Indian Patents. 217404:" PROCESS FOR SEPARATING EPA AND DHA IN MARINE OIL BY LIPASE CATALYSED ESTERIFICATION IOF THE OIL FOLLOWED BY SEPARATION OF EPA AND DHA ENRICHED FRACTIONS"

Title of Invention	" PROCESS FOR SEPARATING EPA AND DHA IN MARINE OIL BY LIPASE CATALYSED ESTERIFICATION IOF THE OIL FOLLOWED BY SEPARATION OF EPA AND DHA ENRICHED FRACTIONS"
Abstract	Marune Oil compositions which contain EPA and DHA as free acids hexyl ester are esterified with ethanol in the presence or a lipase catalysl under essentially organic solvent-free conditions and separated by distillation.

Full Text	This invention relates to the lipase catalysed esterification of marine oils. It is well known in the art to refine oil products of various kinds, including marine oils, with the aid of lipase catalysts whose specificity under the refining conditions employed enhances the recovery of a desired product. 10 Extensive research has been carried out in order to develop lipase-catalysed processes for isolating such commercially important PUFAs as. EPA. (eicosapentaenoic acid, C20:5) and DHA (docosahexaenoic acid, C22:6) from compositions such as fish oils containing them in relatively low concentrations. 15 For example, in PCT/NO95/00050 (WO 95/24459) we disclosed a process for treating an oil composition containing saturated and unsaturaed fatty acids in the form of triglycerides to transesterification reaction conditions with a C1-6 alcohol such as eihanol under substantially anhydrous conditions in the presence of a lipase active to 20 preferentially catalyse the transesterification of the saturated and monounsaturated fatty acids. With the preferred lipases, Pseudomonas sp. Lipase (PSL) and Pseudomanas fluorescens lipase (PFL) it was possible to prepare from marine oil sources concentrates containing more than 70% by weight of the commercially and therapeutically important omega-3 polyunsaturated fatty acids EPA and DHA in the form of glycerides. 25 A number of lipase-catalysed refining processes have utilised glycerol. By way of example, jP 62-91188 (1987); WO91/16W; Int. J. Food Sci. Technol. (1992), 27,73-76, Lie and Molin; Myrnes et al in JAOCS, Vol. 72.. No. 11 (1995), 30 1339-1344; Moore et al in JAOCS, Vol. 73, No. 11 (1996), 1409-1414; McNeil: et al in JAOCS, Vol. 73, No. 11 (1996), 1403-1407; WO96/3758 and WO96/37587 can be mentiond. WO 2004/043894 PCT/NO2003/000364 2 In PCT/NO00/00056 (WO 00/49117) we provided a process for esterifying a marine oil composition containing EPA and OHA as free fatty acids to form a free fatty acid fraction enriched in at least one of these fatty acids as compared to the starting composition, comprising the step of reacting said marine oil composition with glycerol 5 in the presence of a lipase catalyst, Rhizomucor miehei lipase (MML), under reduced pressure and essentially organic solvent-free conditions, and recovering a free fatty acid fraction enriched in at least one of EPA and DHA. Preferably short-path distillation was used to separate the residual free fatty acids from the glyceride mixture. 10 However, it has now become evident that mis strategy based on short-path distillation to separate the residual free fatty acids from the glyceride mixture is not very feasible. This is a result of too high volatility of the shorter chain monoglycerides, which contaminate the distillate to a large extent. 15 We have now discovered that lipase-catalysed processes for preparing concentrates of EPA and DHA by the direct esterification of free fatty acids with methanol or ethanol, or transesterification of Cn & alkyl esters from fish oil (n= 2 - 18) with Cm alcohol (alcoholysis) (m= 1-12; n>m), and subsequent short-path distillation provide high DHA concentrates. These processes are fast and simple reactions offering excellent 20 separation between. EPA and DHA without generating unfavourable monoglycerides in the distillate. The essential features of the processes are defined in the attached patent claims. In a preferred embodiment of the invention the C1-C12 alcohol is ethanol (ethanolysis). 25 Among the C1-C12 alkyl esters, hexyl ester is preferred. The molar ratio of methanol or ethanol to free fatty acids in the starting material in the direct esterification is from 0.5 to 10.0, the preferred ratio is from 0.5 to 3.0, and the most preferred ratio is from 1.0 to 2.0 or even from 1.0 to .1.5. 30 WO 2004/043894 PCT/NO003/000364 3 The molar ralio of Cm alcohols to Cn alkyl esters in the transesterificatiton is from 0.5 to 10.0. the preferred ratio is from 0.5 to 3.0, and the most preferred ratio is from 2.0 to 3.0. 5 The esterifications are conducted at a tempratature of 0°C to 700C, and preferably at a temperature of 20°C to 40°C. The lipase catalysts used in the present invention are immobilized on a carrier. 10 Some Lipases used during the alcohalyses do have the properties that they catalyse the alcoholysis of DHA at a much slower speed than the corresponding alcoholysis of BPA. A preferred lipase having such properties is Rhizomucor mieliei (MML). Other lipases have the property that they catalyse the alcoholysis of both EPA and DHA al a much Slower speed than the corresponding alcoholysis of shorter chain and more saturated 15 fatty acids. Lipases having such properties are Pseudomonas sp. lipase (PSL) and Psedomonas fluorescens lipase (PFL), Direct esterification of fish oil free fatty acids with ethanol by MML is already known from G. G. Haraldsson and B. Kristinsson, j. Am. Oil Chem. Soc. 75: 1551-1556(1998). 20 Scheme 1. Direct esterification of fish oil free fatty acids with ethanul by MML. 25 However, it was not believed that a satisfactory separation of the DHA residual free fatty acids and ethyl esters was possible, by short-path distillation technique. Now we have surprisingly found that Short -path distillation technique can be used highly successfully. This is evident from the results shown in the examples below. 30 WO 2004/043894 PCT/NO2003/000364 4 The present invention furthermore discloses ethanolysis of fish oil hexyl esters by a lipase, and subsequent molecular distillation to separate residual hexyl esters and more volatile ethyl esters. 5 Scheme 2. Ethanolysis of fish oil hexyl esters by lipase (MML). 10 To furtherimprove the recoveries of DHA and the concentration in the product an ethanolysis reaction as described in PCT/NO95/00050 (WO 9524459) can be used as a pre-step before the direct escerification. 15 Scheme 3. Ethanoiysis of fish oil by lipase (MML). Prior to the direct esterification the glyceride mixture needs to be hydrolysed. In order to reduce the bulk of the starting material by half before hydrolysis the ethanolysis 20 reaction of PCT/NO95/00050 (WO 95/24459) is found to be useful. The present invention therefore also discloses, as an alternative process, a two-enzymatic-step reaction starting with an ethanolysis and a subsequent direct esterification, each step followed by concentration by molecular distillation. This two-step reaction is also suitable for oils highly enriched with long-chain monunsaturates, such as Herring oil. 25 The two-step reaction is also applicable and advantageous when fish oil hexyl esters are the starting material. The invention is illustrated by the Examples which follow. WO 2004/043894 PCT/NO2003/000364 5 Starting materials like Sardine oil (SO), Anchovy oil (AO), Herring oil (HO). Cod liver oil (CLO), Tuna oil (TO) and Blue whiting oil (BWO) have been tested. 