JPH0959206A - Production of eicosapentaenoic acid and eicosapentaenoic ester - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain in large quantities at a low cost the subject compound having physiological activities such as anti-thrombogenic and anti-arteriosclerotic effects, without concentration of ω6 fatty acids having function inhibitory effect, by incorporating a fatty acid (ester) mixture with an iso-alkane and acetonitrile followed by layer separation. SOLUTION: A fatty acid (ester) mixture (for example, produced by hydrolyzing fish oil such as cod liver oil, sardine oil or bonito oil with lipase followed by, if needed, esterification) containing (A) eicosapentaenoic acid (ester) is incorporated with (B1 ) an isoalkane such as iso-pentane or iso-dodecane and (B2 ) acetonitrile followed by layer separation to effect concentration of the component A in the B2 layer, and, as necessary, the resultant B2 layer is further newly incorporated with the component B1 followed by layer separation, and this operation is repeated, if needed; thus concentrating the component A in the B2 layer.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing eicosapentaenoic acid and eicosapentaenoic acid ester. More specifically, the present invention provides selective selection from a fatty acid or a mixture of fatty acid esters containing eicosapentaenoic acid or eicosapentaenoic acid ester without concentrating ω6 fatty acid having a function-inhibiting effect on eicosapentaenoic acid. And to a method for producing eicosapentaenoic acid or an eicosapentaenoic acid ester capable of concentrating eicosapentaenoic acid or an eicosapentaenoic acid ester.

[0002]

BACKGROUND OF THE INVENTION With the recent progress of science and technology, the correlation between the structure and function of fatty acids is gradually becoming clear. Especially, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), α-linolenic acid, etc. Ω3
Attention has been focused on the physiologically active action of highly unsaturated fatty acids in the system. When fatty acids or their esters become highly pure, they will exhibit their unique physical properties and physiological actions sharply, and further improve quality levels such as safety, stability, color and odor. It can be used in a high degree in fields related to living organisms such as. Eicosapentaenoic acid has already become a large drug as a therapeutic drug for arteriosclerosis and hyperlipidemia (neutral fat) as ethyl ester, and in the situation where new efficacy is pursued, high purity Therefore, there is a need for a method of mass-producing high-quality products at low cost. ω3 unsaturated fatty acids exhibit excellent physiological actions such as antithrombotic, antiarteriosclerotic, antilipidemic, anti-inflammatory and immune regulation, but competitive inhibition with ω6 unsaturated fatty acids, especially arachidonic acid. It is becoming clear that there is a relationship. Therefore, in order to enhance the physiological action and medicinal effect of EPA, it is essential to increase the purity of EPA and reduce the content of arachidonic acid.
There are many literatures on the inhibitory relationship between EPA and arachidonic acid, but Akira Kumagai's "Medical Medicine of EPA" (Nakayama Shoten, 199).
4), and Tanaka, "Development and Application of Functional Lipids", p. 142 (CMC, 1992) and the like. From a mixture of fatty acids or their esters,
As a conventional technique for concentrating eicosapentaenoic acid or its ester, methods such as a distillation method, a urea addition method, a liquid chromatography, a column chromatography, a supercritical gas extraction method, and a liquid-liquid distribution method have been reported. The distillation method is a separation method based on a difference in boiling point or vapor pressure, and cannot separate substances having a small difference. Fatty acids can separate fatty acids having different carbon numbers, but cannot separate fatty acids having the same carbon number but different double bond numbers or positions. Further, since distillation is subjected to a high temperature heat history, caution is required when it is applied to unsaturated fatty acids having poor thermal stability, particularly highly unsaturated fatty acids (JP-A-6-33088 and JP-B-1-40817). JP-B-5-25870, JP-A-2-268133, JP-A-4-414.
