Composition containing polyunsaturated fatty acid or ester derivative thereof, and method for producing same

Abstract

The present production method provides a method for producing a fatty acid composition that contains polyunsaturated fatty acid (hereinafter referred to as PUFA) or ester derivative thereof. This production method comprises: storing a solvent in a container A to obtain an eluent under an inert gas atmosphere in the container A; purifying a fatty acid mixture containing PUFA or ester derivative thereof by chromatography using this eluent as a mobile phase to obtain a fraction containing PUFA or ester derivative thereof; and concentrating the fraction containing the PUFA or ester derivative thereof to obtain a fatty acid composition containing PUFA or ester derivative thereof.

Description

Composition containing highly unsaturated fatty acid or its ester derivative and method for producing the same

[0001] The present disclosure relates to a fatty acid composition containing a highly unsaturated fatty acid (hereinafter also referred to as PUFA) or an ester derivative thereof, and a method for producing the same.

[0002] Highly unsaturated fatty acids or their ester derivatives are used as raw materials for pharmaceuticals, health foods, and cosmetics. Methods such as precision distillation, silver nitrate complex method, and chromatography are known as methods for obtaining highly pure highly unsaturated fatty acids or their ester derivatives for use in pharmaceuticals, health foods, and cosmetics from natural fats and oils (see, for example, Patent Documents 1 to 4).

[0003] On the other hand, denaturation and / or degradation of compositions containing highly unsaturated fatty acids or their ester derivatives has become a problem. For example, Patent Document 1 describes providing a PUFA or its ester derivative composition having a dissolved oxygen content, peroxide value, acid value, and anisidine value below a certain value in order to solve the problem of preventing and / or delaying the progression of oxidation during the manufacturing process and during the storage period of the product. Patent Document 2 points out the risk that dissolved oxygen in the eluent can lead to denaturation and / or degradation of the PUFA during separation and purification of PUFA or its ester derivative by chromatography using an adsorption resin as a carrier, and describes that to prevent this risk, the eluent can be heated and refluxed or nitrogen or carbon dioxide gas can be blown into the eluent.

[0004] Furthermore, Patent Document 3 describes the use of a channel-type agitator as a reaction vessel in a method for producing PUFA ester derivatives using a silver salt solution, and the carrying out of the production method under low-oxygen conditions to prevent deterioration of the silver salt solution. Patent Document 4 is a patent related to the purification of PUFA ester derivatives by chromatography, including simulated moving bed chromatography, and the examples state that "all chromatographic operations are carried out under an inert gas atmosphere and protected from light."

[0005] Japanese Patent Application Laid-Open No. 2017-114776 Japanese Patent Application Laid-Open No. 61-291540 Japanese Patent Application Laid-Open No. 2019-135307 Special Publication No. 2017-502130

[0006] The present inventors have discovered that in the production of a fatty acid composition containing a highly unsaturated fatty acid or an ester derivative thereof, unless the dissolved oxygen in the mobile phase is suppressed, the oxygen concentration cannot be sufficiently reduced even when a fraction containing a highly unsaturated fatty acid or an ester derivative thereof obtained by chromatography is stored in a container under an inert gas atmosphere, and the highly unsaturated fatty acid or an ester derivative thereof is denatured during solvent removal, in particular resulting in coloration.

[0007] The present disclosure relates to providing a fatty acid composition containing highly unsaturated fatty acids or ester derivatives thereof in which denaturation such as coloring is suppressed by using, as a mobile phase, an eluent obtained by placing the mixture in an inert gas atmosphere in a container in chromatography for purifying a fatty acid mixture containing highly unsaturated fatty acids or ester derivatives thereof.

[0008] In one aspect of the present disclosure, there is provided a method for producing a fatty acid composition containing the following highly unsaturated fatty acid or an ester derivative thereof: In one aspect of the present disclosure, there is provided a fatty acid composition containing the following highly unsaturated fatty acid or an ester derivative thereof:

[0009] [1-1] A method for producing a fatty acid composition containing highly unsaturated fatty acids (hereinafter also referred to as PUFAs) or ester derivatives thereof, comprising: (1) placing a solvent in a container A and obtaining an eluate in the container A under an inert gas atmosphere; (2) purifying a fatty acid mixture containing PUFAs or ester derivatives thereof by chromatography using the eluate as a mobile phase to obtain a fraction containing PUFAs or ester derivatives thereof; and (3) concentrating the fraction containing PUFAs or ester derivatives thereof to obtain a fatty acid composition containing PUFAs or ester derivatives thereof.

[0010] [1-2] The production method according to [1-1], wherein concentrating the fraction comprises recovering the solvent by distillation by reducing pressure and / or heating an eluate contained in the fraction containing the PUFA or an ester derivative thereof. [1-3] The production method according to [1-1] or [1-2], wherein the solvent comprises a solvent obtained by distillation. [1-4] The production method according to any of [1-1] to [1-3], wherein the solvent comprises a solvent recovered from the fraction containing the PUFA or an ester derivative thereof by concentrating the fraction. [1-5] The production method according to any of [1-1] to [1-4], wherein the dissolved oxygen concentration of the eluate in container A is 20 mg / L or less, 15 mg / L or less, or 11 mg / L or less.

[0011] [1-6] The production method according to any one of [1-1] to [1-5], wherein the headspace oxygen concentration in the container A is 10% by volume or less, 5% by volume or less, 4% by volume or less, 3% by volume or less, 2% by volume or less, 1% by volume or less, 0.5% by volume or less, or 0.1% by volume or less. [1-7] The production method according to any one of [1-1] to [1-6], wherein the fraction containing the PUFA or an ester derivative thereof is contained in a container C under an inert gas atmosphere. [1-8] The production method according to [1-7], wherein the headspace oxygen concentration in the container C after containing the fraction is 1.5% by volume or less, 1.0% by volume or less, 0.7% by volume or less, 0.5% by volume or less, 0.3% by volume or less, or 0.1% by volume or less.

[0012] [1-9] The production method according to any one of [1-1] to [1-8], wherein, before purification of the fatty acid mixture, the fatty acid mixture containing the PUFA or an ester derivative thereof is stored under an inert gas atmosphere in a container B. [1-10] The production method according to [1-9], wherein the headspace oxygen concentration of the container B is 1.5% by volume or less, 1.0% by volume or less, 0.7% by volume or less, 0.5% by volume or less, 0.3% by volume or less, or 0.1% by volume or less.

[0013] [1-11] The production method according to any one of [1-1] to [1-10], wherein the fatty acid composition containing the PUFA or an ester derivative thereof is contained in a container D under an inert gas atmosphere. [1-12] The production method according to [1-11], wherein the headspace oxygen concentration of the container D is 0.7% by volume or less, 0.5% by volume or less, 0.3% by volume or less, or 0.1% by volume or less. [1-13] The production method according to any one of [1-1] to [1-12], wherein the inert gases in the containers A, B, C, and D are each independently nitrogen, argon, carbon dioxide, or a combination thereof.

[0014] [1-14] The production method according to any one of [1-1] to [1-13], wherein the inert gas in the container A, container B, container C, and container D is nitrogen. [1-15] The production method according to any one of [1-1] to [1-14], wherein the proportion of PUFA or an ester derivative thereof in all fatty acids or ester derivatives thereof in the fatty acid composition containing the PUFA or an ester derivative thereof is 97% or more, 96% or more, 90% or more, 80% or more, 70% or more, or 50% or more.

[0015] [1-16] The method for producing a fatty acid composition containing the PUFA or an ester derivative thereof according to any one of [1-1] to [1-15], wherein the color b value of the fatty acid composition containing the PUFA or an ester derivative thereof is 3.2 or less, 3 or less, 2.8 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.7 or less. [1-17] The method for producing a fatty acid composition containing the PUFA or an ester derivative thereof according to any one of [1-1] to [1-16], wherein the anisidine value of the fatty acid composition containing the PUFA or an ester derivative thereof is 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less.

[0016] [1-18] The amount of residual eluate in the fatty acid composition containing the PUFA or its ester derivative is 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less, [1-1] to [1-17]. The manufacturing method according to any one of the above.

[0017] [1-19] The production method according to any one of [1-1] to [1-18], wherein the heating temperature for concentrating the fraction is a maximum temperature of 190°C or less, 160°C or less, 140°C or less, 135°C or less, 130°C or less, 120°C or less, or 100°C or less, and / or 40°C or more, 60°C or more, 80°C or more, 100°C or more, 120°C or more, 130°C or more, 135°C or more, 140°C or more, or 160°C or more. [1-20] The production method according to any one of [1-1] to [1-19], wherein the minimum pressure during decompression in concentrating the fraction is 100 Pa or less, 50 Pa or less, 20 Pa or less, 10 Pa or less, 5 Pa or less, 2 Pa or less, or 1 Pa or less, and / or 0.1 Pa or more, 0.2 Pa or more, 0.5 Pa or more, 1 Pa or more, 2 Pa or more, 5 Pa or more, or 10 Pa or more.

[0018] [1-21] The production method according to any one of [1-1] to [1-20], wherein the solvent and eluent are one or a mixture of two or more selected from methanol, ethanol, 2-propanol, acetonitrile, acetone, and hexane, or a mixture of one or more selected from these with water. [1-22] The production method according to any one of [1-1] to [1-21], wherein the purification of the fatty acid mixture uses a silica gel-based adsorbent or a polymer-based adsorbent as a stationary phase. [1-23] The production method according to any one of [1-1] to [1-22], wherein the purification of the fatty acid mixture uses a reversed-phase adsorbent as a stationary phase.

[0019] [1-24] The production method according to any one of [1-1] to [1-23], wherein the concentration of the fraction is performed using one or two or more evaporators, and at least one of the receivers of the evaporators has an internal volume of 10 L or more, 20 L or more, 50 L or more, 100 L or more, 500 L or more, or 1000 L or more, and / or 5000 L or less, 2000 L or less, 1000 L or less, or 500 L or less. [1-25] The production method according to any one of [1-1] to [1-24], wherein the concentration of the fraction is performed using one or more evaporators, and at least one of the evaporators is purged with an inert gas during the concentration of the fraction. [1-26] The production method according to any one of [1-1] to [1-25], wherein the PUFA is one or a combination of two or more selected from EPA, DHA, n-3 DPA, DGLA, and ARA.

