JOURNAL OF OLEO SCIENCE Copyright 2006 by Japan Oil Chemists Society J. Oleo Sci., Vol. 55, No. 10, 537543 (2006) JOS Purification of Monoacylglycerol with Conjugated Linoleic Acid Synthesized through a LipaseCatalyzed Reaction by Solvent Winterization Yomi WATANABE 1, Toshihiro NAGAO 1, Shuji KANATANI 2, Takashi KOBAYASHI 1, Tadamasa TERAI 2 and Yuji SHIMADA 1 1 Osaka Municipal Technical Research Institute (1650 Morinomiya, Jotoku, Osaka 5368553, JAPAN) 2 Department of Applied Chemistry, Osaka Institute of Technology (5161 Oomiya, Asahiku, Osaka 5368585, JAPAN) Accepted May 22, 2006 (received for review April 6, 2006) Abstract: Monoacylglycerol of conjugated linoleic acid (MAGCLA) is synthesized efficiently by esterification of a free fatty acid mixture of CLA (FFACLA) with glycerol using Penicillium camembertii lipase. However, the reaction mixture contained 37 wt% FFAs (the acid value, 614 mg KOH/g), although the acid value of MAG, which can be used as a food additive, is prescribed to be 6 mg KOH/g (the content of FFAs, about 3 wt%). Hence, the removal of FFAs in the reaction mixture by winterization was attempted. After the enzyme reaction, the reaction mixture was separated to the oil and glycerol layers. The oil layer (referred to as Rmix[MAG]) included 21.6 wt% glycerol, and its oil part was composed of 5.2 wt% FFAs, 87.0 wt% MAGs, and 7.8 wt% diacylglycerols (DAGs). Here, oil part means FFAs, MAGs, and DAGs in Rmix[MAG]. Rmix[MAG] was first dissolved in 4 ml hexane per 1 g of the Rmix, and the mixture was kept at 0 for 24 h. The crystallized materials were recovered by centrifugation at 20. The content of FFAs in the precipitates decreased from 5.2 to 2.0 wt%, and the content of MAGs increased from 87.0 to 95.8 wt%. To further remove FFAs, solvent winterization of the precipitates was conducted similarly, resulting in a decrease of the content of FFAs from 2.0 to 0.9 wt% and increase of the content of MAGs from 95.8 to 98.4 wt%. The recovery of MAGs through a series of procedures was 84.7% of the initial content of MAGs in the reaction mixture. These results showed that solvent winterization is very effective for purification of MAGCLA from the reaction mixture obtained by lipasecatalyzed esterification of FFACLA and glycerol. Key words: conjugated linoleic acid, monoacylglycerol, solvent winterization 1 Introduction Monoacylglycerols (MAGs) of saturated and monoenoic fatty acids are used widely as good emulsifiers for foods. They are synthesized industrially by chemical glycerolysis of oils and fats at high temperatures of 210240 (1). Depending on circumstances, the product is further purified by shortpath distillation. But this chemical process cannot be applied to synthesize MAGs of unstable fatty acids. Lipasecatalyzed reactions are effective for synthesis of MAGs of unstable fatty acids (24). Recently, MAG with conjugated linoleic acid (MAGCLA) was reported to be efficiently synthesized by lipasecatalyzed esterification of a free fatty acid (FFA) mixture including CLA (FFACLA) with glycerol at low temperature Correspondence to: Yomi WATANABE, Osaka Municipal Technical Research Institute, 1650 Morinomiya, Jotoku, Osaka 5368553, JAPAN Email: yomi@omtri.city.osaka.jp Journal of Oleo Science ISSN 13458957 print / ISSN 13473352 online J. Oleo Sci., http://jos.jstage.jst.go.jp/en/ Vol. 55, No. 10, 537543 (2006) 537
