Enzymatic synthesis of structured lipid containing arachidonic and palmitic acids

Human milk fat contains 20–25% palmitic acid, and about 70% of the fatty acid is esterified to the 2-position of triglycerides. It was also reported that arachidonic acid (AA) accelerated the growth of preterm infants. Thus, we attempted the synthesis of 1,3-arachidonoyl-2-palmitoyl-glycerol by acidolysis of tripalmitin with AA using 1,3-specific Rhizopus delemar lipase. When a mixture of 10 g tripalmitin/AA (1∶5, w/w) and 0.7 g immobilized Rhizopus lipase was incubated at 40°C for 24 h with stirring, the AA content in glycerides reached 59 mol%. The immobilized lipase could be used five times without a decrease in the extent of acidolysis. Glycerides were extracted from the reaction mixture with n-hexane, and regiospecific analysis was performed. As a result, the AA contents at the 1,3- and 2-positions were 56.9 and 3.2 mol%, respectively. It was therefore confirmed that the fatty acids at the 1,3-positions of triglyceride were exchanged for AA. High-performance liquid chromatography showed that the contents of triarachidonin, 1,3-arachidonoyl-2-palmitoyl-glycerol, and 1(3)-arachidonoyl-2,3(1)-palmitoyl-glycerol were 7.3, 75.9, and 12.4 wt%, respectively.     

Production of structured lipids containing essential fatty acids by immobilizedRhizopus delemar lipase

An attempt was made to produce structured lipids containing essential fatty acid by acidolysis with 1,3-positional specificRhizopus delemar lipase. The lipase was immobilized on a ceramic carrier by coprecipitation with acetone and then was activated by shaking for 2 d at 30°C in a mixture of 5 g safflower or linseed oil, 10 g caprylic acid, 0.3 g water and 0.6 g of the immobilized enzyme. The activated enzyme was transferred into the same amount of oil/caprylic acid mixture without water, and the mixture was shaken under the same conditions as for the activation. By this reaction, 45–50 mol% of the fatty acids in oils were exchanged for caprylic acid, and the immobilized enzyme could be reused 45 and 55 times for safflower and linseed oils, respectively, without any significant loss of activity. The triglycerides were extracted withn-hexane after the acidolysis and then were allowed to react again with caprylic acid under the same conditions as mentioned above. When acidolysis was repeated three times with safflower oil as a starting material, the only products obtained were 1,3-capryloyl-2-linoleoylglycerol and 1,3-capryloyl-2-oleoyl-glycerol, with a ratio of 86∶14 (w/w). Equally, the products from linseed oil were 1,3-capryloyl-2-α-linolenoyl-glycerol, 1,3-caprylol-2-linoleoyl-glycerol, and 1,3-capryloyl-2-oleoly-glycerol (60∶22∶18, w/w/w). All fatty acids at the 1,3-positions in the original oils were exchanged for caprylic acid by the repeated acidolyses, and the positional specificity ofRhizopus lipase was also confirmed to be strict.     

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

TAG (MLM) with medium-chain FA (MCFA) at the 1,3-positions and long-chain FA (LCFA) at the 2-position, and TAG (LMM) with LCFA at the 1(3)-position and MCFA at 2,3(1)-positions are a pair of TAG regioisomers. Large-scale preparation of the two TAG regioisomers was attempted. A commercially available FFA mixture (FFA-CLA) containing 9-cis, 11-trans (9c, 11t)- and 10t,12c-CLA was selected as LCFA, and caprylic acid (C₈FA) was selected as MCFA. The MLM isomer was synthesized by acidolysis of acyglycerols (AG) containing two CLA isomers with C₈FA: A mixture of AG-CLA/C₈ FA (1∶10, mol/mol) and 4 wt% immobilized Rhizomucor miehei lipase was agitated at 30°C for 72 h. The ratio of MLM to total AG was 51.1 wt%. Meanwhile, LMM isomer was synthesized by acidolysis of tricaprylin with FFA-CLA: A mixture of tricaprylin/FFA-CLA (1∶2, mol/mol) and 4 wt% immobilized R. miehei lipase was agitated at 30°C for 24 h. The ratio of LMM to total AG was 51.8 wt%. MLM and LMM were purified from 1,968 and 813 g reaction mixtures by stepwise short-path distillation, respectively. Consequently, MLM was purified to 92.3% with 49.1% recovery, and LMM was purified to 93.2% with 52.3% recovery. Regiospecific analyses of MLM and LMM indicated that the 2-positions of MLM and LMM were 95.1 mol% LCFA and 98.3 mol% C₈ FA, respectively. The results showed that a process comprising lipase reaction and short-path distillation is effective for large-scale preparation of high-purity regiospecific TAG isomers.     