[0020] [1-27] The ester derivative of the PUFA is C of the PUFA １ ~C ６ [1-26] The method for producing a PUFA or an ester derivative thereof, wherein the PUFA or an ester derivative thereof is an alkyl ester derivative. [1-28] The method for producing a PUFA or an ester derivative thereof, wherein the PUFA or an ester derivative thereof is an EPA ethyl ester, a DHA ethyl ester, or a combination thereof. [1-29] The method for producing a PUFA or an ester derivative thereof, wherein the chromatography is HPLC. [1-30] The method for producing a PUFA or an ester derivative thereof, wherein the volume of the vessel A is 50 L or more, 100 L or more, 200 L or more, 500 L or more, 1000 L or more, 2000 L or more, or 4000 L or more, and / or 20000 L or less, 10000 L or less, 4000 L or less, 2000 L or less, or 1000 L or less. [1-31] ​​A fatty acid composition comprising a PUFA or an ester derivative thereof produced by the method for producing a PUFA or an ester derivative thereof, wherein the volume of the vessel A is 50 L or more, 100 L or more, 200 L or more, 500 L or more, 1000 L or more, 2000 L or more, or 4000 L or more, and / or 20000 L or less, 10000 L or less, 4000 L or less, 2000 L or less, or 1000 L or less. [1-32] The production method according to any one of [1-1] to [1-30], wherein a single solvent, or two or less, three or less, four or less, five or less, or ten or less solvents are used as the at least one solvent.

[0021] [2-1] A fatty acid composition comprising a highly unsaturated fatty acid (hereinafter also referred to as PUFA) or an ester derivative thereof, wherein the proportion of PUFA or its ester derivative in all fatty acids or their ester derivatives in the fatty acid composition is 97% or more, 96% or more, 90% or more, 80% or more, 70% or more, or 50% or more, and the color b value of the fatty acid composition is 3.2 or less, 3 or less, 2.8 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.7 or less. [2-2] The fatty acid composition according to [2-1], wherein the anisidine value of the fatty acid composition comprising the PUFA or its ester derivative is 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less.

[0022] [2-3] The fatty acid composition according to [2-1] or [2-2], wherein the amount of residual eluate in the fatty acid composition containing the PUFA or its ester derivative is 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less. [2-4] The fatty acid composition according to any one of [2-1] to [2-3], wherein the PUFA is one or a combination of two or more selected from EPA, DHA, n-3 DPA, DGLA, and ARA.

[0023] [2-5] The ester derivative of the PUFA is C of the PUFA １ ~C ６ [2-6] The fatty acid composition according to any one of [2-1] to [2-5], wherein the PUFA or its ester derivative is an ethyl ester of EPA, an ethyl ester of DHA, or a combination thereof.

[0024] According to the present disclosure, it is possible to produce a fatty acid composition containing a highly unsaturated fatty acid or an ester derivative thereof with reduced discoloration. Furthermore, according to the present disclosure, it is possible to reduce dissolved oxygen in a fatty acid composition containing a highly unsaturated fatty acid or an ester derivative thereof.

[0025] FIG. 1 is a process diagram illustrating this example, showing a method for producing a fatty acid composition containing eicosapentaenoic acid ethyl ester (hereinafter also referred to as EPA-E).

[0026] As used herein, the term "fatty acid" refers to a fatty acid having a molecular weight of about C １２ ~C ２２ Here, "C" refers to aliphatic carboxylic acids of various chain lengths. １２ ~C ２２ The numbers in " refer to the total number of carbon atoms in the carboxylic acid chain. The main chain lengths are C １６ ~C ２２ The structure of a fatty acid can be represented by a simple notation of "X:Y," where X is the total number of carbon atoms in the particular fatty acid and Y is the number of double bonds. For example, a saturated fatty acid with 20 carbon atoms is represented as "C20:0," a monounsaturated fatty acid with 18 carbon atoms is represented as "C18:1," and arachidonic acid can be represented as "C20:4,n-6." The "n-" indicates the position at which the double bond begins, counting from the methyl end of the fatty acid; for example, "n-6" indicates that the double bond begins at the sixth position counting from the methyl end of the fatty acid. This method is well known to those skilled in the art, and fatty acids represented according to this method can be easily identified by those skilled in the art.

[0027] Fatty acids are carboxylic acids with an aliphatic chain, which can be either saturated or unsaturated. Fatty acids are usually produced industrially by hydrolysis of triglycerides or phospholipids from natural sources. Some are also produced synthetically. Regardless of the method of production, purification methods are required to obtain a pure product for food, cosmetic, or industrial use.

[0028] As used herein, the term "highly unsaturated fatty acid" refers to a fatty acid having more than one double bond. As used herein, highly unsaturated fatty acids are sometimes referred to as PUFAs. Highly unsaturated fatty acids can be, for example, fatty acids having 3 to 6 double bonds. Examples of highly unsaturated fatty acids include α-linolenic acid "C18:3,n-3", γ-linolenic acid "C18:3,n-6", dihomo-γ-linolenic acid "C20:3,n-6", arachidonic acid "C20:4,n-6", eicosapentaenoic acid "C20:5,n-3", docosapentaenoic acid "C22:5,n-6", and docosahexaenoic acid "C22:6,n-3". Ester derivatives of highly unsaturated fatty acids as used herein are typically alkyl esters, for example, C １ -C ６ Alkyl ester, or C １ -C ４ It is an alkyl ester. An example of an ester is an ethyl ester.

[0029] <Method for producing a fatty acid composition> One aspect of the present invention is a production method comprising placing a solvent in a container A and obtaining an eluent in the container A under an inert gas atmosphere, purifying a fatty acid mixture containing PUFAs or ester derivatives thereof by chromatography using the eluent as the mobile phase to obtain a fraction containing PUFAs or ester derivatives thereof, and concentrating the fraction containing PUFAs or ester derivatives thereof to obtain a fatty acid composition containing PUFAs or ester derivatives thereof.

[0030] In one embodiment of the present invention, the fatty acid mixture containing PUFAs or their ester derivatives can be obtained from a source containing highly unsaturated fatty acids as constituent fatty acids. For example, the mixture can be obtained from a source containing natural oils and fats, including plant and animal oils and fats, as well as from a source containing oils and fats obtained from microorganisms, including genetically modified plants, animals, and yeasts. Examples of sources include fish oil, algae and microalgae oil, and vegetable oils, such as borage oil, scutellaria oil, and evening primrose oil.

[0031] In one embodiment of the present invention, the fatty acid mixture containing PUFA or its ester derivatives is obtained by subjecting the raw material to distillation.

[0032] In one embodiment of the present invention, the fatty acid mixture containing PUFA or its ester derivative may contain PUFA obtained by decomposing raw material oils and fats into free fatty acids by hydrolysis using water, or ... １ ~C ６ By alcoholysis with alkyl alcohol, C １ ~C ６ It may include ester derivatives decomposed into alkyl esters. １ ~C ６ Examples of alkyl esters include ethyl esters.

[0033] One aspect of the present invention is a production method comprising storing a fatty acid mixture containing PUFAs or ester derivatives thereof in a container B under an inert gas atmosphere before purifying the fatty acid mixture. In one aspect of the present invention, when storing the fatty acid mixture in container B, container B is placed under an inert gas atmosphere. In one aspect of the present invention, container B can be placed under an inert gas atmosphere before, after, or simultaneously with storing the fatty acid mixture. In one aspect of the present invention, it is preferable that the fatty acid mixture is placed in container B after container B has been placed under an inert gas atmosphere.

[0034] Container In one aspect of the present invention, container A is a container for containing and storing an eluent, and can be a tank for containing the eluent. In this specification, a tank for containing an eluent can be referred to as an eluent tank. In one aspect of the present invention, container B is a container for containing and storing a fatty acid mixture containing PUFAs or ester derivatives thereof obtained from a raw material, and can be a tank for containing a fatty acid mixture. In one aspect of the present invention, the fatty acid mixture containing PUFAs or ester derivatives thereof can be a main fraction obtained by continuous rectification. Of container B, the tank for containing the main fraction can be referred to as a main fraction tank.

[0035] In one embodiment of the present invention, the container C is a container for containing and storing a fraction containing PUFAs or ester derivatives thereof obtained by purifying a fatty acid mixture, and may be a tank for containing the fraction. In this specification, the tank for containing the fraction may be referred to as a fraction tank.

[0036] In one embodiment of the present invention, container D is a container for containing and storing a fatty acid composition containing PUFAs or ester derivatives thereof obtained by concentrating the fraction, and may be a tank for containing the fatty acid composition. In this specification, the tank for containing the fatty acid composition may be referred to as a product tank. In this specification, the tank for containing the fatty acid composition may be referred to as a buffer tank.

[0037] In one embodiment of the present invention, the container is purged with an inert gas, thereby placing the solvent, fraction, fatty acid mixture, or fatty acid composition in the container under an inert gas atmosphere. In one aspect of the present invention, the inert gas is not particularly limited as long as it is chemically stable and does not easily affect other elements or compounds. Examples of inert gases include nitrogen, argon, carbon dioxide, and a mixture of two or more of these gases. In one embodiment of the present invention, the inert gas used for the inert gas atmosphere can be nitrogen, argon, or carbon dioxide. In one embodiment of the present invention, the inert gas atmosphere refers to a state in which oxygen, a reactive molecule present in air, is removed and replaced with less reactive nitrogen, argon, or carbon dioxide. In one embodiment of the present invention, the inert gas atmosphere refers to a state in which a gas containing mainly inert gases and having an oxygen concentration of less than 10% by volume is present. In one embodiment of the present invention, purging with an inert gas is performed before or after placing the solvent, fraction, fatty acid mixture, or fatty acid composition in the container.

[0038] In one embodiment of the present invention, the container may be a sealed container, for example, a tank. In one embodiment of the present invention, the container has an inert gas charging device, which is a device for charging an inert gas into the headspace. Examples of inert gas charging devices include a gas seal unit and a breather valve. In one embodiment of the present invention, the container has a space containing gas that is not filled with a solvent, eluent, fatty acid mixture, fraction, or fatty acid composition. In this specification, the space in the container containing gas that is not filled with a solvent, eluent, fatty acid mixture, fraction, or fatty acid composition is referred to as "headspace." In one embodiment of the present invention, the volume of the container is the sum of the volume of the portion filled with the solvent, eluent, fatty acid mixture, fraction, and / or fatty acid composition and the volume of the headspace.

[0039] In one embodiment of the present invention, the headspace volume of the container can be set to an upper limit and / or a lower limit, and can be varied within that range. In one embodiment of the present invention, the upper limit of the headspace volume is the headspace volume at the point when the headspace volume is greatest and the volume of the portion filled with the solvent, eluent, fatty acid mixture, fraction, and / or fatty acid composition is smallest when carrying out the production method of the present invention. In this specification, a container whose headspace volume is at the upper limit may be referred to as a container before storage. This container may also be referred to as a container after transfer. For example, a fraction tank whose headspace volume is at the upper limit may be referred to as a "fraction tank before storage" or a "fraction tank after transfer," respectively.