Y. Watanabe, T. Nagao, S. Kanatani et al. (57) and with dehydration under reduced pressure (8). These reactions achieved about 95% esterification, and the oil part after the reaction was composed of 37 wt% FFAs, 8690 wt% MAGs, and 58 wt% diacylglycerols (DAGs) (58). The acid value of MAG, which can be used as a food additive, is prescribed to be <6 mg KOH/g, but the acid value of the mixture obtained by the enzymatic method is 614 mg KOH/g. Hence, to produce MAG usable as a food additive, FFAs must be removed from the mixture. Removal of FFAs by shortpath distillation is difficult because boiling points of FFAs and MAGs are close. On the other hand, the oil fraction in the reaction mixture was composed of FFAs, MAGs, and DAGs, and the melting point of MAGs was the highest among the three components. In the oil and fat industry, winterization is used widely for crystallization of the high melting point substances in a material (911). This paper shows that solvent winterization is effective for purification of MAGCLA from a mixture obtained by a lipase catalyzedreaction. 2 Materials and Methods 2 1 Materials An FFA mixture containing 9cis, 11trans (9c, 11t) and 10t, 12cCLAs was a commercial product (CLA HG; Nisshin OilliO Group, Ltd., Tokyo) obtained by alkali conjugation of high linoleic safflower oil in propylene glycol. The product contained 38.4 wt% 9c, 11tCLA, 39.5 wt% 10t, 12cCLA, 1.2 wt% 9c, 11c CLA, 1.2 wt% 10c, 12cCLA, 2.7 wt% other CLA isomers, 6.3 wt% palmitic acid, 2.1 wt% stearic acid, 8.4 wt% oleic acid, and 0.2 wt% other fatty acids. This FFA mixture is referred to as FFACLA. The molar amount of FFA was calculated based on the acid value. P. camembertii lipase was obtained from Amano Enzyme Inc. (Aichi). It is an 1,3specific mono and diacylglycerol lipase. One unit (U) of the activity was defined as the amount of enzyme that liberated 1 mmol fatty acid per minute in hydrolysis of monoolein (Tokyo Kasei Kogyo Co. Ltd.; Tokyo) (5). Other chemicals were of analytical grade. 2 2 Enzymatic Esterification of FFAs with Glycerol MAGs and DAGs with CLA were produced in two reaction systems as described previously (5,8). A mixture including MAGs and DAGs as main components was prepared by agitating a 350g mixture of FFA CLA/glycerol (1:2, mol/mol), 1.0 wt% water, and 200 U/gmixture of P. camembertii lipase at 30 for 10 h, followed by agitating for further 24 h with dehydration at 4 mm Hg. After the reaction, the reaction mixture was centrifuged (8,000 g, 5 min), and was separated to the oil and glycerol layers. The oil layer (referred to as Rmix[MAG+DAG]) included 6.5 wt% glycerol, and its oil part was composed of 57.9 wt% MAGs, 37.1 wt% DAGs, 5.0 wt% FFAs. Oil part means FFAs, MAGs, and DAGs in Rmix[MAG+DAG]. Triacylglycerols were scarcely observed. A preferential production of MAGs was conducted using an identical reaction mixture as above at 30 for 48 h with dehydration at 4 mm Hg. The reaction mixture was then centrifuged to separate the oil and glycerol layers. The oil layer included 19.9 wt% glycerol, and its oil part was composed of 87.0 wt% MAGs, 5.9 wt% DAGs, 7.1 wt% FFAs. This oil layer is referred to as Rmix[MAG]. 