5 Experimental procedures Ths bacterial Lipases from Pseudomanas sp. (PSL; Lipase AK) and Pseudomonas fluorescens (PFL; Lipsse PS) were purchased from Amano Bnzyrne inc, The immobolized Rhizomucor miehei (MML; Lypozyme RM IM), Tharmomyces lamiginosa (TLL; Lipozyme TM IM) and Candida antarctica (CAL; Novozym 435) lipases where 10 provided by Novozyme in Denmark. The Sardine oil (14% EPA and 15% DHA), Anchovy oil (18% EPA and 12% DHA), Herring oil (6% EPA and 8% DHA), Tuna oil (6% EPA and 23% DHA), Cod liver oil (9% EPA and 5% t)HA) and Bine whiting oil (11 % EPA and 7% DHA) were all provided by Pronova Biocare. Fatty acid analysis was performed employing a Perkin-elmer 8140 Gas Chromatograph 15 (GC) equipped with a flame ionisation detector (FID). Capillary column was 30 meter DB-225 30N, 0.25 m capillary column from j &W Scientific. The short-path distillation was carried out in a Leyboid KDL 4 still Nuclear magnetic resonance (NMR) spactra were recorded on a Broker AC 250 NMR spectrometer in deuteraied chloroform as solvent Preparative thin-layer chmrnatogcaphy (PLC) was conducted on 20 silica gel plates from Merck (Art 5721). Elution was performed with 80:20:1 mixture of petroleum ether, dieethyl ether: acetic acid- Rhodamin G (Merck) was used to visualise the bands which subsequently were scraped off and methylated. Methyl ester of C19:0 (Sigma) were added to the samples as internal standerds before injection to GC. 25 Hydrolysis of fish oil Fish oil (500 g, 0.55 mol) was added to a solution of sodium hydroxide (190 g, 4.75 mol), water (500 ml) and 96% ethanol (1.7 L). The resulting mixture was allowed to reflux for 30 minutes (until clear coloured Liquid is observed) and then cooled to room temperature, stirring constantly. To neutralise the solution, 6.0 Hhydrochloric acid 30 (870 ml, 10% excess) was carefully added and the resulting mixture transferred to a separatory funnel. The See fatty acids were extracted twice with a 1:1 mixture of petroleum ether and diethyl ether (1.5 L). The organic layer was then washed three WO 2004/043894 PCT/NO2003/000364 6 times with water(1.5 L) and dried over anhydrous magnesium sulphate. The drying agent was filtered off and the solvents removed by evaporation, finishing with high vacuum vaporisation for 2 hours at 50°C. Analysis on analytical TLC, a single spot indicated pure free fatty acids. The colour of the product varied from a yellowish So dark 5 burgundy colour, depending on the fish oil. Direct esterification of fish oil free fatty acids with ethanol Immobilized MML(15 g) was added to a solution of fish oil free fatty acids (300 g, approx. 1.03 mol) and absolute ethanol (143 g, 3.10 mol). The resulting enzyme 10 suspension was gently stirred under nitrogen at 400C until desired conversion was reached. Samples were taken during the reaction and residual amount of free fatty acids detected by titration with 0,02M NaOH in order to monitor the progress of the reaction. Fractionation on was performed by preparative TLC and each lipid fraction was subsequently quantified and analysed. on fatty acid profile by GC. After reaching 15 desired conversion the enzyme was removed by filtration and the excess ethanoi evaporated in vacua. The high DHA concentrate was obtained as residue after short- path distilation of the resulting mixture. Ethanolysis of fish oil by lipase 20 Immobilized MML (20 g) was added to a solution of fish oil (400 g, 0.44 mol) and absolute ethanol (61 g, 1.32 mol). The resulting enzyme suspension was gently stirred under nitrogen at room temperature until desired conversion was reached. Then the enzyme was removed by filtration and the excess ethanol evaporated in vacuo prior to short-path distillation. The progress of the reaction was monitoned by analytical TLC 25 and 1H-NMR. Fractionation was performed by preparative TLC and each lipid fraction was subsequently quantified and analysed on fatty acid profile by GC. Hxanolysis of fish oil by lipase Immobilized CAL (25 g) was added to a solution of fish oil (500 g, 0.55 mol) and i- 30 hexanol (338 g, 3.31 mol). The resulting enzyme suspension was gently stirred under nitrogen 650C until the triacylglycerols had been completely converted to hexyl WO 2004/043894 PCT/NO2003/000364 7 esters, according to analytical TLC and/or 1H-NMR. The enzyme was removed by filtration and the excess hexanol evaporated in vacua. Ethanoysis of fish oil hexyl esters by lipase 5 Immobilized MML (15 g) was added to a solution of fish oil hexyl esters (300 g, 0.80 mol) and absolute ethanol (111 g,2.41 mol). The resulting enzyme suspension was gently stirred under nitrogen at( 40°C until desired conversion was obtained, according to 1H-NMEL The enzyme was removed by filtration and the excess ethanol evaporated in vacua. The high DHA concentrate was obtained as residue after short-path distillation 10 of the resulting mixture. The fatty acid composition of each ester group was determined by single run on GC. Example 1. 15 Direct Esterification. of Fish Oil Free Fatty Asids with Ethanol Sardine oil(SO) The progress of direct esterification reaction of SO free fatty acids, containing 14% EPA and 15% DHA (14/15), with 3 equivalents of ethanol in the presence of MML (5% 20 as based on the weight of free fatty acids) at 400C is displayed in Table 1. Under these conditions the lipase displayed extremely high activity toward the SO free fatty acids. over70% conversion(% efthyl esters) was reached after only 2 hours. After 4 hour reaction the residual free fatty acids contained 49% DHA and 6% EPA in 73% and 10% recoveries, respectivly. In terms of DHA concentration and recoveries the optimal 25 conversion appears to be around 75% conversion. In Table 1 the weight percentage of ethyl esters produced during the progress of the reaction was used directly as a measure of the extent of conversion. 30 WO 2004/043894 PCT/NO2003/000364 8 Excellent results were obtained for direct esterification of SO free fatty acids after 5 separation by short-path distillation. SO free fatty acids were reacted with ethanol in the presence of MML for 4 hours at 40°C to reach 78% conversion. The free fatty acids of ihe reaction mixture comprised 49% DHA and 6% EPA with 75% DHA recoveries. After distillation at 115°C the residue comprised 69% DHA and 9% EPA in 65% and 10% recoveries, respectively (Table 2). The recoveries of DHA were improved by 10 slightly reducing the distillation temperature (see Table 3). We were notable to separate all the ethyl esters from the residual free fatty acids by the distillation. Despite that, we managed to obtain high DHA concentrate of approximately 90% free fatty acids and 10% ethyl esters after short-path distillation at 115°C. The ethyl esters obtained in the residue are highly enriched with DHA like the free fatty acids. Furthermore, the more 15 saturated and shorter-chain free fatty acids are distilled resulting in higher DHA concentration of the residue than for the free fatty acid fraction after the reaction. 