Lipase-catalyzed acidolysis of perilla oil with caprylic acid to produce structured lipids

Structured lipids were synthesized by acidolysis of perilla oil and caprylic acid using two lipases, Lipozyme RM IM from Rhizomucor miehei and Lipozyme TL IM from Thermomyces lanuginosa. Effects of molar ratio, reaction time, reaction temperature, enzyme load, and solvent content on acidolysis reactions were studied. The solvent content ranged from 0.0 (solvent-free) to 85.3%. The results showed that the incorporation increased in parallel with solvent content to 49.0% with Lipozyme RM IM and to 63.8% with Lipozyme TL IM. After 24 h incubation in n-hexane, caprylic acids were incorporated to 48.5 mol% with Lipozyme RM IM and to 51.4 mol% with Lipozyme TL IM, respectively, whereas linolenic acid content was reduced from 61.4 to 31.5 mol% with Lipozyme RM IM and to 28.4 mol% with Lipozyme TL IM, respectively. Lipozyme TL IM showed a higher acyl migration rate than Lipozyme RM IM when acidolysis was performed in the reaction system containing n-hexane as a solvent, whereas the difference in acyl migration between the two lipases in the solvent-free system was negligible.     

Enzymatic synthesis of symmetrical 1,3-diacylglycerols by direct esterification of glycerol in solvent-free system

1,3-Diacylglycerols were synthesized by direct esterification of glycerol with free fatty acids in a solvent-free system. Free fatty acids with relatively low melting points (<45°C) such as unsaturated and medium-chain saturated fatty acids were used. With stoichiometric ratios of the reactants and water removal by evaporation at 3 mm Hg vacuum applied at 1 h and thereafter, the maximal 1,3-diacylglycerol content in the reaction mixture was: 84.6% for 1,3-dicaprylin, 84.4% for 1,3-dicaprin, 74.3% for 1,3-dilinolein, 71.7% for 1,3-dieicosapentaenoin, 67.4% for 1,3-dilaurin, and 61.1% for 1,3-diolein. Some of the system’s parameters (temperature, water removal, and molar ratio of the reactants) were optimized for the production of 1,3-dicaprylin, and the maximal yield reached 98%. The product was used for the chemical synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol. The yield after purification was 42%, and the purity of the triacylglycerol was 98% (both 1,3-dicapryloyl-2-eicosapentaenoylglycerol and 1,2-dicapryloyl-3-eicosapentaenoylglycerol included) by gas chromatographic analysis, of which 90% was the desired structured triacylglycerol (1,3-dicapryloyl-2-eicosapentaenoylglycerol) as determined by silver ion high-performance liquid chromatographic analysis.     

Enzymatic synthesis of position-specific low-calorie structured lipids

An immobilized sn-1,3-specific lipase from Rhizomucor miehei (IM 60) was used to catalyze the interesterification of tristearin (C_18:0) and tricaprin (C_10:0) to produce low-calorie structured lipids (SL). Acceptable product yields were obtained from a 1:1 mole ratio of both triacylglycerols with 10% (w/w of reactants) of IM 60 in 3 mL hexane. The SL molecular species, based on total carbon number, were 44.2% C₄₁ and 40.5% C₄₉, with 3.8 and 11.5% unreacted tristearin C₅₇ and tricaprin C₂₇, respectively, remaining in the product mixture. The best yield of C₄₁ species (44.3%) was obtained with zero added water. Tricaprylin (C_8:0) was also successfully interesterified with tristearin in good yields at 1:1 mole ratio. Products were analyzed by reverse-phase high-performance liquid chromatography with an evaporative light-scattering detector. Reaction parameters, such as substrate mole ratio, enzyme load, time course, added water, reaction media, and enzyme reuse, were also investigated. Hydrolysis by pancreatic lipase revealed the specific fatty acids present at the sn-1,3 positions of SL. Biocatalysis Symposium Paper, presented at the AOCS Annual Meeting & Expo, Seattle, Washington. May 11–14, 1997.     

