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

Abstract

The present process is a method for producing a fatty acid composition containing a polyunsaturated fatty acid (hereinafter referred to as PUFA) or an ester derivative thereof, and is provided, wherein the method includes: storing a solvent in Container A to obtain an eluent under an inert gas atmosphere in Container A; purifying a fatty acid mixture containing PUFA or an ester derivative thereof by chromatography using the eluent as a mobile phase to obtain a fraction containing PUFA or an ester derivative thereof; and concentrating the fraction containing the PUFA or an ester derivative thereof to obtain a fatty acid composition containing the PUFA or an ester derivative thereof.

Description

TECHNICAL FIELD

[0001] This disclosure relates to a fatty acid composition containing a polyunsaturated fatty acid (hereinafter also referred to as PUFA) or an ester derivative thereof, and a method for producing the same.BACKGROUND ART

[0002] Polyunsaturated fatty acids or their ester derivatives are used as raw materials for pharmaceuticals, health foods, and cosmetics. Methods such as precision distillation, a silver nitrate complex method, and chromatography are known as processes for production to obtain high-purity polyunsaturated fatty acids or their ester derivatives for use in pharmaceuticals, health foods, and cosmetics from natural fats and oils (see, for example, PTL 1 to 4).

[0003] On the other hand, there is a problem of alteration and / or deterioration of compositions containing polyunsaturated fatty acids or their ester derivatives. For example, PTL 1 describes the provision of a composition of PUFA or an ester derivative thereof having a dissolved oxygen amount, a peroxide value, an acid value, and an anisidine value that equal to or below certain values in order to solve the problem of preventing and / or delaying the progress of oxidation during the manufacturing process and during the storage period of the product. PTL 2 points out a risk that dissolved oxygen in an eluent can lead to alteration and / or deterioration of PUFA and the like when separating and purifying PUFAs or their ester derivatives by chromatography using an adsorption resin as a carrier, and describes that in order to prevent this risk, the eluent can be used through heating and refluxing or blowing of nitrogen or carbon dioxide gas into the eluent.

[0004] Furthermore, PTL 3 describes the use of a channel-type agitator as a reaction vessel in a process for producing an ester derivative of PUFA using a silver salt solution, and describes carrying out the process under low-oxygen conditions to suppress the deterioration of the silver salt solution.

[0005] PTL4 relates to an ester derivative purification of PUFA by chromatography, including simulated moving bed chromatography and the example describes "All chromatography operations are performed under an inert gas atmosphere while protected from light".CITATION LISTPATENT LITERATURE

[0006] PTL 1: Japanese Patent Laid-Open No. 2017-114776 PTL 2: Japanese Patent Laid-Open No. 61-291540 PTL 3: Japanese Patent Laid-Open No. 2019-135307 PTL 4: Japanese Translation of PCT International Application Publication No. 2017-502130 SUMMARY OF INVENTIONTECHNICAL PROBLEM

[0007] The present inventors have discovered a problem such that in the production of a fatty acid composition containing a polyunsaturated fatty acid or an ester derivative thereof, unless the dissolved oxygen in a mobile phase is suppressed, the oxygen concentration cannot be sufficiently reduced even when a fraction containing a polyunsaturated fatty acid or an ester derivative thereof obtained by chromatography is stored under an inert gas atmosphere in a container, resulting in alteration of the polyunsaturated fatty acid or an ester derivative thereof during solvent removal, particularly a problem of coloring.SOLUTION TO PROBLEM

[0008] The present disclosure relates to providing a fatty acid composition containing a polyunsaturated fatty acid or an ester derivative thereof, in which alteration such as coloring is suppressed by the use of an eluent as a mobile phase obtained by placing it under an inert gas atmosphere in a container during chromatography for purifying the fatty acid mixture containing the polyunsaturated fatty acid or an ester derivative thereof.

[0009] In one aspect of the present disclosure, the following method for producing a fatty acid composition containing a polyunsaturated fatty acid or an ester derivative thereof is provided. In one aspect of the present disclosure the following fatty acid composition containing a polyunsaturated fatty acid or an ester derivative thereof is provided.

[0010] [1-1] A process for producing a fatty acid composition containing a polyunsaturated fatty acid (hereinafter also referred to as PUFA) or an ester derivative thereof, including: (1) storing a solvent in Container A to obtain an eluent under an inert gas atmosphere in Container A; (2) purifying a fatty acid mixture containing a PUFA or an ester derivative thereof by chromatography using the eluent as a mobile phase to obtain a fraction containing the PUFA or an ester derivative thereof; and (3) concentrating the fraction containing the PUFA or an ester derivative thereof to obtain a fatty acid composition containing the PUFA or an ester derivative thereof.

[0011] [1-2] The process according to [1-1], wherein concentrating the fraction includes recovering, as a solvent, the eluent present in the fraction containing the PUFA or an ester derivative thereof by distillation under reduced pressure and / or heating.

[0012] [1-3] The process according to [1-1] or [1-2], wherein the solvent contains a solvent obtained by distillation.

[0013] [1-4] The process according to any one of [1-1] to [1-3], wherein the solvent contains a solvent recovered from the fraction containing the PUFA or an ester derivative thereof in concentrating the fraction.

[0014] [1-5] The process according to any one of [1-1] to [1-4], wherein the dissolved oxygen concentration of the eluent in Container A is 20 mg / L or less, 15 mg / L or less, or 11 mg / L or less.

[0015] [1-6] The process according to any one of [1-1] to [1-5], wherein the headspace oxygen concentration in Container A is 10 vol% or less, 5 vol% or less, 4 vol% or less, 3 vol% or less, 2 vol% or less, 1 vol% or less, 0.5 vol% or less, or 0.1 vol% or less.

[0016] [1-7] The process according to any one of [1-1] to [1-6], wherein the fraction containing the PUFA or an ester derivative thereof is stored under an inert gas atmosphere in Container C.

[0017] [1-8] The process according to [1-7], wherein the headspace oxygen concentration in Container C after the fraction is stored is 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.

[0018] [1-9] The process according to any one of [1-1] to [1-8], wherein the fatty acid mixture containing the PUFA or an ester derivative thereof is stored under an inert gas atmosphere in Container B before the fatty acid mixture is purified.

[0019] [1-10] The process according to [1-9], wherein the headspace oxygen concentration in Container B is 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.

[0020] [1-11] The process according to any one of [1-1] to [1-10], wherein the fatty acid composition containing the PUFA or an ester derivative thereof is stored under an inert gas atmosphere in Container D.

[0021] [1-12] The process according to [1-11], wherein the headspace oxygen concentration in Container D is 0.7 vol% or less, 0.5 vol% or less, 0.3 vol% or less, or 0.1 vol% or less.

[0022] [1-13] The process according to any one of [1-1] to [1-12], wherein the inert gas in Container A, the inert gas Container B, the inert gas in Container C, and the inert gas in Container D are each independently nitrogen, argon, carbon dioxide gas, or a combination thereof.

[0023] [1-14] The process according to any one of [1-1] to [1-13], wherein the inert gas in Container A, the inert gas in Container B, the inert gas in Container C, and the inert gas in Container D are each nitrogen.

[0024] [1-15] The process according to any one of [1-1] to [1-14], wherein the proportion of the PUFA or an ester derivative thereof in all fatty acids or their ester derivatives 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.

[0025] [1-16] The process 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.

[0026] [1-17] The process 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.

[0027] [1-18] The process according to any one of [1-1] to [1-17], wherein the residual amount of eluent in the fatty acid composition containing the PUFA or an ester derivative thereof 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.

[0028] [1-19] The process 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 lower, 160°C or lower, 140°C or lower, 135°C or lower, 130°C or lower, 120°C or lower, or 100°C or lower, 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.

[0029] [1-20] The process according to any one of [1-1] to [1-19], wherein the minimum pressure under reduced pressure when the fraction is concentrated 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.

[0030] [1-21] The process according to any one of [1-1] to [1-20], wherein the solvent and eluent are one type or a mixture of two or more types selected from methanol, ethanol, 2-propanol, acetonitrile, acetone, and hexane, or a mixture of one or more types selected from these with water.

[0031] [1-22] The process according to any one of [1-1] to [1-21], wherein the fatty acid mixture is purified using a silica gel-based adsorbent or a polymer-based adsorbent as a stationary phase.

[0032] [1-23] The process according to any one of [1-1] to [1-22], wherein the fatty acid mixture is purified using a reversed-phase adsorbent as a stationary phase.

[0033] [1-24] The process according to any one of [1-1] to [1-23], wherein the fraction is concentrated 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 1,000 L or more, and / or 5,000 L or less, 2,000 L or less, 1,000 L or less, or 500 L or less.