[0040] In one aspect of the present invention, the lower limit of the headspace volume is the headspace volume at the point when the headspace volume is smallest and the volume of the portion filled with the solvent, eluent, fatty acid mixture, fraction, and / or fatty acid composition is largest when carrying out the production method of the present invention. In this specification, a container whose headspace volume is at the lower limit may be referred to as a container after storage. This container may also be referred to as a container before transfer. For example, a fraction tank whose headspace volume is at the lower limit may be referred to as a "fraction tank after storage" or a "fraction tank before transfer," respectively.

[0041] In one embodiment of the present invention, the upper limit of the headspace volume of container A may be 50 vol.% or more, 70 vol.% or more, 80 vol.% or more, 85 vol.% or more, 87 vol.% or more, 89 vol.% or more, 90 vol.% or more, or 95 vol.% or more of the volume of container A, and / or 100 vol.% or less, 99 vol.% or less, 97 vol.% or less, 95 vol.% or less, 90 vol.% or less, 89 vol.% or less, 85 vol.% or less, 80 vol.% or less, or 70 vol.% or less. In one embodiment of the present invention, the lower limit of the headspace volume of container A may be 10 vol% or more, 20 vol% or more, 30 vol% or more, 50 vol% or more, 70 vol% or more, 80 vol% or more, 85 vol% or more, or 87 vol% or more, and / or 95 vol% or less, 90 vol% or less, 89 vol% or less, 87 vol% or less, 85 vol% or less, 80 vol% or less, 70 vol% or less, or 50 vol% or less of the volume of container A. Also, in one embodiment of the present invention, the volume of container A may be 50 L or more, 100 L or more, 200 L or more, 500 L or more, 1000 L or more, 2000 L or more, or 4000 L or more, and / or 20,000 L or less, 10,000 L or less, 4,000 L or less, 2000 L or less, or 1000 L or less.

[0042] In one embodiment of the present invention, the upper limit of the headspace volume of container B may be 50 vol% or more, 70 vol% or more, 80 vol% or more, 85 vol% or more, 90 vol% or more, or 95 vol% or more, and / or 100 vol% or less, 99.9 vol% or less, 99 vol% or less, 97 vol% or less, 95 vol% or less, 90 vol% or less, 85 vol% or less, 80 vol% or less, or 70 vol% or less of the volume of container B. In one embodiment of the present invention, the lower limit of the headspace volume of container B may be 0 vol% or more, 1 vol% or more, 3 vol% or more, 5 vol% or more, 7 vol% or more, 10 vol% or more, 15 vol% or more, 20 vol% or more, or 30 vol% or more, and / or 70 vol% or less, 50 vol% or less, 30 vol% or less, 20 vol% or less, 15 vol% or less, 10 vol% or less, 7 vol% or less, or 5 vol% or less of the volume of container B. In one embodiment of the present invention, the volume of container B may be 500 L or more, 1000 L or more, 2000 L or more, 5000 L or more, 10,000 L or more, 20,000 L or more, or 50,000 L or more, and / or 200,000 L or less, 100,000 L or less, 50,000 L or less, 20,000 L or less, or 10,000 L or less.

[0043] In one embodiment of the present invention, the upper limit of the headspace volume of container C may be 50 vol.% or more, 70 vol.% or more, 80 vol.% or more, 85 vol.% or more, 90 vol.% or more, or 95 vol.% or more of the volume of container C, and / or 100 vol.% or less, 99.9 vol.% or less, 99 vol.% or less, 97 vol.% or less, 95 vol.% or less, 90 vol.% or less, 85 vol.% or less, 80 vol.% or less, or 70 vol.% or less. In one embodiment of the present invention, the lower limit of the headspace volume of container C may be 0 vol% or more, 1 vol% or more, 3 vol% or more, 5 vol% or more, 7 vol% or more, 10 vol% or more, 20 vol% or more, 30 vol% or more, 35 vol% or more, or 40 vol% or more, and / or 70 vol% or less, 60 vol% or less, 50 vol% or less, 40 vol% or less, 35 vol% or less, 30 vol% or less, 20 vol% or less, 15 vol% or less, or 10 vol% or less of the volume of container C. Furthermore, in one embodiment of the present invention, the volume of container C may be 50 L or more, 100 L or more, 200 L or more, 500 L or more, 1000 L or more, 2000 L or more, or 4000 L or more, and / or 20,000 L or less, 10,000 L or less, 4,000 L or less, 2000 L or less, or 1000 L or less.

[0044] In one embodiment of the present invention, the upper limit of the headspace volume of container D may be 50 vol% or more, 70 vol% or more, 80 vol% or more, 85 vol% or more, 90 vol% or more, or 95 vol% or more, and / or 100 vol% or less, 99.9 vol% or less, 99 vol% or less, 97 vol% or less, 95 vol% or less, 90 vol% or less, 85 vol% or less, 80 vol% or less, or 70 vol% or less of the volume of container D. In one embodiment of the present invention, the lower limit of the headspace volume of container D may be 0 vol% or more, 1 vol% or more, 3 vol% or more, 5 vol% or more, 7 vol% or more, 10 vol% or more, 15 vol% or more, 19 vol% or more, 20 vol% or more, or 30 vol% or more, and / or 70 vol% or less, 50 vol% or less, 30 vol% or less, 20 vol% or less, 19 vol% or less, 15 vol% or less, or 10 vol% or less of the volume of container D. In one embodiment of the present invention, the volume of container D may be 50 L or more, 100 L or more, 200 L or more, 500 L or more, 1000 L or more, 2000 L or more, or 5000 L or more, and / or 20,000 L or less, 10,000 L or less, 5,000 L or less, 2000 L or less, or 1,000 L or less.

[0045] In one aspect of the present invention, the headspace oxygen concentration of a container refers to the concentration of oxygen in the gas contained in the headspace of the container. In one aspect of the present invention, the headspace oxygen concentration of container A can be 10% by volume or less, 5% by volume or less, 4% by volume or less, 3% by volume or less, 2% by volume or less, 1% by volume or less, 0.5% by volume or less, or 0.1% by volume or less. In another aspect of the present invention, the headspace oxygen concentration of container A can be 10% by volume or less, 5% by volume or less, 4% by volume or less, 3% by volume or less, 2% by volume or less, 1% by volume or less, 0.5% by volume or less, or 0.1% by volume or less, and / or 0% by volume or more, 0.01% by volume or more, or 0.05% by volume or more. In one aspect of the present invention, the headspace oxygen concentration of container A can be the headspace oxygen concentration after containing the eluent or the headspace oxygen concentration after transferring the eluent.

[0046] In one embodiment of the present invention, the headspace oxygen concentration of container B can be 1.5 vol% or less, 1.0 vol% or less, 0.7 vol% or less, 0.5 vol% or less, 0.3 vol% or less, or 0.1 vol% or less. In another embodiment of the present invention, the headspace oxygen concentration of container B can be 1.5 vol% or less, 1.0 vol% or less, 0.7 vol% or less, 0.5 vol% or less, 0.3 vol% or less, or 0.1 vol% or less, and / or 0 vol% or more, 0.01 vol% or more, or 0.05 vol% or more. In one embodiment of the present invention, the headspace oxygen concentration of container B can be the headspace oxygen concentration after the fatty acid mixture has been placed in container B or the headspace oxygen concentration after the fatty acid mixture has been transferred.

[0047] In one embodiment of the present invention, the headspace oxygen concentration in container C after the fraction has been collected can be 1.5 vol% or less, 1.0 vol% or less, 0.7 vol% or less, 0.5 vol% or less, 0.3 vol% or less, or 0.1 vol% or less. In other embodiments of the present invention, the headspace oxygen concentration in container C after the fraction has been collected can be 1.5 vol% or less, 1.0 vol% or less, 0.7 vol% or less, 0.5 vol% or less, 0.3 vol% or less, or 0.1 vol% or less, and / or 0 vol% or more, 0.01 vol% or more, or 0.05 vol% or more. In one embodiment of the present invention, the headspace oxygen concentration in container C after the fraction has been transferred can be 0.7 vol% or less, 0.5 vol% or less, 0.3 vol% or less, or 0.1 vol% or less. In other aspects of the invention, the headspace oxygen concentration in container C after fraction transfer can be 0.7 vol.% or less, 0.5 vol.% or less, 0.3 vol.% or less, or 0.1 vol.% or less, and / or 0 vol.% or more, 0.01 vol.% or more, or 0.05 vol.%.

[0048] In one embodiment of the present invention, the headspace oxygen concentration in container D can be 0.7% by volume or less, 0.5% by volume or less, 0.3% by volume or less, or 0.1% by volume or less. In one embodiment of the present invention, the headspace oxygen concentration in container D can be 0.7% by volume or less, 0.5% by volume or less, 0.3% by volume or less, or 0.1% by volume or less, and / or 0% by volume or more, 0.01% by volume or more, or 0.05% by volume. In one embodiment of the present invention, the headspace oxygen concentration in container D can be the headspace oxygen concentration after the fatty acid composition has been placed in container or the headspace oxygen concentration after the fatty acid composition has been transferred.

[0049] The method for measuring the headspace oxygen concentration is not particularly limited, but for example, it can be measured using a residual oxygen meter "Pack Keeper" RO-103KS (manufactured by Iijima Electronics Co., Ltd.) according to the procedure in the instruction manual for the device. In this specification, the above descriptions regarding containers not specified as container A, container B, container C, or container D apply to container A, container B, container C, and container D.

[0050] Eluent In one aspect of the present invention, the method for producing a fatty acid composition comprises placing a solvent in a container A and obtaining an eluent in the container A under an inert gas atmosphere. One aspect of the present invention includes using a solvent that can be used as an eluent before being placed in the container A. In one aspect of the present invention, when the solvent is placed in the container A, the container A is placed under an inert gas atmosphere. In one aspect of the present invention, the container A can be placed under an inert gas atmosphere before, after, or simultaneously with the placement of the solvent. In one aspect of the present invention, it is preferable that the solvent is placed in the container A after the container A has been placed under an inert gas atmosphere.

