Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Rit Fiskideildar 16 (1999) 97-105 The application of lipase for preparing various lipids enriched with omega-3 fatty acids Gudmundur G. Haraldsson Science Institute, University of Iceland Dunhagi 3, IS-107 Reykjavik, Iceland ABSTRACT Immobilized lipases have been employed to generate various lipid classes, including triacylglycerols, phospholipids and non-polar ether lipids, highly enriched with eicosapentaenoic acid and doscosahexaenoic acid. These are long-chain n-3 type polyunsaturated fatty acids, characteristic of marine fat. Lipases are ideally suited as catalysts for transformations involving the highly labile long-chain polyunsaturated fatty acids. The mildness they offer, most certainly protects them from partial destruction of their natural all-cis n-3 framework by oxidation, double-bond migrations or cis-trans isomerization during traditional chemical processes involving extremes of pH and high temperature. The considerable reduction in bulkiness, related to the solvent-free conditions, renders the techniques described a high feasibility for industrialization. Keywords: n-3 polyunsaturated fatty acids, fish oil, lipase, triacylglycerol, phospholipids, ether lipids. INTRODUCTION C14 to C24 of varying degree of unsaturation, from saturated to polyunsaturated. Of the polyunsaturated fatty acids (PUFA), EPA and DHA usually account for between 5 and 15% each, depending on type of fish species, with the total n-3 content usually varying between 20 and 30%. As an example, cod liver oil has roughly 10% each of EPA and DHA, tuna oil is highly enriched with DHA (approximately 5% EPA and 25% DHA) and sardine oil is enriched with EPA (approximately 18% EPA and 12% DHA). Herring oil and capelin oil have lower EPA and DHA content, usually between 5 and 7 % each of EPA and DHA. Seasonal variations and site of catch influence the fatty acid composition of fish and fish oil to a large extent within the same The long-chain n-3 (or omega-3) type polyunsaturated fatty acids are characteristic of marine fat (Ackman 1989). They are produced by phytoplankton and accumulate through the food web in fish, which is unable to biosynthesise them. The most ubiquitous of the n-3 fatty acids in fish are cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), which commonly occur in triacylglycerols (TG; triglycerides) (Hölmer 1989) and phospholipids (PL) (Vaskovsky 1989). The chemical structure of EPA and DHA is shown in Figure 1. Fish oil is virtually pure triacylglycerols, which comprise more than fifty different fatty acids (Ackman 1982). The chain length ranges from Dedicated to Professor Unnsteinn Stefánsson in honour of his contributions to oceonography and education. 97 DHA, more specific methods are required, such as the Corey´s chemical modification, HPLC and, more recently, kinetic resolution by lipase to separate EPA and DHA (Haraldsson et al. 1989). Resynthesis of triacylglycerols or some other natural lipid form, such as ether lipids or phospholipids, is by no means easy by conventional chemical methods. Such traditional esterification methods involve extremes of pH, high temperature and pressure (Haraldsson 1989). Almost certainly, this will partially destroy the all-cis n-3 framework of EPA and DHA by oxidation, cis-trans isomerization, double-bond migrations or polymerization. In order to solve these problems lipases have been introduced to processes involving the highly labile n-3 polyunsaturated fatty acids (Haraldsson and Hjaltason 1992). They offer high efficiency and mildness and their application in organic media is now firmly established (Haraldsson 1992). The application of lipase for preparing various lipid classes highly enriched or homogeneous with EPA and/or DHA is described in this review, which is largely confined to our work at the University of Iceland. This includes triacylglycerols, phospholipids and ether lipids, which are characteristic of shark liver oil. Furthermore, the possibility of employing lipases to produce monoester or free fatty acid concentrates of EPA and DHA by