[0034] [1-25] The process according to any one of [1-1] to [1-24], wherein the fraction is concentrated using one or a plurality of evaporators, and an inert gas is blown through at least one of the evaporators when the fraction is concentrated.

[0035] [1-26] The process according to any one of [1-1] to [1-25], wherein the PUFA is one type or a combination of two or more types selected from EPA, DHA, n-3 DPA, DGLA, and ARA.

[0036] [1-27] The process according to any one of [1-1] to [1-26], wherein the PUFA ester derivative is a C 1 - C 6 alkyl ester derivative of the PUFA.

[0037] [1-28] The process according to any one of [1-1] to [1-27], wherein the PUFA or an ester derivative thereof is an EPA ethyl ester, a DHA ethyl ester, or a combination thereof.

[0038] [1-29] The process according to any one of [1-1] to [1-28], wherein the chromatography is HPLC.

[0039] [1-30] The process according to any one of [1-1] to [1-29], wherein the volume of Container A is 50 L or more, 100 L or more, 200 L or more, 500 L or more, 1,000 L or more, 2,000 L or more, or 4,000 L or more, and / or 20,000 L or less, 10,000 L or less, 4,000 L or less, 2,000 L or less, or 1,000 L or less.

[0040] [1-31] A fatty acid composition containing a PUFA or an ester derivative thereof, which is produced by the process according to any one of [1-1] to [1-30].

[0041] [1-32] The process 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 types of solvents are used as the at least one type of solvent.

[0042] [2-1] A fatty acid composition containing a polyunsaturated fatty acid (hereinafter also referred to as PUFA) or an ester derivative thereof, wherein the proportion of the PUFA or an ester derivative thereof 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.

[0043] [2-2] The fatty acid composition according to [2-1], 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.

[0044] [2-3] The fatty acid composition according to [2-1] or [2-2], wherein the residual amount of eluent in the fatty acid composition containing the PUFA or an ester derivative thereof 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.

[0045] [2-4] The fatty acid composition according to any one of [2-1] to [2-3], wherein the PUFA is one type or a combination of two or more types selected from EPA, DHA, n-3 DPA, DGLA, and ARA.

[0046] [2-5] The fatty acid composition according to any one of [2-1] to [2-4], wherein the PUFA ester derivative is a C 1 -C 6 alkyl ester derivative of the PUFA.

[0047] [2-6] The fatty acid composition according to any one of [2-1] to [2-5], wherein the PUFA or an ester derivative thereof is an ethyl ester of EPA, an ethyl ester of DHA, or a combination thereof.ADVANTAGEOUS EFFECTS OF INVENTION

[0048] According to the present disclosure, a fatty acid composition containing a polyunsaturated fatty acid or an ester derivative thereof with suppressed coloring can be produced. Furthermore, according to the present disclosure, dissolved oxygen in the fatty acid composition containing a polyunsaturated fatty acid or an ester derivative thereof can be suppressed.BRIEF DESCRIPTION OF DRAWING

[0049] [Fig. 1] Fig. 1 is a process diagram for describing the examples, which depicts a process for producing a fatty acid composition containing an eicosapentaenoic acid ethyl ester (hereinafter, also referred to as EPA-E).DESCRIPTION OF EMBODIMENTS

[0050] The term "fatty acid(s)" as used herein refers to aliphatic carboxylic acids with various chain lengths from about C 12 to C 22 . Here, the numbers in "C 12 to C 22 " refer to the total number of carbon atoms in the carboxylic acid chain. The predominant chain length ranges from C 16 to 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 a particular fatty acid and Y is the number of double bonds. For example, a saturated fatty acid with 20 carbon atoms can be designated as "C20:0", a monounsaturated fatty acid with 18 carbon atoms can be designated as "C18:1", and arachidonic acid can be designated as "C20:4, n-6". "n-" indicates the position at which the double bond begins, counting from the methyl end of the fatty acid, e.g., "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 designated according to this method can be easily identified by those skilled in the art.

[0051] Fatty acids are carboxylic acids with aliphatic chains, which are either saturated or unsaturated. Fatty acids are usually produced industrially by hydrolysis of triglycerides or phospholipids derived from natural sources. Some are produced synthetically. Regardless of the process for production, purification methods are required to obtain pure products for foods, cosmetics or industrial uses.

[0052] As used herein, the term "polyunsaturated fatty acid" refers to a fatty acid having more than one double bond. As used herein, polyunsaturated fatty acids can also be referred to as PUFAs. A polyunsaturated fatty acid can be, for example, a fatty acid having 3 to 6 double bonds. Examples of the polyunsaturated fatty acid 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 polyunsaturated fatty acids as used herein are typically alkyl esters, for example C 1 -C 6 alkyl esters, or C 1 -C 4 alkyl esters. Examples of esters include ethyl esters.<Process for producing fatty acid composition>

[0053] One embodiment of the present invention includes a process including: storing a solvent in Container A to obtain an eluent under an inert gas atmosphere in Container A; purifying a fatty acid mixture containing PUFA or an ester derivative thereof by chromatography using the eluent as a mobile phase to obtain a fraction containing the PUFA or an ester derivative thereof; and concentrating the fraction containing the PUFA or an ester derivative thereof to obtain a fatty acid composition containing the PUFA or an ester derivative thereof.Fatty acid mixture

[0054] In one embodiment of the present invention, the fatty acid mixture containing PUFA or an ester derivative thereof can be obtained from a raw material containing the polyunsaturated fatty acid as a constituent fatty acid. For example, it can be obtained from a raw material containing natural oils and fats, including plant and animal oils and fats, and from a raw material containing oils and fats obtained from genetically modified plants, animals and microorganisms including yeast. Examples of the raw materials include fish oils, algal and microalgal oils, and vegetable oils, such as borage oil, Echium plantagineum oil and evening primrose oil.

[0055] In one embodiment of the present invention, the fatty acid mixture containing PUFA or an ester derivative thereof is obtained by subjecting a raw material to distillation.

[0056] In one embodiment of the present invention, the fatty acid mixture containing PUFA or an ester derivative thereof can contain PUFA obtained by decomposing raw-material fats and oils into free fatty acids by hydrolysis using water, or can contain an ester derivative obtained by decomposing raw-material fats and oils into C 1 -C 6 alkyl esters by alcoholysis using a C 1 -C 6 alkyl alcohol. An example of the C 1 -C 6 alkyl ester is an ethyl ester.

[0057] One embodiment of the present invention is a process including storing the fatty acid mixture containing PUFA or an ester derivative thereof under an inert gas atmosphere in Container B before the fatty acid mixture is purified. In one embodiment of the present invention, when the fatty acid mixture is stored in Container B, the inside of Container B is placed under an inert gas atmosphere. In one embodiment of the present invention, the inside of Container B can be placed under an inert gas atmosphere before, after, or simultaneously with storage of the fatty acid mixture. In one embodiment of the present invention, the fatty acid mixture is preferably stored in Container B after Container B is placed under an inert gas atmosphere.Container

[0058] In one embodiment of the present invention, Container A is a container for storing and storing an eluent, and can be a tank for storing an eluent. As used herein, the tank for storing an eluent can be referred to as an eluent tank.

[0059] In one embodiment of the present invention, Container B is a container for storing and storing the fatty acid mixture containing PUFA or an ester derivative thereof obtained from a raw material, and can be a tank for storing the fatty acid mixture. In one embodiment of the present invention, the fatty acid mixture containing PUFA or an ester derivative thereof can be a main fraction obtained by continuous rectification. Among Container B, a tank for storing the main fraction can be referred to as a main fraction tank.

[0060] In one embodiment of the present invention, Container C is a container for storing and storing a fraction containing PUFA or an ester derivative thereof obtained by purifying the fatty acid mixture, and can be a tank for storing the fraction. As used herein, the tank for storing the fraction can be referred to as a fraction tank.

[0061] In one embodiment of the present invention, Container D is a container for storing and preserving the fatty acid composition containing PUFA or an ester derivative thereof obtained by concentrating the fraction, and can be a tank for storing the fatty acid composition. As used herein, the tank for storing the fatty acid composition can be referred to as a product tank. As used herein, the tank for storing the fatty acid composition can be referred to as a buffer tank.

[0062] 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 gas, and a mixed gas of two or more types 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 gas. In one embodiment of the present invention, such a state under an inert gas atmosphere refers to a state in which oxygen, which is a reactive molecule present in the air, is removed and replaced with nitrogen, argon, or carbon dioxide gas, which is less reactive. In one embodiment of the present invention, such a state under an inert gas atmosphere refers to a state in which a gas containing mainly an inert gas and having an oxygen concentration of less than 10 vol% is present. In one embodiment of the present invention, purging with an inert gas is performed before or after the solvent, fraction, fatty acid mixture, or fatty acid composition is stored in the container.

