PROCESS FOR PREPARATION OF TRIGLYCERIDE AND TRIGLYCERIDE COMPOSITION

Abstract: Triglycerides of long-chain fatty acids (e.g. polyunsaturated acids) are prepared in high yield and purity by lipase-catalyzed interesterification of a long-chain free acid or lower alkyl ester thereof with a triglyceride of a short-chain acid. Short-chain free acid or a lower alkyl ester thereof is formed during the reaction, and utilizing its relatively high volatility is removed simultaneously by evaporation.

Description: PROCESS FOR PREPARATION OF TRIGLYCERIDE AND TRIGLYCERIDE COMPOSITION TECHNICAL FIELD The invention relates to a process for the prepara- tion of a triglyceride having at least one long-chain fatty acid (e.g. a polyunsaturated acid) in the molecule. It also relates to a triglyceride composition with a high content of polyunsaturated fatty acid in the triglyceride molecules. BACKGROUND ART It is known that triglycerides of poly-unsaturated fatty acid (PUFA) , such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) , have beneficial medical effects, and within the last decade much attention has been directed to methods of producing triglyceride compositions with a high content of these acids, and particularly a high content of tri¬ glycerides with three such acids in the molecule. PUFA in the form of free fatty acid or lower alkyl (e.g. methyl or ethyl) ester is available in high purity and have been used to prepare triglycerides with high PUFA content. Thus, JP-A 61-43143 (Nisshin Flour Mill et al.) and EP 300,844 (R.F. Azar et al.) describe chemical interester¬ ification of lower alkyl PUFA ester with triacetin or tri- butyrin. Sodium methylate was used as catalyst, and vacuum was used to remove lower alkyl acetate or butyrate formed in the reaction. The former describes production of triglyceride with 90% PUFA content. JP-A 61-246146 (Nissui Seiyaku) describes halogen- ation of PUFA free acid, followed by reaction of PUFA acyl chloride with glycerine. The first step was carried out with oxalyl chloride at 65-90°C for 4 hours, and the second step under reflux for several hours in chloroform in the presence of quinoline or pyridine. The above-mentioned processes use highly reactive chemicals that require special precautions in handling, these reactive chemicals react with part of the labile PUFA acyl groups, and the resulting reaction mixture in each case requires complex purification. JP-A 62-91188 (Nisshin Oil) describes lipase-cata- lyzed production of PUFA glycerides from glycerol and PUFA free acid or ethyl ester, using positionally specific lipase in native form or immobilized on a weakly basic anion exchange resin. It is stated that addition of water is necessary. After reaction and removal of unreacted fatty acid, the glyceride mixture contained at most 86% triglyceride together with at least 14% diglyceride + onoglyceride. The product contained at most 85% polyunsaturated fatty acids together with at least 15% of other fatty acids. It is an object of the invention to provide a simple process, avoiding the use of aggressive chemicals, to produce triglycerides of long-chain fatty acids with a low content of mono- and diglycerides. It is also an object to provide triglyceride compositions with high PUFA content. STATEMENT OF THE INVENTION According to the invention, the objects are met by lipase-catalyzed interesterification of a long-chain free acid or lower alkyl ester thereof with a triglyceride of a short- chain acid. Short-chain free acid or a lower alkyl ester thereof is formed during the reaction, and utilizing its relatively high volatility is removed simultaneously by evapor¬ ation. Accordingly, the invention provides a process for the preparation a.triglyceride having at least one C g+ long- chain fatty acid in the molecule, characterize d by inter- esterification in the presence of a lipase of the corresponding long-chain free