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

Abstract

Provided is a method for producing a fatty acid composition containing a highly unsaturated fatty acid (hereinafter referred to as PUFA) or an ester derivative thereof, the method comprising: accommodating a solvent in a container A, and obtaining an eluent in the container A under an inert gas atmosphere; purifying a fatty acid mixture comprising PUFA or an ester derivative thereof by chromatography using the eluent as a mobile phase to obtain a fraction comprising PUFA or an ester derivative thereof; and concentrating the fraction comprising the PUFA or the ester derivative thereof to obtain a fatty acid composition comprising the PUFA or the ester derivative thereof.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480043664.8 (22) Application Date 2024.06.27 (30) Priority Data 2023-108372 2023.06.30 JP (85) PCT International Application Entering National Phase Date 2025.12.29 (86) PCT International Application Application Data PCT / JP2024 / 023288 2024.06.27 (87) PCT International Application Publication Data WO2025 / 005168 JA 2025.01.02 (71) Applicant: Nissui Co., Ltd. Address: Tokyo, Japan (72) Inventors: Takaaki Kono, Shunsuke Higashisaka, Takeshi Ebi, Yusuke Tateishi, Kuri Ryū, Keishi Matsuda, Seizo Sato, Kiyoyo Furuhata, Hideaki Yamaguchi (74) Patent Agency: Beijing Huisicheng Intellectual Property Agency Co., Ltd. 11444 Patent Attorney: Ming Sun (51) Int.Cl. C11C 1 / 08 (2006.01) (54) Invention Title: Composition Containing Highly Unsaturated Fatty Acids or Ester Derivatives Thereof and Method of Manufacturing Thereof (57) Abstract: This manufacturing method provides a method for manufacturing a fatty acid composition containing highly unsaturated fatty acids (hereinafter referred to as PUFA) or ester derivatives thereof, comprising: receiving a solvent in container A, and in container A An eluent is obtained internally under an inert gas atmosphere; a mixture of fatty acids containing PUFA or its ester derivatives is purified by chromatography using the eluent as the mobile phase to obtain a fraction containing PUFA or its ester derivatives; and the fraction containing PUFA or its ester derivatives is concentrated to obtain a fatty acid composition containing PUFA or its ester derivatives. Claims 3 pages, Description 23 pages, Drawings 1 page, CN 121443710 A 2026.01.30 CN 1 21 44 37 10 A 1. A method for manufacturing a fatty acid composition, characterized in that the fatty acid composition comprises a highly unsaturated fatty acid, i.e., PUFA, or an ester derivative thereof, wherein the manufacturing method comprises: (1) containing a solvent in a container A, and obtaining an eluent inside the container A under an inert gas atmosphere; (2) purifying the fatty acid mixture containing PUFA or an ester derivative thereof by chromatography using the eluent as the mobile phase, to obtain a fraction containing PUFA or an ester derivative thereof; and (3) concentrating the fraction containing PUFA or an ester derivative thereof to obtain a fatty acid composition containing PUFA or an ester derivative thereof. 2. The manufacturing method according to claim 1, wherein the concentration of the fraction comprises: subjecting the eluent contained in the fraction containing PUFA or an ester derivative thereof to reduced pressure and / or heating, and recovering it as a solvent by distillation.3. The manufacturing method according to claim 1 or 2, wherein the solvent comprises a solvent obtained by distillation. 4. The manufacturing method according to any one of claims 1 to 3, wherein the solvent comprises a solvent recovered from a fraction containing PUFA or its ester derivatives through concentration of the fraction. 5. The manufacturing method according to any one of claims 1 to 4, wherein the dissolved oxygen concentration of the eluent inside container A is less than 20 mg / L, less than 15 mg / L, or less than 11 mg / L. 6. The manufacturing method according to any one of claims 1 to 5, wherein the oxygen concentration in the top space of container A is less than 10% by volume, less than 5% by volume, less than 4% by volume, less than 3% by volume, less than 2% by volume, less than 1% by volume, less than 0.5% by volume, or less than 0.1%. 7. The manufacturing method according to any one of claims 1 to 6, wherein the fraction containing PUFA or its ester derivatives is contained inside container C under an inert gas atmosphere. 8. The manufacturing method according to any one of claims 1 to 7, wherein the fraction containing PUFA or its ester derivative is contained inside container C under an inert gas atmosphere, and the oxygen concentration in the top space of container C after containing the fraction 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 manufacturing method according to any one of claims 1 to 8, wherein, before purification of the fatty acid mixture, the fatty acid mixture containing PUFA or its ester derivative is contained inside container B under an inert gas atmosphere. 10. The manufacturing method according to claim 9, wherein the oxygen concentration in the top space of 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 manufacturing method according to any one of claims 1 to 10, wherein the fatty acid composition containing PUFA or its ester derivative is contained inside container D under an inert gas atmosphere. 12. The manufacturing method according to claim 11, wherein the oxygen concentration in the top space of the container D is less than 0.7% by volume, less than 0.5% by volume, less than 0.3% by volume, or less than 0.1% by volume. 13. The manufacturing method according to any one of claims 1 to 12, wherein the inert gas inside the containers A, B, C, and D is independently nitrogen, argon, or carbon dioxide. 14. The manufacturing method according to any one of claims 1 to 13, wherein the inert gas inside the containers A, B, C, and D is nitrogen.15. The manufacturing method according to any one of claims 1 to 14, wherein, in the fatty acid composition comprising PUFA or its ester derivative, the proportion of PUFA or its ester derivative is 97% or more, 96% or more, 90% or more, 80% or more, 70% or more, or 50% or more. (Claims 1 / 3 page 2 CN 121443710 A) 16. The manufacturing method according to any one of claims 1 to 15, wherein the color b value of the fatty acid composition comprising PUFA or its ester derivative is 3.2 or less, 3 or less, 2.8 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.7 or less. 17. The manufacturing method according to any one of claims 1 to 16, wherein the anisidine value of the fatty acid composition comprising PUFA or its ester derivative is 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less. 18. The manufacturing method according to any one of claims 1 to 17, wherein the residual elution dose of the fatty acid composition comprising PUFA or its ester derivative is less than 100 ppm, less than 50 ppm, less than 10 ppm, less than 5 ppm, less than 1 ppm, less than 0.5 ppm, or less than 0.16 ppm. 19. The manufacturing method according to any one of claims 1 to 18, wherein the heating temperature for concentrating the fraction is a maximum temperature of less than 190°C, less than 160°C, less than 140°C, less than 135°C, less than 130°C, less than 120°C, or less than 100°C, and / or more than 40°C, more than 60°C, more than 80°C, more than 100°C, more than 120°C, more than 130°C, more than 135°C, more than 140°C, or more than 160°C. 20. The manufacturing method according to any one of claims 1 to 19, wherein the minimum pressure during the concentration of the fraction under reduced pressure is less than 100 Pa, less than 50 Pa, less than 20 Pa, less than 10 Pa, less than 5 Pa, less than 2 Pa, or less than 1 Pa, and / or more than 0.1 Pa, more than 0.2 Pa, more than 0.5 Pa, more than 1 Pa, more than 2 Pa, more than 5 Pa, or more than 10 Pa. 21. The manufacturing method according to any one of claims 1 to 20, wherein the solvent and eluent are one or more selected from methanol, ethanol, 2-propanol, acetonitrile, acetone, and hexane, or a mixture of one or more selected from these and water. 22. The manufacturing method according to any one of claims 1 to 21, wherein the purification of the fatty acid mixture uses a silica gel-based adsorbent or a polymer-based adsorbent as the stationary phase.23. The manufacturing method according to any one of claims 1 to 22, wherein the purification of the fatty acid mixture uses a reverse-phase adsorbent as the stationary phase. 24. The manufacturing method according to any one of claims 1 to 23, wherein the concentration of the fraction uses one or more evaporators, and at least one of the reservoirs of the evaporators has an internal volume of 10 L or more, 20 L or more, 50 L or more, 100 L or more, 500 L or more, or 1000 L or more, and / or, less than 5000 L, less than 2000 L, less than 1000 L, or less than 500 L. 25. The manufacturing method according to any one of claims 1 to 24, wherein the concentration of the fraction uses one or more evaporators, and at least one of the evaporators is purged with an inert gas during the concentration of the fraction. 26. The manufacturing method according to any one of claims 1 to 25, wherein the PUFA is selected from EPA, DHA, n-3 DPA, DGLA, and ARA, or a combination of two or more of them. 27. The manufacturing method according to any one of claims 1 to 26, wherein the ester derivative of the PUFA is a C1-C6 alkyl ester derivative of PUFA. 28. The manufacturing method according to any one of claims 1 to 27, wherein the PUFA or its ester derivative is EPA ethyl ester, DHA ethyl ester, or a combination thereof. 29. A fatty acid composition, characterized in that it comprises PUFA or its ester derivative obtained by the manufacturing method according to any one of claims 1 to 28. 30. A fatty acid composition, characterized in that it comprises a highly unsaturated fatty acid, i.e., PUFA, or its ester derivative, wherein the proportion of PUFA or its ester derivative in all fatty acids or their ester derivatives in the fatty acid composition is 97% or more, 96% or more, 90% or more, 80% or more, 70% or more, or 50% or more, and the color b value of the fatty acid composition is 3.2 or less, 3 or less, 2.8 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.7 or less. 31. The fatty acid composition of claim 30, wherein the anisidine value of the fatty acid composition comprising PUFA or its ester derivative is 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less. 32. The fatty acid composition of claim 30 or 31, wherein the residual elution dose of the fatty acid composition comprising PUFA or its ester derivative is 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less.33. The fatty acid composition according to any one of claims 30 to 32, wherein the PUFA is selected from one or more of EPA, DHA, n-3 DPA, DGLA, and ARA. 34. The fatty acid composition according to any one of claims 30 to 33, wherein the ester derivative of the PUFA is a C1-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 its ester derivative is an ethyl ester of EPA or an ethyl ester of DHA. Claims 3 / 3 Page 4 CN 121443710 A Composition containing highly unsaturated fatty acids or their ester derivatives and a method for manufacturing the same Technical Field