2 3 Purification of MAGs and DAGs MAGs and DAGs were purified from Rmix[MAG+ DAG]. The Rmix (20 g) was diluted with the same volume of hexane and applied to a silica gel 60 column (160 g; 40 300 mm; Merck, Darmstadt, Germany). DAGs and FFAs were eluted with 1,500 ml hexane/ethyl acetate (80:20, vol/vol), and MAGs were then eluted with 1,000 ml hexane/ethyl acetate (40:60, vol/vol). Organic solvents in the DAG/FFA and MAG fractions were removed with an evaporator. The MAG preparation (yield, 9.8 g) was composed of 98.3 wt% MAGs, 1.3 wt% DAGs, and 0.4 wt% FFAs. FFAs in the DAG/FFA fraction were removed by hexane extraction under alkaline conditions as described previously (12). Hexane in DAGs was removed with an evaporator. The DAG preparation (yield, 5.9 g) was composed of 98.3 wt% DAGs, 1.2 wt% MAGs, and 0.5 wt% FFAs. The unrecovered 4.3 g included glycerol and FFAs in the Rmix, and the loss of the operation. 2 4 Solvent Winterization A standard procedure of solvent winterization was conducted as follows. An oil material including FFAs, MAGs, and DAGs was dissolved in 4 ml hexane per 1 g of the oil material, and was then kept at 0 for 24 h. 538 J. Oleo Sci., Vol. 55, No. 10, 537543 (2006)
Purification of MAGCLA by Winterization The precipitates formed were recovered by centrifugation (8,000 g, 1 min) at 20, and hexane in the precipitates was removed with an evaporator. 2 5 Analyses The contents of MAGs, DAGs, and FFAs were determined with a HewlettPackard 5890 gas chromatograph connected to a DB1ht (0.25 mm 5 m; J&W Scientific, CA, USA). The column temperature was held for 0.5 min at 120. It was then raised from 120 to 280 at 15 /min and from 280 to 370 at 10 /min, and finally held at 370 for 1 min. The injector and detector (FID) temperatures were set at 370 and 390, respectively. Melting points of MAGs, DAGs, and FFAs were measured according to the AOCS standard open tube melting point (slip melting point) method (13). The content of glycerol was analyzed by glycerokinase/pyruvate kinase/lactate dehydrogenase assay system (FKit Glycerol; Boehringer Mannheim, Mannheim, Germany) according to the supplier s protocol. Fig. 1 Temperature for Crystallization of MAGs, DAGs, and FFAs in Hexane. The preparations of MAGs (the content of MAGs, 98.3 wt%), DAGs (the content of DAGs, 98.3 wt%), and FFAs (FFACLA; the content of FFAs, 100 wt%) were used as materials. Each preparation (3 g) was dissolved in 12 ml hexane, and the solutions were kept for 24 h at temperatures of 30 to 20. The precipitates formed were recovered by centrifugation (8,000 g, 1 min) at 20., The amount of FFAs crystallized;, the amount of MAGs crystallized;, the amount of DAGs crystallized. 3 Results and Discussion 3 1 Temperature for Crystallization of MAGs, DAGs, and FFAs in Hexane MAGs and DAGs were synthesized by enzymatic esterification of FFACLA with glycerol, and were purified by silica gel column chromatography as described in Materials and Methods section. Melting points of the MAGs, DAGs, and FFAs (FFACLA) were 34.9, 11.4, and 10.0, respectively. Three grams of the MAGs, DAGs, and FFAs were dissolved separately in 12 ml hexane. The solutions were kept for 24 h at temperatures from 30 to 20, and the precipitates were recovered by centrifugation at 20. The amount of precipitates is shown in Fig. 1. When the MAG preparation was kept below 5, >91 wt% were recovered in the precipitates. Meanwhile, the DAG and FFA preparations did not form any precipitates when keeping at temperatures above 0. These results suggested strongly that only MAGs are crystallized when a mixture of MAGs, DAGs, and FFAs is kept at temperatures of 05 under the condition tested. 