20 WO2004/043894 PCT/NO2003/000364 9 The results for SO were improved by lowering the conversion and the distillation temperature as displayed in Table 3. After 4 hour reaction 75% conversion was obtained. After distillation at 111°C the residue contained 66% DHA in 88% recoveries with DHA/EPA ratio of 4.7. At slightly higher distillation temperature the residue 5 comprised 74% DHA in 75% recovery with a DHA/EPA ratio nearly seven. It should be notified that the DHA recovery after the distillations is based on percent weight of DHA The ethanol content can be reduced to I equivalent resulting in increased reaction time (Table 4), Less lipaae tari also be introduced resulting in cosisiderably lower reaction rate. 15 20 WO 2004/043894 PCT/NO2003/000364 10 Ancholvy Oil(AO) The progress of the direct esterification reaction of AO & free fatty acids comprising 18% EPA and ) 12% DHA (18/12) under identical conditions to the SO is displayed in Table 5. As can be noticed a DHA/EPA ratio of approximately 6:1 was obtained at 82% 5 conversion after 24 hours with EPA comprising 8% and DHA 50%. The DHA recovery was just below 80%. Also after 11 hours, at 79% conversion a DHA/EPA ratio of 5:1 wilh DHA recoveries as high as 84%. Therefore, AO and SO are both highly potential starting materials for making concentrates high in DHA and also, to make concentrates . high in EPA from the ethyl ester fraction if that is of interest. 10 The results for AO are good in terms of DHA concentration and DHA/EPA ratios as 15 displayed in Table 6. Free fatty acids of AO (19/12) were reacted as before to reach 76% conversion in 11 hours. After distillation at 12l°C the residua comprised 61% DHA in only 64% recovery with the DHA/EPA ratio being 5.5. The distillate may possibly be used to make high EPA concentrates by a repeated distillation at lower temperature. As an example a concentrate of 45% EPA and 10% DHA is considered to 20 be a desirable composition for a potential commercial product. 25 WO 2004/043894 PCT/NO2003/000364 11 Herrings Oil(HO) 5 Free fatty acids from herring oil comprising 6% EPA and 8% DHA (6/8) were similarly treated under the direct esterification conditions as described above. The progress of the reaction is displayed in Table 7. The residual free fatty acids after 12 hour reaction contained 37% DHA and 6% EPA with 90% and 13% recoveries, respectively. Free fatty acids from different HO comprising 9% BPA and 9% DHA (9/9) were reacted for 12 hours, to reach 84% conversion, in same way as before, The free fatty acids of 15 the reaction mixture comprised 39% DHA and 8% EPA with 76% DHA recovery. After distillation at 110°C the residue contained 40% DHA and 7% EPA in 68% DHA recovery with a DRA/EPA ratio of almost 6:1 (Table 8). Low DHA concentration results from high contents of long-chain monounsaturated fatty acids of 20% (4%)and 22:1 (37%). This high content of long-chain monounsaturates in HO and Capelin oil 20 renders them less feasible starting material for the process described. A simple urea inclusion of the residual oil may be used to remove most of these monounsaturated fatty acids resulting in a valuable concentrate of DHA. It should be added that HO with its low EPA content is more suitable for obtaining high DHA/EPA natios than SO and AO. WO2004/043894 PCT/NO2003/000364 12 Tuna Oil (TO) 5 The progress of the direct esterification reaction of TO free fatty acids comprising 6% EPA and 23% DHA (6/23) under conditions identical to SO described above is displayed in Table 9 below. After 8 hour reaction conversion of 68% was obtained with the residual free fatty acids comprising 74% DHA and 3% EPA with 83% DHA recovery and a DHA/EPA ratio of 25:1 (Table 9). Clearly, this type of initial EPA/DHA 10 composition of the starting oil is ideal for concentrating DHA. 15 Cod Liver Oil (CLO) The progress of the direct esterification reaction of CLO free fatty acids comprising 9% EPA and 9% DHA (9/9) under similar conditions as described above is displayed in Table 10. Around 79% conversion a DHA/EPA ratio of 5:1 was obtained for the residual free fatty acids with 50% DHA concentration and over 80% recovery. These 20 results are even better than those for SO and AO considering potential DHA recoveries. But in terms of cost, SO and AO are favoured over CLO. It may be of interest to WO 2004/043894 PCT/NO2003/000364 13 compare the results of CLO (9/9) to those of HO (9/9) in light of the feet that CLQ contains far less long-chain monounsaturates (20:1 and 22:1). BlueWhiting Oil(BWO) The progress of the direct esterification reaction of BWO free fatty acids comprising 11% EPA and 7% DHA (11/7) under the conditions described above is displayed in 10 Table 11. Around 73% conversion the residual free fatty acids comprised 24% DHA. in 95% recoveries. EPA was not transferred to ethyl esters as rapidly as expected. Interestingly, and unlike HO, the long-chain monounsaturated free fatty acids were to a much higher extent convetted to ethyl ester. Higher conversion is needed to obtain better separation of BPA and DHA, The reason for the low conversion for BWO is 15 unclear, but several attempts have not resulted in a higher conversion. 20 WO 2004/043894 PCT/NO2003/000364 14 Example 2 Combined Ethanolysis and Direct Esterificafion of Fish Oil A two-step reaction, starting with an ethanolysis ami a subsequent direct esterification, 5 each step followed by molecular distillation, could be used to improve the recoveries of DHA and the concentration. in the product. Prior to the direct esterifcation the glyceride mixture obtained from the ethanolysis needs to be hydrolysed. Therefore, the ethanolysis reaction can be used as a pre-step, reducing the bulk of the starting material by half before hydrolysis. Notice the high recoveries obtained in the ethanolysis at 40°C 10 after separation by distillation (Table 12). Better results were obtained at room temperature as discussed, above and displayed in Tables 13 and 14. The residue from the room temperature reaction comprised 23% DHA and 25% EPA in 97% and 65% recoveries, respectively (Table 13). These results indicate that the DHA recoveries can be improved significantly by the two-step process. Also, there is a dramatic reduction in 15 bulkiness for the hydrolysis reaction, finally, this approach may be suitable for oils highly enriched wife long-chain monounsaturates, such as HO. WO 2004/043894 PCT/NO2003/000364 15 Example 3 5 Ethanolysis of Fish Oil Hexyl Esters Ethanolysis of hexyl esters (HE) from fish oil is an alternative to the previously discribed ethanolysis of fish oil trigtyceridss {Scheme 2). The results indicate that various pases including the Rhizamucor miehei lipase (MML) and the Pseudomonas 10 lipases (PCL and PH.) can be used as well as the recently commercialised Thermomyces latuginosa lipase (TLL) from. Novozyme. Also, it has bsen coonfirmed that molecular distilation is quite suitable to separate residual hexyl ester and the more volatile ethyl ester 15 Candida antarctica lipase (CAL) was used to convert A.O triglycerides into the corresponding hexyl esters in a treatment with hexanol. Treatment of the resulting hexyl ester with ethanol and PSL folloed by molecular distilation of the reaction mixture may afford residual hexyl ester -with approximately 80% of EPA and DHA in a single or in two enzymatic steps. By cocentreting DHA in the hexyl ester not only can we 20 separate the ethyl estets from the hexyl esters but also distil off the more saturated hexyl esters as well.It may be possible lo convert the hexyl esters into ethyl esters either chemically or enzymatically using CAL, Alternatively, it is possible to treat the anchovy oil hexyl esters in ethanolysis using MML that may afford 70% DHA in a single enzymatic step as hexyl esters. They may be further concentrated by an additional 35 MML treatment. From the bulk of the ethyl esters containing most of the EPA it may be possible to purify EPA up to the 95% levels. An alternative two-step approach is based on the ethanolysis of sardine oil to produce a canceotrate of 50% EPA + DHA (30/20) as a glyceride mixture after molecular 30 distillation Treatment of the residual glycerides with hexanol and CAL affords hexyl esters of identical composition. They may either be treated with ethanol and PSL to afford hexyl esters with approximately 80% of EPA and DHA, or ethanol and MML to separate DHA from EPA, followed by further concentration of both EPA and DHA. WO2004/043894 PCT/NO2003/0003640 16 This process may have advantage in that the bulk of fish oil is being treated with ethanol instead of hexanol, which is both easier, less bulky and more feasible from industrial point of view. It must also be frome in mind that very high to excellent recovery of both EPA. and DHA can be expected by that method. 