[0001] This invention relates to fatty acid compositions containing highly unsaturated fatty acids (hereinafter also referred to as PUFA) or their ester derivatives and a method for manufacturing the same. Background Art

[0002] Highly unsaturated fatty acids or their ester derivatives are used as raw materials for pharmaceuticals, health foods, and cosmetics. As a method for producing highly unsaturated fatty acids or their ester derivatives used in pharmaceuticals, health foods, and cosmetics from natural oils in high purity, methods such as precision distillation, silver nitrate complexation, and chromatography are known (see, for example, Patent Documents 1-4).

[0003] On the other hand, the modification and / or deterioration of compositions containing highly unsaturated fatty acids or their ester derivatives have become a problem. For example, Patent Document 1 describes a composition of PUFA or its ester derivatives with a dissolved oxygen content, peroxide value, acid value, and anisidine value below a certain value in order to solve the technical problem of oxidation during the productization process and during the product's shelf life.

[0004] Patent Document 2 describes that when separating and purifying PUFA or its ester derivatives using chromatography with an adsorption resin as a carrier, it is pointed out that dissolved oxygen in the eluent poses a risk of modification and / or deterioration of PUFA, etc. In order to prevent this risk, the eluent can be heated and refluxed, or nitrogen or carbon dioxide can be blown into the eluent.

[0005] Furthermore, Patent Document 3 describes a method for manufacturing PUFA ester derivatives using a silver salt solution, in which a flow-path type stirrer is used as the reaction tank, and the manufacturing method is carried out under low-oxygen conditions to suppress the deterioration of the silver salt solution.

[0006] Patent Document 4 relates to a patent for the purification of PUFA ester derivatives using a chromatographic method including simulated moving bed chromatography, and this embodiment describes the principle that "all chromatographic operations are carried out in the dark under an inert gas atmosphere."

[0007] Prior Art Documents Patent Documents Patent Document 1: Japanese Patent Application Publication No. 2017-114776 Patent Document 2: Japanese Patent Application Publication No. 61-291540 Patent Document 3: Japanese Patent Application Publication No. 2019-135307 Patent Document 4: Japanese Patent Publication No. 2017-502130 Summary of the Invention

[0008] Technical Problem to be Solved by the Invention The inventors have discovered the following technical problem: In the manufacture of fatty acid compositions containing highly unsaturated fatty acids or their ester derivatives, if the dissolved oxygen in the mobile phase is not suppressed, even if the fraction containing highly unsaturated fatty acids or their ester derivatives obtained by chromatography is stored in a container under an inert gas atmosphere, the oxygen concentration cannot be sufficiently reduced, and the highly unsaturated fatty acids or their ester derivatives are modified during desolventization, especially colored.

[0009] Technical Solution for Solving the Technical Problem: The present invention provides a fatty acid composition containing highly unsaturated fatty acids or their ester derivatives, wherein, in a chromatographic method for purifying a mixture of fatty acids containing highly unsaturated fatty acids or their ester derivatives, by using an eluent obtained by placing the container contained in the specification (page 1 / 23, CN 121443710 A) under an inert gas atmosphere as the mobile phase, modification such as coloring can be suppressed.

[0010] In one aspect of the present invention, a method for manufacturing a fatty acid composition containing highly unsaturated fatty acids or their ester derivatives is provided. In one aspect of the present invention, a fatty acid composition is provided containing highly unsaturated fatty acids or their ester derivatives.

[0011] [1-1] A method for manufacturing a fatty acid composition comprising a highly unsaturated fatty acid (hereinafter also referred to as PUFA) or an ester derivative thereof, comprising: (1) receiving a solvent in a container A and obtaining an eluent inside the container A under an inert gas atmosphere; (2) purifying a fatty acid mixture comprising PUFA or an ester derivative thereof by chromatography using the eluent as the mobile phase to obtain a fraction comprising PUFA or an ester derivative thereof; and (3) concentrating the fraction comprising PUFA or an ester derivative thereof to obtain a fatty acid composition comprising PUFA or an ester derivative thereof.

[0012] [1-2] According to the manufacturing method of [1-1], wherein the concentration of the fraction comprises: subjecting the eluent contained in the fraction comprising PUFA or an ester derivative thereof to reduced pressure and / or heating, and recovering it as a solvent by distillation.

[0013] [1-3] According to the manufacturing method of [1-1] or [1-2], wherein the solvent comprises a solvent obtained by distillation.

[0014] [1-4] The manufacturing method according to any one of [1-1] to [1-3], wherein the solvent is a solvent recovered from a fraction containing PUFA or its ester derivatives during the concentration of the fraction.