fatty acid or C-^^ lower alkyl ester thereof with a triglyceride having one or more C 2 -C 6 short-chain fatty acids in the molecule, and by evaporative removal during the reaction of short-chain free acid or lower alkyl ester thereof. The invention also provides a triglyceride composi¬ tion, characterized by at least 95 % by weight (preferably at least 98%) of the fatty acids in the triglyceride molecules being polyunsaturated a °id. DETAILED DESCRIPTION OF THE INVENTION Long-chain fatty acid The process of the invention is well suited for incorporation of C_ 2 fatty acid since the reactant free acid or ester has very low volatility so the loss will be negli¬ gible. In particular, the process can be used for incorporating c 1 8 ~ 22 fatt y acid which may be monounsaturated (e.g. oleic) , diunsaturated (e.g. linoleic) or polyunsaturated (with three or more double bonds) such as linolenic acid, EPA or DHA. Due to the gentle conditions used in the process, by-product formation from these sensitive acids is largely avoided. Thus, the process of the invention can be used to prepare triglyceride compositions with high content of such acid by using a reactant mixture wherein the free acid or lower alkyl ester comprises at least 90%, preferably at least 95% and most preferably at least 98%, of one or more such acids or esters thereof. In this way it is possible to prepare tri- glyceride with such acid in all three positions in good yield. In this connection, it has surprisingly been found that pure EPA or DHA is incorporated particularly fast. Preferably, the long-chain fatty acid reactant is free fatty acid, methyl or ethyl ester; in this case the resulting short-chain acid or ester is very volatile and is easily removed. The long-chain fatty acid or ester may be prepared by known methods, and some are commercially available in high purity, e.g. EPA and DHA as free acid and ethyl ester in 99% purity from Idemitsu Petro Chemical Co., Ltd., Japan. Short-chain fatty acid The short-chain acid present in the reactant tri¬ glyceride is preferably acetic or butyric acid, due to the volatility of the products. It is particularly advantageous to use triglyceride with three short-chain acids in the molecule, especially tributyrin or triacetin. Lipase Positionally specific (1,3-specific) or non-specific lipase may be used. If incorporation of long-chain acyl into all three positions of the triglyceride is desired, it is particularly advantageous to use non-specific lipase to achieve a fast reaction. The lipase must have sufficient stability at the temperature and reaction time to be used. Examples of non-specific lipases are those derived from strains of Candida, especially C. antarctica lipase (WO 88/02775, incorporated herein by reference) , and lipase from C. ru osa (also known as C cylindracea) . It is particularly preferred to use a lipase preparation containing both lipase A and lipase B of C. antarctica described in said reference. Examples of specific lipases are lipase derived from Humicola, especially H. lanuqinosa (WO 89/06278) and recombi- nant Humicola lipase (EP 305,216) and Mucor lipase (EP 140,542) . Immobilized lipase is generally most effective for interesterification. Some immobilized lipases require a certain water content for activation. Depending on the reactants and reaction conditions, water may be given off by the evaporation. To compensate for this, it may be necessary to add water or immobilized lipase step-wise during reaction. An example of a suitable immobilization method is adsorption of lipase on a particulate, macroporous adsorbent (i.e. non-ionic) resin of the acrylic type according to WO 89/02916. An example of a lipase preparation that may be used is SP 382 from Novo Nordisk A/S (mixture of lipases A and B from C. antarctica, immobilized according to WO 89/02916) . Reaction conditions A suitable amount of lipase is generally