5 Anchovy Oil(AO) Like for the ethanolysis of fish oil triglycerides the fatty acid selectivity and activity of MML can be greatly affected by temperature. Thus, MML can be used to concentrate both EPA and DHA at or below 200C, but at 400C EPA is separated from DHA 10 resulting in high DHA concentrates. Anchovy oil hexyl esters comprising 18% EPA and 12% DHA were reacted with 2 equivalents of ethanol in the presence of MML (10% weight of the hexyl esters) for 24 hows at 400C to reach 59% conversion. After removal of the lipase excess ethanol was evaporated and the ethyl ester/hexyl ester (EE/HE) mixture distilled at 135°C at 3 *10-3 mbar. The residue (26% weight) comprised 43% 15 DHA in only 65% recovery. The DHA/EPA ratio-was only 2.2(Table 15). aIn Tables 15 and 16 the conversion of the lipase catalysed reactions is based on mol 20 percentage, whereas the distillation results are based on weight. Interesting results were obtained when the reaction temperature was lowered to 20°C in a similar reaction of Anchovy oil hexyl esters (18/13). After distillation at 1150C tee residue comprised 45% DHA and 30% EPA with 85% and 55% recoveries, respectively 25 (Table 16). 30 WO 2004/043894 PCT/NO2003/000364 17 The Pseudomonas lipases were Tested on a small scale with good results, giving high 5 EPA recovery but considerably lower DHA recovery, especially if the reaction exceeded 50% conversion, The results of the ethanolysis reaction of AO (18/12) with 2 equivalents of ethanol in the presence of PSL and PFL at room temperature is displayed in Table 16. For PFL, after only 44% conversion of sardine oil hexyl esters in 24 hours, the content of 28% EPA and 21% DHA was obtained while 57% conversion for PSL in 10 24 hours yielded in 33% EPA. and 17% DHA 15 Tbe new Novozyme lipase (TLL), immobilized on granular silica gel, was compared to MML. The new lipase was found to be sensitive to ethanol and the activity decreased rapidly with increased temperature, At 20°C both lipases were active and in 24 hours 54% conversion was obtained for MML but only 43% for TLL. The residual hexyl esters of TO, comprising 6% EPA and 28% DHA (6/28), from the TLL reaction 30 contained 8% EPA and 45% DHA. The MML reaction resulted in residual hexyl esters containing 7% EPA and 54% DHA (Table 18). These lipases are obviously similar in fatty acid selectivity but TLL is more sensitive toward ethanol concentration, which makes it inferior to MML. 25 WO 2004/043894 PCT/NO2003/000364 18 5 The results from the ethanolysis of TO hexyl esters (6/28) and ethanol al 40°C are displayed in Table 19, Interestingly, at 400Conly 15% conversion was obtained for TLL and 47% conversion for MML. It is believed that at higher temperature the lipase becomes more sensitive for the polar ethanol and its detrimental effects. For MML, after 47% conversion in 24 hours, the hexyl esters comprised 9% EPA and 49% DHA while 10 only 15% conversion for TLL in 24 hours yielded 33% EPA and 17% DHA. 15 By the present inventon separation of EPA. and DHA. by solvent free direct esterification of fish oil free fatty acids or fish oil hexyl esters and ethanol in the presence of a lipase is successfully obtained. The problems with monoglycerides in the distillate are avoided by the processes according to die present invention. WO 2004/043894 PCT/NO2003/000364 19 C l a i m s 1. A process for separating ethyl or methyl ester fraction enriched in BPA (eicosapentaenoic acid.C20:5) and a free fatty acid fraction enriched in DHA 5 (docosahexaenoic acid, C22:6) obtained from a direct esterification of Fish oil free fatty acids with a ethanol or methanol using lipase, by molecular distillation- 2. A process according to chain 1, wherein the fish oil free fatty acid starting material is obtained by a lipase catalysed alcoholysis of fish oil triglyceridss, a subsequent 10 molecular distillation and hydrolysis of the residual glyceride mixtures. 3. A process for esterifying a marine oil composition containing EPA and DHA as Cn alkyl esters of fatty acids (n = 2-18) to forn (1): a Cn alkyl ester fatty acid fraction [n = 2-18) Enriched in DHA as compared to the starting material and a Cn, alkyl ester fatty 15 acid fraction (m=1-12; n > m) enriched in EPA as compared to the starting material, or (2): a Cn alkyl ester fatty acid fraction (n = 2-18) enriched in both DHA and EPA as compared to the starting material and a Cm alkyl ester fatty acid fraction (m= 1-12; n> m) lower in both DHA and EPA as compared to the starting material comprising the step of reading said marine oil composition with a Cm alcohol (m= 1-12;n>m)in the 20 presence of a lipase catalyst under essentially organic solvent-free conditions, and separating the fractions by molecular distillation. 4. A process according to claim 3, wherein the starting material, C2-C18 alkyl ester, is obtained by a lipase catalysed, alcoholysis of fish oil triglycerides, a subsequent 25 molecular distillation, and alcoholysis of the residual glyceride mixture with a C2-C18 alkyl alcohol. 5. A process according 10 claim 3 and 4, wherein the C2C18 alkyl ester is hcxyl ester. 30 6. A process according to claim 3, wherein the C1-C12 alcohol is ethanol. 7. A process according to claim 3, were said lipase catalyst is Rhizomucor miehei lipase (MML), Thermomyces ianuginosa lipase (TLL), Psedomonas sp. lipase (PSL) or Psedomonas fluorescens lipase (PFL). WO 2004/043894 PCT/NO2003/000364 20 8. A process according to claim 1, wherein the molar ratio of methanol or ethanol free fatty acids in the starting composition is fron 0.5 to 10.0. 9. A process according to claim 8, wherein the molar ratio is from 0.5 to 3.0. 5 10. A process according to claim 8, wherein the molar ratio is from 1.0 to 2.0. 11. A process according to claim 8, wherein the molar ratio is from 0.5 to 1.5. 10 12. A process according to claim 3, wherein the molar ratio of C1-C12alcohol to C2-C18 alkyl ester is from 0.5 to 10.0. 13. A process according to claim 12, wherein The molar ratio is from 0.5 to 3.0. 15 14. A process according to claim 12, wherein the molar ratio is from 2.0 to 3.0. 15. A process according to any preceding claim, wherein the esterification reaction is conducted at a temperature of 00C to 700C. 20 16. A process according to claim 15, wherein the esterification reaction is conducted at a temperature of 200C to 400C. 17. A process according to any preceding claim, wherein said lipase catalyst is immobilized on a carrier. 25 18. A process according to claim 1, wherein said lipase catalyses the alcoholysis of DHA at a much slower speed than the c XXX ponding alcoholysis of EPA. 19. A process according to claim 18, wherein said lipase catalyst is Rhizomucor miehei 30 lipase (MML) or Thermomyces lanuginosa lipase (TLL). Marune Oil compositions which contain EPA and DHA as free acids hexyl ester are esterified with ethanol in the presence or a lipase catalysl under essentially organic solvent-free conditions and separated by distillation.