3 2 Effect of Solvent on Crystallization of MAGs A 3g Rmix[MAG] was dissolved in 12 ml of hexane, acetone, ethanol, and isooctane, which can be used for food processing. Each solution was kept at 0 for 24 h, and the precipitates formed were recovered by centrifugation. The contents of MAGs, DAGs, and FFAs in supernatant and precipitates are shown in Table 1. When hexane and isooctane were used as solvents, precipitates were formed and MAGs were recovered in the precipitates. But acetone and ethanol did not form any precipitates. Hexane was selected because of a popular solvent for food processing. Effect of the amount of the solvent on crystallization of MAGs was next studied. Rmix[MAG] was dissolved in different amounts of hexane, and the mixture was kept at 0 for 24 h. formed were then recovered by centrifugation. The contents of MAGs, DAGs, and FFAs in the precipitate fraction are shown in Table 2. When hexane was not added, Rmix[MAG] was in a solid state and the supernatant fraction was not formed. An increase in the amount of hexane removed efficiently FFAs and DAGs in the supernatant fraction, although the recovery of MAGs in precipitates decreased. In the following experiments, J. Oleo Sci., Vol. 55, No. 10, 537543 (2006) 539
Y. Watanabe, T. Nagao, S. Kanatani et al. the amount of hexane was fixed at 4 ml per 1 g Rmix[MAG]. 3 3 Effect of Temperature on Crystallization of MAGs in Rmix[MAG] A mixture of 3 g Rmix[MAG] and 12 ml hexane was kept for 24 h at temperatures from 20 to 10, and the precipitates formed were recovered by centrifugation. The contents of FFAs, MAGs, and DAGs in the precipitates and the recovery of MAGs are shown in Table 3. The recovery of MAGs in the precipitates was high at the lower temperature, and the content of MAGs decreased a little because of crystallization of DAGs and FFAs. When the mixture was kept at 0, the content of MAGs in the precipitate reached 96.0 wt% and the recovery of MAGs was 89.8%. Based on these results, the temperature for purification of MAGs by winterization was fixed at 0. 3 4 Effect of Contaminants on Crystallization of MAGCLA When MAGCLA was synthesized by esterification of FFACLA with glycerol using P. camembertii lipase, the contents of MAGs, DAGs, and FFAs were not always constant. Hence, effect of the amount of FFAs Table 1 Crystallization of MAGs in Several Solvents. Composition (wt%) a) and DAGs on crystallization of MAGs was studied. FFACLA was added to Rmix[MAG] at the concentration of 7 to 20 wt%, and the mixture was dissolved in 4 ml/gmixture of hexane. After the solution was kept at 0 for 24 h, the precipitates were recovered by centrifugation. The recovery of MAGs in the precipitates and the contents of MAGs, DAGs, and FFAs are shown in Fig. 2. An increase in the amount of FFAs affected a little the content and recovery of MAGs in the precipitates after winterization. The content of DAGs in the precipitates after winterization decreased in proportion Table 2 Amount of hexane a) (ml/g) 0 1 2 4 7 10 Effect of Hexane Amount on Crystallization of MAGs. Composition (wt%) b) FFA MAG DAG 7.1 3.3 2.0 1.6 1.0 87.0 92.1 94.2 95.6 96.2 97.7 5.9 4.6 3.6 2.4 1.3 Recovery of MAG (%) 100 91.3 91.2 90.4 87.2 83.0 Rmix[MAG] (3 g) was dissolved in different amounts of hexane. After keeping the solution at 0 for 24 h, the precipitates formed were recovered by centrifugation (8,000 g, 1 min) at 20. a) The amount (ml) of hexane per 1 g Rmix[MAG]. b) The contents of FFAs, MAGs, and DAGs in the precipitates. Their total content was expressed as 100 wt%. Solvent Fraction FFA MAG DAG None b) Hexane Acetone Ethanol isooctane Rmix[MAG] (3 g) was dissolved in 12 ml organic solvent, and the solutions were kept at 0 for 24 h. From the mixture, the fractions of supernatant and precipitates were separated by centrifugation (8,000 g, 1 min) at 20. Organic solvent in the supernatant and precipitate fractions were removed with an evaporator. a) The total content of MAGs, DAGs, and FFAs in the precipitates was expressed as 100 wt%. b) Rmix[MAG] used as a material. 