kinetic resolution of fatty acids, based on lipase fatty acid selectivity, will also be discussed. Figure 1. The structure of EPA and DHA. species. In lean fish species such as cod, haddock and pollack, the triacylglycerols are largely confined to a large liver, whereas in fatty fish species, such as herring and capelin, the triacylglycerols are confined to their oily flesh. Phospholipids are generally much more highly enriched with EPA and DHA, 20 to 50% of each depending on the type of phospholipids. The total phospholipid content of fish usually remains between 1 and 1.5% as based on total fish wet weight, roughly an order of magnitude lower than the triacylglycerols (Haraldsson et al. 1993). The beneficial health effects of marine fat are now well established and almost exclusively attributed to the n-3 polyunsaturated fatty acids, EPA and DHA in particular (Nelson 1991). Consequently, there are strong demands for their concentrates by the pharmaceutical industry as well as the health food industry for use as food supplements. Triacylglycerols, up to the level of 30 % EPA + DHA, can be prepared directly from fish oils, without splitting the fat, by various methods such as winterization, molecular distillation and solvent crystallization (Haraldsson et al. 1989). Concentration beyond that level is difficult and requires a cleavage of the fatty acids off the acylglycerols, either as free fatty acids or monoesters. This is a consequence of the great combinations of the fatty acids in triacylglycerol oils, where they are more or less randomly distributed, three and three together, bound into the glycerol moiety. Several methods or combination of methods are available for concentrating them to the 50-80% level, including carbon dioxide supercritical fluid extraction, urea complexation, molecular distillation and kinetic resolution by lipase. In order to achieve concentrates beyond the 90% level of EPA and ENRICHMENT OF COD LIVER OIL WITH EPA AND DHA It is relatively easy to concentrate EPA and DHA up to high levels as free fatty acids or ethyl esters. The natural form of these fatty acids in fish oil is triacylglycerols and the big challenge was to provide natural triacylglycerols highly enriched with EPA and DHA, far beyond the 30% level mentioned above. A highly successful solution to that problem was based on treat- 98 ing cod liver oil as triacylglycerols with free fatty acid or monoester concentrates of EPA and DHA in the presence of lipase to effect fatty acid exchange between the natural triacylglycerols and the concentrates (Haraldsson et al. 1989). A 1,3-regiospecific lipase from the fungus Mucor miehei, commercially available in an immobilized form by Novo Nordisk in Denmark, was employed to effect transesterification reactions of cod liver oil with concentrates of EPA and DHA. What 1,3-regiospecificity means for a lipase, is to act with a high preference or exclusively at the primary alcoholic endpositions of the triacylglycerols. The cod liver oil comprised approximately 9-10% each of EPA and DHA, and triacylglycerols highly enriched with n-3 polyunsaturated fatty acids were accomplished, of high purity and in quantitative yields. Interesterification and acidolysis reactions with ethyl ester and free fatty acid concentrates, respectively, both of 85% EPA + DHA content, resulted in triacylglycerols containing 60-65% EPA + DHA and well over 70% total n3 polyunsaturated fatty acids. At that time, this represented by far the highest EPA and DHA enriched triacylglycerol product available. Both reactions were conducted in the absence of any solvent, using 10% dosage of lipase, as based on the weight of fat, at 60-65°C. The reactions are demonstrated in Scheme 1. A three-fold excess of free fatty acids or ethyl esters was used, as based on number of mol equivalents of esters present in the triacylglycerols. Despite the 1,3-regiospecificity of the lipase, the mid-position became enriched to an equal extent as the end-positions. This means that at an equilibrium the fatty acid composition of the triacylglycerols was reflected by the weighted