57, JP-A-4-128250, etc.). The urea addition method is a method of separating fatty acids by utilizing the fact that urea has a property of forming an adduct with a fatty acid having a saturated linear hydrocarbon chain having 7 or more carbon atoms. The separation characteristics of this method are generally effective for separating fatty acids having 3 or less double bonds, but most fatty acids having 4 or more double bonds are saturated linear chains having 7 or more carbon atoms. Since it has no hydrocarbon chain, it does not form a urea adduct. Therefore, C16: 4, C18: 4 contained in fish oil etc.
C20: 4, C20: 5 (eicosapentaenoic acid), C
Since highly unsaturated fatty acids such as 21: 5, C22: 5, C22: 6 (docosahexaenoic acid) do not form urea adducts, separation of these fatty acids is completely impossible. Furthermore, since the urea addition method uses a large amount of urea and a solvent, the production cost is high and the operation is a little complicated (Japanese Patent Publication No. 1-40).
No. 817, Japanese Patent Publication No. 3-47259, Japanese Patent Publication No. 5
-25870 publication). Liquid chromatography is an analytical method and has problems such as low productivity and high equipment cost and running cost as industrial production means (Japanese Patent Laid-Open Nos. 61-37752 and 63-63). 29
5527 publication). Column chromatography is a method of separation based on affinity with an adsorbent, and has problems such as the use of a large amount of solvent, low productivity, and large-scale equipment (Japanese Patent Laid-Open No. 61-197297, JP-A-61-292797). JP-A-63-290845, JP-A-3-16729
No. 4, etc.). Recently, it has been reported that the silver nitrate column method is effective for separating fatty acids with different degrees of unsaturation, but it is problematic as an industrial production means due to the use of expensive silver nitrate. (JP-A-4-103
558, JP-A-4-154896, JP-A-5-159398, etc.). The supercritical gas extraction method is
In recent years, it has been actively studied for the separation of eicosapentaenoic acid and docosahexaenoic acid, but the separation effect depends strongly on the fatty acid composition of the raw materials, so the separation effect is expected unless the composition is adjusted beforehand by another method. Not possible (JP-A-60-214757, JP-A-60-21729)
No. 9, JP-A No. 2-8298, etc.). The supercritical gas extraction method has drawbacks such as high equipment cost and large scale. The liquid-liquid distribution method is a separation method by distributing components between two immiscible solvents. The factors that control the separation effect of this method are the type and amount of the two solvents, the ratio between the solvents, the distribution temperature, the fatty acid composition of the raw materials, and the like, but the key factor is the two solvent types. JP-A 64-83
No. 043 discloses a combination of a hydrocarbon solvent and a polar solvent,
In particular, a method for concentrating highly unsaturated fatty acids by a liquid-liquid partitioning method using a two-solvent system of n-heptane and water-containing acetonitrile is disclosed. In this method, the number of double bonds contained in fish oil or the like is 3 or more. Since all of the unsaturated fatty acids of ω3 are concentrated, ω6 arachidonic acid (C20: 4) and C1 which are function inhibiting fatty acids of EPA which is ω3 fatty acid are concentrated.
6: 3, C16: 4, C18: 3, C18: 4, C2
There is a drawback that 1: 5, C22: 5, C22: 6, etc. are also concentrated at the same time. As described above, the conventional methods for concentrating eicosapentaenoic acid or its ester have various problems, and no method for simply and efficiently concentrating eicosapentaenoic acid or its ester has been found.