[0051] In one embodiment of the present invention, a solvent usable as an eluent has a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less. In another embodiment of the present invention, a solvent usable as an eluent has a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less, and / or 0 mg / L or more, 0.01 mg / L or more, 0.03 mg / L or more, or 0.1 mg / L or more. In one embodiment of the present invention, a solvent that can be used as an eluent is a liquid having a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less, which is one or a mixture of two or more selected from alcohols, ethers, esters, ketones, nitriles, hexanes, and dichloromethane, or a mixture of one or more selected from these with water. In another aspect of the present invention, a solvent usable as an eluent is a liquid having a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less, and / or 0 mg / L or more, 0.01 mg / L or more, 0.03 mg / L or more, or 0.1 mg / L or more. The solvent may be one or a mixture of two or more selected from alcohols, ethers, esters, ketones, nitriles, hexanes, and dichloromethane, or a mixture of one or more selected from these with water. Examples of alcohols include methanol, ethanol, n-propanol, 2-propanol, n-butanol, i-butanol, s-butanol, and t-butanol. In one aspect of the present invention, examples of alcohols include methanol and ethanol. In one embodiment, examples of ethers include methanol. Examples of ethers include diethyl ether, diisopropyl ether, and methyl t-butyl ether. Examples of esters include methyl acetate and ethyl acetate. Examples of ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone.An example of the nitrile is acetonitrile. In one aspect of the present invention, the solvent usable as the eluent is methanol having a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less. In another aspect of the present invention, the solvent usable as the eluent is methanol having a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less, and / or 0 mg / L or more, 0.01 mg / L or more, 0.03 mg / L or more, or 0.1 mg / L or more.

[0052] One embodiment of the present invention is a production method in which the solvent contained in container A includes a solvent obtained by distillation. In one aspect of the present invention, the solvent obtained by distillation can be obtained by recovering the solvent by distillation from a fraction during fraction concentration. In one aspect of the present invention, the solvent obtained by distillation can be obtained by purchasing a distilled solvent and / or by distilling a purchased solvent.

[0053] In one embodiment of the present invention, the solvent contained in the container A includes a solvent recovered from the fraction during concentration of the fraction.

[0054] One aspect of the present invention is a production method in which the concentration of the fraction comprises reducing the pressure and / or heating the eluate contained in the fraction containing the PUFA or its ester derivative, and recovering the solvent by distillation.

[0055] Purification of fatty acid mixture by chromatography In one aspect of the present invention, a method for producing a fatty acid composition comprises purifying a fatty acid mixture containing PUFA or an ester derivative thereof by chromatography using an eluent as the mobile phase to obtain a fraction containing PUFA or an ester derivative thereof.

[0056] As used herein, "chromatography" refers to a method of separating or purifying a mixture in which a substance called a mobile phase passes through the surface or interior of a substance called a stationary phase or carrier. Chromatography is used to analyze and separate mixtures of two or more substances. As used herein, chromatography can be column chromatography, and examples of such chromatography include high turbulence liquid chromatography (high turbulence liquid chromatography), preparative chromatography, high performance liquid chromatography (HPLC), simulated moving bed chromatography, actual moving bed chromatography, and supercritical fluid chromatography.

[0057] As used herein, "chromatographic conditions" refers to various parameters that indicate the conditions under which chromatography is performed. Examples include packing pressure, mobile and stationary phase compositions, slurry concentration, pumping pressure, column temperature, mobile phase temperature, mobile phase gradient, mobile phase flow rate, column type used, detection instrumentation and parameters used, sample preparation protocol used, stationary phase settling time during column preparation and pressure at which settling is performed, and standing time and pressure at which standing is performed. Columns prepared by packing with the stationary phase or commercially available columns pre-packed with the stationary phase can be used.

[0058] In one embodiment of the present invention, the chromatography uses a silica gel-based adsorbent or a polymer-based adsorbent as a stationary phase. The stationary phase can be selected from reversed-phase stationary phases, hydrophilic interaction liquid chromatography stationary phases, acrylamide, silica, phenylhexyl stationary phases, polar alkyl, fluorophenylpropyl, or any stationary phase known in the art of chromatography. Examples of reversed-phase stationary phases include C ３０ , C ２２ , C １８ , C ８ , C ５ , C ４, biphenyl, and fluorophenyl stationary phases are used. In some embodiments, chiral stationary phases are used. The choice of stationary phase will be apparent to those skilled in the art and will depend on the component to be purified by chromatography. A variety of different types of octadecyl silica (ODS) can be used, including fully end-capped, partially end-capped, and base-deactivated. More polar components may require normal-phase stationary phases such as unbonded silica, amino phase, or cyano phase.

[0059] In one embodiment of the present invention, the chromatography uses a reversed-phase adsorbent as the stationary phase. Any adsorbent of the reversed-phase partitioning system can be used without any particular specification, for example, polymer beads such as polystyrene reticulated with divinylbenzene, or C ８ or C １８ Silica gel having alkyl groups bonded thereto, particularly C １８ An ODS column using octadecylsilyl, which is silica gel to which alkyl groups are bonded, can be used.

[0060] In one embodiment of the present invention, the dimensions of the column used are not particularly limited and will depend to some extent on the volume of the fatty acid composition to be purified. The diameter of the column is 1 mm or more, 2 mm or more, 4 mm or more, 8 mm or more, 16 mm or more, 32 mm or more, 64 mm or more, 128 mm or more, 256 mm or more, or 500 mm or more, or 4000 mm or less, 2000 mm or less, 1000 mm or less, 500 mm or less, or 1 to 4000 mm, 2 to 2000 mm, 4 to 1000 mm, 8 to 1000 mm, 16 to 1000 mm, 32 to 500 mm, 30 to 800 mm, or 400 to 800 mm.

[0061] In one aspect of the invention, the length of the column is 5 cm or more, 10 cm or more, 20 cm or more, or 800 cm or less, 400 cm or less, 200 cm or less, 150 cm, 120 cm or less, or 5 to 800 cm, 10 to 400 cm, 20 to 200 cm, 20 to 150 cm, or 20 to 120 cm.

[0062] As used herein, "eluent" refers to a liquid used in chromatography as a mobile phase, which is a phase that contacts the stationary phase and passes through the gaps or surface of the stationary phase. The eluent is used in chromatography to develop and elute components such as PUFAs or their ester derivatives that are adsorbed to a column. As used herein, the eluent is sometimes referred to as the mobile phase.

[0063] In one aspect of the present invention, an eluent is obtained in a container under an inert gas atmosphere and then placed in a column as a mobile phase to develop and elute the PUFA or its ester derivative. The eluent from the column is collected together with the PUFA or its ester derivative as a fraction. The fraction is also called an eluate, and may contain components such as the eluent and the PUFA or its ester derivative.

[0064] In one embodiment of the present invention, the liquid includes a solvent and an eluent. The solvent and / or eluent is one or a mixture of two or more selected from alcohols, ethers, esters, ketones, nitriles, hexanes, and dichloromethane, or a mixture of one or more selected from these with water. Examples of alcohols include methanol, ethanol, n-propanol, 2-propanol, n-butanol, i-butanol, s-butanol, and t-butanol. In one aspect of the present invention, examples of alcohols include methanol and ethanol. In one embodiment, methanol is used. Examples of ethers include diethyl ether, diisopropyl ether, and methyl t-butyl ether. Examples of esters include methyl acetate and ethyl acetate. Examples of ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone. Examples of nitriles include acetonitrile. In one embodiment of the present invention, a single liquid is used as the solvent and eluent. In one embodiment of the present invention, methanol is used as the solvent and eluent. In one embodiment of the present invention, a single liquid or no more than two, no more than three, no more than four, no more than five, or no more than ten liquids are used as the solvent and eluent.

[0065] The eluent may further contain additives, including buffers and pH adjusters. The choice of additive may be determined based on the eluent used, the stationary phase used, and the component to be purified. In some embodiments, the eluent contains an additive selected from one or more of formic acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide. In some embodiments, the eluent may be additive-free.

[0066] In one aspect of the present invention, a solvent is contained in container A. In one embodiment, container A does not contain an eluent, and a solvent is contained therein to be used as the eluent. In one embodiment, before the solvent is contained, container A is placed under an inert gas atmosphere and the solvent is contained in a state containing an eluent, as in another embodiment. In one embodiment, the solvent is contained in container A placed under an inert gas atmosphere. In one embodiment, container A is placed under an inert gas atmosphere before the solvent is contained in container A. In one embodiment, the solvent is contained in container A that is not placed under an inert gas atmosphere. In one embodiment, container A is placed under an inert gas atmosphere after the solvent has been contained therein.

[0067] In one aspect of the present invention, the dissolved oxygen concentration of the solvent contained in container A is, for example, 20 mg / L or less, 15 mg / L or less, or 11 mg / L or less. In another aspect of the present invention, the dissolved oxygen concentration of the solvent contained in container A is, for example, 20 mg / L or less, 15 mg / L or less, or 11 mg / L or less, and / or 0 mg / L or more, 0.01 mg / L or more, 0.03 mg / L or more, or 0.1 mg / L or more.

[0068] In one embodiment of the present invention, the dissolved oxygen concentration of the eluent refers to the concentration of oxygen dissolved in the eluent in container A, and may be 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less. In one embodiment of the present invention, the dissolved oxygen concentration of the eluent refers to the concentration of oxygen dissolved in the eluent in container A, and may be 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less, and / or 0 mg / L or more, 0.01 mg / L or more, 0.03 mg / L or more, or 0.1 mg / L or more. The dissolved oxygen concentration can be measured by a common method using a dissolved oxygen meter.

[0069] In one aspect of the present invention, the solvent contained in container A has an oxygen concentration greater than 20 mg / L. In one embodiment, the oxygen concentration of the solvent contained in container A is greater than 25 mg / L. In one embodiment, an eluent having a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less after containing the solvent is used. In another embodiment, an eluent having a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less after containing the solvent is used. In one aspect of the present invention, the dissolved oxygen concentration of the solvent contained in container A refers to the concentration of oxygen dissolved in the solvent contained in container A, and may be greater than 20 mg / L, greater than 17.5 mg / L, greater than 15 mg / L, greater than 14 mg / L, greater than 13 mg / L, greater than 12 mg / L, or greater than 11 mg / L.

[0070] In one aspect of the present invention, the dissolved oxygen concentration in the solvent contained in container A is 20 mg / L or less, for example, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less. In another aspect of the present invention, the dissolved oxygen concentration in the solvent contained in container A is 20 mg / L or less, for example, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less, and / or 0 mg / L or more, 0.01 mg / L or more, 0.03 mg / L or more, or 0.1 mg / L or more.