average of the initial composition of the cod liver oil triacylglycerols and the concentrates, as well as the ratio between them. Intramolecular non- Scheme 1 enzyme promoted acyl-migration processes were responsible for this, as was established by investigating the fatty acid composition of individual positions of the acylglycerols when the reactions proceeded (Haraldsson and Almarsson 1991). HOMOGENEOUS TRIACYLGLYCEROLS OF EPA AND DHA By the methodology described above, the EPA and DHA fatty acid composition of the triacylglycerol product was determined by a weighted average of the initial fatty acid composition of the cod liver oil triacylglycerols and the composition of the n-3 concentrates. To avoid that limitation and obtain triacylglycerols of composition identical to the concentrates being used, a procedure based on a direct esterification of free fatty acids with glycerol was developed (Haraldsson et al. 1995). This also opened the possibility of synthesising triacylglycerols homogeneous with either EPA or DHA, i.e. 100% EPA or DHA. This had been considered a synthetic challenge and our primary goal, the crown on the triacylglycerol issue. The structure of such homogeneous triacylglycerols of EPA and DHA is depicted in Figure 2. A different lipase, the non-regiospecific yeast lipase from Candida antarctica, also commercially available from Novo Nordisk in Denmark, was highly efficient in generating triacylglycerols 99 Figure 2. The structure of homogeneous triacylglycerols of EPA and DHA. Scheme 2 containing 100% EPA and DHA. This was accomplished by a direct esterification of glycerol with stoichiometric amount of pure EPA and DHA, without any solvent, by stirring at 65°C under vacuum, with a 10% dosage of the immobilized lipase, as based on the weight of substrates. The co-produced water was condensed into a liquid nitrogen cooled trap during the progress of the reaction, thus driving the reaction to completion. This is demonstrated in Scheme 2 for EPA. The resulting triacylglycerols, homogeneous with either EPA or DHA, were afforded in nearly quantitative yields of very high purity. High-field 1H and 13C NMR spectroscopy was found extremely valuable as a probe to monitor the progress of the reactions. It also enabled us to follow the incorporation of EPA and DHA into glycerol to form the various intermediary acylglycerols participating in the direct esterification process, the 1- and 2-monoacylglycerols (MG), 1,2- and Scheme 3 100 Figure 3. The structure of ether lipids homogeneous with EPA and DHA. 1,3-diacylglycerols (DG) and the triacylglycerols (TG). Again, it became evident, despite the non-regio-specificity of the lipase, that acylmigration processes were playing an important role during the process. The overall reaction process is demonstrated in Scheme 3. transesterification reactions with concentrates of EPA and DHA, using Lipozyme in a manner identical to the one previously described for cod liver oil. Direct esterification of the 1-O-alkylsn-glycerols with EPA and DHA resulted in homogeneous ether lipids, when using the Candida antarctica lipase. These reactions are demonstrated in Scheme 4 for EPA. It was interesting to notice that the ether lipids were apparently far less prone to acyl migrations as compared to the triacylglycerols under the transesterification conditions. ETHER LIPIDS HIGHLY ENRICHED WITH EPA AND DHA Non-polar glyceryl ether lipids of the 1-O-alkyl2,3-diacyl-sn-glycerol type are major constituents in liver oil of certain species of shark, such as the Greenland shark (Somniosus microcephalus), making up to 30-60% of the oil (Sargent 1989). They have been claimed to display various beneficial effects on human health (Mangold and Palthauf 1983). Ether lipids, highly enriched with EPA and DHA, were prepared by the lipase catalysis procedures already described in order to possibly combine the claimed beneficial effects of both fish oil and the ether lipids (Haraldsson and Thorarensen 1994). Such ether lipids homogeneous with EPA and DHA are shown in Figure 3. The ether lipids were isolated in a pure state, as