[0063] In one embodiment of the present invention, the container can be a sealed container, for example, a tank. In one embodiment of the present invention, the container has an inert gas filling device, which is a device for filling the head space with an inert gas. Examples of the inert gas filling device include a gas seal unit and a breather valve. In one embodiment of the present invention, the container has a space containing gas, which is not filled with the solvent, eluent, fatty acid mixture, fraction, or fatty acid composition. As used herein, the space in the container containing gas, which is not filled with the solvent, eluent, fatty acid mixture, fraction, and fatty acid composition is referred to as the "head space". In one embodiment of the present invention, the volume of the container is the total volume 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 head space.

[0064] In one embodiment of the present invention, an upper limit and / or a lower limit of the headspace volume of the container is set, and can be allowed to vary within that value range.

[0065] In one embodiment of the present invention, the upper limit of the headspace volume is the headspace volume at the time point that the headspace volume is the largest and the volume of the portion filled with the solvent, eluent, fatty acid mixture, fraction and / or fatty acid composition is the smallest when the process according to the present invention is performed. As used herein, a container whose headspace volume is at the upper limit may be referred to as a container before storage. The 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.

[0066] In one embodiment of the present invention, the lower limit of the headspace volume is the headspace volume at the time point that the headspace volume is the smallest and the volume of the portion filled with the solvent, eluent, fatty acid mixture, fraction and / or fatty acid composition is the largest when the process according to the present invention is performed. As used herein, a container whose headspace volume is at the lower limit may be referred to as a container after storage. The 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.

[0067] In one embodiment of the present invention, the upper limit of the headspace volume of Container A can 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, 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 of the volume of Container A. In one embodiment of the present invention, the lower limit of the headspace volume of Container A can 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. In one embodiment of the present invention, the volume of Container A can be 50 L or more, 100 L or more, 200 L or more, 500 L or more, 1,000 L or more, 2,000 L or more, or 4,000 L or more, and / or 20,000 L or less, 10,000 L or less, 4,000 L or less, 2,000 L or less, or 1,000 L or less.

[0068] In one embodiment of the present invention, the upper limit of the headspace volume of Container B can 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 can 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 can be 500 L or more, 1,000 L or more, 2,000 L or more, 5,000 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.

[0069] In one embodiment of the present invention, the upper limit of the headspace volume of Container C can 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 C. In one embodiment of the present invention, the lower limit of the head space volume of Container C can 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. In one embodiment of the present invention, the volume of Container C can be 50 L or more, 100 L or more, 200 L or more, 500 L or more, 1,000 L or more, 2,000 L or more, or 4,000 L or more, and / or 20,000 L or less, 10,000 L or less, 4,000 L or less, 2,000 L or less, or 1,000 L or less.

[0070] In one embodiment of the present invention, the upper limit of the headspace volume of Container D can 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 can 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 can be 50 L or more, 100 L or more, 200 L or more, 500 L or more, 1,000 L or more, 2,000 L or more, or 5,000 L or more, and / or 20,000 L or less, 10,000 L or less, 5,000 L or less, 2,000 L or less, or 1,000 L or less.

[0071] In one embodiment of the present invention, the headspace oxygen concentration in a container is the concentration of oxygen in the gas present in the headspace of the container.

[0072] In one embodiment of the present invention, the headspace oxygen concentration in Container A can be 10 vol% or less, 5 vol% or less, 4 vol% or less, 3 vol% or less, 2 vol% or less, 1 vol% or less, 0.5 vol% or less, or 0.1 vol% or less. In other embodiments of the present invention, the headspace oxygen concentration in Container A can be 10 vol% or less, 5 vol% or less, 4 vol% or less, 3 vol% or less, 2 vol% or less, 1 vol% or less, 0.5 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 A can be the headspace oxygen concentration after the eluent is stored or the headspace oxygen concentration after the eluent is transferred.

[0073] In one embodiment of the present invention, the headspace oxygen concentration in 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 other embodiments of the present invention, the headspace oxygen concentration in 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 in Container B can be the headspace oxygen concentration after the fatty acid mixture is stored or the headspace oxygen concentration after the fatty acid mixture is transferred.

[0074] In one embodiment of the present invention, the headspace oxygen concentration in Container C after the fraction is stored 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 is stored 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.

[0075] In one embodiment of the present invention, the headspace oxygen concentration in Container C after the fraction is 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 embodiments of the present invention, the headspace oxygen concentration in Container C after the fraction is transferred 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% or less.

[0076] In one embodiment of the present invention, the headspace oxygen concentration in Container D can be 0.7 vol% or less, 0.5 vol% or less, 0.3 vol% or less, or 0.1 vol% or less. In one embodiment of the present invention, the headspace oxygen concentration in Container D 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% or more. 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 is stored or the headspace oxygen concentration after the fatty acid composition is transferred.

[0077] The method for measuring the headspace oxygen concentration is not particularly limited. For example, the headspace oxygen concentration can be measured using a residual oxygen meter "Pack Keeper" RO-103KS (manufactured by Iijima Electronics Corporation) according to the procedure in the instruction manual for the device.

[0078] As used herein, the above descriptions regarding containers not specified as Container A, Container B, Container C, and Container D are applied to Container A, Container B, Container C, and Container D.Eluent

[0079] In one embodiment of the present invention, the process for producing a fatty acid composition includes storing a solvent in Container A to obtain an eluent under an inert gas atmosphere in Container A. One embodiment of the present invention includes an embodiment of using a solvent usable as an eluent before being stored in Container A. In one embodiment of the present invention, when the solvent is stored in Container A, the inside of Container A is placed under an inert gas atmosphere. In one embodiment of the present invention, the inside of Container A can be placed under an inert gas atmosphere before, after, or simultaneously with storage of the solvent. In one embodiment of the present invention, the solvent is preferably stored in Container A after the inside of Container A is placed under an inert gas atmosphere.

[0080] In one embodiment of the present invention, a solvent usable as an eluent is a solvent 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 other embodiments of the present invention, a solvent usable as an eluent is a solvent 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. In one embodiment of the present invention, a solvent usable as an eluent is a liquid that is one type or a mixture of two or more types selected from alcohol, ether, ester, ketone, nitrile, hexanes, and dichloromethane 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, or, a mixture of one or more types selected from these with water. In other embodiments of the present invention, the solvent usable as the eluent is a liquid that is one type or a mixture of two or more types selected from alcohol, ether, ester, ketone, nitrile, hexanes, and dichloromethane 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, or, a mixture of one or more types selected from these with water. Examples of the alcohol 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 the alcohol include methanol and ethanol. In one embodiment, an example thereof is methanol. Examples of the ether include diethyl ether, diisopropyl ether, and methyl t-butyl ether. Examples of the ester include acetic acid methyl ester and acetic acid ethyl ester. Examples of the ketone include acetone, methyl ethyl ketone, and methyl isobutyl ketone. Examples of the nitrile include acetonitrile. In one embodiment 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 other embodiments 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.

[0081] One embodiment of the present invention is a process wherein a solvent that is stored in Container A contains a solvent obtained by distillation. In one aspect of the present invention, the solvent obtained by distillation can be obtained in concentrating a fraction, and then recovering the solvent from the fraction through distillation. In one aspect of the present invention, the solvent obtained by distillation can be obtained by purchasing a distilled solvent and / or by purchasing a solvent and then distilling it.

[0082] One embodiment of the present invention is a process wherein a solvent that is stored in Container A contains a solvent recovered from a fraction in concentrating the fraction.

[0083] One embodiment of the present invention is a process wherein concentrating a fraction includes recovering, as a solvent, an eluent present in the fraction containing PUFA or an ester derivative thereof by distillation under reduced pressure and / or heating.Purification of fatty acid mixture by chromatography

[0084] In one embodiment of the present invention, a process for producing the fatty acid composition includes purifying a fatty acid mixture containing PUFA or an ester derivative thereof by chromatography using an eluent as a mobile phase to obtain a fraction containing PUFA or an ester derivative thereof.

[0085] "Chromatography" as used herein refers to a method for separating or purifying a mixture in the process during which a substance referred to as a mobile phase passes through the surface or interior of a substance referred to as a stationary phase or carrier. Chromatography is used to analyze and separate a mixture of two or more types of substances. As used herein, column chromatography can be performed as chromatography. For example, high turbulence liquid chromatography, preparative chromatography, high performance liquid chromatography (HPLC), simulated moving bed chromatography, real moving bed chromatography, and supercritical fluid chromatography can be performed.

[0086] "Chromatographic conditions" as used herein refers to various parameters that indicate the conditions under which chromatography is performed. Examples thereof include packing pressure, mobile and stationary phase composition, 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, sedimentation time of the stationary phase during column preparation and pressure at which sedimentation is performed, and standing time and pressure at which standing is performed. A column prepared by packing with a stationary phase, or a commercially available column pre-packed with a stationary phase can be used.