[0015] [1-5] According to any one of [1-1] to [1-4], the dissolved oxygen concentration of the eluent inside container A is less than 20 mg / L, less than 15 mg / L, or less than 11 mg / L.

[0016] [1-6] According to any one of [1-1] to [1-5], the oxygen concentration of the top space of container A is less than 10 vol%, less than 5 vol%, less than 4 vol%, less than 3 vol%, less than 2 vol%, less than 1 vol%, less than 0.5 vol%, or less than 0.1 vol%.

[0017] [1-7] According to any one of [1-1] to [1-6], the fraction containing PUFA or its ester derivative is contained inside container C under an inert gas atmosphere.

[0018] [1-8] According to the manufacturing method of [1-7], the oxygen concentration in the top space of the container C containing the fractionated product 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.

[0019] [1-9] According to any one of [1-1] to [1-8], the fatty acid mixture containing PUFA or its ester derivative is contained inside the container B under an inert gas atmosphere before the purification of the fatty acid mixture.

[0020] [1-10] According to the manufacturing method of [1-9], the oxygen concentration in the top space of the 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.

[0021] [1-11] The manufacturing method according to any one of [1-1] to [1-10], wherein the fatty acid composition comprising PUFA or its ester derivative is contained inside container D under an inert gas atmosphere.

[0022] [1-12] The manufacturing method according to [1-11], wherein the oxygen concentration in the top space of container D is 0.7 vol% or less, 0.5 vol% or less, 0.3 vol% or less, or 0.1 vol% or less.

[0023] [1-13] The manufacturing method according to any one of [1-1] to [1-12], wherein the inert gas inside containers A, B, C and D is each independently nitrogen, argon, carbon dioxide or a combination thereof.

[0024] [1-14] The manufacturing method according to any one of [1-1] to [1-13], wherein the inert gas inside the container A, container B, container C and container D is nitrogen.

[0025] [1-15] According to any one of [1-1] to [1-14], the proportion of PUFA or its ester derivative in all fatty acids or ester derivatives of the fatty acid composition containing PUFA or its ester derivative is 97% or more, 96% or more, 90% or more, 80% or more, 70% or more, or 50% or more.

[0026] [1-16] According to any one of [1-1] to [1-15], the color b value of the fatty acid composition containing PUFA or its ester derivative is 3.2 or less, 3 or less, 2.8 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, or 0.7 or less.

[0027] [1-17] The manufacturing method according to any one of [1-1] to [1-16], wherein the anisidine value of the fatty acid composition comprising PUFA or its ester derivative is 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less.

[0028] [1-18] The manufacturing method according to any one of [1-1] to [1-17], wherein the residual elution dose of the fatty acid composition comprising PUFA or its ester derivative is 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less.

[0029] [1-19] The manufacturing method according to any one of [1-1] to [1-18], wherein the heating temperature for concentrating the fraction is a maximum temperature of 190°C or less, 160°C or less, 140°C or less, 135°C or less, 130°C or less, 120°C or less, or 100°C or less, and / or 40°C or more, 60°C or more, 80°C or more, 100°C or more, 120°C or more, 130°C or more, 135°C or more, 140°C or more, or 160°C or more.

[0030] [1-20] The manufacturing method according to any one of [1-1] to [1-19], wherein the minimum pressure during the depressurization of the concentration of the fraction is less than 100 Pa, less than 50 Pa, less than 20 Pa, less than 10 Pa, less than 5 Pa, less than 2 Pa, or less than 1 Pa, and / or more than 0.1 Pa, more than 0.2 Pa, more than 0.5 Pa, more than 1 Pa, more than 2 Pa, more than 5 Pa, or more than 10 Pa.

[0031] [1-21] The manufacturing method according to any one of [1-1] to [1-20], wherein the solvent and eluent are one or a mixture of two or more selected from methanol, ethanol, 2-propanol, acetonitrile, acetone, and hexane, or a mixture of one or more selected from them and water.

[0032] [1-22] The manufacturing method according to any one of [1-1] to [1-21], wherein the purification of the fatty acid mixture uses a silica gel-based adsorbent or a polymer-based adsorbent as the stationary phase.

[0033] [1-23] The manufacturing method according to any one of [1-1] to [1-22], wherein the purification of the fatty acid mixture uses a reverse-phase adsorbent as the stationary phase.

[0034] [1-24] The manufacturing method according to any one of [1-1] to [1-23], wherein the concentration of the fraction uses one or more evaporators, at least one of the reservoirs of the evaporator having an internal volume of 10L or more, 20L or more, 50L or more, 100L or more, 500L or more, or 1000L or more, and / or, less than 5000L, less than 2000L, less than 1000L, or less than 500L.

[0035] [1-25] The manufacturing method according to any one of [1-1] to [1-24], wherein the concentration of the fraction uses one or more evaporators, and at least one of the evaporators is purged with an inert gas during the concentration of the fraction.

[0036] [1-26] The manufacturing method according to any one of [1-1] to [1-25], wherein the PUFA is one or a combination of two or more selected from EPA, DHA, n-3 DPA, DGLA and ARA.

[0037] [1-27] The manufacturing method according to any one of [1-1] to [1-26], wherein the ester derivative of the PUFA is a C1-C6 alkyl ester derivative of the PUFA.

[0038] [1-28] The manufacturing method according to any one of [1-1] to [1-27], wherein the PUFA or its ester derivative is ethyl EPA, ethyl DHA, or a combination thereof. Instruction manual 3 / 23 pages 7 CN 121443710 A

[0039] [1-29] The manufacturing method according to any one of [1-1] to [1-28], wherein the chromatography method is HPLC.

[0040] [1-30] The manufacturing method according to any one of [1-1] to [1-29], wherein the volume of the container A is 50L or more, 100L or more, 200L or more, 500L or more, 1000L or more, 2000L or more, or 4000L or more, and / or, 20000L or less, 10000L or less, 4000L or less, 2000L or less, or 1000L or less.

[0041] [1-31] ​​A fatty acid composition comprising PUFA or its ester derivative obtained by the manufacturing method according to any one of [1-1] to [1-30].

[0042] [1-32] The manufacturing method according to any one of [1-1] to [1-30], wherein, as at least one solvent, a single solvent, or two or fewer, three or fewer, four or fewer, five or fewer, or ten or fewer solvents are used.

[0043] [2-1] A fatty acid composition comprising a highly unsaturated fatty acid (hereinafter also referred to as PUFA) or an ester derivative thereof, wherein the proportion of PUFA or an ester derivative thereof in all fatty acids or ester derivatives of 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.

[0044] [2-2] The fatty acid composition according to [2-1], wherein the anisidine value of the fatty acid composition comprising PUFA or its ester derivative is 15 or less, 10 or less, 5 or less, 1 or less, 0.7 or less, 0.5 or less, 0.3 or less, or 0.25 or less.

[0045] [2-3] The fatty acid composition according to [2-1] or [2-2], wherein the residual elution dose of the fatty acid composition comprising PUFA or its ester derivative is 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less.

[0046] [2-4] The fatty acid composition according to any one of [2-1] to [2-3], wherein the PUFA is selected from one or more of EPA, DHA, n-3 DPA, DGLA, and ARA.