Lipase-catalyzed acidolysis of tristearin with oleic or caprylic acids to produce structured lipids

Two different structured lipids (SL) were synthesized by transesterifying tristearin with caprylic acid (C8∶0) or oleic acid (C18∶1). The objective was to synthesize SL containing stearic acid (C18∶0) at the sn-2 position as possible nutritional and low-calorie fats. The reaction was catalyzed by IM60 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. The range of conditions favorable for synthesizing high yields of C8∶0-containing SL was narrower than for oleic acid. An incubation time of 12–24 h and an enzyme content of 5% (w/w total substrates) favored C8∶0 incorporation. The mole percentage of incorporated C18∶1 did not increase further at enzyme additions greater than 10%. C18∶1 incorporation decreased with the addition of more than 10% water (w/w total substrates) to the tristearin-oleic acid reaction mixture. Increasing the mole ratio of fatty acid (FA) to triacylglycerol increased oleic acid incorporation. The highest C8∶0 incorporation was obtained at a 1∶6 mole ratio of tristearin to FA. Positional analysis confirmed that C18∶0 remained at the sn-2 position of the synthesized SL. The melting profiles of tristearin-caprylic acid and tristearin-oleic acid SL displayed peaks between −20 to 30°C and −20 to 40°C, respectively. Their solid fat contents (∼25%) at 25°C suggest possible use in spreads or for inclusion with other fats in specialized blends.     

Enzymatic modification of triolein: Incorporation of caproic and butyric acids to produce reduced-calorie structured lipids

Lipase-catalyzed acidolysis of triolein with caproic and butyric acids was performed to produce reduced-calorie structured lipids (SL). The SL were obtained by incubating a 1:4:4 mole ratio of triolein, caproic acid, and butyric acid, respectively, with 10% of lipase (w/w of total substrates) in 1.5 mL hexane at 55°C for 24 h. Of nine commercially avaialble lipases screened, IM60, which contains the lipase from Rhizomucor miehei, was the most effective and produced 13 mol% unreacted triolein, 49% disubstituted, and 38% monosubstituted triacylglycerols that contained short-chain fatty acids. The products were analyzed by reverse-phase high performance liquid chromatography with an evaporative light-scattering detector. Reaction parameters studied included time course, temperature, enzyme load, and substrate mole ratio. The yields obtained demonstrate that a structured lipid with long-chain and short-chain fatty acids can be synthesized by using IM60 lipase in organic medium.     

Enrichment of polyunsaturated fatty acids from sardine cannery effluents by enzymatic selective esterification

The sardine canning industry produces vast quantities of effluents that need expensive reprocessing. Their oily component contains valuable n−3 polyunsaturated fatty acids, namely EPA (5,8,11,14,17-eicosapentaenoic acid) and DHA (7,10,13, 16,19-docosahexaenoic acid), up to 10% each. Our aim was to develop a process allowing the recovery of these fatty acids. After removing solid particles, proteins, and peptides from the crude effluent, the obtained oil was hydrolyzed. EPA and DHA were enriched from the recovered free fatty acid fraction by selective enzymatic esterification. Lipases were used as biocatalysts: Lipozyme^TM allowed up to 80% DHA enrichment but gave no EPA enrichment. By immobilizing Candida rugosa lipase on Amberlite IRC50 cation-exchange resin, a 30% EPA enrichment was obtained.     

Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C₂₂ residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.     

Enzymatic synthesis of structured lipids from single cell oil of high docosahexaenoic acid content