[0003]

DISCLOSURE OF THE INVENTION The present invention provides a highly useful eicosapentaenoic acid or its ester from a mixture of fatty acids or its ester without concentrating the ω6 fatty acid component having a function inhibiting property. The object of the present invention is to provide a method for producing a large amount at low cost by a simple method without causing deterioration of the substrate.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a fatty acid ester containing eicosapentaenoic acid or a fatty acid ester containing eicosapentaenoic acid ester is treated with isoalkane and acetonitrile. It was found that eicosapentaenoic acid or eicosapentaenoic acid ester is selectively concentrated in the acetonitrile layer by mixing and then separating the layers, and the present invention has been completed based on this finding. That is, the present invention is characterized in that (1) a fatty acid mixture containing eicosapentaenoic acid is mixed with isoalkane and acetonitrile and then separated into layers, and eicosapentaenoic acid is concentrated into an acetonitrile layer to produce eicosapentaenoic acid. Method, (2) to the fatty acid mixture containing eicosapentaenoic acid, isoalkane and acetonitrile are added and mixed, and then the layers are separated, and a new isoalkane is added to the obtained acetonitrile layer and mixed, and then the layer is separated, A method for producing eicosapentaenoic acid, which comprises concentrating icosapentaenoic acid in an acetonitrile layer, (3) a fatty acid ester mixture containing eicosapentaenoic acid ester, to which isoalkane and acetonitrile are added and mixed, and then the layers are separated to form eicosapentaenoic acid. Beauty salon Method for producing eicosapentaenoic acid ester, characterized in that the concentration of acetonitrile layer,
And, (4) to the fatty acid ester mixture containing eicosapentaenoic acid ester, isoalkane and acetonitrile were added and mixed, and then the layers were separated, and a new isoalkane was added to the obtained acetonitrile layer and mixed, and then the layers were separated. And a method for producing an eicosapentaenoic acid ester, which comprises concentrating an eicosapentaenoic acid ester in an acetonitrile layer.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention can be applied to a fatty acid mixture containing eicosapentaenoic acid and a fatty acid ester mixture containing eicosapentaenoic acid ester. A fatty acid mixture containing eicosapentaenoic acid can be obtained mainly by hydrolysis of fish oil, and examples of the fish oil that gives such a fatty acid mixture include sardine oil, cod liver oil, skipjack oil, herring oil, tuna oil, and saury oil. And so on. Since polyunsaturated fatty acids are likely to be deteriorated when exposed to high temperatures, the lipase decomposition method or the like, which can be performed at around room temperature, can be suitably used for hydrolysis. In the method of the present invention, as the fatty acid ester containing eicosapentaenoic acid ester, methyl ester, ethyl ester, n-propyl ester,
Lower alkyl esters such as isopropyl ester, n-butyl ester, isobutyl ester, sec-butyl ester and tert-butyl ester can be preferably used. Such a fatty acid ester containing eicosapentaenoic acid ester can be obtained by direct esterification of a fatty acid containing eicosapentaenoic acid, or by alcoholysis of an oil or fat having eicosapentaenoic acid as a fatty acid component. In the method of the present invention, a fatty acid ester containing eicosapentaenoic acid or a fatty acid ester containing eicosapentaenoic acid ester is mixed with an immiscible isoalkane and acetonitrile, and the mixture is separated into layers. Concentrate to. An isoalkane is a branched alkane, and isopentane, isohexane, isoheptane, isooctane, isononane, isodecane, isoundecane, isododecane and the like can be preferably used in the method of the present invention.

In the process of the present invention, acetonitrile is used as a solvent immiscible with isoalkane. Acetonitrile can be used in a pure state, or can be used in a state of a mixed solvent of acetonitrile / water, acetonitrile / methanol, acetonitrile / ethanol and the like. In the method of the present invention, the amount of isoalkane and acetonitrile used can be appropriately selected depending on the composition of the raw material fatty acid or its ester, the component to be separated, the separation temperature, the target yield, etc. The total amount of the two solvents with respect to the ester is preferably 1 to 50 times, and more preferably 2 to 20 times by weight ratio. In addition, the ratio of isoalkane and acetonitrile used is 1: by weight.
It is preferably 50 to 50: 1, and 1:20 to 2
It is more preferably 0: 1. In the method of the present invention, the method of mixing and separating the isoalkane containing a fatty acid or a fatty acid ester and acetonitrile is not particularly limited, and either a batch method or a continuous method can be used. In the case of the batch method, a container equipped with a stirrer equipped with a temperature control device is charged with isoalkane, acetonitrile and a fatty acid or a fatty acid ester, and the fatty acid or fatty acid ester is distributed to the isoalkane layer and the acetonitrile layer by stirring, and the mixture is allowed to stand. After the layers are separated, the acetonitrile layer in which eicosapentaenoic acid or its ester is concentrated can be extracted.
In the case of the continuous system, a fatty acid or a fatty acid ester is dissolved in isoalkane, acetonitrile, or both, and a packed column or plate column is used to send acetonitrile with a large specific gravity from the top and isoalkane with a small specific gravity from the bottom. Can be contacted. The continuous countercurrent contact method is advantageous because a high separation effect can be obtained with a small amount of solvent. In continuous mode, centrifugal extractor,
A rotating disk cylinder, a pulsation extraction tower, etc. can also be used.
In the method of the present invention, the liquid-liquid distribution temperature can be appropriately determined depending on the composition of the raw material fatty acid or its ester, the component to be separated, the target yield, the type of the solvent, etc., but is generally at -40 to 40 ° C. It is preferable to carry out. At a low temperature, the concentration effect of eicosapentaenoic acid or its ester is improved, but the yield tends to decrease.