[0071] In one aspect of the invention, the eluent in container A is contained in container A at the same temperature as the chromatography or at a temperature of ±5°C, ±2.5°C, ±2°C or ±1°C, for example room temperature or a temperature higher than room temperature. In one embodiment, room temperature is 20°C to 25°C. Temperatures higher than room temperature are 25°C or higher, 30°C or higher, or 35°C or higher, and / or 60°C or lower, 50°C or lower, or 45°C or lower, or 25-60°C, 30-50°C, or 35-45°C.

[0072] In one embodiment of the present invention, the container A may be a tank containing an eluent used in chromatography. In this specification, a tank containing an eluent may be referred to as an eluent tank.

[0073] The mobile phase may comprise one or more of water, methanol, ethanol, acetonitrile, ethyl acetate, hexanes, dichloromethane, supercritical carbon dioxide, or any other liquid known in the art. The selection of the mobile phase may require consideration of the highly unsaturated fatty acid or its ester derivative to be purified and the stationary phase to be used. In the case of a reversed-phase stationary phase for highly unsaturated fatty acids or their ester derivatives, a polar mobile phase should be selected that is sufficient to develop and elute the highly unsaturated fatty acid or its ester derivative of interest, but not so fast that the development and elution approach the front of the mobile phase.

[0074] In one aspect of the present invention, the chromatography comprises a mobile phase gradient. As used herein, "mobile phase gradient" refers to a change in mobile phase composition over time while the chromatography is being performed. The column can contain mobile phases with different mobile phase compositions that gradually increase or decrease over time while the chromatography is being performed.

[0075] The main purpose of a mobile phase gradient is to elute strongly retained components from the column faster and weakly retained components more slowly, so that the eluted highly unsaturated fatty acids or their ester derivatives produce well-resolved peaks upon detection. For example, in reversed-phase chromatography, starting with a low content of nonpolar mobile phase in the mobile phase allows weakly retained components to be separated. Strongly retained components will either remain on the adsorbent surface at the top of the column or will migrate very slowly. Increasing the amount of nonpolar mobile phase, such as acetonitrile, in the mobile phase allows strongly retained components to migrate faster due to steadily increasing competition for adsorption sites on the stationary phase by the nonpolar mobile phase.

[0076] Thus, in reversed-phase chromatography used for highly unsaturated fatty acids or their ester derivatives, the mobile phase at the start of the chromatography may contain a high percentage of polar mobile phase A, such as water, selected from about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 0%. Mobile phase B may be a mobile phase less polar than mobile phase A. For example, if mobile phase A is water, mobile phase B may be methanol. Mobile phase B would constitute the remaining percentage of the mobile phase. As the chromatography is performed and the mobile phase is eluted through the column, a gradient will result in a gradual increase in the concentration of mobile phase B over time. In some embodiments, a single-component liquid may be used as the mobile phase. In some embodiments, the single-component liquid may be water, methanol, ethanol, acetonitrile, ethyl acetate, hexanes, dichloromethane, supercritical carbon dioxide, or any other liquid known in the art.

[0077] In some embodiments, the rate of increase of B over time can be constant. In some embodiments, there is no gradient and the mobile phase is isocratic during elution. In some embodiments, different rates of increase in the percentage of mobile phase B at different time ranges within the chromatographic conditions can be used. In some embodiments, the mobile phase can be at a specific composition during certain time ranges of the chromatographic conditions and include a gradient during other time ranges.

[0078] The mobile phase delivery system is a pumping device, such as a commercially available chromatography pump, that provides the mobile phase to the column. Such pumps are generally corrosion-resistant and solvent-resistant, offering pulse-free flow, flow rates ranging from 0.1 to 100 L / min, precise flow rate control, and the generation of high pressures up to 6000 psi. Reciprocating pumps consist of a small chamber into which the mobile phase is pumped by the back-and-forth motion of a motor-driven piston. Two check valves, which alternately open and close, control the direction and flow of the mobile phase into and out of the cylinder. Single-piston pumps use specially designed cams to allow very rapid refill times and produce a more continuous flow. The disadvantage of pulsed flow with reciprocating pumps is often overcome by using a pulse damper. The use of a dual-piston pump, with pistons moving out of phase with each other, provides a reasonable solution for pulse-free fluid delivery. Linear velocity in a column represents the rate at which fluid passes through the cross section of the column. Linear velocity can be calculated using the following formula:

[0079] Linear velocity “m / hour” = flow rate “m” ３ / time" / column cross-sectional area "m ２ The linear velocity can be about 0.2 to 20.0 m / hr, about 1.0 to 15.0 m / hr, about 1.0 to 10.0 m / hr, about 1.5 to 10.0 m / hr, or about 2.0 to 9.0 m / hr. In one embodiment, the linear velocity is about 4.0 to 9.0 m / hr.

[0080] In one embodiment of the invention, the chromatography is carried out at room temperature or at a temperature higher than room temperature. In one embodiment, the chromatography is carried out at a temperature higher than room temperature. In one embodiment, room temperature is between 20°C and 25°C.

[0081] In one embodiment of the present invention, the temperature higher than room temperature is 25°C or higher, 30°C or higher, or 35°C or higher, and / or 60°C or lower, 50°C or lower, or 45°C or lower, or 25 to 60°C, 30 to 50°C, or 35 to 45°C.

[0082] As used herein, the term "fraction" refers to a fatty acid mixture containing PUFAs or their ester derivatives that is loaded onto a column and subjected to chromatography, and the eluate received in the column for developing and eluting the fatty acid mixture, and the eluate eluted from the column is collected in small amounts at specific time intervals. The process of obtaining fractions is called fractionation, and during fractionation, the fatty acid composition may vary over time. Different fractions are collected at different time points based on the unique properties of individual components, such as highly unsaturated fatty acids or their ester derivatives, in the fatty acid mixture, for example, differences in their affinity for the stationary phase and / or mobile phase.

[0083] One aspect of the present invention is a production method comprising containing a fraction containing a PUFA or an ester derivative thereof in a container C under an inert gas atmosphere. In one embodiment of the present invention, when the fraction is contained in container C, the container C is placed under an inert gas atmosphere. In one embodiment of the present invention, the container C can be placed under an inert gas atmosphere before, after, or simultaneously with the containing of the fraction. In one embodiment of the present invention, it is preferable that the fraction is contained in container C after container C has been placed under an inert gas atmosphere.

[0084] In one embodiment of the present invention, the container C may be a tank for storing fractions obtained by chromatography. In this specification, a tank for storing fractions may be referred to as a fraction tank.

[0085] Concentration of Fractions In one aspect of the present invention, the method for producing a fatty acid composition comprises concentrating the fraction containing PUFAs or ester derivatives thereof obtained by chromatography to obtain a fatty acid composition containing PUFAs or ester derivatives thereof.

[0086] Concentrating the fraction involves distilling the eluate by reducing pressure and / or heating the fraction containing the PUFA or its ester derivative and the eluate, and the PUFA or its ester derivative and the eluate are separated during the concentration. In one aspect of the present invention, the fraction can be subjected to one or more evaporators during the concentration of the fraction. In one aspect of the present invention, the eluate separated by distillation during the concentration of the fraction can be recovered as a solvent and reused for producing a fatty acid composition. The solvent separated and recovered during the concentration of the fraction is placed in container A, and the eluate can be obtained in container A under an inert gas atmosphere.

[0087] In one embodiment of the present invention, the evaporator used in concentrating the fraction includes an eluent evaporator and / or a thin film evaporator. Examples of the eluent evaporator include a single-effect evaporator, a multi-chamber evaporator, a natural circulation evaporator, a forced circulation evaporator, a falling thin film evaporator, an ascending thin film evaporator, and combinations thereof. In one aspect of the present invention, examples of the natural circulation evaporator include an external heating type and a calandria type.

[0088] In one embodiment of the present invention, any type of evaporator can be used to concentrate the fraction, including, for example, a thin film evaporator, a single evaporator, a multiple-effect evaporator, a multi-chamber evaporator, a natural circulation evaporator, a forced circulation evaporator, a falling thin film evaporator, an ascending thin film evaporator, and combinations thereof. In one aspect of the present invention, examples of the natural circulation evaporator include an external heating type and a calandria type.

[0089] In one embodiment of the present invention, the internal volume of at least one of the receivers of the evaporator used in concentrating the fraction is 10 L or more, 20 L or more, 50 L or more, 100 L or more, 500 L or more, or 1000 L or more, and / or 5000 L or less, 2000 L or less, 1000 L or less, or 500 L or less.

[0090] In one embodiment of the present invention, at least one of the evaporators used in concentrating the fractions is blown with an inert gas during the concentration of the fractions. Examples of the inert gas include nitrogen, argon, carbon dioxide, or a combination thereof. In one embodiment, nitrogen is used. In one embodiment of the present invention, the evaporator blown with an inert gas is an eluent evaporator.

[0091] In this specification, "inert gas blowing" means supplying an inert gas to an apparatus or container such as an evaporator for the purpose of gas replacement. The amount and speed of the inert gas supplied are not particularly limited. Nitrogen blowing is one form of inert gas blowing. Note that "purging" means blowing until the gas is replaced with an inert gas by inert gas blowing.

[0092] As used herein, "residual eluate amount" refers to the concentration of eluate contained in the fatty acid composition containing PUFAs or ester derivatives thereof obtained by concentrating the fraction. In one aspect of the present invention, the fatty acid composition containing PUFAs or ester derivatives thereof obtained by concentrating the fraction contains an eluate of 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less. In other aspects of the present invention, the fatty acid composition contains an eluate of 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less, and / or 0.001 ppm or more, 0.003 ppm or more, or 0.01 ppm or more.

[0093] In one embodiment of the present invention, the fraction can be concentrated at a maximum heating temperature of 190°C or less, 160°C or less, 140°C or less, 135°C or less, 130°C or less, 120°C or less, or 100°C or less, and / or 40°C or more, 60°C or more, 80°C or more, 100°C or more, 120°C or more, 130°C or more, 135°C or more, 140°C or more, or 160°C or more.

[0094] In one embodiment of the present invention, the concentration of the fraction can be carried out at a minimum pressure during decompression of 100 Pa or less, 50 Pa or less, 20 Pa or less, 10 Pa or less, 5 Pa or less, 2 Pa or less, or 1 Pa or less, and / or 0.1 Pa or more, 0.2 Pa or more, 0.5 Pa or more, 1 Pa or more, 2 Pa or more, 5 Pa or more, or 10 Pa or more. In this specification, the "minimum pressure during decompression" refers to the pressure at the highest vacuum level during decompression.