in the range 0.5 - 10 BlU/g (typically 1-5 BlU/g) of reactant mixture (BIU = Batch Interesterification Unit, see WO 89/06278) by use of immobilized lipase, or 50 - 500 LU/g of oil (LU = Lipase Unit, see WO 88/02775) by use of native (non-immobilized) lipase. It is preferable to use the two reactants in the stoichiometric ratio or with a moderate excess (e.g. 0-50%, especially 0-20%) of the long-chain fatty acid reactant. And it is preferable to let the reaction continue until at least 70% (particularly more than 90, especially more than 95%, e.g. more than 98%) of the short-chain acid in the triglyceride molecule has been replaced with long-chain acid. This makes it possible to prepare triglyceride of long-chain fatty acid in good yield and purity. It is generally not necessary to use a pH buffer or an organic solvent in the process. A temperature of 40-80°C, especially 60-80"C, is generally suitable for the reaction and the evaporation. The reaction time will generally be below 72 hours. Removal of volatile acid or ester The removal by evaporation of volatile fatty acid (ester) may be done continuously from a stirred tank. Reactants may be added batch wise, semi-batch wise or continuously. The lipase may be immobilized and can be reused. Alternatively, the reaction may occur in two or more steps, and evaporation can be done between the steps. Each process step can be made in a stirred tank, or immobilized lipase can be used continuously in a fixed bed. The evaporation is most conveniently done under vacuum, e.g. below 200 Pa and especially below 20 Pa. EXAMPLES EXAMPLES 1 - 4 Preparation of triσlvcerides containing 20-85% EPA/DHA Immobilized lipase derived from Candida antarctica (SP-382 from Novo-Nordisk A/S; activity approx. 30 BlU/g; 0.82- 0.86g; 10 % moisture content) was added to a mixture of tributyrin (98-99% from Aldrich; 6.6 mmol) and fatty acid ethyl ester concentrate (19.8 mmol) of varying composition, as indi¬ cated below. The mixture was gently stirred at 65°C on a magnetic stirrer hot-plate under a continuous vacuum of 0.1 mm Hg. The volatile ethyl butyrate product was continuously condensed into a liquid nitrogen cooled trap, which could be separated and weighed regularly during the process by discon¬ necting the reaction by replacing the vacuum with dry nitrogen or argon atmosphere. After 72 hours the reaction was discontinued, hexane added and the enzyme separated off by filtration. After hexane removal in vacuo on rotary evaporator the crude reaction product was afforded. Hydrolysis was found to occur only to a minimum extent as indicated by titration, which demonstrated less than 1% free fatty acid content of the product and further established by latroscan studies. The product was freed from the remaining ethyl esters and purified by the aid of preparatory High Performance Liquid Chromato- graphy (HPLC) eluting with 10% ether in pentane or hexane to afford 100% pure triglycerides as established by latroscan studies. Capillary Gas Liquid Chromatography (GLC) analysis in each case showed fatty acid composition almost identical to the original ethyl esters. The ethylbutyrate nature of the trapped component was established by Nuclear Magnetic Resonance (NMR) . There was little or no sign of water coming over. The following equation was used to calculate the % incorporation of fatty acids into tributyrin from the weight measurements: %incorporation -100% This was deduced from the following equation: %incorp. = 1/3- [# of eq. of entrapped ethyl butyrate/ # of eq. of tributyrin used] • 100% = 1/3- [Wt ethyl butyrate/ M,Wt ethyl butyratel/ [ Wt tributyrir M,wt tributyrin^ ' 100 ' = 1/3- [Wt ethyl ' 100 ' - V3 • [ t ethyl butγrat( -.t tr , butγr - n ] ■ [302.4/116.2] • 100% = 0.867- [Wt ethyl -100% The following results were found: Ex. 1 Ex. 2 Ex. 3 Ex. 4 %EPA 10 29 38 55 %DHA 10 30 28 33 % incorp.* 24 hrs in. 80 81 82 87 max. 83 85 86 91 % incorp.