In the method of the present invention, the fatty acid or fatty acid ester containing concentrated eicosapentaenoic acid or its ester can be recovered from the separated acetonitrile layer by distilling acetonitrile off under reduced pressure. In the method of the present invention, for a fatty acid ester containing a fatty acid containing eicosapentaenoic acid or an eicosapentaenoic acid ester concentrated by liquid-liquid partitioning between isoalkane and acetonitrile, by repeating liquid-liquid partitioning with isoalkane and acetonitrile. , The concentration of eicosapentaenoic acid or its ester can be increased. Further, by adding a new isoalkane to the acetonitrile layer containing concentrated eicosapentaenoic acid or its ester and repeating liquid-liquid distribution, the total fatty acid or fatty acid ester of eicosapentaenoic acid or its ester in the acetonitrile layer is occupied. The rate can be increased. Alternatively, liquid-liquid partitioning is performed with isoalkane and acetonitrile, and then liquid-liquid partitioning is repeated by adding new acetonitrile to the isoalkane layer, and eicosapentaenoic acid or its ester dissolved in the isoalkane layer is extracted. The yield of icosapentaenoic acid or its ester can be increased. According to the liquid-liquid partitioning using isoalkane and acetonitrile of the method of the present invention, only ω3 type fatty acid such as eicosapentaenoic acid and docosahexaenoic acid or its ester is concentrated in the acetonitrile layer, and ω6 type fatty acid such as arachidonic acid and linoleic acid. , Unsaturated fatty acids such as palmitoleic acid and oleic acid, saturated fatty acids such as palmitic acid and stearic acid or their esters are all concentrated in the isoalkane layer. In the conventional separation method by liquid-liquid distribution, ω3 type fatty acids such as eicosapentaenoic acid and docosahexaenoic acid are mixed with ω3 such as arachidonic acid.
The 6 series fatty acids were also concentrated on the polar solvent side, and the ω3 series fatty acids and the ω6 series fatty acids could not be separated. ω6
Since the system fatty acids have a function-inhibiting property with respect to the ω3 type fatty acids, they have been a major obstacle to the high-level utilization of eicosapentaenoic acid in biomedical fields such as pharmaceuticals, cosmetics and foods. According to the method of the present invention, high-purity eicosapentaenoic acid or eicosapentaenoic acid ester containing no ω6 fatty acid can be easily produced. In the liquid-liquid partitioning using isoalkane and acetonitrile of the method of the present invention, when the ω3 type fatty acid or fatty acid ester and other fatty acid or fatty acid ester containing the ω6 type coexist, the ω3 type fatty acid or fatty acid ester is present in the acetonitrile layer. Other fatty acids or fatty acid esters containing the ω6 system are concentrated and concentrated in the isoalkane layer. However, in the case of a mixture having a large content of ω3 fatty acid or fatty acid ester and a small content of other fatty acid or fatty acid ester, eicosapentaenoic acid or eicosapentaenoic acid ester is selectively concentrated in the acetonitrile layer, Since ω3 fatty acids or fatty acid esters other than eicosapentaenoic acid or eicosapentaenoic acid ester are concentrated in the isoalkane layer together with other fatty acids or fatty acid esters, finally eicosapentaenoic acid or eicosapentaenoic acid ester can be isolated purely. It is possible to separate all fatty acids or fatty acid esters other than eicosapentaenoic acid or eicosapentaenoic acid.