[0087] In one embodiment of the present invention, 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 embedded alkyl, fluorophenylpropyl, or any stationary phase known in the art of chromatography. Examples of reversed-phase stationary phase include C 30 , C 22 , C 18 , C 8 , C 5 , C 4 , biphenyl, and fluorophenyl stationary phases. In one embodiment, a chiral stationary phase is used. The selection of the stationary phase will be apparent to those skilled in the art and can depend on the components to be purified by chromatography. A variety of different types of octadecyl silica (hereinafter, also referred to as ODS), including fully end-capped, partially end-capped, and base deactivated, can be used. More polar components can require normal phase stationary phases such as non-bonded silica, amino phase, or cyano phase.

[0088] In one embodiment of the present invention, chromatography uses a reversed-phase adsorbent as a stationary phase. Any reversed-phase distribution adsorbent can be used without any particular specification. For example, polymer beads such as polystyrene reticulated with divinylbenzene, or silica gels bonded with C 8 or C 18 alkyl groups, or an ODS column using particularly octadecylsilyl, which is silica gel bonded with C 18 alkyl groups, can be used.

[0089] In one embodiment of the present invention, the dimension of the column used is 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 4,000 mm or less, 2,000 mm or less, 1,000 mm or less, 500 mm or less, or ranges from 1 to 4,000 mm, 2 to 2,000 mm, 4 to 1,000 mm, 8 to 1,000 mm, 16 to 1,000 mm, 32 to 500 mm, 30 to 800 mm, or 400 to 800 mm.

[0090] In one embodiment of the present 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 or less, 120 cm or less, or ranges from 5 to 800 cm, 10 to 400 cm, 20 to 200 cm, 20 to 150 cm, or 20 to 120 cm.

[0091] As used herein, "eluent" refers to a liquid used as a mobile phase in chromatography, which is a phase that passes through the gaps or surface of the stationary phase while being in contact with the stationary phase. In chromatography, the eluent is used to develop and elute components such as PUFA or an ester derivative thereof that is adsorbed to a column. The eluent as used herein is sometimes referred to as the mobile phase.

[0092] In one embodiment of the present invention, the eluent is obtained under an inert gas atmosphere in a container and then stored in a column as a mobile phase to develop and elute PUFA or an ester derivative thereof. The eluent leaving the column is recovered as a fraction together with PUFA or an ester derivative thereof. The fraction is also referred to as an eluate and can contain components such as the eluent and PUFA or an ester derivative thereof.

[0093] In one embodiment of the present invention, liquids include a solvent and an eluent. The solvent and / or the eluent is one type or a mixture of two or more types selected from alcohol, ether, ester, ketone, nitrile, hexanes, and dichloromethane, or a mixture of one or more types selected from these with water. Examples of the alcohol 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 the alcohol include methanol and ethanol. In one embodiment, an example thereof is methanol. Examples of the ether include diethyl ether, diisopropyl ether, and methyl t-butyl ether. Examples of the ester include acetic acid methyl ester and acetic acid ethyl ester. Examples of the ketone include acetone, methyl ethyl ketone, and methyl isobutyl ketone. Examples of the nitrile include acetonitrile. In one embodiment of the present invention, a single liquid is used as the solvent and the eluent. In one embodiment of the present invention, methanol is used as the solvent and the eluent. In one embodiment of the present invention, a single liquid or two or less, three or less, four or less, five or less, or ten or less types of liquids are used as the solvent and eluent.

[0094] The eluent can further contain additives, including buffers and pH adjusters. The selection of the additives may be determined based on the eluent used, the stationary phase used, and the components to be purified. In some embodiments, the eluent contains an additive selected from one or more types of formic acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide. In some embodiments, the eluent may be additive-free.

[0095] In one aspect of the present invention, a solvent is stored in Container A. In one embodiment, Container A does not contain an eluent, and a solvent is stored therein and used as an eluent. In one embodiment, specifically in other embodiments before a solvent is stored, the solvent is stored in a state where the inside of Container A is placed under an inert gas atmosphere and the eluent is contained. In one embodiment, a solvent is stored in Container A placed under an inert gas atmosphere. In one embodiment, the inside of Container A is placed under an inert gas atmosphere before a solvent is stored in Container A. In one embodiment, a solvent is stored in Container A that is not under an inert gas atmosphere. In one embodiment, the inside of Container A is placed under an inert gas atmosphere after a solvent is stored therein.

[0096] In one aspect of the present invention, the dissolved oxygen concentration of a solvent stored in Container A is, for example, 20 mg / L or less, 15 mg / L or less, or 11 mg / L or less. In other aspects of the present invention, the dissolved oxygen concentration of a solvent stored 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.

[0097] In one embodiment of the present invention, the dissolved oxygen concentration of an eluent is the concentration of oxygen dissolved in the eluent in Container A, and can 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 an eluent is the concentration of oxygen dissolved in the eluent in Container A, and can 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 general method using a dissolved oxygen meter.

[0098] In one aspect of the invention, a solvent to be stored in Container A has an oxygen concentration greater than 20 mg / L. In one embodiment, the oxygen concentration of a solvent to be stored 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 the solvent is stored is used. In other embodiments, 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, 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 after the solvent is stored is used. In one aspect of the present invention, the dissolved oxygen concentration of a solvent to be stored in Container A is the concentration of oxygen dissolved in the solvent to be stored in Container A, and can 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.

[0099] In one aspect of the present invention, the concentration of oxygen dissolved in a solvent to be stored in Container A is 20 mg / L or less, e.g., 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 other aspects of the present invention, the concentration of oxygen dissolved in a solvent to be stored in Container A is 20 mg / L or less, e.g., 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.

[0100] In one aspect of the present invention, an eluent in Container A is stored in Container A at the same temperature as that for chromatography or at such temperature ±5°C, ±2.5°C, ±2°C or ±1°C, e.g., room temperature or a temperature higher than room temperature. In one embodiment, room temperature ranges from 20°C to 25°C. 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 ranges from 25°C to 60°C, 30°C to 50°C, or 35°C to 45°C.

[0101] In one embodiment of the present invention, Container A can be a tank for storing an eluent used in chromatography. As used herein, a tank for storing an eluent may be referred to as an eluent tank.

[0102] The mobile phase can contain 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 can require consideration of a polyunsaturated fatty acid or an ester derivative thereof to be purified and a stationary phase to be used. Regarding a reversed-phase stationary phase for the polyunsaturated fatty acid or an ester derivative thereof, a polar mobile phase that should be selected is sufficient to develop and elute a target polyunsaturated fatty acid or an ester derivative thereof, but develops and elutes not so fast that it approaches the front of the mobile phase.

[0103] In one embodiment of the present invention, chromatography includes a mobile phase gradient. As used herein, "mobile phase gradient" refers to a change in mobile phase composition over time while chromatography is being performed. A column can store mobile phases with different mobile phase compositions that gradually increase or decrease in percentage over time while chromatography is being performed.

[0104] The main purpose of the mobile phase gradient is to elute the strongly retained components on the column faster while eluting the weakly retained components slower so that the eluted polyunsaturated fatty acid or an ester derivative thereof produces well-resolved peaks upon detection. For example, in reversed-phase chromatography, starting with a low content of the non-polar mobile phase in the mobile phase allows the weakly retained components to be separated. The strongly retained components will either remain on the adsorbent surface at the top of the column or migrate very slowly. Increasing the amount of non-polar mobile phase, such as acetonitrile in the mobile phase allows the strongly retained components to migrate faster due to the steadily increasing competition for adsorption sites on the stationary phase by the non-polar mobile phase.

[0105] Thus, in reversed-phase chromatography used for the polyunsaturated fatty acid or an ester derivative thereof, the mobile phase at the start of the chromatography can contain a high percentage 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% of polar mobile phase A, e.g., water. Mobile phase B can be a mobile phase that is less non-polar than mobile phase A. For example, if mobile phase A is water, mobile phase B can be methanol. Mobile phase B will make up the remaining percentage of the mobile phase. As chromatography is performed and the mobile phase is eluted through the column, the gradient will result in a gradual increase in the concentration of mobile phase B over time. In some embodiments, a single-composition liquid can also be used as the mobile phase. In some embodiments, the single-composition liquid can be water, methanol, ethanol, acetonitrile, ethyl acetate, hexanes, dichloromethane, supercritical carbon dioxide, or any other liquid known in the art.

[0106] In one embodiment, the rate of increase of B over time can be constant. In one embodiment, there is no gradient and the mobile phase is of a constant composition during elution. In one embodiment, different rates of increase in the percentage of mobile phase B at different time ranges of the chromatographic conditions can be used. In one embodiment, the mobile phase can be of a specific composition during specific time ranges of the chromatographic conditions and include a gradient during other time ranges.