was established by high-field NMR, from shark liver oil concentrates using preparative HPLC. There are three major fatty alcohol constituents present in the ether moiety, C16:0, C18:0 and C18:1, the last one being the most abundant, with EPA and DHA making up only 0.4 and 2.5% of the fatty acid composition of the ether lipids acyl counterparts, respectively. They were converted into pure 1-O-alkyl-sn-glycerols by sodium methoxide catalyzed methanolysis. Ether lipids highly enriched with either EPA or DHA or both EPA and DHA were obtained by PHOSPHOLIPIDS HIGLY ENRICHED WITH EPA AND DHA Phospholipids are major constituents of cell membranes and play essential roles in biochemistry and physiology of the cell functions (Mead et al. 1986). Phospholipids in fish and marine species are highly enriched with the long-chain n-3 type polyunsaturated fatty acids. 40-50% content of EPA and DHA is not uncommon in some phospholipid classes in fish (Haraldsson et al. 1993). The n-3 polyunsaturated fatty acids presumably play significant roles in adjusting the membrane integrity and functions at the lower temperatures, by adding to the membrane fluidity and mobility as a result of their higher unsaturation. Among the phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are by far the most abundant in fish flesh, especially the former one, which commonly makes up 50-60% of the total phospholipid content. The composition of individual phospholipid classes is remarkably similar among fish species as is the characteristic fatty acid composition of each class (Haraldsson et al. 1993). 101 Scheme 4 Lecithins of plant and vegetable origin are popular as health supplements. They are highly enriched with n-6 fatty acids, which characterise vegetable oil in a similar manner as do the n-3 fatty acid fish oils. Purified fish lecithins, which are phospholipids highly enriched with n-3 polyunsaturated fatty acids, on the other hand, are not available on the market at all. Unlike plant or vegetable lecithins, they are by no means readily obtainable, since tedious extraction procedures from fish are required (Haraldsson et al. 1993). We therefore decided to attempt the preparation of such phospolipids, highly enriched with EPA and DHA, from the more readily available plant or animal lecithins (Haraldsson and Thorarensen 1995). Figure 4 shows the structure of phospatidylcholine (PC) homogeneous with EPA and DHA. Pure phosphatidylcholine was obtained from egg yolk after purification by preparative HPLC and was treated under the acidolysis reaction conditions described above for cod liver oil, using the Mucor miehei lipase and pure EPA. As might be anticipated, the rate of the reaction involving the phospholipids, possessing the zwitterionic head group, was much lower as compared to the natural triacylglycerol substrates. Large quantities of lipase were therefore required, which again resulted in high extent of hydrolysis sidereaction. Optimal reaction conditions offered a mixture of phospholipids of approximately 40% each of the desired phosphatidylcholine and lysophosphatidylcholine (LPC) and 20%of glycerol phosphatidylcholine (GPC). In LPC one of the acyl moiety has been hydrolysed and in glycerol phosphatidylcholine both acyl groups have been hydrolysed (see Scheme 5). When pure EPA was used, both the PC and the LPC fractions were highly enriched with EPA, especially the LPC fraction, 58 and 69%, respectively. This is by far the highest enrichment of n-3 polyunsaturated fatty acids into phospholipids reported in the literature. More detailed information about the nature of Figure 4. The structure of phosphatidylcholine homogeneous with EPA and DHA. 102 Scheme 5 the acidolysis reaction was afforded by 31P NMR spectroscopy analysis (Haraldsson and Thorarensen 1995). The results revealed the complex role played by water in terms of compromising the lipase activity, hydrolysis side-reactions, reaction rate and extent of incorporation under the non-aqueous reaction conditions. The rather complicated reaction process is demonstrated in Scheme 5. As before, acyl migrations were