* 48 hrs in. 90 86 90 91 max. 93 90 94 95 % incorp.* 72hrs min. 92 92 94 93 max. 96 96 97 97 * The incorporation was determined by mass measurements of the entrapped product. The minimum incorporation was based on the assumption that all the moisture had escaped from the immobilized lipase. These results show that essentially complete incor¬ poration of fatty acids with a high content of PUFA, is possible with tributyrin, using stoichiometric amounts of the reactants. EXAMPLE 5 The preparation of triglycerides containing 99% EPA Immobilized lipase SP 382 (0.65 g; 10% moisture- content) was added to a mixture of tributyrin (99% from 5 Aldrich; 1.48 g, 4.89 mmol) and 99% EPA ethyl ester concentrate (M.wt. 330.5 g/mol; 4,87 g, 14.7 mmol). The mixture was gently stirred at 65 β C on a magnetic stirrer hot-plate under a continous vacuum of 0.5 - 0.1 mmHg. The volatile ethyl butyrate product was continuously condensed into a liquid nitrogen 10 cooled trap, which could be separated and weighed regularly during the process by disconnecting the reaction by replacing the vacuum with dry nitrogen or argon atmosphere. After 72 hours the reaction was discontinued, hexane added and the enzyme separated off by filtration. After solvent removal .in 15 vacuo on rotary evaporator the crude reaction product was afforded (4.43 g, 95%). Hydrolysis was found to occur only to a minimum extent as indicated by titration, which demonstrated less than 1% free fatty acid content of the product (0.7%) and further established by iatroscan studies. The weight 20 measurements indicated 95-98% incorporation, iatroscan studies 97% and NMR spectorscopy 95%. The product was freed from the remaining ethyl esters and purified by the aid of prep. HPLC eluting with 10% ether in hexane to afford 100% pure triglycerides (3.80 g, 86% recovery equivalent to 82% overall 25 yield) as established by iatroscan studies. NMR spectroscopy indicated very pure material with only t ced of the butyric acid moiety left in the triglycerides. f250 MHz -hi NMR (CDC1 3 ) : S 5.41-5.26 (m, 31 H, =C-H and -CH 2 -CH-CH 2 -) , 4.30 (dd,J = 11.90 Hz, J = 4.34 Hz, 2 30 H, -CH 2 -CH-CH 2 -) , 4.14 (dd,J = 11.90 Hz, J = 5.93 Hz, 2 H, - CH 2 -CH-CH 2 -) , 2.90-2.78 (m, 24 H, =C-CH 2 -C=) , 2.33 (t,J = 7.34 Hz, 2H, 00C-CH 2 -) , 2.32 (t,J = 7.36 Hz, 4 H, OOC-CH 2 ~) , 2.15- 2.01 (m, 12 H, -CH 2 -CH 2 -C=) , 1.75-1.61 (m, 6H, =CH-CH 2 -CH 3 ) , and 0.97 ppm (t,J = 7.52 Hz, 9 H, -CH 3 ) . 13 C NMR (COC1-. ) : δ 35 172.9(s), 172.6(s), 132.0(d), 128,9(d), 128.7(d), 128.5(d), 128.2(d), 128.1(d), 128.1(d), 128.0(d), 127.8(d), 127.0(d), 68.9(d), 62.1(t), 33.5(t), 33.3(t), 26.4(t), 25.6(t), 25.6(t), 25.5(t), 24.7(t), 24.7(t), 24.6(t), 20.5(t) and 14.2 ppm(q) , IR (neat liquid): v maχ 3020 (vs,C=C-H) , 2970 (S, CH 3 ) , 2935 (s, CH 2 ), 2875 (s, CH 3 ), 2850 (w, CH 2 ), 1745 (vs, C=0) and 1645 cm " 1 (ms, C=C) . ] The following results were obtained: Time % incorporation hours min.* max.* NMR 2 53 57 64 4 67 70 73 8 80 83 82 24 90 93 91 48 94 97 93 72 95 98 95 * The incorporation was determined by mass measurements of the entrapped product. The minimum incorporation was based on the assumption that all the moisture had escaped from the immobilized lipase. EXAMPLE 6 The preparation of triglycerides containing 99% DHA Immobilized lipase SP 382 (0.65 g; 10% moisture- content) was added to a mixture of tributyrin (99% from Aldrich; 1.38 g, 4.56 mmol) and 99% DHA ethyl ester concentrate (M.wt. 356.6 g/mol; 4,87 g, 13.6 mmol). The mixture was gently stirred at 65°C on a magnetic stirrer hot-plate under a continous vacuum of 0.5 - 0.1 mmHg. The volatile ethyl butyrate product was continuously condensed into a liquid nitrogen cooled trap, which could be separated and weighed regularly during the process by disconnecting the reaction by replacing the vacuum with dry nitrogen or argon atmosphere. After 72 hours the