[0107] The mobile phase delivery system is a pumping device, such as a commercially available chromatography pump that provides a mobile phase to a column. Such pumps are generally corrosion and solvent resistant, provide pulse-free flow, flow rates ranging from 0.1 to 100 L / min, precise control of flow rate, and generate high pressure up to 6000 psi. Reciprocating pumps are composed of small chambers in which a mobile phase is stored through pumping by the back and forth movement of a motor-driven piston. Two check valves that alternately open and close control the direction and flow of the mobile phase into and out of the cylinder. Single piston pumps allow very rapid refill times using specially designed cams, creating a more continuous flow. The disadvantage of pulsed flow produced by reciprocating pumps is often overcome by the use of pulse dampers. The use of dual piston pumps, which operate with pistons moving out of phase with each other, provides a reasonable solution for pulse-free fluid delivery. The linear velocity in a column represents the speed at which the fluid passes through the cross section of the column. Linear velocity is a value that can be calculated using the following formula:

[0108] Linear velocity "m / hour" = flow rate "m 3< / hour" / 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.

[0109] In one embodiment of the present invention, chromatography is performed at room temperature or at a temperature higher than room temperature. In one embodiment, chromatography is performed at a temperature higher than room temperature. In one embodiment, room temperature is between 20°C and 25°C.

[0110] 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 ranges from 25°C to 60°C, 30°C to 50°C, or 35°C to 45°C.

[0111] As used herein, "fraction" refers to, of a fatty acid mixture containing PUFA or an ester derivative thereof that is loaded onto a column and subjected to chromatography, and an eluent that is stored in the column to develop and elute the fatty acid mixture, the eluate that is eluted from the column and then collected in small portions at specific time intervals. The process of obtaining fractions is referred to as fractionation, and in fractionation, the composition of fatty acids can vary over time. Different fractions are collected at different time points based on the unique properties of individual components, such as polyunsaturated fatty acids or their ester derivatives, in the fatty acid mixture, such as differences in their affinity for the stationary phase and / or mobile phase.

[0112] One aspect of the present invention is a process that includes storing a fraction containing PUFA or an ester derivative thereof under an inert gas atmosphere in Container C. In one embodiment of the present invention, when the fraction is stored in Container C, the inside of Container C is placed under an inert gas atmosphere. In one embodiment of the present invention, the inside of Container C can be placed under an inert gas atmosphere before, after, or simultaneously with storage of the fraction. In one embodiment of the present invention, preferably the fraction is stored in Container C after Container C is placed under an inert gas atmosphere.

[0113] In one embodiment of the present invention, Container C can be a tank for storing a fraction obtained by chromatography. As used herein, a tank for storing a fraction may be referred to as a fraction tank.Concentration of fraction

[0114] In one embodiment of the present invention, the process for producing a fatty acid composition includes concentrating a fraction containing PUFA or an ester derivative thereof obtained by chromatography to obtain the fatty acid composition containing PUFA or an ester derivative thereof.

[0115] Concentrating a fraction includes subjecting the fraction containing PUFA or an ester derivative thereof and an eluent to reduced pressure and / or heating to distill the eluent, thereby separating the PUFA or an ester derivative thereof from the eluent through the concentration. In one embodiment of the present invention, a fraction can be subjected to one or a plurality of evaporators when the fraction is concentrated. In one embodiment of the present invention, an eluent separated by distillation during the concentration of the fraction can be recovered as a solvent and reused for the production of the fatty acid composition. The solvent separated and recovered when the fraction is concentrated is stored in Container A, and the eluent can be obtained under an inert gas atmosphere in Container A.

[0116] In one embodiment of the present invention, an example of the evaporator used when the fraction is concentrated is an eluent evaporator and / or a thin film evaporator. Examples of the eluent evaporator include a single evaporator, a multiple effect evaporator, a multi-chamber evaporator, a natural circulation evaporator, a forced circulation evaporator, a falling thin film evaporator, a rising thin film evaporator, and a combination of these. In one aspect of the present invention, examples of the type of natural circulation evaporator include an external heating type and a calandria type.

[0117] In one embodiment of the present invention, any type of evaporator can be used to concentrate the fraction, such as 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 rising thin film evaporator, or a combination of these. In one aspect of the present invention, examples of the type of the natural circulation evaporator include an external heating type and a calandria type.

[0118] In one embodiment of the present invention, the internal volume of at least one of the receivers of the evaporator used to concentrate the fraction is 10 L or more, 20 L or more, 50 L or more, 100 L or more, 500 L or more, or 1,000 L or more, and / or 5,000 L or less, 2,000 L or less, 1,000 L or less, or 500 L or less.

[0119] In one embodiment of the present invention, at least one of the evaporators used to concentrate the fraction is blown with an inert gas when the fraction is concentrated. Examples of the inert gas include nitrogen, argon, carbon dioxide gas, or a combination thereof. In one embodiment, an example thereof is nitrogen. In one embodiment of the present invention, an example of the evaporator that is blown with an inert gas is an eluent evaporator.

[0120] As used herein, "inert gas blow" refers to supplying an inert gas to an apparatus or container such as an evaporator for the purpose of replacing the gas. There are no particular limitations on the amount or speed of the inert gas supplied. Nitrogen blow is one form of inert gas blow. Purging refers to blowing until the gas is replaced with an inert gas by inert gas blowing.

[0121] As used herein, "residual amount of an eluent" refers to the concentration of the eluent present in the fatty acid composition containing PUFA or an ester derivative thereof obtained through concentration of the fraction. In one embodiment of the present invention, the fatty acid composition containing PUFA or an ester derivative thereof obtained through concentration of a fraction contains 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 of an eluent. In other embodiments of the present invention, the fatty acid composition contains 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 of an eluent.

[0122] In one embodiment of the present invention, the fraction can be concentrated at a maximum heating temperature of 190°C or lower, 160°C or lower, 140°C or lower, 135°C or lower, 130°C or lower, 120°C or lower, or 100°C or lower, 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.

[0123] In one embodiment of the present invention, the fraction can be concentrated with a minimum pressure under reduced pressure 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. As used herein, the "minimum pressure under reduced pressure" refers to the pressure at the highest degree of vacuum under reduced pressure.

[0124] One embodiment of the present invention is a process that includes storing the fatty acid composition containing PUFA or an ester derivative thereof under an inert gas atmosphere in Container D. In one embodiment of the present invention, when the fatty acid composition is stored in Container D, the inside of Container D is placed under an inert gas atmosphere. In one embodiment of the present invention, the inside of Container D can be placed under an inert gas atmosphere before, after, or simultaneously with storage of the fatty acid composition. In one embodiment of the present invention, the fatty acid composition is preferably stored in Container D after Container D is placed under an inert gas atmosphere.

[0125] In one embodiment of the present invention, Container D can be a tank for storing the fatty acid composition containing PUFA or an ester derivative thereof that has been concentrated through concentration of the fraction. In one embodiment of the present invention, the tank for storing the fatty acid composition containing PUFA or an ester derivative thereof may be referred to as a product tank.<Fatty acid composition>

[0126] As used herein, the "color b value" refers to the value of b* in the L*a*b color system. In the L*a* b color system, L represents lightness, and a* and b* represent hue and saturation. Among them, b* represents a hue ranging from blue to yellow, and when b* is 0, it is an achromatic color, and the more positive the value, the more yellowish it is, and the more negative the value, the more blueish it is. The L*a* b color system is also referred to as CIE LAB. The color b value can be measured by a general method using a colorimeter. As used herein, the colorimeter is also referred to as a color difference meter.

[0127] In one embodiment of the present invention, the color b value of the fatty acid composition containing PUFA or an ester derivative thereof can 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 embodiments of the present invention, the color b value of the fatty acid composition can 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 embodiment of the present invention, the color b value of the fatty acid composition containing PUFA or an ester derivative thereof can be 0 or more, 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.

[0128] As used herein, "anisidine value" refers to a numerical value obtained by colorimetrically quantifying carbonyl compounds using p-anisidine for color development. This method measures the yellow color tone produced by the reaction of aldehydes, which are degradation products of fats and oils, with an anisidine reagent in the presence of acetic acid, at an absorbance of 350 nm, whereby measurement can be performed by a method described as the official method (2:4,26-81) in the Standard Methods for the Analysis of Fats, Oils, and Related Materials. For example, a UV-visible spectrophotometer can be used for the measurement. In one embodiment of the present invention, the anisidine value of the fatty acid composition containing 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 embodiments of the present 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.

