Modification of Oils and Fats to Produce Structured Lipids

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1 JOURNAL OF OLEO SCIENCE Copyright 2005 by Japan Oil Chemists Society JOS REVIEW Modification of Oils and Fats to Produce Structured Lipids Ruby TRIVEDI and R.P. SINGH Deptt. of Oil & Paint Technology, Harcourt Butler Technological Institute (Kanpur , INDIA) Edited by T. Itoh, Kitasato Univ., and accepted April 11, 2005 (received for review February 14, 2005) Abstract: Awareness regarding the harmful effects of excessive oils and fats intake is universal. Consequently, health conscious individuals are modifying their dietary habits and eating less fat, as high fat intake specially saturated fat is associated with increased risk for obesity, high blood cholesterol, coronary heart disease, cancer, and arteriosclerosis. Although oils and fats are not only the concentrated source of energy but also the source of essential fatty acids (EFA). Taking into account the drawbacks and benefits of oils and fats, the production of Structured Lipids (SLs) containing especial fatty acids has been now a days attracting more and more attention. Key words:structured lipid, S:M:P, essential fatty acid, lipase, SFA, PUFA, MUFA, MCFA, cholesterol Introduction Oils and fats play a very important role in human diet, besides providing calories they are also the source of essential fatty acids, vitamins (A, D, E & K) etc. However, these days, attempts are being made to reduce oils & fats from food products. These concerns are mainly due to cholesterol and its link to obesity and other diseases, but some people are constantly in a dilemma as medical research has linked dietary fats with such maladies as cancer, heart disease and stroke. The over consumption of fats has been declared one of the major dietary health concerns in the United States by the surgeon general (1). As a result, there is an increased awareness by consumers of their needs to reduce the intake of calories derived from fats. There is so much information around us regarding fats that are healthy and otherwise sometimes it gets confusing. This is especially so since our understanding of dietary fats is inadequate. We are constantly in a dilemma regarding so many aspects surrounding our fat intake. Questions like - Are fat necessary? How much fat should we eat? Which fat will protect us against heart disease? Besides quantity is quality of fats is equally important? In this review paper an easy attempt has been made to address all these questions. Oils and fats are triglycerides of fatty acids, these fatty acids may be saturated (SFA), monounsaturated (MUFA) or polyunsaturated (PUFA). Let us first consider the effect of SFA on human health in detail. Saturated fatty acids (SFA) are the precursors of Bile acids so its intake is essential but these are associated with certain drawbacks. SFA being the precursors of cholesterol are implicated in the cause of heart disease. The underlying feature of most heart diseases is the thickening of inner walls of the arteries by deposits that contain fats, cholesterol, connective tissue and calcium. Low density lipoprotein (LDL) transport cholesterol that has been synthesized in the liver from SFA to body cells. Increase in the level of LDL- cholesterol therefore Correspondence to: R.P. SINGH, Director, Harcourt Butler Technological Institute, Kanpur , INDIA rpshbti@redifmail.com rub_tri@yahoo.co.in Journal of Oleo Science ISSN print / ISSN online 423

2 R. Trivedi and R.P. Singh enhances the risk of coronary heart disease. In this context LDL-cholesterol is considered as Bad cholesterol (2) and these are the factors for cardiovascular diseases. However, all the saturated fatty acids are not hypercholesterolemic as the Medium chain saturated fatty acids (MCFA) (C 6 to C 10 ) enter the portal system and directly transported to liver where it is metabolized in carnitine; independent pathway to generate quick energy and it give low calorie in comparison to long chain SFA. MCFA have immense medicinal and nutritional importance (3-6). As increase intake of SFA predisposes us towards heart diseases, so it is important to scale down the intake of SFA and substitute it with polyunsaturated fatty acids (PUFA). Almost we all are aware about the benefits of PUFA i. e. these are essential fatty acids being precursors of prostaglandins, but on the other hand its excessive intake is also related with some physiological disorders with which general people are not aware. 1. The permeability of cellular membranes to materials such as drugs, foreign bodies, bacteria or viruses may be affected. 