[0047] [2-5] The fatty acid composition according to any one of [2-1] to [2-4], wherein the ester derivative of the PUFA is a C1 to C6 alkyl ester derivative of the PUFA.

[0048] [2-6] The fatty acid composition according to any one of [2-1] to [2-5], wherein the PUFA or its ester derivative is an ethyl ester of EPA, an ethyl ester of DHA, or a combination thereof.

[0049] Effects of the Invention According to the present invention, it is possible to manufacture a fatty acid composition containing highly unsaturated fatty acids or their ester derivatives with suppressed coloring. In addition, according to the present invention, it is possible to suppress dissolved oxygen in a fatty acid composition containing highly unsaturated fatty acids or their ester derivatives. Brief Description of the Drawings

[0050] FIG1 is a process diagram illustrating this embodiment, showing a process diagram of a method for manufacturing a fatty acid composition containing ethyl eicosapenoate (hereinafter also referred to as EPA-E). Detailed Description of the Embodiments

[0051] In this specification, the term "fatty acid" refers to aliphatic carboxylic acids of various chain lengths of about C12 to C22.Here, the numbers in "C12~C22" refer to the total number of carbon atoms in the chain of the carboxylic acid. The main chain length is C16~C22. The structure of fatty acids can be represented by the simple expression "X:Y". Here, X is the total number of carbon atoms in a specific fatty acid, and Y is the number of double bonds. For example, a saturated fatty acid with 20 carbon atoms can be expressed as "C20:0", a monovalent unsaturated fatty acid with 18 carbon atoms can be expressed as "C18:1", and arachidonic acid can be expressed as "C20:4, n-6". "n-" indicates the starting position of the double bond, counting from the methyl end of the fatty acid. For example, if it is "n-6", it means that the starting position of the double bond is the 6th position counting from the methyl end of the fatty acid. This method is known to those skilled in the art, and those skilled in the art can easily determine the fatty acid expressed according to this method.

[0052] The fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated. Fatty acids are typically manufactured industrially through the hydrolysis of triglycerides or phospholipids derived from natural sources. Some fatty acids are manufactured synthetically. Regardless of the manufacturing method, purification is required to obtain pure products for food, cosmetic, or industrial use.

[0053] In this specification, the term "highly unsaturated fatty acid" refers to a fatty acid having more than one double bond. In this specification, highly unsaturated fatty acids are sometimes also referred to as PUFAs. Highly unsaturated fatty acids, for example, can be fatty acids having 3 to 6 double bonds. Examples of highly unsaturated fatty acids include: α-linolenic acid (C18:3,n-3), γ-linolenic acid (C18:3,n-6), dihoxy-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 these highly unsaturated fatty acids are typically alkyl esters, such as C1-C6 alkyl esters or C1-C4 alkyl esters. Ethyl esters are an example of esters.

[0054] <Method for manufacturing fatty acid composition> As one aspect of the present invention, the following manufacturing method can be described, which includes: containing a solvent in a container A, obtaining an eluent inside the container A under an inert gas atmosphere; purifying a fatty acid mixture containing PUFA or its ester derivative by using chromatography with the eluent as the mobile phase to obtain a fraction containing PUFA or its ester derivative; and concentrating the fraction containing PUFA or its ester derivative to obtain a fatty acid composition containing PUFA or its ester derivative.

[0055] In one aspect of the invention, the fatty acid mixture can be obtained from a raw material containing highly unsaturated fatty acids as constituting fatty acids, resulting in a fatty acid mixture containing PUFA or its ester derivatives. For example, it can be obtained from a raw material containing natural oils and fats containing plant and animal oils and fats, or from a raw material containing oils and fats obtained from genetically recombinant plants, animals, and microorganisms containing yeast. Examples of raw materials include fish oil, algae and microalgae oils, and plant oils such as borage oil, echium oil, and evening primrose oil.

[0056] In one aspect of the invention, the fatty acid mixture containing PUFA or its ester derivatives is obtained by distilling the raw material.

[0057] In one aspect of the invention, the fatty acid mixture containing PUFA or its ester derivatives may contain PUFA obtained by hydrolyzing the raw oil into free fatty acids using water, or may contain ester derivatives obtained by alcoholysis of the raw oil into C1-C6 alkyl esters using C1-C6 alkyl alcohols. Examples of C1-C6 alkyl esters include ethyl esters.

[0058] As one aspect of the present invention, a manufacturing method comprising: placing a fatty acid mixture containing PUFA or its ester derivative in a container B under an inert gas atmosphere before purification of the fatty acid mixture. In one aspect of the present invention, when the fatty acid mixture is placed in container B, the interior of container B is placed under an inert gas atmosphere. In one aspect of the present invention, the interior of container B may be placed under an inert gas atmosphere before, after, or simultaneously with the placement of the fatty acid mixture. In one aspect of the present invention, it is preferable to place the fatty acid mixture in container B after placing it under an inert gas atmosphere.

[0059] In one aspect of the present invention, container A refers to a container for containing and storing eluent, which may be a can for containing eluent. In this specification, a can for containing eluent may be referred to as an eluent can. Instruction manual 5 / 23 page 9 CN 121443710 A

[0060] In one aspect of the present invention, container B refers to a container for receiving and storing a fatty acid mixture containing PUFA or its ester derivatives obtained from the raw materials, and may be a tank for storing the fatty acid mixture. In one aspect of the present invention, the fatty acid mixture containing PUFA or its ester derivatives may be the main fraction obtained by continuous distillation. In container B, the tank for storing the main fraction may be called a main fraction tank.

[0061] In one aspect of the present invention, container C refers to a container for receiving and storing a fraction containing PUFA or its ester derivatives obtained by purifying the fatty acid mixture, and may be a tank for storing the fraction. In this specification, the tank for storing the fraction may be called a fraction tank.

[0062] In one aspect of the present invention, container D refers to a container for receiving and storing a fatty acid composition containing PUFA or its ester derivatives obtained through fractionation concentration, and may be a tank for receiving the fatty acid composition. In this specification, the tank for receiving the fatty acid composition may be called a product tank. In this specification, the tank for receiving the fatty acid composition may be called a buffer tank.

[0063] In one aspect of the present invention, the solvent, fractions, fatty acid mixture, or fatty acid composition in the container is placed in an inert gas atmosphere by purging the container with an inert gas. In one aspect of the present invention, the inert gas is not particularly limited as long as it is a chemically stable gas that does not easily affect other elements or compounds. Examples of inert gases include nitrogen, argon, carbon dioxide, and mixtures of two or more of them. In one aspect of the present invention, the inert gas used for the inert gas atmosphere may be nitrogen, argon, or carbon dioxide. In one aspect of the present invention, an inert gas atmosphere means a state in which readily reactive molecules present in the air, i.e., oxygen, are removed and replaced with less reactive nitrogen, argon, or carbon dioxide. In one aspect of the invention, an inert gas atmosphere refers to a state in which a gas containing mainly inert gases with an oxygen concentration of less than 10% by volume is present. In one aspect of the invention, purging with an inert gas is performed before or after the container is filled with solvents, fractions, fatty acid mixtures, or fatty acid compositions.