[0129] The fatty acid composition obtained by the process in one embodiment of the present invention can contain one type of polyunsaturated fatty acid or an ester derivative thereof or a combination of two or more types of polyunsaturated fatty acids or their ester derivatives 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. The ester derivative can be a C 1 -C 6 alkyl ester derivative, such as a methyl, ethyl, propyl, butyl, pentyl, or hexyl ester. In one embodiment of the present invention, the fatty acid composition contains an EPA ethyl ester, a DHA ethyl ester, or a combination thereof. In one embodiment of the present invention, the fatty acid composition contains an EPA ethyl ester. In one embodiment of the present invention, the fatty acid composition can contain one type of polyunsaturated fatty acid or an ester derivative thereof or a combination of two or more types of polyunsaturated fatty acids or their ester derivatives selected from 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-gamma-linolenic acid, eicosatrienoic acid, stearidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, ozubondo acid, sardine acid, tetracosa pentaenoic acid, herring 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, henicosylic acid, behenic acid, tricosyl acid, lignoceric acid, pentacosylic acid, cerotic acid, carboceric acid, montanic acid, nonacosylic acid, melissic acid, hentriacontyl acid, lacceroic acid, psyllic acid, geddic acid, celloplastic acid, hexatriacontylic acid, heptatriacontylic acid, octatriacontylic acid, nonatriacontylic acid, and tetracontylic acid.

[0130] In one embodiment of the present invention, the fatty acid composition contains a polyunsaturated fatty acid or an ester derivative thereof 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.

[0131] Certain ranges are given 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 near or approximately the number preceded by the term. In determining whether a number is near or approximately a specifically recited number, an unrecited number that is near or approaching the recited number can be a number that provides a substantial equivalent amount to the specifically recited number in the context in which it is given. In one embodiment, "about" can refer to the number (to which it refers) ±5%, ±2.5%, ±2%, or ±1%.

[0132] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. All methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, and representative exemplary methods and materials are described herein.

[0133] Where a range of values is provided, it is understood that each value between the upper and lower limits of that range, and every other stated or intervening value in that stated range, are encompassed within the scope of the present 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 present invention. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, which are also encompassed within the scope of the present invention, and when one or both subjected to every specifically excluded limitations are included, ranges excluding either or both of limitations included are also included in the present invention. As used herein, unless otherwise specified, "%" refers to "wt% (% by weight)".

[0134] Specific embodiments described herein can naturally vary. Hence, the present invention is not limited to such specific embodiments. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention is considered limited only by the appended claims.

[0135] As used herein, singular expressions should be understood to include the plural, unless otherwise specified. Thus, singular articles (e.g., in the English language, "a," "an," "the," etc.) should be understood to include the plural, unless otherwise specified.

[0136] A UV / Vis absorbance detector composed 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 nm to 360 nm. A tungsten lamp can be used as the light source for the visible range of 360 nm to 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.

[0137] A photodiode array (hereinafter, also referred to as PDA) detector is an ultraviolet / visible absorbance detector that allows for very rapid collection of data over a selected spectral range. Absorbance spectral data for each chromatographic peak can be collected and stored. The stored data can be compared to the 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 of the non-separable components are likely to be different.

[0138] Fluorescence detectors are useful in 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 detection of the emission radiation, typically separated by a grid at a 90 degree angle to the excitation beam. The number of fluorescent species can be increased by post-column derivatization of the eluted compounds with special reagents, or by pre-column derivatization of the sample itself.

[0139] Refractive index detectors respond to almost all solutes. They are also referred to as RI detectors. The difference in the refractive index of the reference mobile phase relative to the column effluent results in the detection of the 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 a detection limit observed with absorbance detectors.

[0140] Conductivity detectors provide high sensitivity detection of all chargeable components. The detectors can be used with LC systems for simple and reliable detection of anions, cations, metals, organic acids and surfactants down to ppb levels. Addition of chemical suppressors between the column and the conductivity detector serves to reduce the conductivity of the eluate, allowing the use of gradient elution and determination of ppb levels with minimal baseline drift. For typical determination of anions at low levels, the eluate is converted to its weakly ionized low conductivity acid, reducing background noise. For example, Na 2 CO 3 is converted to carbonic acid. At the same time, the anions of the components are converted to their corresponding high conductivity acids, increasing the signal of the components relative to each other. For example, NaCl is converted to HCl.

[0141] As used herein, the "area %" which represents the proportion of each fatty acid in a composition, is determined by identifying the peaks of each component in a chart obtained by analyzing the composition using gas chromatography with a hydrogen flame ionization detector (hereinafter also referred to as GC-FID), determining the peak area of each fatty acid using the Agilent ChemStation integration algorithm, and expressing the content ratio of the component of that peak based on the ratio of each peak area to the total sum of the peak area of the fatty acids. In the field of oil chemistry, area % is used as almost synonymous with wt%. See the Standard Methods for Analysis of Fats, Oils and Related Materials, 2013 Edition, 2.4.2.1-2013, Fatty Acid Composition (FID Constant Temperature Gas Chromatography Method) and 2.4.2.2-2013, Fatty Acid Composition (FID Temperature Programmed Gas Chromatography Method) established by the Japan Oil Chemists' Society (hereinafter also referred to as JOCS). The analytical conditions for gas chromatography (hereinafter also referred to as GC) are as follows.GC-FID measurement conditions

[0142] 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 about 30 minutes Split ratio: 1:100 Make-up gas: Nitrogen 30 mL / min. Area measurement range: Approximately 2.5 times the retention time of eicosapentaenoic acid ethyl ester from the solvent peak Sample injection volume: 1.0 µL Analysis time: Approximately 75 minutes

[0143] In one embodiment of the present invention, an internal standard can be used during analysis by gas chromatography. An internal standard can be added to a sample as a reference marker to determine the relative retention time of an analyte with respect 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, 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 for use as a reference for the unknown analyte sample to be quantified.EXAMPLES

[0144] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, "%" refers to "wt% (% by weight)" unless otherwise specified.

[0145] In the examples of the present application, the following measuring instruments were used. · Headspace oxygen concentration: Measured using a residual oxygen meter "Pack Keeper" RO-103KS (manufactured by Iijima Electronics Corporation) according to the procedures in the instruction manual for the device. · Dissolved oxygen concentration in methanol: Measured using a dissolved oxygen meter: FOR-21 (manufactured by Automatic system Research Co., Ltd.) according to the procedure in the instruction manual for the device. · Color b value: Measured using color and turbidity simultaneous measuring instrument: TZ-6000 (manufactured by NIPPON DENSHOKU INDUSTRIES CO., LTD.), C light source, 2 degree visual field, and a 20 mm test tube, according to the procedure in the instruction manual for the instrument. As used herein, the color b value represents the b* value in the L* a*b* color system / CIE LAB. · Anisidine value: Measured using an ultraviolet-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 (Metrohm AG) 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 gas chromatography. The headspace and GC analytical conditions are as follows. Headspace device conditions

[0146] Headspace sampler: G4557A (Agilent Technologies) Equilibration temperature in vial: constant temperature around 100°C Equilibration time in vial: 20 minutes Injection line temperature: 150°C Carrier gas: Helium Pressurization pressure: 103 kPa Pressurization time: 0.01 min. Sample injection volume: 1.0 mL GC-FID measurement conditions

[0147] 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: Keep at about 40°C for 5 minutes → Increase at 30°C / min → Keep at about 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: Adjust so that the retention time of methanol is about 4.3 minutes Split ratio: 1:10 Make-up gas: Nitrogen 25 mL / min. Vial capacity: 10 mL Vial agitation level: 5 Analysis time: Approximately 16.7 minutes <Method for preparing main fraction>

[0148] Fish oil was ethyl-esterified by a conventional method to prepare a fish oil ethyl ester having 16 area % or more of EPA-E in the fatty acid ethyl ester. The fish oil ethyl ester was continuously rectified using a multi-stage rectification apparatus under the conditions of a column top pressure of 26.7 Pa or less and a temperature of 190°C or lower, 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 having a higher volatility than that of the main fraction, and is mainly composed of ethyl esters of fatty acids having less than C 20 . The main fraction is mainly composed of a C 20 fatty acids ethyl ester, and has 70 area % or more of EPA-E in the fatty acid ethyl ester. The residual fraction is a fraction having a lower volatility than that of the main fraction, and is mainly composed of fatty acid ethyl esters having C 21 or more.