Full Text

This invention relates to the lipase catalysed esterification of marine oils.
It is well known in the art to refine oil products of various kinds, including marine oils,
with the aid of lipase catalysts whose specificity under the refining conditions employed
enhances the recovery of a desired product.
10
Extensive research has been carried out in order to develop lipase-catalysed processes
for isolating such commercially important PUFAs as. EPA. (eicosapentaenoic acid,
C20:5) and DHA (docosahexaenoic acid, C22:6) from compositions such as fish oils
containing them in relatively low concentrations.
15
For example, in PCT/NO95/00050 (WO 95/24459) we disclosed a process for treating
an oil composition containing saturated and unsaturaed fatty acids in the form of
triglycerides to transesterification reaction conditions with a C1-6 alcohol such as eihanol
under substantially anhydrous conditions in the presence of a lipase active to
20 preferentially catalyse the transesterification of the saturated and monounsaturated fatty
acids. With the preferred lipases, Pseudomonas sp. Lipase (PSL) and Pseudomanas
fluorescens lipase (PFL) it was possible to prepare from marine oil sources concentrates
containing more than 70% by weight of the commercially and therapeutically important
omega-3 polyunsaturated fatty acids EPA and DHA in the form of glycerides.
25
A number of lipase-catalysed refining processes have utilised glycerol.
By way of example, jP 62-91188 (1987); WO91/16W; Int. J. Food Sci. Technol.
(1992), 27,73-76, Lie and Molin; Myrnes et al in JAOCS, Vol. 72.. No. 11 (1995),
30 1339-1344; Moore et al in JAOCS, Vol. 73, No. 11 (1996), 1409-1414; McNeil: et al in
JAOCS, Vol. 73, No. 11 (1996), 1403-1407; WO96/3758 and WO96/37587 can be
mentiond.

WO 2004/043894 PCT/NO2003/000364
2
In PCT/NO00/00056 (WO 00/49117) we provided a process for esterifying a marine oil
composition containing EPA and OHA as free fatty acids to form a free fatty acid
fraction enriched in at least one of these fatty acids as compared to the starting
composition, comprising the step of reacting said marine oil composition with glycerol
5 in the presence of a lipase catalyst, Rhizomucor miehei lipase (MML), under reduced
pressure and essentially organic solvent-free conditions, and recovering a free fatty acid
fraction enriched in at least one of EPA and DHA. Preferably short-path distillation was
used to separate the residual free fatty acids from the glyceride mixture.
10 However, it has now become evident that mis strategy based on short-path distillation to
separate the residual free fatty acids from the glyceride mixture is not very feasible.
This is a result of too high volatility of the shorter chain monoglycerides, which
contaminate the distillate to a large extent.
15 We have now discovered that lipase-catalysed processes for preparing concentrates of
EPA and DHA by the direct esterification of free fatty acids with methanol or ethanol,
or transesterification of Cn & alkyl esters from fish oil (n= 2 - 18) with Cm alcohol
(alcoholysis) (m= 1-12; n>m), and subsequent short-path distillation provide high
DHA concentrates. These processes are fast and simple reactions offering excellent
20 separation between. EPA and DHA without generating unfavourable monoglycerides in
the distillate. The essential features of the processes are defined in the attached patent
claims.
In a preferred embodiment of the invention the C1-C12 alcohol is ethanol (ethanolysis).
25 Among the C1-C12 alkyl esters, hexyl ester is preferred.
The molar ratio of methanol or ethanol to free fatty acids in the starting material in the
direct esterification is from 0.5 to 10.0, the preferred ratio is from 0.5 to 3.0, and the
most preferred ratio is from 1.0 to 2.0 or even from 1.0 to .1.5.
30

WO 2004/043894 PCT/NO003/000364
3
The molar ralio of Cm alcohols to Cn alkyl esters in the transesterificatiton is from 0.5 to
10.0. the preferred ratio is from 0.5 to 3.0, and the most preferred ratio is from 2.0 to
3.0.
5 The esterifications are conducted at a tempratature of 0°C to 700C, and preferably at a
temperature of 20°C to 40°C.
The lipase catalysts used in the present invention are immobilized on a carrier.
10 Some Lipases used during the alcohalyses do have the properties that they catalyse the
alcoholysis of DHA at a much slower speed than the corresponding alcoholysis of BPA.
A preferred lipase having such properties is Rhizomucor mieliei (MML). Other lipases
have the property that they catalyse the alcoholysis of both EPA and DHA al a much
Slower speed than the corresponding alcoholysis of shorter chain and more saturated
15 fatty acids. Lipases having such properties are Pseudomonas sp. lipase (PSL) and
Psedomonas fluorescens lipase (PFL),
Direct esterification of fish oil free fatty acids with ethanol by MML is already known
from G. G. Haraldsson and B. Kristinsson, j. Am. Oil Chem. Soc. 75: 1551-1556(1998).
20

Scheme 1. Direct esterification of fish oil free fatty acids with ethanul by MML.
25 However, it was not believed that a satisfactory separation of the DHA residual free
fatty acids and ethyl esters was possible, by short-path distillation technique. Now we
have surprisingly found that Short -path distillation technique can be used highly
successfully. This is evident from the results shown in the examples below.
30

WO 2004/043894 PCT/NO2003/000364
4
The present invention furthermore discloses ethanolysis of fish oil hexyl esters by a
lipase, and subsequent molecular distillation to separate residual hexyl esters and more
volatile ethyl esters.
5

Scheme 2. Ethanolysis of fish oil hexyl esters by lipase (MML).
10 To furtherimprove the recoveries of DHA and the concentration in the product an
ethanolysis reaction as described in PCT/NO95/00050 (WO 9524459) can be used as a
pre-step before the direct escerification.

15
Scheme 3. Ethanoiysis of fish oil by lipase (MML).
Prior to the direct esterification the glyceride mixture needs to be hydrolysed. In order
to reduce the bulk of the starting material by half before hydrolysis the ethanolysis
20 reaction of PCT/NO95/00050 (WO 95/24459) is found to be useful. The present
invention therefore also discloses, as an alternative process, a two-enzymatic-step
reaction starting with an ethanolysis and a subsequent direct esterification, each step
followed by concentration by molecular distillation. This two-step reaction is also
suitable for oils highly enriched with long-chain monunsaturates, such as Herring oil.
25
The two-step reaction is also applicable and advantageous when fish oil hexyl esters are
the starting material.
The invention is illustrated by the Examples which follow.

WO 2004/043894 PCT/NO2003/000364
5
Starting materials like Sardine oil (SO), Anchovy oil (AO), Herring oil (HO). Cod liver
oil (CLO), Tuna oil (TO) and Blue whiting oil (BWO) have been tested.
5 Experimental procedures
Ths bacterial Lipases from Pseudomanas sp. (PSL; Lipase AK) and Pseudomonas
fluorescens (PFL; Lipsse PS) were purchased from Amano Bnzyrne inc, The
immobolized Rhizomucor miehei (MML; Lypozyme RM IM), Tharmomyces lamiginosa
(TLL; Lipozyme TM IM) and Candida antarctica (CAL; Novozym 435) lipases where
10 provided by Novozyme in Denmark. The Sardine oil (14% EPA and 15% DHA),
Anchovy oil (18% EPA and 12% DHA), Herring oil (6% EPA and 8% DHA), Tuna oil
(6% EPA and 23% DHA), Cod liver oil (9% EPA and 5% t)HA) and Bine whiting oil
(11 % EPA and 7% DHA) were all provided by Pronova Biocare.
Fatty acid analysis was performed employing a Perkin-elmer 8140 Gas Chromatograph
15 (GC) equipped with a flame ionisation detector (FID). Capillary column was 30 meter
DB-225 30N, 0.25 m capillary column from j &W Scientific. The short-path
distillation was carried out in a Leyboid KDL 4 still Nuclear magnetic resonance
(NMR) spactra were recorded on a Broker AC 250 NMR spectrometer in deuteraied
chloroform as solvent Preparative thin-layer chmrnatogcaphy (PLC) was conducted on
20 silica gel plates from Merck (Art 5721). Elution was performed with 80:20:1 mixture of
petroleum ether, dieethyl ether: acetic acid- Rhodamin G (Merck) was used to visualise
the bands which subsequently were scraped off and methylated. Methyl ester of C19:0
(Sigma) were added to the samples as internal standerds before injection to GC.
25 Hydrolysis of fish oil
Fish oil (500 g, 0.55 mol) was added to a solution of sodium hydroxide (190 g, 4.75
mol), water (500 ml) and 96% ethanol (1.7 L). The resulting mixture was allowed to
reflux for 30 minutes (until clear coloured Liquid is observed) and then cooled to room
temperature, stirring constantly. To neutralise the solution, 6.0 Hhydrochloric acid
30 (870 ml, 10% excess) was carefully added and the resulting mixture transferred to a
separatory funnel. The See fatty acids were extracted twice with a 1:1 mixture of
petroleum ether and diethyl ether (1.5 L). The organic layer was then washed three