[0064] In one aspect of the invention, the container may be a sealed container, such as a can. In one aspect of the invention, the container has a device for sealing the inert gas into the top space, i.e., an inert gas sealing device. Examples of inert gas sealing devices include, for example, a gas sealing unit and a breather valve. In one aspect of the invention, the container has a space containing gas that is not filled with solvents, eluents, fatty acid mixtures, fractions, or fatty acid compositions. In this specification, the space in the container that is not filled with solvents, eluents, fatty acid mixtures, fractions, or fatty acid compositions and contains gas is referred to as the "top space". In one embodiment of the invention, the volume of the container is the sum of the volume of the portion filled with solvent, eluent, fatty acid mixture, fractions and / or fatty acid composition, and the volume of the headspace.

[0065] In one embodiment of the invention, the headspace volume of the container may be set with an upper limit and / or a lower limit and may vary within the range of such values.

[0066] In one embodiment of the invention, the upper limit of the headspace volume refers to the headspace volume at the moment when the headspace volume is at its maximum and the volume of the portion filled with solvent, eluent, fatty acid mixture, fractions and / or fatty acid composition is at its minimum during the implementation of the manufacturing method of the invention.In this specification, a container with a top space volume of the upper limit is sometimes referred to as a container before storage. Additionally, this container is sometimes referred to as a container after transfer. For example, if it is a fractionation tank with a top space volume of the upper limit, it is sometimes referred to as a "fractionation tank before storage" and a "fractionation tank after transfer," respectively.

[0067] In one embodiment of the invention, the lower limit of the top space volume refers to the top space volume at the moment when the top space volume is minimum and the volume of the portion filled with solvent, eluent, fatty acid mixture, fraction, and / or fatty acid composition is maximum during the implementation of the manufacturing method involved in the invention. In this specification, a container with a top space volume of the lower limit is sometimes referred to as a container after storage. Additionally, this container is sometimes referred to as a container before transfer. For example, if it is a fractionation tank with a top space volume of the lower limit, it is sometimes referred to as a "fractionation tank after storage" and a "fractionation tank before transfer," respectively.

[0068] In one embodiment of the present invention, the upper limit of the top space volume of container A can be 50% or more, 70% or more, 80% or more, 85% or more, 87% or more, 89% or more, 90% or more, or 95% or more, and / or less than 100% or less, less than 99% or less, less than 97% or less, less than 95% or less, less than 90% or less, less than 89% or less, less than 85% or less, less than 80% or less, or less than 70% or less. In one embodiment of the present invention, the lower limit of the top space volume of container A can be 10% or more, 20% or more, 30% or more, 50% or more, 70% or more, 80% or more, 85% or more, or 87% or more, and / or, less than 95% or less, less than 90% or less, less than 89% or less, less than 87% or less, less than 85% or less, less than 80% or less, less than 70% or less, or less than 50% or less. Additionally, in one embodiment of the present invention, the volume of container A can be 50L or more, 100L or more, 200L or more, 500L or more, 1000L or more, 2000L or more, or 4000L or more, and / or, less than 20000L, less than 10000L, less than 4000L, less than 2000L, or less than 1000L.

[0069] In one embodiment of the present invention, the upper limit of the top space volume of container B can be 50% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more, and / or, less than 100%, less than 99.9%, less than 99%, less than 97%, less than 95%, less than 90%, less than 85%, less than 80%, or less than 70% of the volume of container B.In one embodiment of the present invention, the lower limit of the top space volume of container B can be 0% or more, 1% or more, 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, or 30% or more, and / or, less than 70% or less, less than 50% or less, less than 30% or less, less than 20% or less, less than 15% or less, less than 10% or less, less than 7% or less, or less than 5% or less. Additionally, in one embodiment of the present invention, the volume of container B can be 500L or more, 1000L or more, 2000L or more, 5000L or more, 10000L or more, 20000L or more, or 50000L or more, and / or, less than 200000L, less than 100000L, less than 50000L, less than 20000L, or less than 10000L.

[0070] In one embodiment of the present invention, the upper limit of the top space volume of container C can be 50% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more, and / or, less than 100%, less than 99.9%, less than 99%, less than 97%, less than 95%, less than 90%, less than 85%, less than 80%, or less than 70% of the volume of container C. In one embodiment of the present invention, the lower limit of the top space volume of container C can be 0% or more, 1% or more, 3% or more, 5% or more, 7% or more, 10% or more, 20% or more, 30% or more, 35% or more, or 40% or more, and / or, less than 70% or less, less than 60% or less, less than 50% or less, less than 40% or less, less than 35% or less, less than 30% or less, less than 20% or less, less than 15% or less, or less than 10% or less. Furthermore, in one embodiment of the present invention, the volume of container C can be 50L or more, 100L or more, 200L or more, 500L or more, 1000L or more, 2000L or more, or 4000L or more, and / or, less than 20000L, less than 10000L, less than 4000L, less than 2000L, or less than 1000L.

[0071] In one embodiment of the present invention, the upper limit of the top space volume of container D can be 50% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more, and / or, less than 100%, less than 99.9%, less than 99%, less than 97%, less than 95%, less than 90%, less than 85%, less than 80%, or less than 70% of the volume of container D.In one embodiment of the present invention, the lower limit of the top space volume of container D can be 0% or more, 1% or more, 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 19% or more, 20% or more, or 30% or more, and / or, less than 70% or less, less than 50% or less, less than 30% or less, less than 20% or less, less than 19% or less, less than 15% or less, or less than 10% or less, as per specification page 7 / 23, CN 121443710 A. In addition, in one aspect of the present invention, the volume of container D can be 50L or more, 100L or more, 200L or more, 500L or more, 1000L or more, 2000L or more, or 5000L or more, and / or, less than 20000L, less than 10000L, less than 5000L, less than 2000L, or less than 1000L.

[0072] In one aspect of the present invention, the oxygen concentration in the top space of the container refers to the concentration of oxygen in the gas contained in the top space of the container.

[0073] In one aspect of the present invention, the oxygen concentration in the top space of container A can be less than 10% by volume, less than 5% by volume, less than 4% by volume, less than 3% by volume, less than 2% by volume, less than 1% by volume, less than 0.5% by volume, or less than 0.1% by volume. In another aspect of the present invention, the oxygen concentration in the top space of container A may be less than 10% by volume, less than 5% by volume, less than 4% by volume, less than 3% by volume, less than 2% by volume, less than 1% by volume, less than 0.5% by volume, or less than 0.1% by volume, and / or more than 0% by volume, more than 0.01% by volume, or more than 0.05% by volume. In one aspect of the present invention, the oxygen concentration in the top space of container A may be the oxygen concentration in the top space after receiving the eluent or the oxygen concentration in the top space after transferring the eluent.

[0074] In one aspect of the present invention, the oxygen concentration in the top space of container B may be less than 1.5% by volume, less than 1.0% by volume, less than 0.7% by volume, less than 0.5% by volume, less than 0.3% by volume, or less than 0.1% by volume. In another aspect of the invention, the oxygen concentration in the headspace of container B can be less than 1.5% by volume, less than 1.0% by volume, less than 0.7% by volume, less than 0.5% by volume, less than 0.3% by volume, or less than 0.1% by volume, and / or more than 0% by volume, more than 0.01% by volume, or more than 0.05% by volume. In one aspect of the invention, the oxygen concentration in the headspace of container B can be the oxygen concentration in the headspace after containing the fatty acid mixture or the oxygen concentration in the headspace after transferring the fatty acid mixture.