[0149] The main fraction can be prepared by referring to the methods described in, for example, Japanese Patent Laid-Open Nos. 04-041457, 04-128250, and 05-222392.<HPLC purification method>

[0150] Purified EPA-E was produced from the main fraction according to the process diagram shown in Fig. 1. Specifically, the main fraction in the main fraction tank was purified by HPLC using the eluent in the eluent tank, and the obtained fraction was stored in a fraction tank. The fraction was concentrated using an eluent evaporator, and then the obtained intermediate EPA-E was stored in a buffer tank. The intermediate EPA-E was further concentrated using a thin film evaporator, and then the obtained purified EPA-E was stored in a product tank. The main fraction contains 70 area % or more of EPA-E in the fatty acid ethyl esters, and the purified EPA-E contains 96 area % or more of EPA-E in the fatty acid ethyl esters.

[0151] All the devices in this process diagram were closed systems except for the eluent tank. The headspaces of the main fraction tank, fraction tank, and product tank, other than the depressurized eluent evaporator, buffer tank, and thin film evaporator, were each purged with nitrogen using a gas seal unit and a breather valve. As used herein, the main fraction is included in the fatty acid mixture containing PUFA or an ester derivative thereof, and the intermediate EPA-E and purified EPA-E are included in the fatty acid composition containing PUFA or an ester derivative thereof.

[0152] The eluent tank was kept at a temperature between 38°C and 42°C, and the volume ratio of methanol used as the eluent to the headspace was set to range from 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.

[0153] The temperature of the main fraction tank was set to range from 39.5°C to 40.5°C, and the volume ratio of the main fraction to the headspace was set to range from 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.

[0154] In the HPLC, the main fraction (a) transferred from the main fraction tank was purified at a temperature between 38°C and 42°C using methanol (b) in the eluent tank as an eluent and ODS as a stationary phase, and the fraction (c) containing 96 area% or more of EPA-E in fatty acid ethyl esters was stored in the fraction tank.

[0155] The temperature of the fraction tank was not regulated, and was gradually decreased from approximately 40°C. The volume ratio of the fraction to the headspace was set to range from 5:95 to 65:35. Here, the volume ratio is "fraction:headspace". The fraction tank had been purged with nitrogen, and the gas in the headspace was released using the installed gas seal unit and breather valve (G) when the fraction was received from HPLC, and nitrogen was blown in when the fraction was transferred to the eluent evaporator, purging the headspace of the fraction tank with nitrogen.

[0156] After adding DL-α-tocopherol in an amount of 0.05% relative to EPA-E to the stored fraction (d), demethanolization was performed using an eluent evaporator. Demethanolization in the eluent evaporator was carried out by blowing in a small amount of nitrogen while setting the degree of vacuum to range from 35.8 kPa to 36.2 kPa in terms of absolute pressure, gradually increasing the temperature between 41°C and 42°C, and finally to 130°C to 133°C, and the intermediate EPA-E (e) with a residual amount of methanol of approximately 500 ppm was stored in a buffer tank. Meanwhile, methanol (f) liquefied after vacuum distillation was recovered into the eluent tank.

[0157] The buffer tank was set to have the degree of vacuum of 35.8 kPa to 36.2 kPa in terms of absolute vacuum and a temperature between 130°C and 133°C. The volume ratio of intermediate EPA-E to the headspace was set to range from 6:94 to 9:91. Here, the volume ratio is "intermediate EPA-E:headspace". Meanwhile, methanol (h) liquefied after vacuum distillation was recovered into the eluent tank.

[0158] The stored intermediate EPA-E (g) was further de-methanolized using a thin-film evaporator. The further demethanolization in the thin-film evaporator was carried out with the degree of vacuum set to range from 1 to 20 Pa in terms of absolute pressure and at a temperature between 110°C and 127°C, until the residual amount of methanol was 5 ppm or less. DL-α-tocopherol was added to this to make a total amount of 0.2% relative to the EPA-E, and the resultant was stored in a product tank as purified EPA-E (i). Meanwhile, a small amount of methanol (j) liquefied after vacuum distillation was extracted into an eluent receiver as waste solvent.

[0159] The product tank was set at a temperature between 20°C and 22°C, and the volume ratio of purified EPA-E to the headspace was set to range from 0:100 to 81:19. Here, the volume ratio is "purified EPA-E:headspace." The product tank had been purged with nitrogen, and the headspace of the product tank was purged with nitrogen by releasing gas from the headspace when the purified EPA-E was received using the installed gas seal unit and breather valve (G), and nitrogen blowing was performed when the purified EPA-E was transferred.Example 1: Effect of oxygen concentration in eluent tank on the amount of dissolved oxygen in eluent

[0160] In Example 1a, methanol obtained by liquefaction after vacuum distillation was dropped into an eluent tank that had been subjected to purging by nitrogen blowing to result in a headspace oxygen concentration of 0.00 vol%, and the headspace oxygen concentration and the amount of dissolved oxygen in methanol, which was the eluent at that time, were measured. In Example 1b, the headspace oxygen concentration was set to 12.50 vol%, and the rest of the experiment was carried out in the same manner as in Example 1a.

[0161] In this example, the temperature for measuring the amount of dissolved oxygen in methanol was set at 23°C, and the measurement time from inserting the dissolved oxygen meter sensor into methanol to start the measurement until reading the value on the dissolved oxygen meter was set to 15 minutes. The results are shown in Table 1. From this example, it can be seen 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. [Table 1]Table 1 Effects of the presence or absence of nitrogen purging of eluent tank on headspace oxygen concentration and the amount of dissolved oxygen in solventHeadspace oxygen concentration (vol%)Amount of dissolved oxygen in methanol (mg / L)Example 1a0.0010.09Example 1b12.5024.17 Example 2 Oxygen concentrations in fraction tank depending on the presence or absence of nitrogen purging of eluent tankExample 2a Perform nitrogen purging of eluent tank

[0162] Methanol obtained by vacuum distillation was dropped into the eluent tank purged by blowing nitrogen into the eluent tank subjected in advance to nitrogen purging and then stored as the eluent. The methanol was passed through HPLC and stored in a nitrogen purged fraction tank. The volume ratio of methanol, which is a fraction in the fraction tank before storage, to the headspace was set at 5:95, and the volume ratio of the same in the fraction tank after storage was set at 65:35. Thereafter, methanol, which is a fraction, was transferred while blowing nitrogen to return to the volume ratio of 5:95 before storage. Here, such volume ratio is "fraction:headspace".

[0163] The cycle of storing the fraction and transferring it while blowing nitrogen was repeated 10 times, and changes in oxygen concentration in the fraction tank were measured. The results are shown in Table 2.Example 2b No nitrogen purging of eluent tank

[0164] Methanol obtained by vacuum distillation was dropped into an eluent tank in which the head space was filled with air without nitrogen blowing, and stored as an eluent. The methanol was passed through 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 those in Example 2a. The results are shown in Table 2. This example shows that the headspace oxygen concentration in the fraction tank can be reduced by purging the eluent tank with nitrogen. It also shows that, without nitrogen purging, oxygen enters together with methanol upon storage, and that the change in oxygen concentration becomes almost constant when the storage and transfer cycle is repeated five or more times. [Table 2]Table 2 Effects of the presence or absence of nitrogen purging of eluent tank on oxygen concentration in fraction tankNitrogen purging of eluent tankCycleHeadspace oxygen concentration in eluent tank (vol%)Headspace oxygen concentration in fraction tank (vol%)After containingAfter transferExample 2aPresentBefore start0.110.0310.070.200.0820.030.080.0330.030.010.0140.000.00-0.0150.07-0.01-0.036-0.030.02-0.0270.010.040.0181.630.010.0290.010.03-0.02100.00-0.020.01Example 2bAbsentBefore startOxygen concentration in air0.0810.560.2021.090.4831.390.5641.620.6051.850.6961.870.7271.790.7281.810.7591.580.74101.880.77* Vented to atmosphere via a vent tube. Example 3 Headspace oxygen concentration in eluent tank and color b value of intermediate EPA-E in buffer tank

[0165] Changes in the color b value of intermediate EPA-E in the buffer tank subjected to HPLC purification and evaporation of the eluent, depending on the presence or absence of nitrogen purging of the eluent tank, were confirmed.

[0166] The main fraction stored in the nitrogen-purged main fraction tank was purified by HPLC using methanol stored as an eluent in the eluent tank, and a fraction in which the proportion of eicosapentaenoic acid ethyl ester in the fatty acid composition was 96 area % or more was stored in a nitrogen-purged fraction tank. DL-α-tocopherol was added to the stored fraction in an amount of 0.05% relative to EPA-E, and removal of methanol was performed using an eluent evaporator, and the resultant was stored in a tank as intermediate EPA-E with a residual amount of methanol of about 500 ppm.

[0167] In this example, the volume ratio of the fraction to the head space was set to range from 5:95 to 65:35, and the cycle of storage and transfer was repeated in the same manner as in Example 2. Here, the volume ratio is "fraction:head space."

[0168] After the cycle of storage and transfer in / to the fraction tank was repeated 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.