WO 2004/043894 PCT/NO2003/000364
6
times with water(1.5 L) and dried over anhydrous magnesium sulphate. The drying
agent was filtered off and the solvents removed by evaporation, finishing with high
vacuum vaporisation for 2 hours at 50°C. Analysis on analytical TLC, a single spot
indicated pure free fatty acids. The colour of the product varied from a yellowish So dark
5 burgundy colour, depending on the fish oil.
Direct esterification of fish oil free fatty acids with ethanol
Immobilized MML(15 g) was added to a solution of fish oil free fatty acids (300 g,
approx. 1.03 mol) and absolute ethanol (143 g, 3.10 mol). The resulting enzyme
10 suspension was gently stirred under nitrogen at 400C until desired conversion was
reached. Samples were taken during the reaction and residual amount of free fatty acids
detected by titration with 0,02M NaOH in order to monitor the progress of the reaction.
Fractionation on was performed by preparative TLC and each lipid fraction was
subsequently quantified and analysed. on fatty acid profile by GC. After reaching
15 desired conversion the enzyme was removed by filtration and the excess ethanoi
evaporated in vacua. The high DHA concentrate was obtained as residue after short-
path distilation of the resulting mixture.
Ethanolysis of fish oil by lipase
20 Immobilized MML (20 g) was added to a solution of fish oil (400 g, 0.44 mol) and
absolute ethanol (61 g, 1.32 mol). The resulting enzyme suspension was gently stirred
under nitrogen at room temperature until desired conversion was reached. Then the
enzyme was removed by filtration and the excess ethanol evaporated in vacuo prior to
short-path distillation. The progress of the reaction was monitoned by analytical TLC
25 and 1H-NMR. Fractionation was performed by preparative TLC and each lipid fraction
was subsequently quantified and analysed on fatty acid profile by GC.
Hxanolysis of fish oil by lipase
Immobilized CAL (25 g) was added to a solution of fish oil (500 g, 0.55 mol) and i-
30 hexanol (338 g, 3.31 mol). The resulting enzyme suspension was gently stirred under
nitrogen 650C until the triacylglycerols had been completely converted to hexyl

WO 2004/043894 PCT/NO2003/000364
7
esters, according to analytical TLC and/or 1H-NMR. The enzyme was removed by
filtration and the excess hexanol evaporated in vacua.
Ethanoysis of fish oil hexyl esters by lipase
5 Immobilized MML (15 g) was added to a solution of fish oil hexyl esters (300 g, 0.80
mol) and absolute ethanol (111 g,2.41 mol). The resulting enzyme suspension was
gently stirred under nitrogen at( 40°C until desired conversion was obtained, according
to 1H-NMEL The enzyme was removed by filtration and the excess ethanol evaporated
in vacua. The high DHA concentrate was obtained as residue after short-path distillation
10 of the resulting mixture. The fatty acid composition of each ester group was determined
by single run on GC.
Example 1.
15 Direct Esterification. of Fish Oil Free Fatty Asids with Ethanol
Sardine oil(SO)
The progress of direct esterification reaction of SO free fatty acids, containing 14%
EPA and 15% DHA (14/15), with 3 equivalents of ethanol in the presence of MML (5%
20 as based on the weight of free fatty acids) at 400C is displayed in Table 1. Under these
conditions the lipase displayed extremely high activity toward the SO free fatty acids.
over70% conversion(% efthyl esters) was reached after only 2 hours. After 4 hour
reaction the residual free fatty acids contained 49% DHA and 6% EPA in 73% and 10%
recoveries, respectivly. In terms of DHA concentration and recoveries the optimal
25 conversion appears to be around 75% conversion. In Table 1 the weight percentage of
ethyl esters produced during the progress of the reaction was used directly as a measure
of the extent of conversion.
30

WO 2004/043894 PCT/NO2003/000364
8

Excellent results were obtained for direct esterification of SO free fatty acids after
5 separation by short-path distillation. SO free fatty acids were reacted with ethanol in the
presence of MML for 4 hours at 40°C to reach 78% conversion. The free fatty acids of
ihe reaction mixture comprised 49% DHA and 6% EPA with 75% DHA recoveries.
After distillation at 115°C the residue comprised 69% DHA and 9% EPA in 65% and
10% recoveries, respectively (Table 2). The recoveries of DHA were improved by
10 slightly reducing the distillation temperature (see Table 3). We were notable to separate
all the ethyl esters from the residual free fatty acids by the distillation. Despite that, we
managed to obtain high DHA concentrate of approximately 90% free fatty acids and
10% ethyl esters after short-path distillation at 115°C. The ethyl esters obtained in the
residue are highly enriched with DHA like the free fatty acids. Furthermore, the more
15 saturated and shorter-chain free fatty acids are distilled resulting in higher DHA
concentration of the residue than for the free fatty acid fraction after the reaction.

20

WO2004/043894 PCT/NO2003/000364
9
The results for SO were improved by lowering the conversion and the distillation
temperature as displayed in Table 3. After 4 hour reaction 75% conversion was
obtained. After distillation at 111°C the residue contained 66% DHA in 88% recoveries
with DHA/EPA ratio of 4.7. At slightly higher distillation temperature the residue
5 comprised 74% DHA in 75% recovery with a DHA/EPA ratio nearly seven. It should be
notified that the DHA recovery after the distillations is based on percent weight of DHA

The ethanol content can be reduced to I equivalent resulting in increased reaction time
(Table 4), Less lipaae tari also be introduced resulting in cosisiderably lower reaction
rate.
15

20

WO 2004/043894 PCT/NO2003/000364
10
Ancholvy Oil(AO)
The progress of the direct esterification reaction of AO & free fatty acids comprising 18%
EPA and ) 12% DHA (18/12) under identical conditions to the SO is displayed in Table
5. As can be noticed a DHA/EPA ratio of approximately 6:1 was obtained at 82%
5 conversion after 24 hours with EPA comprising 8% and DHA 50%. The DHA recovery
was just below 80%. Also after 11 hours, at 79% conversion a DHA/EPA ratio of 5:1
wilh DHA recoveries as high as 84%. Therefore, AO and SO are both highly potential
starting materials for making concentrates high in DHA and also, to make concentrates .
high in EPA from the ethyl ester fraction if that is of interest.
10

The results for AO are good in terms of DHA concentration and DHA/EPA ratios as
15 displayed in Table 6. Free fatty acids of AO (19/12) were reacted as before to reach
76% conversion in 11 hours. After distillation at 12l°C the residua comprised 61%
DHA in only 64% recovery with the DHA/EPA ratio being 5.5. The distillate may
possibly be used to make high EPA concentrates by a repeated distillation at lower
temperature. As an example a concentrate of 45% EPA and 10% DHA is considered to
20 be a desirable composition for a potential commercial product.
25

WO 2004/043894 PCT/NO2003/000364
11

Herrings Oil(HO)
5 Free fatty acids from herring oil comprising 6% EPA and 8% DHA (6/8) were similarly
treated under the direct esterification conditions as described above. The progress of the
reaction is displayed in Table 7. The residual free fatty acids after 12 hour reaction
contained 37% DHA and 6% EPA with 90% and 13% recoveries, respectively.