The lipase-catalyzed acidolysis of a single-cell oil (SCO) containing docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) with caprylic acid (CA) was investigated. The targeted products were structured lipids containing CA residues at the sn-1 and -3 positions and a DHA or DPA residue at the sn-2 position of glycerol. Rhizomucor miehei lipase (RML) and Pseudomonas sp. KWI-56 lipase (PSL) were used as the biocatalysts. When PSL was used > 60 mol% of total SCO fatty acids (FA) were exchanged with CA, with DHA and DPA as well as the other saturated FA being exchanged. The content of the triacylglycerols (TG) containing two CA and one DHA or DPA (number of carbon atoms = 41, i.e., C₄₁) residue was high (36%), and the isomer with the desired configuration (unsaturated FA residue at the sn-2 position) represented 77–78% of C₄₁. In the case of RML, CA content reached only 23 mol% in the TG. A large amount of DHA and DPA residues remained unexchanged with RML, so that the resulting oil was rich in TG species containing two or three DHA or DPA residues (46%). TG C₄₁ amounted to 22%, almost all of which had the desired configuration. This result suggested that the difference in the degree of acidolysis by the two enzymes was due to their different selectivity toward DHA and DPA, as well as the difference in their positional specificities.     

Lipase-catalyzed synthesis of structured triacylglycerides from 1,3-diacylglycerides

A new method for the lipase-catalyzed synthesis of structured TAG (ST) is described. First, sn1,3-dilaurin or-dicaprylin were enzymatically synthesized using different published methods. Next, these were esterified at the sn2-position with oleic acid or its vinyl ester using different lipases. Key to successful enzymatic synthesis of ST was the choice of a lipase with appropriate FA specificity, i.e., one that does not act on the FA already present in the sn1,3-DAG, but that at the same time exhibits high selectivity and activity toward the FA to be introduced. Reactions were performed in the presence of organic solvents or in solvent-free systems under reduced pressure. With this strategy, mixed ST containing the desired compounds 1,3-dicaprylol-2-oleyl-glycerol or 1,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at 87 and 78 mol% yield, respectively, using immobilized lipases from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C. However, regiospecific analysis with porcine pancreatic lipase indicated that in CyOCy, 25.7% caprylic acid and in LaOLa 11.1% lauric acid were located at the sn2-position. Oleic acid vinyl ester was a better acyl donor than oleic acid. Esterification of sn1,3-DAG and free oleic acid gave very low yield (<20%) of ST in a solvent system and moderate yield (>50%) in a solvent-free system under reduced pressure.     

Structured lipids: Lipase-catalyzed interesterification of tricaproin and trilinolein

Structured lipids were synthesized by interesterification of trilinolein and tricaproin with sn-1,3-specific (IM 60) and nonspecific (SP 435) lipases. The interesterification reaction was performed by incubating a 1:2 mole ratio of trilinolein and tricaproin in 3 mL hexane at 45°C for the IM 60 lipase from Rhizomucor miehei, and at 55°C for the SP 435 lipase from Candida antarctica. Reaction products were analyzed by reverse-phase high-performance liquid chromatography with an evaporative light-scattering detector. The fatty acids at the sn-2 position were identified after pancreatic lipase hydrolysis and analysis with a gas chromatograph. IM 60 lipase produced 53,5 mol% dicaproyllinolein (total carbon number = C33) and 22.2% monocaproyldilinolein (C45). SP 435 lipase produced 41% C33 and 18% C45. When caproic acid was used in place of tricaproin as the acyl donor, the IM 60 lipase produced 62.9% C33. The effects of variation in mole ratio, temperature, added water, solvent polarity, and time course on the interesterification reaction were also investigated. In the absence of organic solvent, IM 60 lipase produced 52.3% C33.     

Enzymatic synthesis of steryl esters of polyunsaturated fatty acids

Steryl esters of long-chain fatty acids have water-holding properties, and polyunsaturated fatty acids (PUFA) have various physiological functions. Because steryl ester of PUFA can be expected to have both features, we attempted to synthesize steryl esters of PUFA by enzymatic methods. Among lipases used, Pseudomonas lipase was the most effective for the synthesis of cholesteryl docosahexaenoate. When a mixture of cholesterol/docosahexaenoic acid (3:1, mol/mol), 30% water, and 3000 units/g of lipase was stirred at 40°C for 24 h, the esterification extent attained 89.5%. Under the same reaction conditions, cholesterol, cholestanol, and sitosterol were also esterified efficiently with docosahexaenoic, eicosapentaenoic, arachidonic, and γ-linolenic acids.     