7.1 29.3 1.9 7.2 7.0 29.6 1.8 87.0 46.9 96.1 86.3 86.7 48.0 96.0 5.9 23.8 2.0 6.5 6.3 22.4 Table 3 Temperature ( ) 20 10 5 0 5 10 Effect of Temperature on Crystallization of MAGs. Composition (wt%) a) FFA MAG DAG 2.4 2.1 2.1 1.8 1.9 1.9 94.1 95.2 95.4 96.0 96.0 95.9 3.5 2.7 2.5 2.1 Recovery of MAG (%) 96.3 95.8 92.0 89.8 80.2 37.1 Rmix[MAG] (3 g) was dissolved in 12 ml hexane, and the solution was kept at 0 for 24 h. The precipitates were recovered by centrifugation (8,000 g, 1 min) at 20. a) The contents of MAGs, DAGs, and FFAs in the precipitates. Their total content was expressed as 100 wt%. 540 J. Oleo Sci., Vol. 55, No. 10, 537543 (2006)
Purification of MAGCLA by Winterization to that before winterization. In addition, the content of FFAs in the precipitates after winterization increased in proportion to that before winterization, and the content was <3 wt% (acid value, 6 mg KOH/g) when the content before winterization was <12 wt%. An effect of DAGs on crystallization of MAGs was next studied. A mixture of Rmix[MAG]/Rmix[MAG+ DAG], in which the content of DAGs in the oil part was 6 to 37 wt%, was dissolved in hexane. After the solution was kept at 0 for 24 h, the contents of MAGs, DAGs, and FFAs in the precipitates were determined (Fig. 3). The content of MAGs decreased with increasing the content of DAGs in the material before winterization, and was >90 wt% when the content of DAGs before winterization was <30 wt%. The recovery of MAGs in the precipitates also decreased with increasing the content of DAGs before winterization, and was >90% when the content of DAGs was <10 wt%. The content of FFAs before winterization was <7 wt%; thus, the solvent winterization decreased the content of FFAs in the precipitates to <2 wt%. 3 5 Purification of MAGCLA from Reaction Mixture by Solvent Winterization The results described above showed that MAGs in the reaction mixture can be purified to 95 wt% with >90% recovery when the contents of contaminants, FFAs and DAGs, are <12 and <10 wt%, respectively. A lipasecatalyzed esterification of FFACLA with glycerol efficiently produced MAGs. The oil fraction in the reaction mixture is composed of 37 wt% FFAs, 8690 wt% MAGs, and 58 wt% DAGs, showing that MAGs can be purified from the reaction mixture. Hence, Fig. 2 Effect of the Amount of FFAs on Crystallization of MAGs. FFACLA was added to Rmix[MAG] (FFAs, 7.1 wt%; MAGs, 87.0 wt%; DAGs, 5.9 wt%) at the concentration of 7 to 20 wt%. The mixture (3 g) was dissolved in 12 ml hexane, and then was kept at 0 for 24 h. The precipitates formed were recovered by centrifugation (8,000 g, 1 min) at 20, and the contents of FFAs, MAGs, and DAGs in the precipitates were analyzed. The horizontal axis shows the content of FFAs in the material before winterization., The content of FFAs in the material before winterization;, the content of FFAs in precipitates after winterization;, the content of MAGs in the material