[0008]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the examples, the fatty acid composition was analyzed by gas chromatography for the fatty acid after methyl esterification of the fatty acid, and the content of the eicosapentaenoic acid ester and the eicosapentaenoic acid ester having similar retention time was shown. . The conditions of gas chromatography are as follows. Gas chromatograph: Shimadzu GC-17A Column: DB-WAX (J & W), 30m x 0.25mm
X 0.25 μm Film Carrier gas (He) flow rate: 1 ml / min Sample concentration: 100 mg / ml isooctane Injection amount: 0.2 μl Injection temperature: 240 ° C. Column temperature: 210 ° C. Detector temperature: 240 ° C. Detector: FID Split ratio: 50: 1 Example 1 In a liquid-liquid distribution separation tank, ethyl eicosapentaenoate 67.
9% by weight, ethyl docosahexaenoate 2.8% by weight, ethyl arachidonate 3.7% by weight, ethyl oleate 1.2
% Of fatty acid ethyl ester, 0.7% by weight of ethyl arachiate, 1 double bond of ω11 to 7 and 4.8% by weight of ethyl ester of fatty acid having 20 carbon atoms.
kg, 4.0 kg of isooctane and 4.0 kg of acetonitrile
And the liquid-liquid distribution temperature is 20 ° C while vigorously stirring and mixing.
The stirring and mixing was continued for 20 minutes. Then, the mixture was allowed to stand for 10 minutes to separate the layers, and then the acetonitrile layer was separated, and acetonitrile was distilled off by a rotary evaporator to obtain 0.30 kg of fatty acid ethyl ester. As a result of analyzing this fatty acid ethyl ester by gas chromatography,
Its composition is ethyl eicosapentaenoate 78.8% by weight, ethyl docosahexaenoate 3.4% by weight, ethyl arachidonate 2.1% by weight, ethyl oleate 0.6% by weight, ethyl arachiate 0.0% by weight, One double bond of ω11 to 7, ethyl ester of fatty acid having 20 carbon atoms 1.3
% By weight. Example 2 Except for changing the liquid-liquid distribution temperature in Example 1 to -10 ° C, the same operation as in Example 1 was repeated to obtain 0.20 kg of fatty acid ethyl ester from the acetonitrile layer. The composition of this fatty acid ethyl ester was as follows: ethyl eicosapentaenoate 82.1% by weight, ethyl docosahexaenoate 3.5.
% By weight, ethyl arachidonate 2.2% by weight, ethyl oleate 0.2% by weight, ethyl arachinate 0.0% by weight, ω
It was one double bond of 11 to 7 and 0.6% by weight of ethyl ester of fatty acid having 20 carbon atoms. Example 3 The amount of solvent used in Example 2 was 2.5 kg of isooctane.
And the same operation as in Example 1 was repeated except that the amount of acetonitrile was changed to 5.5 kg, and 0.39 kg of fatty acid ethyl ester was obtained from the acetonitrile layer. The composition of this fatty acid ethyl ester is ethyl eicosapentaenoate 8
1.6% by weight, ethyl docosahexaenoate 3.3% by weight,
Ethyl arachidonate 1.9% by weight, ethyl oleate 0.1.
3% by weight, ethyl arachiate 0.0% by weight, ω11 to 7
Was one double bond and was 0.8% by weight of an ethyl ester of a fatty acid having 20 carbon atoms. Comparative Example 1 Instead of 4.0 kg of isooctane in Example 1, n-
The same operation as in Example 1 was repeated except that 4.0 kg of heptane was used to obtain 0.26 kg of fatty acid ethyl ester from the acetonitrile layer. The composition of this fatty acid ethyl ester is 80.1% by weight of ethyl eicosapentaenoate,
Ethyl docosahexaenoate 3.4% by weight, ethyl arachidonate 4.4% by weight, ethyl oleate 0.8% by weight, ethyl arachiate 0.0% by weight, double bond 1 of ω11 to 7
2.7% by weight of ethyl ester of C20 fatty acid
Met. The results of Examples 1 to 3 and Comparative Example 1 are collectively shown in Table 1.

[0009]

[Table 1]

