUFA content and yield depend on the solvent to FFA ratio (solvent/FFA) as well as the storage temperature. It was observed that a decrease in the storage temperature resulted in an increase in the GLA content and a slight decrease in the GLA yield.17 At a storage temperature of -20 °C, both the GLA content and yield remain fairly constant for solvent/FFA higher than 50 mL/g. If the storage temperature is -40 °C, the GLA content and yield will remain constant only when solvent/FFA is higher than 60 mL/g. The object of the first-stage operation is to obtain a high GLA yield (>90%) without sacrificing the GLA content. As can be seen from Table 1, a storage temperature of -40 °C is preferred because both high GLA content (66.1%) and yield (93.0%) were obtained. A higher storage temperature of -20 °C in the first stage resulted in a lower GLA content of 32.7%, which in turn resulted in a GLA content of 93.7% and a GLA yield of 87.8% after the second-stage operation. With -40 °C as the first-stage storage temperature, the corresponding GLA content and yield after the secondstage operation are 93.9% and 92.4%, respectively. A storage temperature lower than -40 °C is difficult to operate because this is already close to the melting point of acetonitrile, which is -45 °C. Second-Stage Solvent Crystallization. After the first-stage operation, most saturated and monounsaturated fatty acids have been removed, as can be seen from Table 1. A solvent with a higher hydrophobicity and a lower melting point is required for the secondstage operation if linoleic acid (18:2) is to be separated from GLA (18:3). In this work, a mixture of 30% acetonitrile and 70% acetone was used as the solvent for the second-stage operation. A storage temperature of -80 °C was chosen based on the principle that a lower operation temperature gives better results,17 and a temperature lower than -80 °C is difficult to operate because acetone has a melting point of -90 °C. Figure 1 shows the effects of the solvent to FFA ratio on the GLA content and yield in both solid and liquid phases in the second-stage operation, using FFA obtained from the first stage with a GLA content of 66.1% as the substrate. As solvent/FFA increases, the GLA content in the liquid phase remains fairly constant, while the GLA yield increases rapidly. At solvent/FFA ) 90 mL/ g, a GLA content of 93.9% with a corresponding yield of 92.4% (an overall yield of 86.0% for the two-stage process) can be obtained. Both the GLA content and
Ind. Eng. Chem. Res., Vol. 40, No. 17, 2001 3783 Table 2. Compositions of the Liquid Phase Recovered from the Solid Phase of the Second-Stage Operation content (wt %)
Figure 1. Effects of solvent/FFA on the GLA content and yield in liquid and solid phases after the second-stage operation (solvent ) 30% acetonitrile + 70% acetone, storage at -80 °C for 24 h), using FFA obtained from the first stage with a GLA content of 66.1% as the substrate.
Figure 2. Effects of solvent/FFA on the GLA content and yield in the liquid phase after the second-stage operation (solvent ) 30% acetonitrile + 70% acetone, storage at -80 °C for 24 h), using FFA obtained from the first stage with a GLA content of 32.7% as the substrate.
yield in the solid phase decrease rapidly with increasing solvent/FFA. Figure 2 shows the effects of solvent/FFA on the GLA content and yield of the liquid phase in the second-stage operation, using FFA obtained from the first stage with a GLA content of 32.7% as the substrate. Again, the GLA content remains fairly constant while the GLA yield increases rapidly with increasing solvent/ FFA. From the results shown in Figures 1 and 2, it is clear that, in order to obtain both high GLA content and yield, it is desirable to choose first-stage operation conditions which result in as high values of the GLA content and yield in the first stage as possible. Recovery of GLA from the Solid Phase. The solid phase resulting from the first-stage operation usually contains less than 5% GLA and was discarded. The solid phase from the second-stage operation contains ca. 10% GLA and was recovered by applying the second-stage operation on the solid phase. Table 2 shows compositions of the liquid phase recovered from the solid phase before and after the second-stage operation. A GLA content of 77.0% was obtained. In addition, more than half (59.6%) of the GLA in the solid phase was recovered. There are several works reported in the literature concerning the purification of GLA from natural sources. FFAs with a GLA content of ca. 90% can be achieved. However, all reported GLA yields are less than 70%.11,12,18,19 As shown in Table 1, the GLA content in the FFA derived from saponified borage oil can be increased from 23.4% to 93.9% with an overall yield of 86.0% by the two-stage process developed in this work.
FFA
initiala
afterb
16:0 18:0 18:1 18:2 18:3, GLA 20:1 22:1 yield of GLA (%)
0.30 ( 0.00 NDc 11.17 ( 0.03 75.02 ( 0.11 11.75 ( 0.12 1.41 ( 0.01 0.35 ( 0.02 100
0.08 ( 0.01 ND 2.72 ( 0.21 19.88 ( 0.97 77.04 ( 1.19 0.23 ( 0.00 0.07 ( 0.01 59.64 ( 0.34
a Solid phase from the second-stage operation shown in Table 1 (second stageb). b Solvent ) 30% acetonitrile + 70% acetone, solvent/FFA ) 30 mL/g, storage temperature ) -80 °C. c Not detected.