reaction was discontinued, hexane added and the enzyme separated off by filtration. After solvent removal jLn vacuo on rotary evaporator the crude reaction product was afforded (4.40 g, 94%). Hydrolysis was found to occur only to a minimum extent as indicated by titration, which demonstrated about 1% free fatty acid content of the product (1.2%) and further established by iatroscan studies. The weight measurements indicated 95-99% incorporation, iatroscan 96%, but NMR spectorscopy indicated 94%. The product was freed from the remaining ethyl esters and purified by the aid of prep. HPLC eluting with 10% ether in hexane to afford 100% pure triglycerides (3.56 g, 81% recovery equivalent to 76% overall yield) as established by iatroscan studies. NMR spectroscopy indicated very pure material with only traces of the butyric acid moiety left in the triglycerides. [250 MHz -kl NMR fCDCl 3 ) : δ 5.44-5.25 (m, 37 H, =C-H and -CH 2 -CH-CH 2 -) , 4.30 (dd,J = 11.90 Hz, J = 4.36 Hz, 2 H, -CH 2 -CH-CH 2 -) , 4.15 (dd,J = 11.90 Hz, J = 5.89 Hz, 2 H, - CH 2 -CH-CH 2 -) , 2.90-2.79 (m, 30 H, =C-CH 2 -C=) , 2.39-2.38 (m, A 2 B 2 , 12 H, =CH-CH 2 -CH 2 -C00H) , 2.13-2.02 (m, 6 H, =CH-CH 2 ~ CH 3 ), and 0.97 ppm (t,J = 7.53 Hz, 9 H, -CH 3 ) . 13 C NMR fCDCl 3 ) : <9 172.5(s), 172.l(s), 132.0(d), 129.5(d), 128.5(d), 128.3(d), 128.3(d), 128.2(d), 128.2(d), 128.0(d), 127.9(d), 127.8(d), 127.6(d), 127.0(d), 69.0(d), 62.2(t), 34.0(t), 33.8(t), 25.6(t), 25.6(t), 25.6(t), 25.6(t), 25.5(t), 22.6(t), 20.5(t) and 14.2 ppm(q) , IR (neat liquid): v maχ 3020 (vs,C=C-H), 2970 (ε, CH 3 ), 2930 (S, CH 2 ) , 2870 (s, CH 3 ) , 2850 (w, CH 2 ) , 1750 (vs, C=0) and 1650 cm -1 (ms, C=C) . ] The following results were obtained: Time % incorporation hours min.* max. NMR 2 32 35 33 4 63 67 63 8 77 81 78 24 91 95 89 48 94 98 94 72 95 99 94 * The incorporation was determined by mass measurements of the entrapped product. The minimum incorporation was based on the assumption that all the moisture had escaped from the immobilized lipase. EXAMPLE 7 Preparation of triglycerides with reuse of lipase Immobilized lipase (as in Ex. 1-4; 8.25g) was added to a mixture of tributyrin (20.Og; 66.1 mmol) and ethyl esters of cod liver oil (9% EPA and 9% DHA; M.wt. 313.1 g/mol; 62.lg; 199 mmol) . The mixture was gently stirred at 65°C under a continuous vacuum of 0.1 mm Hg. The volatile ethyl butyrate product was condensed into a liquid nitrogen cooled trap, which was weighed regularly during the progress of the reaction. After 72 hours the reaction was discontinued and the lipase directly separated off without an organic solvent by filtration under dry nitrogen by the aid of a pressure equalized funnel equipped with a sintered glass filter plate and inlets/outlets to the nitrogen and the vacuum lines to aid the filtration, which was controlled by teflon key stopcocks. The oil was collected for further analysis. The immobilized lipase was reintroduced into the reaction vessel. This was repeated 4 times, reusing the same immobilized lipase. The following results were obtained: Run % incorporation no . max. * 1 93 2 86 3 85 4 87 5** 89 * As determined by mass measurements of the entrapped product after 72 hours. ** Results for the moisture-free lipase EXAMPLE 8 Preparation of EPA/DHA triglyceride using triacetin Example 1 was repeated, but with triacetin instead of tributyrin, and with 10% excess of ethyl esters. Water was observed to go off rapidly, so for calculation of incorpora¬ tion, all water present was assumed to be included in the trapped weight. After 45 hours 72% incorporation had occurred, and after 69 hours 83%. The results show that a high degree of incorporation can be achieved with triacetin, but water tends to go off (presumably due to formation of an azeotrope with ethyl acetate) , so stepwise addition of lipase may be advantageous to compensate for enzyme deactivation (due to water loss) .