As can be seen from Table 1, ethyl icosapentaenoate and docosahexaenoate, which are ethyl esters of ω3 fatty acids, are concentrated and concentrated by separating the fatty acid ethyl ester using isooctane and acetonitrile as solvents. Is rising, but ω
The concentration of ethyl arachidonic acid, which is an ethyl ester of 6-type fatty acid, decreased, and the concentrations of ω9 fatty acid, one double bond of ω11 to 7 and ethyl ester of fatty acid having no double bond also decreased. There is. from this result,
It was found that the ethyl ester of ω3 fatty acid can be selectively concentrated by separating the layers using isooctane and acetonitrile as solvents. On the other hand, in Comparative Example 1 in which the fatty acid ethyl ester was separated into layers using n-heptane and acetonitrile as solvents, ethyl eicosapentaenoate and ethyl docosahexaenoate, which are ethyl esters of ω3 fatty acids, were concentrated and the concentration was increased. Has ω3 fatty acid function inhibition
Ethyl arachidonic acid, which is an ethyl ester of 6-type fatty acid, is also concentrated and its concentration is increasing. Further, Comparative Example 1
In the result, the concentration effect and yield of eicosapentaenoic acid are lower than those of Example 1 of the present invention. Example 4 The procedure of Example 2 was repeated, and the obtained acetonitrile layer was concentrated to a weight of 1.0 kg and isooctane 0.8
Liquid addition was performed at 0 ° C. by adding kg. When the acetonitrile layer was sampled in a small amount and the acetonitrile was evaporated, it was found that the acetonitrile layer contained 0.06 kg of fatty acid ethyl ester. Analysis of this fatty acid ethyl ester revealed that its composition was 87.8% by weight of ethyl eicosapentaenoate, 3.6% by weight of ethyl docosahexaenoate, 1.2% by weight of ethyl arachidonic acid, and 0.0% by weight of ethyl oleate. The amount of ethyl arachiate was 0.0% by weight, and the amount of ethyl ester of fatty acid having 20 carbon atoms of ω11 to 7 was 0.0% by weight. Example 5 The acetonitrile layer obtained in Example 4 has a weight of 0.30 k.
Concentrate to g and add 0.24 kg of isooctane to 1
Liquid-liquid partition was performed at 0 ° C., and 0.03 kg of fatty acid ethyl ester was obtained from the acetonitrile layer. The composition of this fatty acid ethyl ester is ethyl eicosapentaenoate 9
0.4% by weight, ethyl docosahexaenoate 3.8% by weight,
Ethyl arachidonate 0.8% by weight, ethyl oleate 0.0.
0% by weight, ethyl arachiate 0.0% by weight, ω11 to 7
It was 0.0% by weight of ethyl ester of fatty acid having 20 carbon atoms. The results of Examples 4 to 5 are shown in Table 2 together with the results of Example 2.

[0011]

[Table 2]

As can be seen from Table 2, by separating the fatty acid ethyl ester using isooctane and acetonitrile as a solvent and adding new isooctane to the obtained acetonitrile layer to separate the layers, the ω3 type fatty acid Ethyl ester ethyl eicosapentaenoate and ethyl docosahexaenoate were concentrated and their concentrations were increased, while ethyl arachidonate, an ethyl ester of ω6 fatty acid, was reduced in concentration, and further ω9 fatty acids and ω11-7 fatty acids were used. Further, since the ethyl ester of fatty acid having no double bond is not contained, it was found that the ethyl ester of ω3 fatty acid can be selectively concentrated by repeating the phase separation using isooctane and acetonitrile as the solvent. Example 6 5.5 kg of acetonitrile was added to the isooctane layer obtained in Example 3, and the second acetonitrile extraction was performed by liquid-liquid partitioning at −10 ° C. to obtain 0.28 kg of fatty acid ethyl ester from the acetonitrile layer. The composition of this fatty acid ethyl ester was as follows: ethyl eicosapentaenoate 80.0% by weight, ethyl docosahexaenoate 3.3% by weight, ethyl arachidonic acid 2.1% by weight, ethyl oleate 0.3% by weight, ethyl arachiate 0.3%. 0% by weight, ω1
The content was 1 double bond of 1 to 7 and 0.8% by weight of ethyl ester of fatty acid having 20 carbon atoms. The results of Example 3 and Example 6 are collectively shown in Table 3.

[0013]

[Table 3]

From the results shown in Table 3, after the layers were separated using isooctane and acetonitrile as solvents, acetonitrile was further added to the isooctane layer to separate the layers, and ethyl eicosapentaenoate was extracted from the acetonitrile layer than in the case of the first separation. Although the concentration of ω3 is slightly low, the ethyl ester of ω3 fatty acid is concentrated in the same manner as the first time,
It has been found that a mixture with a reduced concentration of other fatty acid ethyl esters is obtained. Example 7 Ethyl eicosapentaenoate 88.3% by weight, ethyl docosahexaenoate 2.5% by weight, ethyl arachidonate 2.
5% by weight, 4 double bonds, ethyl ester of ω3 fatty acid having 18 carbon atoms 4.6% by weight, 5 double bonds, 21 carbon atoms
1.0 kg of fatty acid ethyl ester containing 0.9 wt% of ethyl ester of ω3 fatty acid and 5 double bonds and 0.3 wt% of ethyl ester of ω3 fatty acid having 22 carbon atoms, 4.0 kg of isooctane and 4.0 ml of acetonitrile. 0 kg was added, and the acetonitrile layer was separated by the same operation as in Example 1 at a liquid-liquid distribution temperature of -20 ° C to obtain 0.38 kg of fatty acid ethyl ester from this acetonitrile layer. The composition of this fatty acid ethyl ester is 93.2 ethyl eicosapentaenoate.
% By weight, ethyl docosahexaenoate 2.0% by weight, ethyl arachidonate 1.0% by weight, 4 double bonds, 1 carbon atom
3.5% by weight of omega-3 fatty acid ethyl ester, 5 double bonds, 21 omega-3 fatty acid ethyl ester.
3% by weight and 5 double bonds, 0.0% by weight of ethyl ester of ω3 fatty acid having 22 carbon atoms. The results are shown in Table 4.