before winterization;, the content of MAGs in precipitates after winterization;, the content of DAGs in the material before winterization;, the content of DAGs in precipitates after winterization;, the recovery of MAGs in precipitates after winterization. Fig. 3 Effect of the Amount of DAGs on Crystallization of MAGs. Rmix[MAG] (FFAs, 7.1 wt%; MAGs, 87.0 wt%; DAGs, 5.9 wt%) and Rmix[MAG + DAG] (FFAs, 5.0 wt%; MAGs, 57.9 wt%; DAGs, 37.1 wt%) were mixed at different ratios. The mixture (3 g) was dissolved in 12 ml hexane, and then was kept at 0 for 24 h. The precipitates generated were recovered by centrifugation (8,000 g, 1 min) at 20, and the contents of FFAs, MAGs, and DAGs in the precipitates were analyzed. The horizontal axis shows the content of DAGs in the material before winterization., The content of FFAs in the material before winterization;, the content of FFAs in precipitates after winterization;, the content of MAGs in the material before winterization;, the content of MAGs in precipitates after winterization;, the content of DAGs in the material before winterization;, the content of DAGs in precipitates after winterization;, the recovery of MAGs in precipitates after winterization. J. Oleo Sci., Vol. 55, No. 10, 537543 (2006) 541
Y. Watanabe, T. Nagao, S. Kanatani et al. Table 4 Purification of MAGs by Repeated Solvent Winterization. Weight (g) Composition (wt%) a) Recovery of MAGs Procedure Total Glycerol FFA MAG DAG (%) Oil material b) Winterization (1) c) Winterization (2) c) 30.0 24.8 23.1 6.5 5.6 5.5 5.2 2.0 0.9 87.0 95.8 98.4 7.8 0.7 100 89.9 84.7 a) The total content of MAGs, DAGs, and FFAs was expressed as 100 wt%. b) Reaction mixture obtained by esterification of FFACLA with glycerol was centrifuged at 8,000 g for 5 min, and was separated to the oil and glycerol layers. The oil layer including 21.6 wt% glycerol was used as an oil material. c) The oil material was dissolved in 4 ml/g of hexane, and the solution was kept at 0 for 24 h. The precipitates formed were recovered by centrifugation (8,000 g, 1 min) at 20, and the solvent was removed with an evaporator. MAGs was purified actually from the reaction mixture. A mixture of FFACLA/glycerol (1:2, mol/mol), 1.0 wt% water, and 200 U/g P. camembertii lipase was agitated at 30 /4 mm Hg for 48 h. After the reaction, the mixture was centrifuged and separated to the oil and glycerol layers. The oil layer contained 21.6 wt% glycerol, and the contents of FFAs, MAGs, and DAGs in the oil part were 5.2, 87.0, and 7.8 wt%, respectively. Purification of MAGs from the reaction mixture is summarized in Table 4. The reaction mixture (30 g) was dissolved in 120 ml hexane, and the solution was kept at 0 for 24 h. The winterization removed FFAs and DAGs in the supernatant. The content of FFAs in the precipitates decreased from 5.2 to 2.0 wt%, and the content of MAGs increased from 87.0 to 95.8 wt%. To further remove FFAs, solvent winterization of the precipitates was conducted similarly, resulting in a decrease of the content of FFAs from 2.0 to 0.9 wt% and an increase of the content of MAGs from 95.8 to 98.4 wt%. The recovery of MAGs through a series of procedures was 84.7% of the initial content of MAGs in the reaction mixture. 