Hence, the GLA content obtained is comparable to the best results available in the literature. However, the GLA yield obtained in this work is considerably higher than those reported in the literature. Results of preliminary studies show that the method developed in this work is equally effective in the concentration of docosahexaenoic acid (DHA; C22:6 n3) from DHASCO, a single cell oil. It is moderately effective in the concentration of arachidonic acid (ARA; C20:4 n6) from ARASCO, also a single cell oil. However, it performs poorly in the concentration of R-linolenic acid (ALA; C18:3 n3) from linseed oil. The method developed in this work is simpler; no enzyme-catalyzed reaction is required. Only simple physical processes such as cooling, solid-liquid separation, and solvent evaporation are involved. Because the processes are mostly operated under a very low temperature environment, the degradation of thermal labile PUFA can be kept at a minimum. Conclusions A two-stage, low-temperature solvent fractional crystallization process was applied for the concentration of GLA from saponified borage oil. The GLA content was raised from 23.4% to 66.1% in the first stage with a corresponding yield of 93%. The GLA content was further increased to 93.9% in the second-stage operation with a corresponding yield of 92.4%. The overall yield (based on the GLA content in borage oil) was 86%. Acknowledgment The financial support by the grant from the National Science Council of Taiwan (NSC89-2214-E-011-005) is gratefully acknowledged. Literature Cited (1) Sardessai, V. M. Nutritional Role of Polyunsaturated Fatty Acids. J. Nutr. Biochem. 1992, 3, 154. (2) Wu, D.; Meydani, S. N. Gamma-Linolenic Acid and Immune Function. In Gamma-Linolenic Acid: Metabolism and Its Roles in Nutrition and Medicine; Huang, Y. S., Mills, D. E., Eds.; AOCS Press: Champaign, IL, 1996. (3) Scott, J. Fish and Evening Primrose Oils: Gaining Medical Recognition. Curr. Ther. Res. 1989, 45. (4) Barber, A. J. Evening Primrose Oil: A Panacea? Pharmaceut. J. 1988, 240, 723. (5) Jantti, J.; Seppala, E.; Vapaatalo, H.; Isomaki, H. Evening Primrose Oil and Olive Oil in Treatment of Rheumatoid Arthritis. Clin. Rheumatol. 1989, 8, 238. (6) Haagsma, N.; van Gent, C. M.; Luten, J. B.; de Jong, R.; van Doorn, W. E. Preparation of an ω 3 Fatty Acid Concentrate from Cod Liver Oil. J. Am. Oil Chem. Soc. 1982, 59, 117.
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(7) Teramoto, M.; Matsuyama, H.; Ohnishi, N.; Uwagawa, S.; Nakai, K. Extration of Ethyl and Methyl Esters of Polyunsaturated Fatty Acids with Aqueous Silver Nitrate Solutions. Ind. Eng. Chem. Res. 1994, 33, 341. (8) Kubata, F.; Goto, M.; Nashio, F. Separation of Polyunsaturated Fatty Acids with Silver Nitrate Using a Hollow-Fiber Membrane Extractor. Sep. Sci. Technol. 1997, 32, 1529. (9) Yokochi, T.; Usita, M. T.; Kamisaka, Y.; Nakahara, T.; Suzuki, O. Increase in the γ-Linolenic Acid Content by Solvent Winterization of Fungal Oil Extracted from Mortierella Genus. J. Am. Oil Chem. Soc. 1990, 67, 846. (10) Arai, M.; Fukuda, H.; Morikawa, H. Selective Separation of γ-Linolenic Acid Ethyl Ester Using Y-Zeolite. J. Ferment. Technol. 1987, 65, 569. (11) Shimada, Y.; Sugihara, A.; Shibahiraki, M.; Fujita, H.; Nakano, H.; Nagao, T.; Terai, T.; Tominaga, Y. Purification of γ-linolenic Acid from Borage Oil by a Two-Step Enzymatic Method. J. Am. Oil Chem. Soc. 1997, 74, 1465. (12) Shimada, Y.; Sakai, N.; Sugihara, A.; Fugita, H.; Honda, Y.; Tominaga, Y. Large Scale Purification of γ-Linolenic Acid by Selective Esterification Using Rhizopus delemar Lipase. J. Am. Oil Chem. Soc. 1998, 75, 1539. (13) Brown, J. B. Fractional Solvent Crystallization. J. Am. Oil Chem. Soc. 1955, 32, 646.
(14) Uksila, E.; Lehtinen, I. Crystallization of Linseed Oil Fatty Acids from Acetonitrile. Acta Chem. Scand. 1966, 20, 1645. (15) Yamauchi, K.; Tanabe, T.; Kinoshita, M. Trimethylsulfonium Hydroxide: A New Methylating Agent. J. Org. Chem. 1979, 44, 638. (16) Sonntag, N. O. V. Separation of Fatty Acids. In Fatty Acids; Pryde, E. V., Ed.; AOCS Press: Champaign, IL, 1979. (17) Chen, T. C.; Ju, Y. H. Polyunsaturated Fatty Acid Concentrate from Borage and Linseed Oil Fatty Acids. J. Am. Oil Chem. Soc. 2001, 78, 485. (18) Huang, F. C.; Ju, Y. H.; Huang, C. W. Enrichment of γ-Linolenic Acid from Borage Oil via Lipase-Catalyzed Reactions. J. Am. Oil. Chem. Soc. 1997, 74, 977. (19) Syed Rahmatullah, M. S. K.; Shukla, V. K. S.; Mukherjee, K. D. Enrichment of γ-Linolenic Acid from Evening Primrose Oil and Borage Oil via Lipase Catalyzed Esterification. J. Am. Oil Chem. Soc. 1994, 71, 563.
Received for review January 3, 2001 Accepted May 29, 2001 IE010024U