[0015]

[Table 4]

From the results shown in Table 4, when the ethyl ester of fatty acid containing a large amount of ethyl ester of ω3 fatty acid containing ethyl eicosapentaenoate was separated by isooctane and acetonitrile, the ω6 fatty acid in the acetonitrile layer was separated. Although the concentration of arachidonic acid is decreased, the concentration of ethyl esters of ω3 fatty acids other than ethyl eicosapentaenoate is also reduced, and ethyl icosapentaenoate is particularly strong against acetonitrile among the ethyl esters of ω3 fatty acids. It can be seen that it has an affinity and is selectively enriched. Example 8 18.1% by weight of ethyl eicosapentaenoate, 6.9% by weight of ethyl docosahexaenoate, ethyl arachidonate 1.
Ethyl ester of fish oil fatty acid 1.0 containing 2% by weight, 17.9% by weight of ethyl palmitate, 8.1% by weight of ethyl palmitoleate and 12.9% by weight of ethyl oleate.
to kg, isooctane 4.0 kg and acetonitrile 4.0
kg was added, and the acetonitrile layer was separated by operating in the same manner as in Example 1 at a liquid-liquid distribution temperature of -10 ° C to obtain 0.12 kg of fatty acid ethyl ester from the acetonitrile layer. The composition of this fatty acid ethyl ester was as follows: ethyl eicosapentaenoate 39.4% by weight, ethyl docosahexaenoate 14.9% by weight, ethyl arachidonate 1.1% by weight, ethyl palmitate 5.0% by weight, ethyl palmitoleate 4. 2% by weight
And ethyl oleate 4.3% by weight. The results are shown in Table 5.

[0017]

[Table 5]

From the results shown in Table 5, by separating the ethyl ester of fish oil fatty acid with isooctane and acetonitrile, ethyl eicosapentaenoate and docosahexaenoate, which are ethyl esters of ω3 fatty acid, were concentrated and the concentration was increased. However, it can be seen that the concentration of the ethyl ester of a fatty acid other than the ω3 fatty acid is lowered. Example 9 97.9% by weight of eicosapentaenoic acid, 0.8% by weight of docosahexaenoic acid, 0.8% by weight of arachidonic acid, 5 double bonds, 0.3% by weight of ω3 fatty acid having 21 carbon atoms and 5 double bonds And 1.0 kg of fatty acid containing 0.2% by weight of ω3 fatty acid having 22 carbon atoms, 4.0 kg of isooctane and 4.0 kg of acetonitrile were added, and the same operation as in Example 1 was carried out at a liquid-liquid distribution temperature of -20 ° C. The acetonitrile layer was separated and 0.47 kg of fatty acid was obtained from this acetonitrile layer. The composition of this fatty acid is 99.5% by weight of eicosapentaenoic acid, 0.3% by weight of docosahexaenoic acid, 0.2% by weight of arachidonic acid, 5 double bonds, 0.0% by weight of ω3 fatty acid having 21 carbon atoms and 2% by weight. 5 heavy bonds, ω3 fatty acid with 22 carbon atoms 0.0
% By weight. The results are shown in Table 6.

[0019]

[Table 6]