2. Early ageing of certain types of cells, skin cells in particular may occur. 3. Imbalance may be caused between different prostaglandin Since dietary fat with high PUFA content may be as harmful as fat with low PUFA content, a balance between PUFA and SFA in the dietary fats need to be struck but P/S ratio as a criterion for the nutritional assessment of fats is insufficient, because of the presence of monounsaturated fatty acids (MUFA) in significant quantities in almost all oils. MUFA have not yet been implicated in any type of disorder; apart from the supplying energy they also have the cushioning effect on the negative qualities of SFA and PUFA. These types of findings about the effects of individual fatty acids has prompted Oil Technologists & Nutritionists to search for an ideal oil which has the S:M:P ratio of 1:1:1 recommended by World Health Organization. However, no natural oil or fat is ideal in this respect. Therefore there is a need to modify the available edible oils to produce Structured Lipids (SLs). In a broader sense Structured Lipids will include any lipid that have been restructured to change the fatty acid position or modified to change the fatty acid composition (7-9). SLs are being produced mainly by interesterification or by genetic modification. Interesterification may be performed by chemical or enzymatic means. Now a days enzymatic interesterification is more popular in comparison to chemical interesterification because enzymes (Lipases) offer several advantages (10-13) over chemical interesterification such as lipases offer specificity means these are stereo specific and substrate specific, so according to need it attacks on the particular site of the triglyceride (14, 15), these are Eco-friendlybiodegradable; the contribution of lipases to BOD in the waste stream is negligible; the catalytic efficiency is very high; immobilized enzymes are reusable; mild conditions are required; and waste is reduced (16). Lipases are usually thought of as being hydrolytic enzymes in common other hydrolases; however, they operate in a reverse mechanism in which they can, under appropriate circumstances catalyze the reverse reactions i. e. esterification and transacylation (17-19). SLs incorporating MCFA (Capric, caprylic, caproic) are being used in the medical fields as a fat for nutritional feeding (20). In fact, Sandoz nutrition, a Minnieapolis, Minnesota (21) launched a product called IMPACT containing SLs made (by interesterification) from a high lauric acid oil and a high linoleic acid oil. Product IMPACT is especially beneficial for patients who have or at risk of developing hyper metabolism - induced immune suppression. During periods of metabolic stress caused by trauma, sepsis or major surgery, patients appear to have special nutritional needs like IMPACT. Medium-chain triglycerides (MCT) are mainly utilized as a nutritional supplement for patients who suffer from malasorption caused by intestinal resection and also as a component for infant feeding formulas (22-24). It is also used as a solvent or carrier of lipophilic nutrients or drugs, such as vitamin K (25) and phospholipids (26). Proctor and Gamble Companies have developed Caprenin (20, 21) as a reduced calorie fat with 5 cal/g, it is a randomly SLs containing behenic acid C 22:0 and two medium chain fatty acids caprylic and capric acids. Uniliver Scientists have launched a product called Betapol, (20) which is human milk substitute for infants, the main constituents of Betapol is O-P-O. Loaders Croklon, a Uniliver company is also developing other SLs, particularly with long chain PUFA to increase DHA and EPA contents (21). 424

3 Modification of Oils and Fats to Produce Structured Lipids Carl-Erik-head of the Deptt of Biochemistry and Nutrition at the Technical University of Denmark in Lyngby, Denmark has set up a pilot plant using immobilized enzymes for producing targeted SLs with PUFA at the Sn-2 position. The operation which uses a 1,3- specific lipase from Novo Nordisk in Bagasvaerd, Denmark, produces SLs in quantities ranging from 10 to 100 g. Such SLs offer a potential treatment for persons with malasorption problems, such as cystic fibrosis. The lipase reaction however, is expensive and difficult to scale up Certainly cost is the hurdle. Thus if we can demonstrate the health benefits outweigh economic considerations, these may become viable. Vivienne V. Yankah (27) synthesized two different SLs by transesterifying tristearin with caprylic acid or oleic acid. The objective was to synthesize SLs containing stearic acid at the Sn-2 position as possible nutritional and low calorie fats. The