[0095] One aspect of the present invention is a production method comprising containing a fatty acid composition containing a PUFA or an ester derivative thereof in a container D under an inert gas atmosphere. In one aspect of the present invention, when the fatty acid composition is contained in the container D, the container D is placed under an inert gas atmosphere. In one aspect of the present invention, the container D can be placed under an inert gas atmosphere before, after, or simultaneously with containing the fatty acid composition. In one aspect of the present invention, it is preferable that the fatty acid composition is contained in the container D after the container D has been placed under an inert gas atmosphere.

[0096] In one embodiment of the present invention, vessel D may be a tank containing a fatty acid composition containing PUFAs or ester derivatives thereof concentrated through concentration of the fraction. In one embodiment of the present invention, the tank containing the fatty acid composition containing PUFAs or ester derivatives thereof may be referred to as a product tank.

[0097] <Fatty Acid Composition> In this specification, the "color b value" refers to the L ＊ a ＊ b in the b color system ＊ It means the value of L ＊ a ＊In the b color system, L represents lightness, ＊ and b ＊ represents hue and saturation. ＊ represents a hue ranging from blue to yellow, and when b* is 0, the color is achromatic. As the value increases in the positive direction, the color becomes more yellowish, and as the value increases in the negative direction, the color becomes more blueish. ＊ a ＊ The b color system is also called CIE LAB. The color b value can be measured by a common method using a colorimeter. In this specification, the colorimeter is also called a color difference meter.

[0098] In one aspect of the present invention, the color b value of the fatty acid composition comprising a PUFA or an ester derivative thereof may be 3.2 or less, 3 or less, 2.8 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.7 or less. In other aspects of the present invention, the color b value of the fatty acid composition may be 3.2 or less, 3 or less, 2.8 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.7 or less, and / or -0.3 or more, -0.1 or more, 0 or more, 0.1 or more, 0.3 or more. In one aspect of the present invention, the color b value of the fatty acid composition comprising a PUFA or an ester derivative thereof may be 0 or more, and may be 0 to 3.2, 0 to 3, 0 to 2.8, 0 to 2.5, 0 to 2, 0 to 1.5, 0 to 1, or 0 to 0.7.

[0099] As used herein, the term "anisidine value" refers to a value determined by colorimetric quantification of carbonyl compounds using p-anisidine for color development. The yellow color produced by the reaction of aldehydes, a degradation product of fats and oils, with an anisidine reagent in the presence of acetic acid is measured by measuring the absorbance at 350 nm. This method can be measured by the method described as an official method in the Standard Methods for the Analysis of Fats, Oils, and Related Materials (2.4.26-81). For example, an ultraviolet-visible spectrophotometer can be used for the measurement. In one aspect of the present invention, the anisidine value of a fatty acid composition containing a PUFA or an ester derivative thereof can be 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less. In other aspects of the invention, the anisidine value of the fatty acid composition can be 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less, and / or 0 or more, 0.001 or more, 0.003 or more, or 0.01 or more.

[0100] The fatty acid composition obtained by the production method in one embodiment of the present invention can contain one highly unsaturated fatty acid or an ester derivative thereof selected from eicosapentaenoic acid (hereinafter also referred to as EPA), n-3 docosapentaenoic acid (hereinafter also referred to as n-3 DPA), docosahexaenoic acid (hereinafter also referred to as DHA), dihomo-γ-linolenic acid (hereinafter also referred to as DGLA), arachidonic acid (hereinafter also referred to as ARA), stearidonic acid, C18:3, C19:4, C20:4, and C21:5, or a combination of two or more highly unsaturated fatty acids or ester derivatives thereof. １ ~C ６It may be an alkyl ester derivative, such as a methyl, ethyl, propyl, butyl, pentyl, or hexyl ester. In one embodiment of the invention, the fatty acid composition comprises EPA ethyl ester, DHA ethyl ester, or a combination thereof. In one embodiment of the invention, the fatty acid composition comprises EPA ethyl ester. In one aspect of the invention, the fatty acid composition comprises crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, mead acid, dihomo-γ-linolenic acid, eicosatrienoic acid, stearidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, ozubondo acid, sardine acid, tetracosapentaenoic acid, herring acid, acid), propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, carboceric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontylic acid, lacceric acid, silicic acid, geddic acid The fatty acid may comprise one highly unsaturated fatty acid or an ester derivative thereof selected from the group consisting of hexatriacontylic acid, celloplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, and tetracontylic acid, or a combination of two or more highly unsaturated fatty acids or ester derivatives thereof.

[0101] In one embodiment of the present invention, the fatty acid composition contains highly unsaturated fatty acids or their ester derivatives in a proportion of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more, and / or less than 97%, less than 98%, less than 99.0%, less than 99.95%, or less than 99.99% of the total fatty acid composition.

[0102] Certain ranges are provided herein by numerical values ​​preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes as well as a number that is close to or approximately the number preceded by the term. In determining whether a number is close to or approximately a specifically stated number, an unstated number that is close to or approaching the stated number can be a number that provides a substantial equivalent of the specifically stated number in the context in which it is given. In some embodiments, about can refer to ±5%, ±2.5%, ±2%, or ±1% of the number to which it refers.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0104] Where a range of values ​​is provided, it is understood that each value between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the scope of the invention. Unless the context clearly dictates otherwise, to the tenth of a unit of each value between the upper and lower limits of that range, is encompassed within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also encompassed within the scope of the invention, and where one or both limits are included, subject to any specifically excluded limits, ranges excluding either or both of those included limits are also included in the invention. As used herein, unless otherwise specified, "%" means "% by weight."

[0105] The present invention is not limited to the particular embodiments described herein, as such embodiments may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is intended to be limited only by the appended claims.

[0106] In this specification, singular expressions should be understood to include the plural unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural unless otherwise specified.

[0107] A UV / Vis absorbance detector consisting of a scanning spectrophotometer with grating optics can be used. A deuterium lamp can be used as the light source for the ultraviolet range of 190-360 nm. A tungsten lamp can be used as the light source for the visible range of 360-800 nm. The independent or combined use of a deuterium lamp and / or a tungsten lamp provides a simple means of detecting absorbing species as they emerge from the column.

[0108] Photodiode array (PDA) detectors are ultraviolet / visible absorbance detectors that allow for very rapid collection of data across a selected spectral range. Absorbance spectral data for each chromatographic peak can be collected and stored. The stored data can be compared to spectra of pure standards from a library. Spectra obtained with a PDA detector are useful in identifying components that have overlapping peaks and are difficult to separate, since the characteristic spectra for each unresolved component are likely to be different.

[0109] Fluorescence detectors are useful for detecting compounds that exhibit chemiluminescent properties such as fluorescence or phosphorescence. They are at least an order of magnitude more sensitive than UV absorbance detectors. Fluorescence is observed by detecting grating-separated emission radiation, typically at a 90-degree angle to the excitation beam. The number of fluorescent species can be enhanced by post-column derivatization of the eluted compounds with special reagents, or by pre-column derivatization of the sample itself.

[0110] Refractive index detectors respond to almost all solutes. They are also called RI detectors. The difference in the refractive index of the reference mobile phase relative to the column effluent results in the detection of separated components as peaks on the chromatogram. Due to its extremely high sensitivity to the mobile phase, this detector cannot be used without sufficient pulse attenuation in the LC pump, and it is also not suitable for gradient applications due to the changing mobile phase composition. The detection limit is usually lower than that observed with absorbance detectors.

[0111] Conductivity detectors provide highly sensitive detection of all chargeable components. They can be used with LC systems for simple and reliable detection of anions, cations, metals, organic acids, and surfactants down to ppb levels. The addition of a chemical suppressant between the column and the conductivity detector serves to reduce the conductivity of the eluate, allowing the use of gradient elution and ppb-level determination with minimal baseline drift. For typical determination of low levels of anions, the eluate is converted to its weakly ionized, low-conductivity acid, reducing background noise. For example, Na ２ CO ３ At the same time, the anions of the components are converted to their corresponding high conductivity acids, increasing the component signal relative to the anions, e.g., NaCl to HCl.

[0112] In this specification, the "area %" representing the proportion of each fatty acid in a composition refers to a chart obtained by analyzing the composition using gas chromatography with a flame ionization detector (hereinafter also referred to as GC-FID). The peaks of each component are identified in the chart, and the peak area of ​​each fatty acid is calculated using the Agilent ChemStation integration algorithm. The ratio of each peak area to the sum of the fatty acid peak areas indicates the content ratio of that peak component. In the field of oil chemistry, area % is used as almost synonymous with weight %. See the Japan Oil Chemists' Society (JOCS) Standard Methods for Analysis of Fats and Oils, 2013 Edition, 2.4.2.1-2013, Fatty Acid Composition (FID Constant Temperature Gas Chromatography) and 2.4.2.2-2013, Fatty Acid Composition (FID Programmed Temperature Gas Chromatography). The analytical conditions for gas chromatography (hereinafter also referred to as GC) are as follows:

[0113] GC-FID measurement conditions: GC: 7890B or 8890 (Agilent Technologies); Detector: FID; Column: 30 m x 0.25 mm, 0.25 μm film (capillary column equivalent to USP PHASE G16); Column temperature: Constant temperature around 200°C; Detector temperature: Constant temperature around 300°C; Sample inlet temperature: Constant temperature around 300°C; Carrier gas: Helium; Flow rate: Adjusted so that the retention time of eicosapentaenoic acid ethyl ester is approximately 30 minutes; Split ratio: 1:100; Makeup gas: Nitrogen 30 mL / min; Area measurement range: From after the solvent peak to approximately 2.5 times the retention time of eicosapentaenoic acid ethyl ester; Sample injection volume: 1.0 μL; Analysis time: Approximately 75 minutes

[0114] In one aspect of the present invention, an internal standard can be used during gas chromatography analysis.The internal standard can be added to a sample as a reference marker to determine the relative retention time of an analyte relative to the internal standard, or to aid in the quantification of the analyte.The internal standard can be appropriately selected by those skilled in the art to be a compound that is very similar to, but not identical to, the target analyte, for example, a deuterated derivative of the target analyte.When used for quantification purposes, the internal standard can then be used for calibration by plotting the ratio of the analyte signal to the internal standard signal as a function of the standard analyte concentration, where the standard is a sample of known concentration prepared by those skilled in the art to be used as a reference for the unknown analyte sample to be quantified.

[0115] 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 following examples, "%" means "% by weight" unless otherwise specified.