believed to play important roles in involving the mid-position in the overall process. Recently, we described a highly efficient process to concentrate EPA together with DHA by lipase-catalyzed ethanolysis of fish oil (Haraldsson et al. 1997; Breivik et al. 1997). Pseudomonas lipases were observed to convert the bulk of the unwanted saturated and monounsaturated fatty acids of the fish oil triacylglycerols into ethyl esters. The n-3 polyunsaturated fatty acids including both EPA and DHA, on the other hand, remained attached to the residual acylglycerols, mainly as mono- and diacylglycerols, but also triacylglycerols, depending upon the extent of conversion. This is demonstrated in Scheme 6. No solvents were required and only stoichiometric amounts of ethanol were required. By that method it became easy to obtain concentration levels of 50% EPA + DHA in the residual acylglycerol mixture with very high EPA and DHA recovery in sardine oil comprising 15% EPA and 10% DHA. It was interesting to observe that the Pseudomonas lipases displayed preference for DHA as compared to EPA, which is most unusual (Haraldsson et al. 1997). In a further development of the work we became interested in discriminating between EPA EPA AND DHA CONCENTRATES FROM FISH OILS BY LIPASE The use of lipases as catalysts for producing monoester or free fatty acid concentrates of EPA and DHA from fish oil, as an alternative to conventional chemical procedures, has also been investigated. Such work is based on a wide range of fatty acid selectivity among the various commercially available lipases. Many lipases do not tolerate the long-chain n-3 fatty acids very well as substrates. Lipases that do so usually display preference for EPA as compared to DHA. 103 Scheme 6 and DHA in fish oil instead of concentrating both EPA and DHA by lipase promoted kinetic resolution. Different requirements for the lipase selectivity were therefore made and a lipase strongly discriminating between EPA and DHA was needed. In order to avoid complications related to regioselectivity of the lipase and nonhomogeneous distribution of EPA and DHA into various positions of the triacylglycerols (Christie 1986) a different approach was required. That approach was based on a direct esterification of free fatty acids from fish oil, which is demonstrated in Scheme 7. The immobilized Mucor miehei lipase was observed to convert the bulk of the fatty acids present, including EPA, into ethyl esters, leaving the more reluctant DHA unaffected in the residual free fatty acids (Haraldsson and Kristinsson 1998). Free fatty acids from various fish oils were treated under the direct esterification reaction conditions, using stoichiometric amounts of ethanol at room temperature, in the absence of a solvent. This modification of the lipase catalysed separation of EPA and DHA was much more efficient and much faster than the corresponding ethanolysis reaction involving fish oil triacylglycerols. As an example, when tuna oil free fatty acids of 6% EPA and 23% DHA composition were directly esterified with ethanol, the residual free fatty acids comprised 74% DHA and only 3% EPA. The recovery of both DHA into the residual free fatty acid fraction and EPA into the ethyl ester product remained very high, 83 and 87%, respectively. Based on these results, there are reasons to believe that in terms of separating EPA and DHA, lipase can be used as a powerful alternative to traditional chemical techniques by a two-step or a multi-step approach. ACKNOWLEDGEMENT Lysi hf. in Reykjavik Iceland, Novo Nordisk AS in Bagsvaerd Denmark, Norsk Hydro AS in Porsgrunn Norway, Pronova Biocare AS in Sandefjord Norway, The Icelandic Government Science Fund and The Scheme 7 University of Iceland Research Fund are acknowledged for financial support. Freygarður Þorsteinsson, Páll A. Höskuldsson, Snorri Þ. Sigurðsson, Örn Almars- 104 Haraldsson, G.G., P.A. Höskuldsson, S.