[0075] In one embodiment of the present invention, the oxygen concentration in the top space of the container C after fractionation can be less than 1.5% by volume, less than 1.0% by volume, less than 0.7% by volume, less than 0.5% by volume, less than 0.3% by volume, or less than 0.1% by volume. In another embodiment of the present invention, the oxygen concentration in the top space of the container C after fractionation can be less than 1.5% by volume, less than 1.0% by volume, less than 0.7% by volume, less than 0.5% by volume, less than 0.3% by volume, or less than 0.1% by volume, and / or more than 0% by volume, more than 0.01% by volume, or more than 0.05% by volume.

[0076] In one embodiment of the present invention, the oxygen concentration in the top space of the container C after fractionation can be less than 0.7% by volume, less than 0.5% by volume, less than 0.3% by volume, or less than 0.1% by volume. In another aspect of the present invention, the oxygen concentration in the headspace of container C after transfer fractionation may be less than 0.7 vol%, less than 0.5 vol%, less than 0.3 vol%, or less than 0.1 vol%, and / or more than 0 vol%, more than 0.01 vol%, or 0.05 vol%.

[0077] In one aspect of the present invention, the oxygen concentration in the headspace of container D may be less than 0.7 vol%, less than 0.5 vol%, less than 0.3 vol%, or less than 0.1 vol%. In one aspect of the present invention, the oxygen concentration in the headspace of container D may be less than 0.7 vol%, less than 0.5 vol%, less than 0.3 vol%, or less than 0.1 vol%, and / or more than 0 vol%, more than 0.01 vol%, or 0.05 vol%. In one aspect of the present invention, the oxygen concentration in the headspace of container D may be the oxygen concentration in the headspace after receiving the fatty acid composition or the oxygen concentration in the headspace after transferring the fatty acid composition.

[0078] The method for measuring the oxygen concentration in the headspace is not particularly limited. For example, a residual oxygen meter "Pack Keeper" RO-103KS (manufactured by Iijima Electronics Co., Ltd.) can be used, and the measurement can be performed according to the steps in the instruction manual of the device.

[0079] In this specification, the above description regarding containers not specifically designated as container A, container B, container C, and container D applies to containers A, B, C, and D.

[0080] Eluent In one aspect of the present invention, the method for manufacturing a fatty acid composition includes: containing a solvent in container A, and obtaining an eluent inside container A under an inert gas atmosphere. As one aspect of the present invention, the solvent is used before being contained in container A. In one aspect of the present invention, when containing the solvent in container A, the interior of container A is placed under an inert gas atmosphere.In one embodiment of the present invention, the interior of container A may be placed in an inert gas atmosphere before, after, or simultaneously with the placement of the solvent. In one embodiment of the present invention, it is preferable to place the interior of container A in an inert gas atmosphere before placing the interior of container A in container A.

[0081] In one embodiment of the present invention, the solvent that can be used as an eluent is a solvent with a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less. In another aspect of the invention, the solvent that can be used as an eluent is a solvent with a dissolved oxygen concentration of less than 20 mg / L, less than 17.5 mg / L, less than 15 mg / L, less than 14 mg / L, less than 13 mg / L, less than 12 mg / L, or less than 11 mg / L, and / or more than 0 mg / L, more than 0.01 mg / L, more than 0.03 mg / L, or more than 0.1 mg / L. In one aspect of the invention, the solvent that can be used as an eluent is a liquid selected from one or more of alcohols, ethers, esters, ketones, nitriles, hexanes, and dichloromethane, or a mixture of two or more of them, or a mixture of one or more of them with water, with a dissolved oxygen concentration of less than 20 mg / L, less than 17.5 mg / L, less than 15 mg / L, less than 14 mg / L, less than 13 mg / L, less than 12 mg / L, or less than 11 mg / L. In another aspect of the invention, the solvent that can be used as the eluent is a liquid selected from one or more of alcohols, ethers, esters, ketones, nitriles, hexanes, and dichloromethane, or a mixture of two or more of them, or a mixture of one or more of them with water, 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. Examples of alcohols include methanol, ethanol, n-propanol, 2-propanol, n-butanol, isobutanol, sec-butanol, and tert-butanol. In one aspect of the invention, examples of alcohols include methanol and ethanol. In one aspect, methanol is mentioned. Examples of ethers include diethyl ether, diisopropyl ether, and methyl tert-butyl ether. Examples of esters include methyl acetate and ethyl acetate. Examples of ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone. Examples of nitriles include acetonitrile. In one embodiment of the invention, a solvent that can be used as an eluent is methanol with a dissolved oxygen concentration of 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less.In another aspect of the present invention, the solvent that can be used as an eluent is methanol with 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.

[0082] As one aspect of the present invention, a method for manufacturing a solvent contained in container A comprising a solvent obtained by distillation can be provided. As one aspect of the present invention, the solvent obtained by distillation can be recovered from the fraction by distillation during the concentration of the fraction. As one aspect of the present invention, the solvent obtained by distillation can be obtained by purchasing distilled solvent, and / or by distillation after purchasing solvent.

[0083] As one aspect of the present invention, a method for manufacturing a solvent contained in container A comprising a solvent recovered from the fraction during the concentration of the fraction can be provided.

[0084] As one aspect of the present invention, a manufacturing method is provided in which the concentration of the fraction includes: subjecting the eluent contained in the fraction containing PUFA or its ester derivatives to reduced pressure and / or heating, and recovering it as a solvent by distillation.

[0085] Purification of fatty acid mixtures by chromatography A method for manufacturing a fatty acid composition according to one aspect of the present invention includes: purifying a fatty acid mixture containing PUFA or its ester derivatives by using chromatography with the eluent as the mobile phase to obtain a fraction containing PUFA or its ester derivatives.

[0086] In this specification, "chromatography" refers to a method for separating or purifying a mixture by passing a substance called the mobile phase through the surface or interior of a substance called the stationary phase or carrier. Chromatography is used for the analysis and separation of mixtures of two or more substances. (Page 9 / 23, CN 121443710 A) In this specification, as a chromatographic method, column chromatography can be performed, such as high turbulence liquid chromatography (HLC), preparative chromatography, high performance liquid chromatography (HPLC), simulated moving bed chromatography, real moving bed chromatography, and supercritical fluid chromatography.

[0087] In this specification, "chromatographic conditions" refers to various parameters that represent the conditions under which the chromatographic method is performed. Examples include packing pressure, composition of mobile and stationary phases, slurry concentration, delivery pressure, column temperature, mobile phase temperature, mobile phase gradient, mobile phase flow rate, column type used, detector and parameters used, sample preparation scheme used, settling time of stationary phase during column preparation, pressure at which settling is performed, settling time, and pressure at which settling is performed.The column can be a column prepared by packing a stationary phase or a commercially available column pre-packed with a stationary phase.

[0088] In one aspect of the invention, the chromatography uses a silica-based adsorbent or a polymer-based adsorbent as the stationary phase. The stationary phase can be selected from reversed-phase stationary phases, hydrophilic interaction liquid chromatography stationary phases, acrylamide, silica, phenyl-hexyl stationary phases, polar embedded alkyl, fluorophenylpropyl, or all stationary phases known in the art of chromatography. Examples of reversed-phase stationary phases include C30, C22, C18, C8, C5, C4, biphenyl, and fluorophenyl stationary phases. In one aspect, a chiral stationary phase is used. The choice of stationary phase will be apparent to those skilled in the art and can depend on the component to be purified by chromatography. Various types of octadecyl silica (hereinafter also referred to as ODS) can be used, including fully endcapped, partially endcapped, and base-deactivated substances. Components with higher polarity require a normal-phase stationary phase, such as unbonded silica, an amino phase, or a cyano phase.