[0116] In the examples of this application, the following measuring instruments were used. Headspace oxygen concentration: Measured using a residual oxygen meter "Pack Keeper" RO-103KS (manufactured by Iijima Electronics Co., Ltd.) according to the instructions in the instruction manual for the instrument. Dissolved oxygen concentration in methanol: Measured using a dissolved oxygen meter: FOR-21 (manufactured by Automatic System Research Co., Ltd.) according to the instructions in the instruction manual for the instrument. Color b value: Measured using a color and turbidity simultaneous measuring instrument: TZ-6000 (manufactured by Nippon Denshoku Industries Co., Ltd.) with a C light source, a 2-degree field of view, and a 20 mm test tube according to the instructions in the instruction manual for the instrument. In this specification, the color b value refers to the L * a * b * Color Space / CIE LAB b * The values ​​represent the values ​​of the above. Anisidine value: Measured using a UV-visible spectrophotometer, V-730 (DS type) (manufactured by JASCO Corporation), according to the procedure described in USP401 Anisidine Value. Acid value: Measured using an automatic titrator, 876 Dosimat (manufactured by Metrohm), according to the procedure described in USP401 Acid Value. Fatty acid composition and isomer analysis: Measured using GC-FID. The analytical conditions for gas chromatography are as described separately. Residual amount of methanol: The residual amount of methanol was analyzed using headspace gas chromatography (hereinafter also referred to as HS-GC), which combines the headspace method and a gas chromatograph. The analytical conditions for headspace and GC are as follows:

[0117] Headspace device conditions Headspace sampler: G4557A (Agilent Technologies) Equilibration temperature in vial: constant temperature around 100°C Equilibration time in vial: 20 min Injection line temperature: 150°C Carrier gas: Helium Pressurization pressure: 103 kPa Pressurization time: 0.01 min Sample injection volume: 1.0 mL

[0118] GC-FID measurement conditions: GC: 7890B or 6850A (Agilent Technologies) Detector: FID Column: 30 m x 0.25 mm, 0.50 μm film (capillary column equivalent to USP PHASE G16) Column temperature: Hold at approximately 40°C for 5 minutes → Heat at 30°C / min → Hold at approximately 240°C for 5 minutes Sample inlet temperature: Constant temperature around 250°C Detector temperature: Constant temperature around 280°C Carrier gas: Helium Flow rate: Adjusted so that the methanol retention time was approximately 4.3 minutes Split ratio: 1:10 Makeup gas: Nitrogen 25 mL / min Vial volume: 10 mL Vial stirring level: 5 Analysis time: Approximately 16.7 minutes

[0119] <Method for preparing main fraction> Fish oil was ethyl-esterified by a conventional method to prepare a fish oil ethyl ester having EPA-E of 16 area % or more in the fatty acid ethyl ester. The fish oil ethyl ester was continuously rectified using a multi-stage rectification apparatus under conditions of a column top pressure of 26.7 Pa or less and a temperature of 190°C or less, and fractionated into an initial fraction, a main fraction, and a residual fraction to obtain the main fraction. Here, the initial fraction is a fraction with a higher volatility than the main fraction, and C 20 The main components are ethyl esters of fatty acids less than C. 20 The main component is ethyl ester of fatty acid, and EPA-E in the fatty acid ethyl ester is 70% or more by area. The residue is a fraction with lower volatility than the main fraction, and C 21 The main components are the ethyl esters of the above fatty acids.

[0120] The preparation of the main fraction can be carried out with reference to the methods described in, for example, Japanese Patent Application Laid-Open Nos. 04-041457, 04-128250, and 05-222392.

[0121] <HPLC Purification Method> Purified EPA-E was produced from the main fraction according to the process diagram shown in Figure 1. That is, the main fraction in the main fraction tank was purified by HPLC using the eluent in the eluent tank, and the obtained fractions were placed in fraction tanks. The fractions were concentrated using an eluent evaporator to obtain intermediate EPA-E, which was placed in a buffer tank. The intermediate EPA-E was further concentrated using a thin-film evaporator to obtain purified EPA-E, which was placed in a product tank. The main fraction had EPA-E in the fatty acid ethyl esters at 70 area % or more, and the purified EPA-E had EPA-E in the fatty acid ethyl esters at 96 area % or more.

[0122] All of the equipment in this process diagram was a closed system except for the eluent tank. The headspaces of the main fraction tank, fraction tank, and product tank, excluding the reduced-pressure eluent evaporator, buffer tank, and thin-film evaporator, were purged with nitrogen using gas seal units and breather valves. In this specification, the main fraction is included in the fatty acid mixture containing PUFA or its ester derivatives. The intermediate EPA-E and purified EPA-E are included in the fatty acid composition containing PUFA or its ester derivatives.

[0123] The temperature of the eluent tank was set to 38°C to 42°C, and the volume ratio of methanol used as the eluent to the headspace was set to 11:89 to 13:87. Here, the volume ratio is "eluent:headspace." The vent pipe (V) of the eluent tank was opened to fill the headspace with air, or nitrogen was blown through the vent pipe (V) to purge the headspace with nitrogen.

[0124] The temperature of the main fraction tank was 39.5°C to 40.5°C, and the volume ratio of the main fraction to the headspace was 5:95 to 90:10. Here, the volume ratio is "main fraction:headspace." The main fraction tank was purged with nitrogen, and the gas in the headspace was released using the installed gas seal unit and breather valve (G) when the main fraction was received, and nitrogen was blown in when the main fraction was transferred to the HPLC, purging the headspace of the main fraction tank with nitrogen.

[0125] In HPLC, the main fraction (a) transferred from the main fraction tank was purified at a temperature of 38°C to 42°C using methanol (b) from the eluent tank as the eluent and ODS as the stationary phase, and fraction (c) containing 96% or more by area of ​​EPA-E in fatty acid ethyl esters was stored in a fraction tank.

[0126] The fraction tank was not temperature-controlled and the temperature was gradually decreased from approximately 40°C. The volume ratio of fraction to headspace was set to 5:95 to 65:35. Here, the volume ratio is "fraction:headspace." The fraction tank was purged with nitrogen, and the gas in the headspace was released using the gas seal unit and breather valve (G) installed in the tank when receiving fractions from the HPLC, and nitrogen was blown in when transferring fractions to the eluent evaporator, purging the headspace of the fraction tank with nitrogen.

[0127] DL-α-tocopherol was added to the stored fraction (d) in an amount of 0.05% relative to the EPA-E, and the methanol was then removed using an eluent evaporator. The methanol removal in the eluent evaporator was carried out by blowing a small amount of nitrogen under an absolute vacuum of 35.8 kPa to 36.2 kPa, gradually increasing the temperature from 41°C to 42°C, and finally to 130°C to 133°C. This intermediate, EPA-E (e), with a residual methanol content of approximately 500 ppm, was stored in a buffer tank. Meanwhile, the liquefied methanol (f) obtained after vacuum distillation was recovered in the eluent tank.

[0128] The buffer tank was maintained at an absolute vacuum of 35.8 kPa to 36.2 kPa and a temperature of 130°C to 133°C. The volume ratio of intermediate EPA-E to headspace was 6:94 to 9:91. Here, the volume ratio is "intermediate EPA-E:headspace." Meanwhile, the methanol (h) liquefied after vacuum distillation was recovered in the eluent tank.

[0129] The stored intermediate EPA-E (g) was further de-methanolized using a thin-film evaporator. The de-methanolization in the thin-film evaporator was carried out at an absolute vacuum of 1 to 20 Pa and a temperature of 110 to 127°C until the residual methanol content was 5 ppm or less. DL-α-tocopherol was added to this product in a total amount of 0.2% based on the EPA-E, and the product was stored in a product tank as purified EPA-E (i). Meanwhile, the small amount of methanol (j) liquefied after vacuum distillation was withdrawn into an eluent receiver as waste solvent.

[0130] The product tank was kept at a temperature of 20°C to 22°C, and the volume ratio of purified EPA-E to headspace was 0:100 to 81:19. Here, the volume ratio was "purified EPA-E:headspace." The product tank was purged with nitrogen, and the gas in the headspace was released using the installed gas seal unit and breather valve (G) when the purified EPA-E was received, and nitrogen was blown in when the purified EPA-E was transferred, purging the headspace of the product tank with nitrogen.

[0131] Example 1: Effect of oxygen concentration in eluent tank on the amount of dissolved oxygen in the eluent In Example 1a, methanol obtained by vacuum distillation and liquefaction was dropped into an eluent tank that had been purged with nitrogen blow to a headspace oxygen concentration of 0.00% by volume, and the headspace oxygen concentration and the amount of dissolved oxygen in the methanol eluent were measured. In Example 1b, the headspace oxygen concentration was set to 12.50% by volume, and the other procedures were the same as in Example 1a.

[0132] In this example, the temperature for measuring the amount of dissolved oxygen in methanol was 23°C, and the measurement time from when the dissolved oxygen meter sensor was placed in the methanol to when the measurement was started until the value on the dissolved oxygen meter was read was 15 minutes. The results are shown in Table 1. This example shows that lowering the headspace oxygen concentration in the eluent tank by purging the eluent tank with nitrogen led to a reduction in the amount of dissolved oxygen in the eluent.

[0133]

[0134] Example 2 Oxygen Concentration in Fraction Tank with and without Nitrogen Purging of Eluent Tank Example 2a With Nitrogen Purging of Eluent Tank Methanol obtained by vacuum distillation was added dropwise to an eluent tank purged by blowing nitrogen into the eluent tank and stored as an eluent. The methanol was passed through an HPLC and stored in a nitrogen-purged fraction tank. The volume ratio of methanol, which is the fraction in the fraction tank, to the headspace before storage was 5:95, and the volume ratio of the fraction tank after storage was 65:35. Thereafter, the methanol, which is the fraction, was transferred while blowing nitrogen to return the volume ratio to 5:95 before storage. Here, these volume ratios are "fraction:headspace."

[0135] This cycle of storing the fractions and transferring them while blowing nitrogen was repeated 10 times, and the oxygen concentration in the fraction tank was measured over time. The results are shown in Table 2.

[0136] Example 2b: No nitrogen purge of eluent tank. Methanol obtained by vacuum distillation was added dropwise to an eluent tank whose headspace was filled with air without nitrogen blowing, and stored as the eluent. The methanol was passed through an HPLC and stored in a nitrogen-purged fraction tank. In this example, the conditions other than the nitrogen purging of the eluent tank were the same as in Example 2a. The results are shown in Table 2. This example demonstrates that nitrogen purging of the eluent tank can reduce the oxygen concentration in the headspace of the fraction tank. It can be seen that without nitrogen purging, oxygen enters the fraction tank along with the methanol during storage, and that the change in oxygen concentration becomes approximately constant when the storage and transfer cycle is repeated five or more times.