4 Conclusion Winterization in hexane was very effective for purification of MAGCLA from the reaction mixture which was obtained by lipasecatalyzed esterification of FFA CLA with glycerol. But the winterization remained glycerol in the precipitates (purified MAG fraction). If necessary, the glycerol can be removed by a conventional shortpath distillation which is adopted industrially for purification of MAGs produced by a chemical process. If the MAG preparation obtained by the single winterization of reaction mixture is further purified by shortpath distillation, the content of FFAs in the purified MAG preparation will become 2.0 wt% (acid value, about 4 mg KOH/g). Because the standard of MAG as a food additive is <6 mg KOH/g, winterization in hexane may be used industrially for purification of MAGCLA which was synthesized by a lipasecatalyzed esterification. References 1. N.O.V. SONNTAG, New Developments in the Fatty Acid Industry in America, J. Am. Oil Chem. Soc., Vol. 61, 229232 (1984). 2. B. AHA, M. BERGER, B. JAKOB, G. MACHMÜLLER, C. WALDINGER and M.P. SCHNEIDER, LipaseCatalyzed Synthesis of Regioisomerically Pure Mono and Diglycerides, in Enzymes in Lipid Modification, (U.T. BORNSCHEUER, ed.), WileyVCH, Weinheim, pp. 100115 (2000). 3. G.P. MCNEILL, S. SHIMIZU and T. YAMANE, HighYield Enzymatic Glycerolysis of Fats and Oils, J. Am. Oil Chem. Soc., Vol. 68, 15 (1991). 4. S. YAMAGUCHI and T. MASE, HighYield Synthesis of Monoglyceride by Mono and Diacylglycerol Lipase from Penicillium camembertii U150, J. Ferment. Bioeng., Vol. 72, 162167 (1991). 5. Y. WATANABE, Y. SHIMADA, Y. YAMAUCHISATO, M. KASAI, T. YAMAMOTO, K. TSUTSUMI, Y. TOMINAGA and A. SUGIHARA, Synthesis of MAG of CLA with Penicillium camembertii Lipase, J. Am. Oil Chem. Soc., Vol. 79, 891896 (2002). 6. Y. WATANABE, Y. YAMAUCHISATO, T. NAGAO, T. YAMAMOTO, K. TSUTSUMI, A. SUGIHARA and Y. SHI 542 J. Oleo Sci., Vol. 55, No. 10, 537543 (2006)
Purification of MAGCLA by Winterization MADA, Production of MAG of CLA in a SolventFree System at Low Temperature with Candida rugosa Lipase, J. Am. Oil Chem. Soc., Vol. 80, 909914 (2003). 7. Y. WATANABE, Y. YAMAUCHISATO, T. NAGAO, T. YAMAMOTO, K. OGITA and Y. SHIMADA, Production of Monoacylglycerol of Conjugated Linoleic Acid by Esterification followed by Dehydration at Low Temperature Using Penicillium camembertii Lipase, J. Mol. Catal. B: Enzym., Vol. 27, 249254 (2004). 8. Y. WATANABE, Y. YAMAUCHISATO, T. NAGAO, S. NEGISHI, T. TERAI, T. KOBAYASHI and Y. SHIMADA, Production of MAG of CLA by Esterification with Dehydration Using Penicillium camembertii Lipase, J. Am. Oil Chem. Soc., Vol. 82, 619623 (2005). 9. T. YOKOCHI, M.T. USITA, Y. KAMISAKA, T. NAKAHARA and O. SUZUKI, Increase in the glinolenic Acid Content by Solvent Winterization of Fungal Oil Extracted from Mortierella Genus, J. Am. Oil Chem. Soc., Vol. 67, 846851 (1990). 10. W. HAMM, Trends in Edible Oil Fractionation, Trends Food Sci. Technol., Vol. 6, 121126 (1995). 11. J.C. LÓPEZMARTÍNEZ, P. CAMPRAMADRID and J.L. GUILGUERRERO, glinolenic Acid Enrichment from Borago officinalis and Echium fastuosum Seed Oils and Fatty Acids by Low Temperature Crystallization, J. Biosci. Bioeng., Vol. 97, 294298 (2004). 12. Y. SHIMADA, N. FUKUSHIMA, H. FUJITA, Y. HONDA, A. SUGIHARA and Y. TOMINAGA, Selective Hydrolysis of Borage Oil with Candida rugosa Lipase: Two Factors Affecting the Reaction, J. Am. Oil Chem. Soc., Vol. 75, 15811586 (1998). 13. Official Methods and Recommended Practices of the American Oil Chemists Society, (D. FIRESTONE, ed.), 5th edn., AOCS, Champaign, 1998, AOCS Official Method Cc 325. J. Oleo Sci., Vol. 55, No. 10, 537543 (2006) 543