reaction was catalyzed by Sn-1, 3-specific immobilized lipase from Rhizomucor miehei in the presence of n-hexane. The effects of reaction parameters affecting the incorporation of caprylic acid into tristearin were compared with those for incorporating oleic acid into tristearin. For all parameters studied, oleic acid incorporation was higher than caprylic acid. Their solid fat contents at 25 suggest possible use in spreads or for inclusion with other fats in specialized blends. Vimon Seriburi (28) synthesized SLs using triolein and stearic acid with Sn-1,3- specific lipase from Rhizomucor miehei and non-specific lipase from Candida antarctica. The optimal reaction temperature for both enzymes was 55 at a mole ratio of triolein to acyl donor of 1:2, equilibrium were reached at 8 h for Rhizomucor miehei and 24 h for Candida antarctica. With Candida antarctica the softest fat was produced after 18 h of incubation and the hardest after 30 h. For Rhizomucor miehei system 18 h of incubation gave the most plastic fat. Toshihiro Nagao (29) produced human milk substitute (O-P-O) from tri-palmitin (PPP) and oleic acid (OA) using Fusarium heterosporum lipases. A mixture of PPP/OA (1:2 w/w) and 8% immobilized lipase catalyst was incubated at 50 for 24 h with shaking at 130 rmp. The acidolysis reached 50% under these conditions. X. Xu. (30) of Deptt of Biotechnology, Technical University of Denmark, Denmark investigated the application of membrane technology to the enzymatic production of specific SLs. Reaction experiments were carried out in a membrane reactor between MCT and n- 3 PUFA from fish oil using lipase from Rhizomucor miehei. The incorporation of PUFA into MCT was increased by about 15% in 90 h reaction by simultaneous separation of the released MCFA, compared to no separation under the same reaction conditions. It has thus clearly been demonstrated that membrane assisted separation improved the incorporation of acyl donors into oils beyond the reaction equilibrium defined by the original substrate concentration. He also produced specific SLs by enzymatic interesterification of Rape seed oil and capric acid. Zaida Zainal (31) has performed enzymatic interesterification of palm-stearin and palm kernel olein. At 60 the interesterification with lipase from Rhizomucor miehei can be completed in 5 h. Results showed that interesterification was effective in producing solid fats with less than 0.5% trans content and he reported that the slip melting point of physical blend was 40 while after interesterification it was drop to Ki-Teak Lee (32) enzymatically synthesized SLs from tricaprylin and eicosapentaenoic/ docosahexaenoic acids. Tricaprylin was mixed with n-3 PUFA in a 1:2 molar ratio and tranesterified by incubating at 55 in n-hexane with lipase from Candida antarctica (10% by wt of total substrate) in a 125 ml Erlenmeyer flask as the bioreactor. After several batches of reaction the products were pooled and hexane was evaporated. Short path distillation was used for purification of synthesized SLs. The distillation conditions were 1.1 torr and 170 at a feed flow rate of 3 ml/min. Upto 240 g SL was isolated and deacidified by alkaline extraction or ethanolwater solvents. He also synthesized SLs by two immobilized lipases from tricaprin and trilinolein (33). In-Hwan Kim (34) synthesized SLs by the acidolysis of Corn Oil by Caprylic acid in supercritical carbon dioxide with lipase from Rhizomucor miehei. The effects of temperature and pressure on the reaction were studied. He also performed the lipase catalyzed incorporation of conjugated linoleic acid into tricaprylin (35). He used three commercial lipases from Rhizomucor miehei, Candida antarctica and Pseudomonas Cepacia as biocatalyst for the interesterification of conjugated Linoleic (CLA) ethyl ester and tricaprylin. Lipase from Candida antarctica as compared to Rhizomucor miehei and Pseudomonas Cepacia showed the highest degree of CLA incorporation into tricaprylin. 425