[0089] In one aspect of the invention, the chromatography uses a reversed-phase adsorbent as the stationary phase. Any reversed-phase adsorbent can be used without particular specification; for example, polymer beads such as polystyrene networked with divinylbenzene can be used, or C8 or C18 alkyl-bonded silica gel, particularly C18 alkyl-bonded silica gel, i.e., octadecylsilyl-based ODS columns.

[0090] In one aspect of the invention, the column size used is not particularly limited and depends to some extent on the volume of the fatty acid composition to be purified. The diameter of the column is 1mm or more, 2mm or more, 4mm or more, 8mm or more, 16mm or more, 32mm or more, 64mm or more, 128mm or more, 256mm or more, or 500mm or more, or less than 4000mm, less than 2000mm, less than 1000mm, less than 500mm, or 1-4000mm, 2-2000mm, 4-1000mm, 8-1000mm, 16-1000mm, 32-500mm, 30-800mm, or 400-800mm.

[0091] 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 5 to 800 cm, 10 to 400 cm, 20 to 200 cm, 20 to 150 cm, or 20 to 120 cm.

[0092] In this specification, "eluent" refers to a liquid used in chromatography as a mobile phase that contacts the stationary phase and passes through its gaps or surfaces. The eluent is used in chromatography to develop and elute components such as PUFA or its ester derivatives adsorbed on the column.In this specification, the eluent is sometimes referred to as the mobile phase.

[0093] In one aspect of the invention, the eluent is obtained in a container under an inert gas atmosphere and then contained as the mobile phase in a column to develop and elute PUFA or its ester derivatives. The eluent flowing out of the column is recovered as a fraction along with the PUFA or its ester derivatives. The fraction, also called the eluent, may contain components such as the eluent and PUFA or its ester derivatives.

[0094] In one aspect of the invention, the liquid comprises a solvent and an eluent. The solvent and / or the eluent is one or a mixture of two or more selected from alcohols, ethers, esters, ketones, nitriles, hexanes and dichloromethane, or a mixture of one or more selected from them and water. Examples of alcohols include methanol, ethanol, n-propanol, 2-propanol, n-butanol, isobutanol, sec-butanol and tert-butanol. In one aspect of the invention, examples of alcohols include methanol and ethanol. In one embodiment, methanol is mentioned on page 10 / 23 of the specification, CN 121443710 A. Examples of ethers include diethyl ether, diisopropyl ether, and methyl tert-butyl ether. Examples of esters include methyl acetate and ethyl acetate. Examples of ketones include acetone, methyl ethyl ketone, and methyl isobutyl ketone. Examples of nitriles include acetonitrile. In one embodiment of the invention, a single liquid is used as both the solvent and the eluent. In one embodiment of the invention, methanol is used as both the solvent and the eluent. In one embodiment of the invention, a single liquid, or two or fewer, three or fewer, four or fewer, five or fewer, or ten or fewer liquids are used as both the solvent and the eluent.

[0095] The eluent may also contain additives containing buffers and pH adjusters. The selection of additives may be determined based on the eluent used, the stationary phase used, and the component to be purified. In some embodiments, the eluent contains one or more additives selected from formic acid, ammonium formate, trimethylamine, ammonia, and ammonium hydroxide. In some embodiments, the eluent may not contain additives.

[0096] In one aspect of the invention, the solvent is contained in container A. In one embodiment, container A does not contain eluent, but contains the solvent as an eluent. In another embodiment, prior to containing the solvent, container A is placed in an inert gas atmosphere, and the solvent is contained in a state containing the eluent. In one embodiment, the solvent is contained inside container A placed in an inert gas atmosphere. In one embodiment, container A is placed in an inert gas atmosphere before containing the solvent. In one embodiment, the solvent is contained in container A not placed in an inert gas atmosphere. In one embodiment, container A is placed in an inert gas atmosphere after containing the solvent.

[0097] In one aspect of the invention, the dissolved oxygen concentration of the solvent contained in container A is, for example, less than 20 mg / L, less than 15 mg / L, or less than 11 mg / L.In another aspect of the invention, the dissolved oxygen concentration of the solvent contained in container A is, for example, 20 mg / L or less, 15 mg / L or less, or 11 mg / L or less, and / or, 0 mg / L or more, 0.01 mg / L or more, 0.03 mg / L or more, or 0.1 mg / L or more.

[0098] In one aspect of the invention, the dissolved oxygen concentration of the eluent is the concentration of oxygen dissolved in the eluent inside container A, and may be 20 mg / L or less, 17.5 mg / L or less, 15 mg / L or less, 14 mg / L or less, 13 mg / L or less, 12 mg / L or less, or 11 mg / L or less. In one aspect of the invention, the dissolved oxygen concentration of the eluent is the concentration of oxygen dissolved in the eluent inside container A, which can be less than 20 mg / L, less than 17.5 mg / L, less than 15 mg / L, less than 14 mg / L, less than 13 mg / L, less than 12 mg / L, or less than 11 mg / L, and / or more than 0 mg / L, more than 0.01 mg / L, more than 0.03 mg / L, or more than 0.1 mg / L. The dissolved oxygen concentration can be measured using a dissolved oxygen meter by common methods.

[0099] In one aspect of the invention, the oxygen concentration of the solvent contained in container A exceeds 20 mg / L. In one aspect, the oxygen concentration of the solvent contained in container A exceeds 25 mg / L. In one method, an eluent with 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 solvent absorption is used. In another method, an eluent with 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 solvent absorption is used. In one aspect of the present invention, the dissolved oxygen concentration of the solvent contained in container A is the concentration of oxygen dissolved in the solvent contained in container A, which may be greater than 20 mg / L, greater than 17.5 mg / L, greater than 15 mg / L, greater than 14 mg / L, greater than 13 mg / L, greater than 12 mg / L, or greater than 11 mg / L.

[0100] In one aspect of the present invention, the dissolved oxygen concentration in the solvent contained in container A is less than 20 mg / L, for example, less than 17.5 mg / L, less than 15 mg / L, less than 14 mg / L, less than 13 mg / L, less than 12 mg / L, or less than 11 mg / L.In another aspect of the invention, the oxygen concentration dissolved in the solvent contained in container A is less than 20 mg / L, for example, less than 17.5 mg / L, less than 15 mg / L, less than 14 mg / L, less than 13 mg / L, less than 12 mg / L, or less than 11 mg / L, and / or more than 0 mg / L, more than 0.01 mg / L, more than 0.03 mg / L, or more than 0.1 mg / L.

[0101] In one aspect of the invention, the eluent inside container A is contained inside container A at a temperature the same as that used in chromatography or ±5°C, ±2.5°C, ±2°C, or ±1°C, for example, at room temperature or above room temperature. In one embodiment, room temperature is 20°C to 25°C. Temperatures above room temperature are 25°C or higher, 30°C or higher, or 35°C or higher, and / or, below 60°C, below 50°C or below 45°C, or, 25–60°C, 30–50°C, or 35–45°C.