Study of TAG ethanolysis to 2-MAG by immobilized Candida antarctica lipase and synthesis of symmetrically structured TAG

Regiospecific ethanolysis of homogenous TAG with immobilized Candida antarctica lipase (Novozym 435) was studied using trioleoylglycerol (TO) as a model substrate. Optimization of the reactant weight ratio revealed that the 2-MAG reaction yield increased when a larger amount of ethanol was used. These results suggested that Novozym 435 showed strict regiospecificity in an excess amount of ethanol. The process optimization (reaction temperature and reactant molar ratio) and a study of lipase specificity for various substrates were performed. Under the optimized conditions (ethanol/TO molar ratio=77∶1 and 25°C), 2-monooleoylglycerol (2-MO) was obtained in more than 98% content among glycerides of the reaction mixture and approximately 88% reaction yield in 4 h. The above reaction conditions were applied for ethanolysis of tridocosahexaenoylglycerol, trieicosapentaenoylglycerol, triarachidonoylglycerol, tri-α-linolenoylglycerol, and trilinoleoylglycerol. Reaction yields ranging from 71.9 to 93.7% were obtained in short reaction times (2.5 to 8 h). Purified (>98%) 2-MO and 2-monodocosahexaenoylglycerol (2-MD) were reesterified with caprylic acid by immobilized Rhizomucor miehei lipase (Lipozyme IM) to afford symmetrical structured TAG. At a stoichiometric ratio of 2-MAG/caprylic acid, 25°C and 2–5 mm Hg vacuum, the glyceride composition of the esterification mixture was approximately 95% 1,3-dicapryloyl-2-oleoylglycerol (COC) at 4 h, and 96% 1,3-dicapryloyl-2-docosahexaenoylglycerol (CDC) at 4 h, and 96% 1,3-dicapryloyl-2-docosahexaenoylglycerol (CDC) at 8 h. The regioisomeric purity of both COC and CDC was 100%.     

Comparison of acyl donors for lipase-catalyzed production of 1,3-dicapryloyl-2-eicosapentaenoylglycerol

Synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol (CEC) catalyzed by Lipozyme IM (immobilized Rhizomucor miehei lipase) was performed by interesterification of trieicosapentaenoylglycerol (EEE) with caprylic acid (CA) (acidolysis) and EEE with ethyl caprylate (EtC) (interesterification). Both methods involved two steps: (i) transesterification at an optimized water content and temperature for the high yield conversion of the substrate to CEC, 1-capryloyl-2-eicosapentaenoylglycerol (CEOH) and 2-eicosapentaenoylglycerol (OHEOH), and (ii) reesterification of CEOH and OHEOH to CEC by water removal under reduced pressure. Interesterification had clear advantages over acidolysis. The reaction rates for interesterification were higher and the reaction times shorter. The final yield of CEC by interesterification was higher, and the extent of acyl migration, indicated by the tricapryloylglycerol content, was lower. The disadvantage of the higher price of EtC used for interesterification (approximately 10 times higher than the price of CA) was overcome by synthesizing it directly in the same reaction vessel prior to the interesterification step. EtC was rapidly synthesized by esterification of CA with ethanol in high yield (92% obtained in 2.5 h). The amount of water added to the reaction mixture and the reaction temperature influenced the yields of CEC, CEOH, and OHEOH in the transesterification step for both interesterification and acidolysis methods. The regioisomeric purity of CEC was 100% for both methods at temperatures of 40°C or less. The highest yield of CEC (81%) was obtained for the interesterification of EEE with EtC, formed directly in the same reaction vessel, at a CA/EEE molar ratio of 20∶1 and 30°C.     

Lipase-catalyzed modification of borage oil: Incorporation of capric and eicosapentaenoic acids to form structured lipids

Two immobilized lipases, nonspecific SP435 from Candida antarctica and sn-1,3 specific IM60 from Rhizomucor miehei, were used as biocatalysts for the restructuring of borage oil (Borago officinalis L.) to incorporate capric acid (10:0, medium-chain fatty acid) and eicosapentaenoic acid (20:5n-3) with the free fatty acids as acyl donors. Transesterification (acidolysis) reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The fatty acid profiles of the modified borage oil were different from that of unmodified borage oil. Higher incorporation of 20:5n-3 (10.2%) and 10:0 (26.3%) was obtained with IM60 lipase, compared to 8.8 and 15.5%, respectively, with SP435 lipase. However, SP435 lipase was able to incorporate both 10:0 and 20:5n-3 fatty acids at the sn-2 position, but the IM60 lipase did not. Solvents with log P values between 3.5 and 4.5 supported the acidolysis reaction better than those with log P values between −0.33 and 3.0.     