4 R. Trivedi and R.P. Singh By hydrolysis with pancreatic lipase it was found that lipase from Rhizomucor miehei and Pseudomonas Cepacia exhibited high selectivity for the Sn-1, 3 position of the triacylglycerol early in the interesterification, with small extent of incorporation of CLA into the Sn-2 position, probably due to acyl migration, at later reaction times. Lydia B. Fomuso (36) synthesized SLs by the interesterification of tricaproin and trilinolein with lipases from Rhizomucor miehei and Candida antarctica. The interesterification was performed by incubating a 1:2 mole ratio of trilinolein and tricaproin in 3 ml hexane at 45 for lipase from Rhizomucor miehei and at 55 for lipase from Candida antarctica. Reaction products were analyzed by reversed phase HPLC with an evaporative light scattering detector. Lipase Rhizomucor miehei produced 53.5 mol% dicaproyllinolein (C33) and 22.2% monocaproyllinolein (C45). Lipase from Candida antarctica produced 41% C33 and 18% C45. When caproic acid was used in place of tricaproin as the acyl donor, the Rhizomucor miehei produced 62.9% C33 and in absence of organic solvent, it produced 52.3% C33. Moussata (37) investigated the ability of lipase PS 30 (Pseudomonas sp.) to modify the fatty acid profile of melon seed oil by incorporation of oleic acid. Hirokawa (38) studied trans-esterification of oils and/or fats with lipase as a catalyst. A mixture of higholeic sunflower oil, stearic acid and hexane was passed through a column filled with lipase. Ainsworth (39) interesterified cocoa butter, milk fat and a blend of milk-fat / canola oil using lipase from Rhizomucor miehei. Marangoni (40) studied the lipase-catalyzed interesterification of tripalmitin and triolein in reversed micelles using canola lecithin which allowed successful modification of triolein and tripalmitin to yield a fat of intermediate properties between the two initial substrates. Negishi (41) reported transesterification with powder lipase, of sp. Alcaligenes in the absence of water. Transesterification of palm oil with rapeseed oil with lipase PS-30 was shown. Yoon (42) studied the effect of substrate solubility in interesterification with triolein and behenic acid (BA) or Et Behenate (EB) by immobilized lipase in supercritical CO 2. The results showed that incorporation of behenoyl group into triolein was much higher with EB as a substrate than with BA to give final product (BOB). Meijer (43) studied the effect of interesterified fat on blood lipids, blood enzymes and homeostasis parameter, these effects were studied on 60 healthy humans. The experimental results showed the interesterified fat in the diet of healthy people does not influence fasting, blood lipid, blood enzymes and / or homeostasis parameters in any adverse way, when compared with noninteresterified fat with the same fatty acid composition. Ghosh (44) studied the interesterification reactions with different proportions of blends of some saturated fats like sal, mango kernel, mowrah, palmstearin with rice bran oil catalyzed by Mucor miehei lipase. On the basis of slip melting point, solid fat content at different temperatures it has been observed that some interesterified products are suitable for use as margarine fat base and some are suitable as vanaspati-like fats. Ming (45) investigated the enzymatic trans-esterification of palm stearin -palm kernel olein (40:60) in a solvent free system to improve the physical and melting characteristics of solid fats. Results indicated that enzymatic trans-esterification was able to produce fat mixtures with substantially lower M. P. by repositioning the fatty acids of triglycerides in the higher melting range to form lower or middle melting components. Recently he synthesized structured lipids of 1, 3-dioleoyl-2- palmitoylglycerol from palm oil using lipase from Candida antarctica under vacuum (46). X. Xu. (47) carried out the pilot batch production of specific SLs by lipase catalyzed interesterification of refined fish oil and capric acid. It may be noted that specific SLs have been reported to have beneficial effects on immune function, nitrogen balance, and improve lipid clearance from the blood stream (48-52). Free fatty acids liberated from food during absorption are metabolized more easily if they are medium- or short- chain i.e. C 10 or below, and 2-MAG can be absorbed directly. Therefore essential or desired FA are most efficiently utilized from the Sn-2 position in acylglycerols. A laboratory- scale continuous reactor (54) was constructed by H. Mu. for production of specific SLs containing essential fatty acids and MCFA in the Sn-2 and Sn-1, 3- positions respectively. Longer reaction time resulted higher yield, but was accompanied by increased acyl migration. Increased reaction temperature and content of MCFA in the initial reaction sub- 426