Free fatty acids from different HO comprising 9% BPA and 9% DHA (9/9) were reacted
for 12 hours, to reach 84% conversion, in same way as before, The free fatty acids of
15 the reaction mixture comprised 39% DHA and 8% EPA with 76% DHA recovery. After
distillation at 110°C the residue contained 40% DHA and 7% EPA in 68% DHA
recovery with a DRA/EPA ratio of almost 6:1 (Table 8). Low DHA concentration
results from high contents of long-chain monounsaturated fatty acids of 20% (4%)and
22:1 (37%). This high content of long-chain monounsaturates in HO and Capelin oil
20 renders them less feasible starting material for the process described. A simple urea
inclusion of the residual oil may be used to remove most of these monounsaturated fatty
acids resulting in a valuable concentrate of DHA. It should be added that HO with its
low EPA content is more suitable for obtaining high DHA/EPA natios than SO and AO.

WO2004/043894 PCT/NO2003/000364
12

Tuna Oil (TO)
5 The progress of the direct esterification reaction of TO free fatty acids comprising 6%
EPA and 23% DHA (6/23) under conditions identical to SO described above is
displayed in Table 9 below. After 8 hour reaction conversion of 68% was obtained with
the residual free fatty acids comprising 74% DHA and 3% EPA with 83% DHA
recovery and a DHA/EPA ratio of 25:1 (Table 9). Clearly, this type of initial EPA/DHA
10 composition of the starting oil is ideal for concentrating DHA.

15 Cod Liver Oil (CLO)
The progress of the direct esterification reaction of CLO free fatty acids comprising 9%
EPA and 9% DHA (9/9) under similar conditions as described above is displayed in
Table 10. Around 79% conversion a DHA/EPA ratio of 5:1 was obtained for the
residual free fatty acids with 50% DHA concentration and over 80% recovery. These
20 results are even better than those for SO and AO considering potential DHA recoveries.
But in terms of cost, SO and AO are favoured over CLO. It may be of interest to

WO 2004/043894 PCT/NO2003/000364
13
compare the results of CLO (9/9) to those of HO (9/9) in light of the feet that CLQ
contains far less long-chain monounsaturates (20:1 and 22:1).

BlueWhiting Oil(BWO)
The progress of the direct esterification reaction of BWO free fatty acids comprising
11% EPA and 7% DHA (11/7) under the conditions described above is displayed in
10 Table 11. Around 73% conversion the residual free fatty acids comprised 24% DHA. in
95% recoveries. EPA was not transferred to ethyl esters as rapidly as expected.
Interestingly, and unlike HO, the long-chain monounsaturated free fatty acids were to a
much higher extent convetted to ethyl ester. Higher conversion is needed to obtain
better separation of BPA and DHA, The reason for the low conversion for BWO is
15 unclear, but several attempts have not resulted in a higher conversion.

20

WO 2004/043894 PCT/NO2003/000364
14
Example 2
Combined Ethanolysis and Direct Esterificafion of Fish Oil
A two-step reaction, starting with an ethanolysis ami a subsequent direct esterification,
5 each step followed by molecular distillation, could be used to improve the recoveries of
DHA and the concentration. in the product. Prior to the direct esterifcation the glyceride
mixture obtained from the ethanolysis needs to be hydrolysed. Therefore, the
ethanolysis reaction can be used as a pre-step, reducing the bulk of the starting material
by half before hydrolysis. Notice the high recoveries obtained in the ethanolysis at 40°C
10 after separation by distillation (Table 12). Better results were obtained at room
temperature as discussed, above and displayed in Tables 13 and 14. The residue from the
room temperature reaction comprised 23% DHA and 25% EPA in 97% and 65%
recoveries, respectively (Table 13). These results indicate that the DHA recoveries can
be improved significantly by the two-step process. Also, there is a dramatic reduction in
15 bulkiness for the hydrolysis reaction, finally, this approach may be suitable for oils
highly enriched wife long-chain monounsaturates, such as HO.

WO 2004/043894 PCT/NO2003/000364
15

Example 3
5 Ethanolysis of Fish Oil Hexyl Esters
Ethanolysis of hexyl esters (HE) from fish oil is an alternative to the previously
discribed ethanolysis of fish oil trigtyceridss {Scheme 2). The results indicate that
various pases including the Rhizamucor miehei lipase (MML) and the Pseudomonas
10 lipases (PCL and PH.) can be used as well as the recently commercialised Thermomyces
latuginosa lipase (TLL) from. Novozyme. Also, it has bsen coonfirmed that molecular
distilation is quite suitable to separate residual hexyl ester and the more volatile ethyl
ester
15 Candida antarctica lipase (CAL) was used to convert A.O triglycerides into the
corresponding hexyl esters in a treatment with hexanol. Treatment of the resulting hexyl
ester with ethanol and PSL folloed by molecular distilation of the reaction mixture
may afford residual hexyl ester -with approximately 80% of EPA and DHA in a single
or in two enzymatic steps. By cocentreting DHA in the hexyl ester not only can we
20 separate the ethyl estets from the hexyl esters but also distil off the more saturated hexyl
esters as well.It may be possible lo convert the hexyl esters into ethyl esters either
chemically or enzymatically using CAL, Alternatively, it is possible to treat the
anchovy oil hexyl esters in ethanolysis using MML that may afford 70% DHA in a
single enzymatic step as hexyl esters. They may be further concentrated by an additional
35 MML treatment. From the bulk of the ethyl esters containing most of the EPA it may be
possible to purify EPA up to the 95% levels.
An alternative two-step approach is based on the ethanolysis of sardine oil to produce a
canceotrate of 50% EPA + DHA (30/20) as a glyceride mixture after molecular
30 distillation Treatment of the residual glycerides with hexanol and CAL affords hexyl
esters of identical composition. They may either be treated with ethanol and PSL to
afford hexyl esters with approximately 80% of EPA and DHA, or ethanol and MML to
separate DHA from EPA, followed by further concentration of both EPA and DHA.

