Enzymatic synthesis of high‐purity structured lipids with caprylic acid at 1,3‐positions and polyunsaturated fatty acid at 2‐position

Abstract

We attempted to synthesize high-purity structured triacylglycerols (TAG) with caprylic acid (CA) at the 1,3-positions and a polyunsaturated fatty acid (PUFA) at the 2-position by a two-step enzymatic method. The first step was synthesis of TAG of PUFA (TriP), and the second step was acidolysis of TriP with CA. Candida antarctica lipase was effective for the first reaction. When a reaction medium of PUFA/glycerol (3:1, mol/mol) and 5% immobilized Candida lipase was mixed for 24 h at 40°C and 15 mm Hg, syntheses of TAG of γ-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids reached 89, 89, 88, and 83%, respectively. In these reactions, the lipase could be used for at least 10 cycles without significant loss of activity. In the second step, the resulting trieicosapentaenoin was acidolyzed at 30°C for 48 h with 15 mol parts CA using 7% of immobilized Rhizopus delemar lipase. The CA content in the acylglycerol fraction reached 40 mol%. To increase the content further, the acylglycerols were extracted from the reaction mixture with n-hexane and were allowed to react again with CA under conditions similar to those of the first acidolysis. After three successive acidolysis reactions, the CA content reached 66 mol%. The content of dicapryloyl-eicosapentaenoyl-glycerol reached 86 wt% of acylglycerols, and the ratio of 1,3-dicapryloyl-2-eicosapentaenoyl-glycerol to 1(3),2-dicapryloyl-3(1)-eicosapentaenoyl-glycerol was 98:2 (w/w). In this reaction, the lipase could be used for at least 20 cycles without significant loss of activity. Repeated acidolysis of the other TriP with CA under similar conditions synthesized 1,3-dicapryloyl-2-γ-linolenoyl-glycerol, 1,3-dicapryloyl-2-arachidonoyl-glycerol, and 1,3-dicapryloyl-2-docosahexaenoyl-glycerol in yields of 58, 87, and 19 wt%, respectively.Paper no. J9783 in JAOCS 78, 611-616 (June 2001).Polyunsaturated fatty acids (PUFA) have various physiological functions. Eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) play a role in the prevention of a number of human diseases (1-3). The ethyl ester of EPA has been used in the treatment of arteriosclerosis obliterans and hyperlipemia (4), and oil containing 50% DHA has been used as a health food (5). γ-Linolenic acid (18:3n-6; GLA) shows the physiological functions of modulating atopic eczema (6) and rheumatoid arthritis (7), and arachidonic acid (20:4n-6; AA) and DHA are known to be important for preterm infants (8,9). A structured triacylglycerol (TAG) containing mediumchain fatty acids at the 1,3-positions and a long-chain fatty acid at the 2-position (MLM-type) is absorbed into intestinal mucosa more rapidly than natural oils composed of only longchain fatty acids (LLL-type) (10,11). These reports have focused a good deal of attention on the physiological activity of MLM-type structured lipids. To evaluate the nutraceutical effects, the ability to synthesize high-purity MLM-type structured lipid on a laboratory scale is necessary. It was recently reported that 1,3-capryloyl-2-eicosapentaenoy...

Enzymatic Synthesis of Medium- and Long-Chain Triacylglycerols (MLCT): Optimization of Process Parameters Using Response Surface Methodology

“…MLCT that contained medium-chain fatty acids (MCFA, C6-C10) and long-chain fatty acids (LCFA, C12-C24) in the same triacylglycerol (TAG) are the latest structured lipids being focused nowadays. Most of the MLCT works reported were designed to provide the most effective means of delivery of the desired fatty acid (FA) for nutritive purposes, and for targeting specific disease and metabolic conditions (Fomuso and Akoh 1997;Kawashima et al 2001Kawashima et al , 2004Mu et al 2001;Aoyama 2003;Kim and Akoh 2005;Mu and Porsgaard 2005). The FA and their position in the TAG determine the functional and physical properties, metabolic rate, and health benefits of the structured lipid.…”

“…Enzymatic interesterification with sn-1,3-specific lipases has been used by industry for the production of cocoa butter-like fats, human milk fat substitutes, and so on (Xu 2000a). The most commonly used method for the production of MLCT is acidolysis, using a regiospecific lipase to incorporate the MCFA into the primary positions of TAG (Fomuso et al 1997;Kawashima et al 2001Kawashima et al , 2004Mu et al 2001;Kim and Akoh 2005). The acidolysis reaction proceeds in two steps.…”

Preparation of regioisomers of structured TAG consisting of one mole of CLA and two moles of caprylic acid

“…Two TAG regioisomers, which have the same FA composition and carry a functional FA at the 1(3)-and 2-position, are necessary in order to study this subject. The two regioisomers have been synthesized by transesterification of long-chain TAG (LLL) with medium-chain FA (MCFA) using a 1,3-position-specific lipase and by transesterification of medium-chain TAG (MMM) with long-chain FA (LCFA) (1)(2)(3)(4)(5)(6)(7)(8)(9). The products can reportedly be purified by HPLC (10,11).…”

Synthesis of regioisomerically pure triacylglycerols containing n ‐3 very long‐chain polyunsaturated fatty acids

“…Interestingly, the sn-1(3)-positionally specific lipase RML (Lipozyme RM IM) can also be used for the synthesis of YYY from glycerol. Spontaneous isomerization of the initially formed 1,3-DAG to 1,2-DAG, followed by further lipase catalyzed esterification at the 3-position has been suggested as the mechanism for YYY synthesis using RML [48,49]. [43,50,51].…”