Claims: CLAIMS 1. A process for the preparation of a triglyceride having at least one C 8+ long-chain fatty acid in the molecule, characterized by interesterification in the presence of a 5 lipase of the long-chain free fatty acid or ^-^ lower alkyl ester thereof with a triglyceride having one or more C 2 -C 8 short-chain fatty acids in the molecule, and by evaporative removal during the reaction of short-chain free acid or lower alkyl ester thereof. 102. A process according to Claim 1 wherein the long- chain fatty acid is C 12+ , preferably mono-, di- or poly¬ unsaturated C lg _ 22 , and most preferably eicosapentaenoic or docosahexaenoic acid. 3. A process according to a preceding claim, wherein the 15 free acid or lower alkyl ester initially present comprises at least 90%, preferably at least 95% and most preferably at least 98%, of one or more long-chain acids or esters thereof. 4. A process according to a preceding claim for prepar¬ ing a triglyceride three long-chain acids in the molecule. 205. A process according to a preceding claim, using a stoichiometric excess of 0-50% (preferably 0-20%) of the long chain fatty acid or ester relative to the short-chain fatty acid in the triglyceride molecule. 6. A process according to Claim 5, wherein the reaction 25 is continued until the at least 70% by weight, preferably at least 90%, more preferably at least 95% and most preferably at least 98% of the short-chain acid in the triglyceride is replaced. 7. A process according to a preceding claim, wherein the reactant triglyceride has three short-chain acids in the molecule, and is preferably tributyrin or triacetin. 8. A process according to a preceding claim, wherein the 5 reactant free acid or ester is a free fatty acid or a methyl or ethyl ester. 9. A process according to a preceding claim, wherein the lipase is positionally non-specific, and is preferably derived from a strain of Candida, most preferably C. antarctica. 1010. A process according to a preceding claim, wherein the lipase is in immobilized form, preferably immobilized by adsorption on a particulate, macroporous adsorbent resin of the acrylic type. 11. A process according to a preceding claim, wherein the 15 removal of acid ester is done under vacuum, preferably below 1 mm Hg, and preferably at 40-80°C. 12. A triglyceride composition, characterized by at least 95 % by weight (preferably at least 98%) of the fatty acids in the triglyceride molecules being polyunsaturated C 18 _ 22 acid. 2013. A composition according to Claim 12, wherein the polyunsaturated acid is eicosapentaenoic acid, docosahexaenoic acid or a mixture of these.

Inventors: HANSEN TOMAS TAGE, HARALDSSON GUDMUNDUR G, HJALTASON BALDUR, SVANHOLM HANNE, HANSEN, TOMAS, TAGE, HARALDSSON, GUDMUNDUR, G., HJALTASON, BALDUR, SVANHOLM, HANNE
Applicants: LYSI HF, NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1990
Main classification: C12P7
IPC codes: C07C69, C11C3, C12P7