WO2004/043894 PCT/NO2003/0003640
16
This process may have advantage in that the bulk of fish oil is being treated with
ethanol instead of hexanol, which is both easier, less bulky and more feasible from
industrial point of view. It must also be frome in mind that very high to excellent
recovery of both EPA. and DHA can be expected by that method.
5
Anchovy Oil(AO)
Like for the ethanolysis of fish oil triglycerides the fatty acid selectivity and activity of
MML can be greatly affected by temperature. Thus, MML can be used to concentrate
both EPA and DHA at or below 200C, but at 400C EPA is separated from DHA
10 resulting in high DHA concentrates. Anchovy oil hexyl esters comprising 18% EPA and
12% DHA were reacted with 2 equivalents of ethanol in the presence of MML (10%
weight of the hexyl esters) for 24 hows at 400C to reach 59% conversion. After removal
of the lipase excess ethanol was evaporated and the ethyl ester/hexyl ester (EE/HE)
mixture distilled at 135°C at 3 *10-3 mbar. The residue (26% weight) comprised 43%
15 DHA in only 65% recovery. The DHA/EPA ratio-was only 2.2(Table 15).

aIn Tables 15 and 16 the conversion of the lipase catalysed reactions is based on mol
20 percentage, whereas the distillation results are based on weight.
Interesting results were obtained when the reaction temperature was lowered to 20°C in
a similar reaction of Anchovy oil hexyl esters (18/13). After distillation at 1150C tee
residue comprised 45% DHA and 30% EPA with 85% and 55% recoveries, respectively
25 (Table 16).
30

WO 2004/043894 PCT/NO2003/000364
17

The Pseudomonas lipases were Tested on a small scale with good results, giving high
5 EPA recovery but considerably lower DHA recovery, especially if the reaction
exceeded 50% conversion, The results of the ethanolysis reaction of AO (18/12) with 2
equivalents of ethanol in the presence of PSL and PFL at room temperature is displayed
in Table 16. For PFL, after only 44% conversion of sardine oil hexyl esters in 24 hours,
the content of 28% EPA and 21% DHA was obtained while 57% conversion for PSL in
10 24 hours yielded in 33% EPA. and 17% DHA

15 Tbe new Novozyme lipase (TLL), immobilized on granular silica gel, was compared to
MML. The new lipase was found to be sensitive to ethanol and the activity decreased
rapidly with increased temperature, At 20°C both lipases were active and in 24 hours
54% conversion was obtained for MML but only 43% for TLL. The residual hexyl
esters of TO, comprising 6% EPA and 28% DHA (6/28), from the TLL reaction
30 contained 8% EPA and 45% DHA. The MML reaction resulted in residual hexyl esters
containing 7% EPA and 54% DHA (Table 18). These lipases are obviously similar in
fatty acid selectivity but TLL is more sensitive toward ethanol concentration, which
makes it inferior to MML.
25

WO 2004/043894 PCT/NO2003/000364
18

5 The results from the ethanolysis of TO hexyl esters (6/28) and ethanol al 40°C are
displayed in Table 19, Interestingly, at 400Conly 15% conversion was obtained for
TLL and 47% conversion for MML. It is believed that at higher temperature the lipase
becomes more sensitive for the polar ethanol and its detrimental effects. For MML, after
47% conversion in 24 hours, the hexyl esters comprised 9% EPA and 49% DHA while
10 only 15% conversion for TLL in 24 hours yielded 33% EPA and 17% DHA.

15 By the present inventon separation of EPA. and DHA. by solvent free direct
esterification of fish oil free fatty acids or fish oil hexyl esters and ethanol in the
presence of a lipase is successfully obtained. The problems with monoglycerides in the
distillate are avoided by the processes according to die present invention.

WO 2004/043894 PCT/NO2003/000364
19
C l a i m s
1. A process for separating ethyl or methyl ester fraction enriched in BPA
(eicosapentaenoic acid.C20:5) and a free fatty acid fraction enriched in DHA
5 (docosahexaenoic acid, C22:6) obtained from a direct esterification of Fish oil free fatty
acids with a ethanol or methanol using lipase, by molecular distillation-
2. A process according to chain 1, wherein the fish oil free fatty acid starting material is
obtained by a lipase catalysed alcoholysis of fish oil triglyceridss, a subsequent
10 molecular distillation and hydrolysis of the residual glyceride mixtures.
3. A process for esterifying a marine oil composition containing EPA and DHA as Cn
alkyl esters of fatty acids (n = 2-18) to forn (1): a Cn alkyl ester fatty acid fraction [n =
2-18) Enriched in DHA as compared to the starting material and a Cn, alkyl ester fatty
15 acid fraction (m=1-12; n > m) enriched in EPA as compared to the starting material, or
(2): a Cn alkyl ester fatty acid fraction (n = 2-18) enriched in both DHA and EPA as
compared to the starting material and a Cm alkyl ester fatty acid fraction (m= 1-12; n>
m) lower in both DHA and EPA as compared to the starting material comprising the
step of reading said marine oil composition with a Cm alcohol (m= 1-12;n>m)in the
20 presence of a lipase catalyst under essentially organic solvent-free conditions, and
separating the fractions by molecular distillation.
4. A process according to claim 3, wherein the starting material, C2-C18 alkyl ester, is
obtained by a lipase catalysed, alcoholysis of fish oil triglycerides, a subsequent
25 molecular distillation, and alcoholysis of the residual glyceride mixture with a C2-C18
alkyl alcohol.
5. A process according 10 claim 3 and 4, wherein the C2C18 alkyl ester is hcxyl ester.
30 6. A process according to claim 3, wherein the C1-C12 alcohol is ethanol.
7. A process according to claim 3, were said lipase catalyst is Rhizomucor miehei lipase
(MML), Thermomyces ianuginosa lipase (TLL), Psedomonas sp. lipase (PSL) or
Psedomonas fluorescens lipase (PFL).

WO 2004/043894 PCT/NO2003/000364
20
8. A process according to claim 1, wherein the molar ratio of methanol or ethanol free
fatty acids in the starting composition is fron 0.5 to 10.0.
9. A process according to claim 8, wherein the molar ratio is from 0.5 to 3.0.
5
10. A process according to claim 8, wherein the molar ratio is from 1.0 to 2.0.
11. A process according to claim 8, wherein the molar ratio is from 0.5 to 1.5.
10 12. A process according to claim 3, wherein the molar ratio of C1-C12alcohol to C2-C18
alkyl ester is from 0.5 to 10.0.
13. A process according to claim 12, wherein The molar ratio is from 0.5 to 3.0.
15 14. A process according to claim 12, wherein the molar ratio is from 2.0 to 3.0.
15. A process according to any preceding claim, wherein the esterification reaction is
conducted at a temperature of 00C to 700C.
20 16. A process according to claim 15, wherein the esterification reaction is conducted at
a temperature of 200C to 400C.
17. A process according to any preceding claim, wherein said lipase catalyst is
immobilized on a carrier.
25
18. A process according to claim 1, wherein said lipase catalyses the alcoholysis of
DHA at a much slower speed than the c XXX ponding alcoholysis of EPA.
19. A process according to claim 18, wherein said lipase catalyst is Rhizomucor miehei
30 lipase (MML) or Thermomyces lanuginosa lipase (TLL).

Marune Oil compositions which contain EPA and DHA as free acids hexyl ester are esterified with ethanol in the
presence or a lipase catalysl under essentially organic solvent-free conditions and separated by distillation.

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Patent Number

217404

Indian Patent Application Number

00876/KOLNP/2005

PG Journal Number

13/2008

Publication Date

28-Mar-2008

Grant Date

26-Mar-2008

Date of Filing

12-May-2005

Name of Patentee

PRONOVA BIOCARE AS

Applicant Address

LYSAKER TORG 8, N-1327, USALER, NORWAY.

Inventors:

#	Inventor's Name	Inventor's Address
1	HARLADSSON GUDMUNDUR G	KLYFJASEL 14, IS-109 REYKJAVIK, ICELAND.
2	HALLDORSSON ARNAR	FORNHAGI 13, IS-107 REYKJAVIK, ICELAND.
3	THORSTAD OLAV	SOLVIVEIEN 11-N-3940 PORSGRUNN, NORWAY.

PCT International Classification Number

C07C 67/03

PCT International Application Number

PCT/NO2003/000364

PCT International Filing date

2003-10-31

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	20025456	2002-11-14	Norway