[0137]

[0138] Example 3 Headspace oxygen concentration in the eluent tank and color b value of intermediate EPA-E in the buffer tank Changes in the color b value of intermediate EPA-E in the buffer tank after HPLC purification and evaporation of the eluent were confirmed, depending on whether or not the eluent tank was purged with nitrogen.

[0139] The main fraction stored in the nitrogen-purged main fraction tank was purified by HPLC using the methanol stored in the eluent tank as the eluent. Fractions with eicosapentaenoic acid ethyl ester content of 96 area % or more in the fatty acid composition were stored in nitrogen-purged fraction tanks. DL-α-tocopherol was added to the stored fraction in an amount of 0.05% relative to EPA-E, and the methanol was removed using an eluent evaporator. The resulting intermediate EPA-E with a residual methanol content of approximately 500 ppm was stored in a tank. In this example, the volume ratio of fraction to headspace was set to 5:95 to 65:35, and the storage and transfer cycle was repeated as in Example 2. Here, the volume ratio is "fraction:headspace."

[0140] After repeating the cycle of storing and transferring fractions to the fraction tank five or more times, the headspace oxygen concentration in the eluent tank and the color b value of the intermediate EPA-E in the buffer tank were measured. The results are shown in Table 3. This example demonstrates that the color difference (color b value) of the intermediate EPA-E can be reduced by using the eluent in the eluent tank that had been purged with nitrogen.

[0141]

[0142] Example 4 Nitrogen purging of the eluent tank and physical properties of purified EPA-E The effects of nitrogen purging of the eluent tank on the color b value, anisidine value, and acid value of purified EPA-E in the product tank after HPLC purification, eluent evaporation, and thin-film evaporation were confirmed.

[0143] Intermediate EPA-E was stored in a buffer tank in the same manner as in Example 3. This intermediate EPA-E was further demethanolized using a thin-film evaporator, and then DL-α-tocopherol was added to the intermediate EPA-E to adjust the total amount to 0.2% based on the EPA-E, and the resulting mixture was stored as purified EPA-E in a nitrogen-purged product tank.

[0144] The results of measuring the physical properties and fatty acid composition of purified EPA-E with and without nitrogen purging of the eluent tank are shown in Table 4. This example shows that the color b value and anisidine value of purified EPA-E can be reduced by using a nitrogen-purged eluent. On the other hand, there was almost no difference in the acid value, residual methanol amount, or isomer ratio.

[0145]

[0146] V: Vent pipe G: Gas seal unit and breather valve a: Main fraction b: Eluent (methanol) c: Fraction d: Saved fraction e: Intermediate EPA-E f: Methanol liquefied after distillation under reduced pressure g: Saved intermediate EPA-E h: Methanol liquefied after distillation under reduced pressure i: Purified EPA-E j: Small amount of methanol liquefied after distillation under reduced pressure

Claims

1. A method for producing a fatty acid composition containing highly unsaturated fatty acids (hereinafter referred to as PUFAs) or their ester derivatives, (1) Place the solvent in container A and obtain the eluent in container A under an inert gas atmosphere. (2) Purify a fatty acid mixture containing PUFA or its ester derivative by chromatography using the eluent as the mobile phase to obtain a fraction containing PUFA or its ester derivative, and (3) Concentrate the fraction containing the PUFA or its ester derivative to obtain a fatty acid composition containing the PUFA or its ester derivative. A manufacturing method that includes this.

2. The manufacturing method according to claim 1, wherein the concentration of the fraction includes recovering the eluent contained in the fraction containing the PUFA or its ester derivative as a solvent by distillation under reduced pressure and / or heating.

3. The manufacturing method according to claim 1 or 2, wherein the solvent includes a solvent obtained by distillation.

4. The manufacturing method according to claim 1 or 2, wherein the solvent includes a solvent recovered from a fraction containing PUFA or its ester derivative during the concentration of the fraction.

5. The manufacturing method according to claim 1 or 2, wherein the dissolved oxygen concentration of the eluent in the container A is 20 mg / L or less, 15 mg / L or less, or 11 mg / L or less.

6. The manufacturing method according to claim 1 or 2, wherein the headspace oxygen concentration of the container A is 10% by volume or less, 5% by volume or less, 4% by volume or less, 3% by volume or less, 2% by volume or less, 1% by volume or less, 0.5% by volume or less, or 0.1% by volume or less.

7. The manufacturing method according to claim 1 or 2, wherein the fraction containing the PUFA or its ester derivative is contained in a container C under an inert gas atmosphere.

8. The manufacturing method according to claim 1 or 2, wherein the fraction containing the PUFA or its ester derivative is contained in a container C under an inert gas atmosphere, and the oxygen concentration in the headspace of the container C after the fraction is contained is 1.5% by volume or less, 1.0% by volume or less, 0.7% by volume or less, 0.5% by volume or less, 0.3% by volume or less, or 0.1% by volume or less.

9. The manufacturing method according to claim 1 or 2, wherein, before the purification of the fatty acid mixture, the fatty acid mixture containing the PUFA or its ester derivative is contained in a container B under an inert gas atmosphere.

10. The manufacturing method according to claim 9, wherein the headspace oxygen concentration of the container B is 1.5% by volume or less, 1.0% by volume or less, 0.7% by volume or less, 0.5% by volume or less, 0.3% by volume or less, or 0.1% by volume or less.

11. The manufacturing method according to claim 1 or 2, wherein the fatty acid composition containing the PUFA or its ester derivative is contained in a container D under an inert gas atmosphere.

12. The manufacturing method according to claim 11, wherein the headspace oxygen concentration of the container D is 0.7 volume% or less, 0.5 volume% or less, 0.3 volume% or less, or 0.1 volume% or less.

13. The manufacturing method according to claim 1 or 2, wherein the inert gas in containers A, B, C, and D is independently nitrogen, argon, or carbon dioxide.

14. The manufacturing method according to claim 1 or 2, wherein the inert gas in containers A, B, C, and D is nitrogen.

15. The manufacturing method according to claim 1 or 2, wherein the proportion of PUFA or its ester derivative in the total fatty acids or their ester derivatives in the fatty acid composition containing the PUFA or its ester derivative is 97% or more, 96% or more, 90% or more, 80% or more, 70% or more, or 50% or more.

16. The manufacturing method according to claim 1 or 2, wherein the color b value of the fatty acid composition containing the PUFA or its ester derivative is 3.2 or less, 3 or less, 2.8 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.7 or less.

17. The manufacturing method according to claim 1 or 2, wherein the anisidine value of the fatty acid composition containing the PUFA or its ester derivative is 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less.

18. The manufacturing method according to claim 1 or 2, wherein the amount of residual eluent in the fatty acid composition containing the PUFA or its ester derivative is 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less.

19. The manufacturing method according to claim 1 or 2, wherein the heating temperature for concentrating the fraction is a maximum of 190°C or less, 160°C or less, 140°C or less, 135°C or less, 130°C or less, 120°C or less, or 100°C or less, and / or 40°C or higher, 60°C or higher, 80°C or higher, 100°C or higher, 120°C or higher, 130°C or higher, 135°C or higher, 140°C or higher, or 160°C or higher.

20. The manufacturing method according to claim 1 or 2, wherein the minimum pressure during depressurization in the concentration of the fraction is 100 Pa or less, 50 Pa or less, 20 Pa or less, 10 Pa or less, 5 Pa or less, 2 Pa or less, or 1 Pa or less, and / or 0.1 Pa or more, 0.2 Pa or more, 0.5 Pa or more, 1 Pa or more, 2 Pa or more, 5 Pa or more, or 10 Pa or more.

21. The manufacturing method according to claim 1 or 2, wherein the solvent and eluent are one selected from methanol, ethanol, 2-propanol, acetonitrile, acetone, and hexane, or a mixture of two or more of these, or a mixture of one or more selected from these and water.

22. The manufacturing method according to claim 1 or 2, wherein the purification of the fatty acid mixture is performed using a silica gel-based adsorbent or a polymer-based adsorbent as the stationary phase.

23. The manufacturing method according to claim 1 or 2, wherein the purification of the fatty acid mixture is performed using a reversed-phase adsorbent as the stationary phase.

24. The manufacturing method according to claim 1 or 2, wherein the concentration of the fraction is performed using one or more evaporators, and the internal volume of at least one of the receivers of the evaporators is 10 L or more, 20 L or more, 50 L or more, 100 L or more, 500 L or more, or 1000 L or more, and / or 5000 L or less, 2000 L or less, 1000 L or less, or 500 L or less.

25. The manufacturing method according to claim 1 or 2, wherein the concentration of the fraction is performed using one or more evaporators, and at least one of the evaporators is blown with inert gas during the concentration of the fraction.

26. The manufacturing method according to claim 1 or 2, wherein the PUFA is one selected from EPA, DHA, n-3 DPA, DGLA, and ARA, or a combination of two or more of these.

27. The ester derivative of the aforementioned PUFA is C of PUFA １ ~C ６ The method for producing an alkyl ester derivative according to claim 1 or 2.

28. The method for producing the product according to claim 1 or 2, wherein the PUFA or its ester derivative is EPA ethyl ester, DHA ethyl ester, or a combination thereof.

29. A fatty acid composition comprising a PUFA or an ester derivative thereof, produced by the manufacturing method described in claim 1 or 2.

30. A fatty acid composition comprising highly unsaturated fatty acids (hereinafter referred to as PUFA) or their ester derivatives, wherein the proportion of PUFA or its ester derivative in the total fatty acids or their ester derivatives in the fatty acid composition is 97% or more, 96% or more, 90% or more, 80% or more, 70% or more, or 50% or more, and the color b value of the fatty acid composition is 3.2 or less, 3 or less, 2.8 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.7 or less.

31. The fatty acid composition according to claim 30, wherein the anisidine value of the fatty acid composition comprising the PUFA or its ester derivative is 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less.

32. The fatty acid composition according to claim 30 or 31, wherein the amount of residual eluent in the fatty acid composition containing the PUFA or its ester derivative is 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less.

33. The fatty acid composition according to claim 30 or 31, wherein the PUFA is one selected from EPA, DHA, n-3 DPA, DGLA, and ARA, or a combination of two or more thereof.

34. The ester derivative of the aforementioned PUFA is C of PUFA １ ~C ６ The fatty acid composition according to claim 30 or 31, wherein the derivative is an alkyl ester derivative.

35. The fatty acid composition according to claim 30 or 31, wherein the PUFA or its ester derivative is an ethyl ester of EPA or an ethyl ester of DHA.