Þ. Sigurdsson, F. Þorsteinsson, and S. Guðbjarnason 1989. The preparation of triglycerides highly enriched with ω-3 polyunsaturated fatty acids via lipase catalyzed interesterification, Tetrahedron Lett. 30: 1671-1674. Haraldsson, G.G. and B. Kristinsson 1998. Separation of eicosapentaenoic acid and docosahexaenoic acid in fish oil by kinetic resolution using lipase. J. Am. Oil Chem. Soc. 75: 1551-1556. Haraldsson, G.G., B. Kristinsson and S. Guðbjarnason 1993. The fatty acid composition of various lipid classes in several species of fish caught in Icelandic waters. INFORM 4: 535. Haraldsson, G.G., B. Kristinsson, R. Sigurðardottir, G.G. Guðmundsson and H. Breivik 1997. The preparation of concentrates of eicosapentaenoic acid and docosahexaenoic acid by lipase-catalyzed transesterification of fish oil with ethanol. J. Am. Oil Chem. Soc. 74: 1419-1424. Haraldsson, G.G. and A. Thorarensen 1994. The generation of glyceryl ether lipids highly enriched with eicosapentaenoic acid and docosahexaenoic acid by lipase. Tetrahedron Letters 35: 7681-7684. Haraldsson, G.G. and A. Thorarensen 1995. The generation of phospholipids highly enriched with eicosapentaenoic acid and docosahexaenoic acid by lipase. In G.G. Haraldsson, S. Guðbjarnason and G. Lambertsen (Eds). Proceedings of the 18th Nordic Lipid Symposium, Reykjavik, Iceland, 1995, Lipidforum, Bergen, pp. 62-66. Hölmer, G. 1989. Triglycerides. In R.G. Ackman (Ed.), Marine Biogenic Lipids, Fats and Oils, Vol. I, CRC Press, Inc., Boca Raton, Florida, pp. 139-173. Mangold, H.K. and F. Palthauf (Eds.) 1983. Ether Lipids. Biochemical and Biomedical Aspects, Academic Press, Inc., New York, 439 pp. Mead, J.F., R.B. Alfin-Slater, D.R. Howton and G. Popják 1986. Lipids. Chemistry, Biochemistry and Nutrition, Plenum Press, New York, 486 pp. Nelson, G.J. (Ed.) 1991. Health Effects of Dietary Fatty Acids, American Oil Chemist´s Society, Champaign, Illinois, 274 pp. Sargent, J.R. 1989. Ether-linked glycerides in marine animals. In R.G. Ackman (Ed.), Marine Biogenic Lipids, Fats and Oils, Vol. I, CRC Press, Inc., Boca Raton, Florida, pp. 175-197. Vaskovsky, V.E. 1989. Phospholipids. In R.G. Ackman (Ed.), Marine Biogenic Lipids, Fats and Oils, Vol. I, CRC Press, Inc., Boca Raton, Florida, pp. 199-242. son, Birgir Örn Guðmundsson, Björn Kristinsson, Atli Thorarensen, Guðmundur G. Guðmundsson, Ragnheiður Sigurðardóttir, Sigmundur Guðbjarnason, Baldur Hjaltason, Tomas T. Hansen, Harald Breivik and Sigríður Jónsdóttir are gratefully thanked for their contributions to the work described in this paper. REFERENCES Ackman, R.G. 1989. Fatty acids. In R.G. Ackman (Ed.), Marine Biogenic Lipids, Fats and Oils, Vol. I, CRC Press, Inc., Boca Raton, Florida, pp. 103-137. – 1982. Fatty acid composition of fish oils. In S.M. Barlow and M.E. Stansby (Eds.), Nutritional Evaluation of Long-Chain Fatty Acids in Fish Oil, Acad. Press, New York, pp. 25-88. Breivik, H., G.G. Haraldsson and B. Kristinsson 1997. The preparation of highly purified concentrates of EPA and DHA. J. Am. Oil Chem. Soc. 74: 14251429. Christie, W.W. 1986. The positional distributions of fatty acids in triglycerides. In R.J. Hamilton and J.B. Rossell (Eds.), Analysis of Oils and Fats, Elsevier, London, pp. 313-339. Haraldsson, G.G. 1989. The application of lipases for modification of fats and oils, including marine oils. In M.N. Voigt and R. Bhotta (Eds.), Advances in Fisheries Technology for Increased Profitability, Technomic Publishing Co., Inc., Pennsylvania, pp. 337-357. Haraldsson, G.G. 1992. The Application of Lipases in Organic Synthesis. In S. Patai (Ed.), The Chemistry of the Functional Groups, Supplement B2: The Chemistry of Acid Derivatives, Vol. 2, John Wiley and Sons, Chichester, pp. 1395-1473. Haraldsson, G.G. and Ö. Almarsson 1991. Studies on the positional specificity of lipase from Mucor miehei during interesterification reactions of cod liver oil with n-3 polyunsaturated fatty acid and ethyl ester concentrates. Acta Chemica Scandinavica 45: 723-730. Haraldsson, G.G., B.Ö. Gudmundsson and Ö. Almarsson 1995. The synthesis of homogeneous triglycerides of eicosapentaenoic acid and docosahexaenoic acid by lipase. Tetrahedron 51: 941-952. Haraldsson, G.G. and B. Hjaltason 1992. Using biotechnology to modify marine lipids. INFORM 3: 626-629. 105