[0169] This example shows that the color difference (color b value) of the intermediate EPA-E can be reduced by using the eluent in the nitrogen-purged eluent tank. [Table 3]Table 3 Effects of the presence or absence of nitrogen purging of eluent tank on the color b value of intermediate EPA-ErunNitrogen purging of eluent tankHeadspace oxygen concentration in eluent tank (vol%)Color b value of intermediate EPA-E in buffer tank1Present0.220.4920.230.2230.180.194Absent19.42.81 Example 4 Nitrogen purging of eluent tank and physical properties of purified EPA-E

[0170] The effects of the presence or absence 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 subjected to HPLC purification, eluent evaporation, and thin-film evaporation were confirmed.

[0171] Intermediate EPA-E was stored in a buffer tank according to the same procedure as in Example 3. This intermediate EPA-E was further de-methanolized using a thin-film evaporator, and DL-α-tocopherol was added to adjust the total amount to 0.2% relative to EPA-E, and the resultant was stored as purified EPA-E in a nitrogen-purged product tank.

[0172] The physical properties and fatty acid composition of purified EPA-E depending on the presence or absence of nitrogen purging of the eluent tank were measured, and the results are shown in Table 4.

[0173] This example shows that the color b value and anisidine value of purified EPA-E can be reduced by using a nitrogen purged eluent, while there was almost no difference in the acid value, residual amount of methanol, or proportion of isomers. [Table 4]Table 4 Effects of the presence or absence of nitrogen purging of eluent tank on the physical properties of purified EPA-ErunNitrogen purgingColor b valueAnisidine valueAcid valueResidual amount of methanol(ppm)1Present0.640.230.17≤0.16*20.150.120.15≤0.16*30.150.110.15≤0.16*40.200.110.16≤0.16*5Absent3.230.900.15≤0.16*63.661.060.15≤0.16* runNitrogen purgingEPA-E (%)Isomer A (%)Isomer B+B' (%)Isomer C (%)Isomer D+E (%)1Present97.990.220.190.110.29297.990.200.200.110.30397.990.200.190.110.30497.970.210.190.110.305Absent98.030.210.180.110.30698.040.200.180.110.29 * ≤0.16: Residual amount of methanol was equal to or less than the detection limit, 0.16 ppm or less. REFERENCE SIGNS LIST

[0174] V: Vent pipe G: Gas seal unit and breather valve a: Main fraction b: Eluent (methanol) c: Fraction d: Stored fraction e: Intermediate EPA-E f: Methanol liquefied after vacuum distillation g: Stored intermediate EPA-E h: Methanol liquefied after vacuum distillation i: Purified EPA-E j: A small amount of methanol liquefied after vacuum distillation

Claims

1. A process for producing a fatty acid composition containing a polyunsaturated fatty acid (hereinafter also referred to as PUFA) or an ester derivative thereof, comprising: (1) storing a solvent in Container A to obtain an eluent under an inert gas atmosphere in Container A; (2) purifying a fatty acid mixture containing a PUFA or an ester derivative thereof by chromatography using the eluent as a mobile phase to obtain a fraction containing the PUFA or an ester derivative thereof; and (3) concentrating the fraction containing the PUFA or an ester derivative thereof to obtain a fatty acid composition containing the PUFA or an ester derivative thereof.

2. The process according to claim 1, wherein concentrating the fraction comprises recovering, as a solvent, the eluent present in the fraction containing the PUFA or an ester derivative thereof by distillation under reduced pressure and / or heating.

3. The process according to claim 1 or 2, wherein the solvent contains a solvent obtained by distillation.

4. The process according to any one of claims 1 to 3, wherein the solvent contains a solvent recovered from the fraction containing the PUFA or an ester derivative thereof in concentrating the fraction.

5. The process according to any one of claims 1 to 4, wherein the dissolved oxygen concentration of the eluent in Container A is 20 mg / L or less, 15 mg / L or less, or 11 mg / L or less.

6. The process according to any one of claims 1 to 5, wherein the headspace oxygen concentration in Container A is 10 vol% or less, 5 vol% or less, 4 vol% or less, 3 vol% or less, 2 vol% or less, 1 vol% or less, 0.5 vol% or less, or 0.1 vol% or less.

7. The process according to any one of claims 1 to 6, wherein the fraction containing the PUFA or an ester derivative thereof is stored under an inert gas atmosphere in Container C.

8. The process according to any one of claims 1 to 7, wherein the fraction containing the PUFA or an ester derivative thereof is stored under an inert gas atmosphere in Container C and the headspace oxygen concentration in Container C after the fraction is stored is 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.

9. The process according to any one of claims 1 to 8, wherein the fatty acid mixture containing the PUFA or an ester derivative thereof is stored under an inert gas atmosphere in Container B before the fatty acid mixture is purified.

10. The process according to claim 9, wherein the headspace oxygen concentration in Container B is 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.

11. The process according to any one of claims 1 to 10, wherein the fatty acid composition containing the PUFA or an ester derivative thereof is stored under an inert gas atmosphere in Container D.

12. The process according to claim 11, wherein the headspace oxygen concentration in Container D is 0.7 vol% or less, 0.5 vol% or less, 0.3 vol% or less, or 0.1 vol% or less.

13. The process according to any one of claims 1 to 12, wherein the inert gas in Container A, the inert gas in Container B, the inert gas in Container C, and the inert gas in Container D are each independently nitrogen, argon, or carbon dioxide gas.

14. The process according to any one of claims 1 to 13, wherein the inert gas in Container A, the inert gas in Container B, the inert gas in Container C, and the inert gas in Container D are each nitrogen.

15. The process according to any one of claims 1 to 14, wherein the proportion of the PUFA or an ester derivative thereof in all fatty acids or their ester derivatives 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.

16. The process according to any one of claims 1 to 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.

17. The process according to any one of claims 1 to 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.

18. The process according to any one of claims 1 to 17, wherein the residual amount of eluent in the fatty acid composition containing the PUFA or an ester derivative thereof 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 process according to any one of claims 1 to 18, wherein the heating temperature for concentrating the fraction is a maximum temperature of 190°C or lower, 160°C or lower, 140°C or lower, 135°C or lower, 130°C or lower, 120°C or lower, or 100°C or lower, 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 process according to any one of claims 1 to 19, wherein the minimum pressure under reduced pressure when the fraction is concentrated 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 process according to any one of claims 1 to 20, wherein the solvent and eluent are one type or a mixture of two or more types selected from methanol, ethanol, 2-propanol, acetonitrile, acetone, and hexane, or a mixture of one or more types selected from these with water.

22. The process according to any one of claims 1 to 21, wherein the fatty acid mixture is purified using a silica gel-based adsorbent or a polymer-based adsorbent as a stationary phase.

23. The process according to any one of claims 1 to 22, wherein the fatty acid mixture is purified using a reversed-phase adsorbent as a stationary phase.

24. The process according to any one of claims 1 to 23, wherein the fraction is concentrated using one or a plurality of 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 1,000 L or more, and / or 5,000 L or less, 2,000 L or less, 1,000 L or less, or 500 L or less.

25. The process according to any one of claims 1 to 24, wherein the fraction is concentrated using one or a plurality of evaporators, and an inert gas is blown through at least one of the evaporators when the fraction is concentrated.

26. The process according to any one of claims 1 to 25, wherein the PUFA is one type or a combination of two or more types selected from EPA, DHA, n-3 DPA, DGLA, and ARA.

27. The process according to any one of claims 1 to 26, wherein the PUFA ester derivative is a C1 to C6 alkyl ester derivative of the PUFA.

28. The process according to any one of claims 1 to 27, wherein the PUFA or an ester derivative thereof is an EPA ethyl ester, a DHA ethyl ester, or a combination thereof.

29. A fatty acid composition comprising a PUFA or an ester derivative thereof, which is produced by the process according to any one of claims 1 to 28.

30. A fatty acid composition comprising a polyunsaturated fatty acid (hereinafter also referred to as PUFA) or an ester derivative thereof, wherein the proportion of the PUFA or an ester derivative thereof 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.

31. The fatty acid composition according to claim 30, wherein the anisidine value of the fatty acid composition comprising 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.

32. The fatty acid composition according to claim 30 or 31, wherein the residual amount of eluent in the fatty acid composition comprising the PUFA or an ester derivative thereof 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 any one of claims 30 to 32, wherein the PUFA is one type or a combination of two or more types selected from EPA, DHA, n-3 DPA, DGLA, and ARA.

34. The fatty acid composition according to any one of claims 30 to 33, wherein the PUFA ester derivative is a C1 to C6 alkyl ester derivative of the PUFA.

35. The fatty acid composition according to any one of claims 30 to 34, wherein the PUFA or an ester derivative thereof is an ethyl ester of EPA or an ethyl ester of DHA.

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