As can be seen from Table 6, the concentration of eicosapentaenoic acid increased and the concentration of arachidonic acid decreased, but the concentration of docosahexaenoic acid, which is a ω3 fatty acid, also decreased, and the carbon number was 21 and The ω3 fatty acid having 22 carbon atoms is not included. From these results, when the concentration of eicosapentaenoic acid is high, eicosapentaenoic acid has a particularly strong affinity for acetonitrile among the ω3 type fatty acids in the separation layer of isooctane and acetonitrile, and ω3 other than eicosapentaenoic acid is concentrated. It can be seen that the fatty acids in the system tend to be excluded from the acetonitrile layer. Example 10 Eicosapentaenoic acid 25.4% by weight, docosahexaenoic acid 7.5% by weight, arachidonic acid 2.3% by weight, 5 double bonds, ω3 fatty acid having 22 carbon atoms 2.4% by weight, stearic acid 4. To 1.0 kg of fatty acid containing 3% by weight and 15.1% by weight of oleic acid, 4.0 kg of isooctane and 4.0 kg of acetonitrile were added, and the liquid-liquid distribution temperature was 20 ° C.
The acetonitrile layer was separated in the same manner as in 1. to obtain 0.27 kg of fatty acid from this acetonitrile layer. The composition of this fatty acid is as follows: eicosapentaenoic acid 40.8% by weight, docosahexaenoic acid 14.2% by weight, arachidonic acid 1.9% by weight, 5 double bonds, C22 fatty acid ω3 fatty acid 2.7% by weight, stearin. Acid was 1.4% by weight and oleic acid was 8.0% by weight. The results are shown in Table 7.

[0021]

[Table 7]

From the results in Table 7, the concentration of ω3 fatty acids, eicosapentaenoic acid, docosahexaenoic acid, and five double bonds, and ω3 fatty acid having 22 carbon atoms were all increased by the separation using isooctane and acetonitrile. However, the concentration of all the other fatty acids, including arachidonic acid, which is a ω6 fatty acid, has decreased, so the separation of ω3 by isooctane and acetonitrile
It can be seen that the fatty acids in the system are selectively concentrated. Example 11 Eicosapentaenoic acid 13.9% by weight, docosahexaenoic acid 8.8% by weight, arachidonic acid 1.1% by weight, palmitic acid 17.1% by weight, palmitoleic acid 9.7% by weight and oleic acid 13.7% by weight. 1.0% fish oil fatty acid containing
In addition, 3.0 kg of isohexane and 4.0 kg of acetonitrile
Was added, and the acetonitrile layer was separated at the liquid-liquid distribution temperature of -10 ° C in the same manner as in Example 1, and 0.15 kg of fatty acid was obtained from this acetonitrile layer. The composition of this fatty acid is 33.2% by weight of eicosapentaenoic acid, 15.8% by weight of docosahexaenoic acid, 1.0% by weight of arachidonic acid, 8.1% by weight of palmitic acid, 6.9% by weight of palmitoleic acid and 7% of oleic acid. It was 0.3% by weight. The results are shown in Table 8.

[0023]

[Table 8]

From the results shown in Table 8, even in the phase separation using isohexane and acetonitrile, the ω3 fatty acids eicosapentaenoic acid and docosahexaenoic acid are concentrated and the concentration is increased, and the ω6 fatty acid arachidone is increased. It can be seen that the concentration of all other fatty acids, including acids, is reduced.

[0025]

According to the method of the present invention, a fatty acid ester containing eicosapentaenoic acid or a fatty acid ester containing an ester of eicosapentaenoic acid can be used to obtain eicosapentaenoic acid or its ester free from ω6 fatty acids having a function inhibiting property. By concentrating, high-purity eicosapentaenoic acid or its ester can be produced.

Claims (4)

[Claims] 1. A method for producing eicosapentaenoic acid, which comprises adding an isoalkane and acetonitrile to a fatty acid mixture containing eicosapentaenoic acid, mixing and separating the layers, and concentrating the eicosapentaenoic acid into an acetonitrile layer. 2. A fatty acid mixture containing eicosapentaenoic acid is mixed with isoalkane and acetonitrile, and the mixture is separated into layers. Then, a new isoalkane is added to the obtained acetonitrile layer and the mixture is separated, and the mixture is separated into layers. A method for producing eicosapentaenoic acid, which comprises concentrating icosapentaenoic acid in an acetonitrile layer. 3. A method for producing an eicosapentaenoic acid ester, characterized in that a fatty acid ester mixture containing an eicosapentaenoic acid ester is mixed with isoalkane and acetonitrile and then separated into layers to concentrate the eicosapentaenoic acid ester into an acetonitrile layer. Method. 4. An operation in which isoalkane and acetonitrile are added to a fatty acid ester mixture containing eicosapentaenoic acid ester and mixed, and then the mixture is separated into layers, and a new isoalkane is added to the obtained acetonitrile layer and mixed, and then separated into layers. A method for producing eicosapentaenoic acid ester, comprising concentrating eicosapentaenoic acid ester in an acetonitrile layer.