5 Modification of Oils and Fats to Produce Structured Lipids strate improved the incorporation of MCFA and the yield of the desired TAG in the products. Increasing the water content from 0.03 to 0.11% (w/w substrate) in the reaction substrate had almost no effect on either the incorporation or the migration of MCFA or on the resulting composition of TAG products and their free fatty acid content. Yuehua H. (55) studied enzymatic synthesis of acylglycerol directly from glycerol and a w-3 fatty acid concentration, prepared from seal blubber oil, in organic solvents; seven lipases were used as biocatalyst for esterification. Lipase LP-401-AS from chromobacterium viscosum showed the highest activity for esterification. The maximal degree of acylglycerol synthesis reached was 94.3%, the conc of monoacylglycerols, diacylglycerols, and triacylglycerols were 13.8, 43.1, and 37.4% respectively. Therefore acylglycerols containing predominantly w-3 fatty acid concentrates may be easily synthesized directly via their reaction with glycerol. G. P. McNeil (56) synthesized SLs by monoglycerides of erucic acid (C 22:1, D 13 ) with caprylic acid (C 8:0 ) by using lipases with the intention of synthesizing a triglyceride that contains two molecules of caprylic acid and one molecule of erucic acid (Caprucin). He used lipases from Geotricum Candidum (GC-20) and Pseudomonas cepacia (PS-30). When the lipase PS 30 was used a yield of approximately 37% caprucin was obtained, together with a complex mixture of di-and triglycerides that resulted from the random transesterification of the erucic acid. The fatty acid specific lipase GC-20 promoted minimal transesterification of erucic acid and resulted in a yield of 75% caprucin and approx 10% interesterified products. Lipase from Candida rugosa exhibited a similar, although less pronounced specificity to that from lipase GC-20 and promoted more transesterification of erucic acid. Optimum conditions for lipase GC-20 were at 50 and an initial water content of 5.5%. After the reaction erucic was converted to behenic acid by hydrogenation, thereby converting caprucin into caprenin, a commercially available low calorie triglyceride. Consumption of fish oil or fish oil rich in n-3 PUFA has been linked to cardiovascular and immune functions (57) and may mediate inflammatory events. Not everyone likes to eat fish, but most people used vegetable oils in food preparations, therefore, vegetable oils are considered as alternative molecules for carrying n-3 PUFA. Kuan-hsiang Huang incorporated EPA & DHA into soyabean oil using two immobilized lipases from Rhizomucor miehei and Candida antarctica. He also reported that gamma linolenic acid (GLA), a precursor of arachidopnic acid, possesses physiological functions of modulating immune and inflammatory response. Highly purified GLA is desired both as an ingredient of cosmetics. In this work, urea fractionation and lipasecatalyzed reactions were employed for the enrichment of borage oil (58). He also optimized and scale up the enzymatic synthesis of structured lipids using RSM (59). Tsuneo Yamane (60) made an attempt to further increase the content of n-3 PUFA of fish oil by lipase catalyzed acidolysis of fish oil and n-3 PUFA. A bioreactor system was constructed composed of a water jacketed packed bed column and a substrate reservoir with a circulation pipeline between the packed bed column and the reservoir. By keeping the temperature of the reservoir at -10 (for the first 20 h) followed by -20 (for the subsequent 40 h) during the batch acidolysis, crystals of free fatty acids appeared, which were removed intermittently by a cotton plug packed in the tip of the outlet pipe in the reservoir. The n-3 PUFA content of triacylglycerol fraction increased a further 10% by the reduced temperature of the reservoir. P. Forssell (61) studied the optimum conditions for enzymatic transesterification of rapeseed oil and lauric acid in a fixed bed reactor without using any solvent. A small amount of water was dissolved in the substrate mixture to maintain the activity of the enzyme at as high a level as possible. For maximum yield, the transesterification was performed with a residence time of about 20 min at water content in the range of 0.1 to 0.2% and at the lowest possible temperature. One of the earliest processes was the manufacture of cocoa butter substitutes (Macrae 1983) (62). The fat has a high level of palmitic and stearic acids giving it its essential property of displaying sharp melting transition just below 37. It can be made synthetically by acyltransfer reactions between stearic acid and palm oil or sunflower oil. It is also being produced from PKO, Dhupa, Mahua, and Kokum etc. Genetic engineering (63) is seen as a powerful tool to extend the ability of geneticists and plant breeders to modify functional properties of oilseed crops. However, while conventional plant breeding involves selecting 427