Production of structured lipids by lipase-catalyzed acidolysis in supercritical carbon dioxide: Effect on acyl migration

Structured lipids were synthesized by the acidolysis of corn oil by caprylic acid in supercritical carbon dioxide (SCCO₂) with Lipozyme RM IM from Rhizomucor miehei. The effects of pressure and temperature on the reaction were studied. To compare the degrees of acyl migration in the SCCO₂ and solvent-free reaction systems, the effects of reaction time on the degree of acyl migration were also studied. The highest mole percentage incorporation of caprylic acid (62.2 mol%) occurred at 24.13 MPa in SCCO₂. The overall incorporation of caprylic acid in the SCCO₂ system remained higher than that in the solvent-free system at every temperature tested. This trend was observed more clearly at lower temperatures (35–55°C) than at higher temperatures (65–75°C). Acyl migration with both reaction systems was low, with a negligible difference between them up to 12 h, after which the degree of acyl migration in the solvent-free system increased rapidly with time up to 24 h compared with the SCCO₂ system.     

Assessment of lipase- and chemically catalyzed lipid modification strategies for the production of structured lipids

The purpose of the present study was to devise a two-step reaction to produce partial glycerides, which would subsequently be used as substrates in both lipase-catalyzed and chemically catalyzed esterification reactions with caprylic acid. The yields and kinetics of these two-step reactions were compared to established lipase-catalyzed acidolysis and transesterification as well as to chemical transesterification reactions. Acyl migration did not occur during the hydrolysis or short-path distillation steps in the preparation of free fatty acid-free partial glycerides for esterification reactions. No significant differences in final yields (59.9% to 82.8% w/w of total triacylglycerols) of new structured lipids were detected among lipase-catalyzed (24 h) and chemically catalyzed (5 h) reactions; however, the yield of new structured triacylglycerols (TAG) after 2 h was lower for acidolysis than for the other lipase-catalyzed reactions (P≤0.05). Since no differences in final yields were detected among the reactions, chemical esterification using hydrolyzed oil could represent the best synthetic option, since it offers the advantage of positional distribution control associated with lipase-catalyzed reactions as well as rapid reaction times associated with chemically catalyzed reactions. Attempts to evaluate the positional distribution of caprylic acid using allyl magnesium bromide (Grignard reagent) were hampered by production of unknown species, which prevented accurate determination of the concentration of some key fatty acids.     

Utilization of reaction medium-dependent regiospecificity of Candida antarctica lipase (Novozym 435) for the synthesis of 1,3-dicapryloyl-2-docosahexaenoyl (or eicosapentaenoyl) glycerol

A highly efficient enzymatic method for the synthesis of regioisomerically pure 1,3-dicapryloyl-2-docosahexaenoyl glycerol (CDC) in two steps was established. 2-Monoglyceride (2-MG) formation by ethanolysis of tridocosahexaenoylglycerol (DDD) with immobilized Candida antarctica lipase (Novozym 435) as catalyst was the key step of the synthesis. CDC was finally obtained by reesterification of 2-MG with ethylcaprylate (EtC) catalyzed by Rhizomucor miehei lipase (Lipozyme IM). The regiospecificity of Novozym 435 depended on the type of reaction and the initial composition of the reaction medium. It displayed strict 1,3-regiospecificity for ethanolysis at a high excess of ethanol in the reaction mixture although it displayed no regiospecificity in transesterification and esterification reactions. The highest yield of CDC (85.4%) was obtained by ethanolysis at a 4∶1 weight ratio of ethanol/DDD for 6 h followed by reesterification at a 20∶1 molar ratio of EtC/initial DDD for 1.5 h. The regioisomeric purity of CDC was 100%. Good results were obtained also for the synthesis of 1,3-dicapryloyl-eicosapentaenoylglycerol (CEC) by the same method: 84.2% yield and 99.8% regioisomeric purity at the same reactant ratios as above. The yield of the reesterification step and the regioisomeric purity of the product were influenced by the molar ratio of the reactants for both CDC and CEC syntheses: higher excess of EtC favored higher yields and regioisomeric purity of the products.