6 R. Trivedi and R.P. Singh amongst existing variability or randomly induced mutations, the new technologies require detailed understanding or lipid biochemistry in order to isolate specific gene sequences. Calgene Inc. (20) of Davis, California has taken another approach to creating SLs; that of using bioengineering (genetics) to produce them in plants. Currently its high laurate canola marketed under the name of Laurical, is a targeted SLs created by genetically inserting the lauroyl- APC thioesterases enzyme from the seed of the California bay tree into canola, which normally does not contain lauric fatty acids. Bioengineered canola containing 40% laurate content is now available and being used in confectionery coatings, coffee whiteners, whipped toppings, and center (filling) fats. Potential uses will include medical applications and infant formulas Hoy said, noting that PUFA in the Sn- 2 position appear to offer health advantages for visual and brain development particularly in infants born prematurely. Bioengineering to create SLs is more cost effective then synthetically producing them through enzymatic interesterification. Certainly during the first few years of commercialization bioengineered SLs will be costly. Later on, however, as production numbers increase, they will have cost advantages over synthetically produced materials and as these SLs are plant derived, they give warm and fuzzy feeling in marketing. Bistrian and his colleagues published findings in the Annals of surgery in 1996 from an internal feeding study involving post surgical cancer patients. In this study, those receiving the Structured Lipid formula that contained n-3 fatty acids experienced improved long chain fatty acid absorption and less gastrointestinal complications and infections. They noted that the data suggested improved liver and renal function due to the use of SLs. Enzyme biotechnology is continuously providing new, modified & better enzymes, but for industrial scale production of SLs cost concern is still there which restrict the industrial scale development in this area especially in India? The main stumbling block, particularly to using SLs in foods for the general public, is cost. We ll need some new technologies to bring the cost down. Fats and oils produced from microbes are known as single cell oils/ fats and are raw materials for the fats and oils industries in making value added specialty products like cocoa butter equivalents and medicinal oils (64). Mason said In order to gain acceptance for novel foods (SLs), we will have to overcome certain reluctance both from industrialists and consumers (20). References 1. K.S. DOBSON, K.D. WILLIAMS and C.J. BORIACK, The Preparation of Polyglycerol Esters Suitable as Low-Caloric Fat Substitute, J. Am. Oil Chem. Soc., Vol. 70, (1993). 2. V.K. BABAYAN, Medium Chain Length Fatty Acid Esters and Their Medical and Nutritional Applications, J. Am. Oil Chem. Soc., Vol. 58, 49A-51A (1981). 3. D.Y. KWON, H.N. SONG and S.H. YOON, Synthesis of Medium-Chain Glycerides by Lipase in Organic Solvent, J. Am. Oil Chem. Soc., Vol. 73, (1996). 4. S. GHOSE and D.K. BHATTACHARYA, Medium Chain Fatty Acid-Rich Glycerides by Chemical and Lipase Catalyzed Polyester-Monoester Interchange Reaction, J. Am. Oil Chem. Soc., Vol. 74, (1997). 5. A Health Oil for the Next Millennium, inform, Vol. 11(1), (2000). 6. C.C. AKOH, Structured Lipids-Enzymatic Approach, inform, Vol. 6, (1995). 7. F.D. GUNSTONE, Movements towards Tailor-Made Fats, Prog. Lipid Res., Vol. 37, (1998). 8. A.B. CHRISTOPHE (ed.), Structural Modified Food Fats: Synthesis, Biochemistry and Use, AOCS Press, Champaign, p. 246 (1994). 9. A.R. MACRAE, J. Am. Oil Chem. Soc., Vol. 60, 243A (1983). 10. F. ERGAN, M. TRANI and G. ANDRE, Biotech. Bioeng., Vol. 35, 195 (1990). 11. M.M. HOQ, T. YAMANE, S. SHIMIZU, T. FUNADA and S. ISHIDA, J. Am. Oil Chem. Soc., Vol. 61, 776 (1984). 12. G.P. McNEILL, S. SHIMIZU and T. YAMANE, J. Am. Oil Chem. Soc., Vol. 67, 779 (1990). 13. N.O.V. SONNTAG, in Bailey s Industrial Oil and Fat Products, Vol. 2, 4th edn, (1982). 14. C.C. AKOH and R.V. SISTA, Enzymatic Modification of Borage Oil: Incorporation of Eicosapentaenoic Acid, J. Food Lipids, Vol. 2, (1995). 15. S. RAY and D.K. BHATTACHARYA, Comparative Nutritional Study of Enzymatically and Chemically Interesterified Palm Oil Products, J. Am. Oil Chem. Soc., Vol. 72, (1994). 16. P. QUINLAN and S. MOORE, Modification of Triglycerides by Lipases: Process Technology and its Application to the Production of Nutritionally Improved Fats, inform, Vol. 4, (1993). 17. K.-T. LEE and C.C. AKOH, Immobilized Lipase - Catalyzed Production of Structured Lipids with Eicosapentaenoic Acid at Specific Positions, J. Am. Oil Chem. Soc., Vol. 73,

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