[0102] In one embodiment of the invention, container A may be a tank for holding the eluent used in chromatography. In this specification, the tank for holding the eluent is sometimes referred to as an eluent tank.

[0103] The mobile phase may contain one or more of water, methanol, ethanol, acetonitrile, ethyl acetate, hexane, dichloromethane, supercritical carbon dioxide, or all other liquids known in the art. The selection of the mobile phase needs to take into account the highly unsaturated fatty acid or its ester derivative to be purified and the stationary phase used. In the reversed-phase stationary phase relative to the highly unsaturated fatty acid or its ester derivative, a polar mobile phase should be selected that is sufficient for developing and eluting the highly unsaturated fatty acid or its ester derivative, but whose development and elution are not too rapid as they approach the tip of the mobile phase.

[0104] In one aspect of the invention, the chromatography includes a mobile phase gradient.

[0105] In this specification, "mobile phase gradient" refers to the change in the composition of the mobile phase over time during the performance of the chromatography. A mobile phase with different mobile phase compositions, increasing or decreasing over time as a percentage during the performance of the chromatography, can be contained in the column.

[0106] The main purpose of the mobile phase gradient is to dissolve the strongly retained components in the column more quickly and the weakly retained components more slowly, so that the dissolved highly unsaturated fatty acids or their ester derivatives produce sufficiently separated peaks upon detection. For example, in reversed-phase chromatography, starting with a low concentration of a nonpolar mobile phase allows for the separation of weakly retained components. Strongly retained components may remain on the adsorbent surface at the top of the column or move very slowly. Increasing the amount of a nonpolar mobile phase, such as acetonitrile, in the mobile phase allows for faster movement of strongly retained components because the competition for adsorption sites relative to the stationary phase increases steadily.

[0107] Therefore, in reversed-phase chromatography for highly unsaturated fatty acids or their ester derivatives, the mobile phase at the start of the chromatography may comprise a high percentage of polar mobile phase A, such as water, selected from about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, or about 0%. Mobile phase B may be a mobile phase that is less polar than mobile phase A. For example, in the case where mobile phase A is water, mobile phase B may be methanol. Mobile phase B constitutes the remaining percentage of the mobile phase. When the chromatography is performed, as the mobile phase dissolves through the column, the gradient result causes the concentration of mobile phase B to increase over time. In some embodiments, a single-component liquid may also be used as the mobile phase. In some embodiments, the single-component liquid may be water, methanol, ethanol, acetonitrile, ethyl acetate, hexanes, dichloromethane, supercritical carbon dioxide, or all other liquids known in the art.

[0108] In one embodiment, the rate of increase of B over time may be constant. In one approach, there is no gradient, and the mobile phase has a constant composition during dissolution. In another approach, the percentage increase rate of mobile phase B in different time ranges under chromatographic conditions can be used. In yet another approach, the mobile phase can have a specific composition within a specific time range under chromatographic conditions, and contain a gradient within other time ranges.

[0109] The mobile phase delivery system is a pump device, such as a commercially available chromatographic pump, which supplies the mobile phase to the column. Such pumps are typically corrosion-resistant and solvent-resistant, providing pulsation-free flow, flow rates in the range of 0.1 to 100 L / min, accurate flow rate control, and generating high pressures up to 6000 psi. Reciprocating pumps contain a small chamber in which the mobile phase is received by the pump through the back-and-forth movement of a motor-driven piston. Two check valves that open and close alternately control the direction and flow of the mobile phase into and out of the cylinder. Single-piston pumps use specially designed cams to achieve very rapid replenishment times, producing more continuous flow. The disadvantages of pulsation flow caused by reciprocating pumps can often be overcome by using pulse dampers. The use of a dual-piston pump, which operates with pistons that move in phases different from each other (page 12 / 23, CN 121443710 A), provides a reasonable solution for pulse-free fluid transport. The linear velocity in the column represents the velocity of the fluid through the column cross-section. The linear velocity is a value that can be calculated using the following mathematical formula:

[0110] Linear velocity “m / hour” = Flow rate “m³ / hour” / Column cross-sectional area “m²”. The linear velocity can be approximately 0.2–20.0 m / hour, approximately 1.0–15.0 m / hour, approximately 1.0–10.0 m / hour, approximately 1.5–10.0 m / hour, or approximately 2.0–9.0 m / hour. In one embodiment, the linear velocity is approximately 4.0–9.0 m / hour.

[0111] In one aspect of the invention, the chromatography is performed at room temperature or above room temperature. In another aspect, the chromatography is performed at a temperature above room temperature. In another aspect, the room temperature is 20°C to 25°C.

[0112] In one aspect of the invention, the temperature above room temperature is 25°C or higher, 30°C or higher, or 35°C or higher, and / or, 60°C or lower, 50°C or lower, or 45°C or lower, or, 25 to 60°C, 30 to 50°C, or 35 to 45°C.

[0113] In this specification, "fraction" refers to a substance obtained by collecting small amounts of the eluent dissolved from the column at specific intervals in a mixture of fatty acids containing PUFA or its ester derivatives loaded on a column and adsorbed onto the chromatogram, and in an eluent used to develop and elute the fatty acid mixture and collect it on the column. The process of obtaining fractions is called fractionation, in which the composition of fatty acids may change over time. At different time points, different fractions are collected based on the unique properties of each component in the fatty acid mixture, such as highly unsaturated fatty acids or their ester derivatives (e.g., differences in affinity with the stationary phase and / or mobile phase).

[0114] As one aspect of the invention, a manufacturing method is provided, comprising: containing a fraction comprising PUFA or its ester derivative in an inert gas atmosphere inside a container C. In one aspect of the invention, the container C is placed in an inert gas atmosphere when containing the fraction. In one aspect of the invention, the container C may be placed in an inert gas atmosphere before, after, or simultaneously with the containing of the fraction. In one aspect of the invention, it is preferable to contain the fraction inside the container C after placing the container C in an inert gas atmosphere.

[0115] In one aspect of the invention, the container C may be a tank for containing fractions obtained by chromatography. In this specification, the tank for containing fractions is sometimes referred to as a fraction tank.

[0116] Concentration of Fractions A method for manufacturing a fatty acid composition according to one aspect of the present invention includes: concentrating a fraction containing PUFA or its ester derivative obtained by chromatography to obtain a fatty acid composition containing PUFA or its ester derivative.

[0117] Concentration of the fraction includes: distilling the eluent by subjecting the fraction containing PUFA or its ester derivative and an eluent under reduced pressure and / or heating, wherein the PUFA or its ester derivative is separated from the eluent during concentration. In one aspect of the present invention, the fraction may be added to one or more evaporators during concentration. In one aspect of the present invention, the eluent separated by distillation during concentration of the fraction can be recovered as a solvent and reused in the manufacture of the fatty acid composition. The solvent separated and recovered during concentration of the fraction is stored in container A, and the eluent can be obtained inside container A under an inert gas atmosphere.

[0118] In one aspect of the invention, the evaporator used for fraction concentration may include an eluent evaporator and / or a thin-film evaporator. Examples of eluent evaporators include single-evaporation devices, multi-effect evaporation devices, multi-chamber evaporation devices, natural circulation evaporators, forced circulation evaporators, falling film evaporators, rising film evaporators, and combinations thereof. In one aspect of the invention, examples of natural circulation evaporators include external heating and calandria systems.

[0119] In one aspect of the invention, any type of evaporator can be used for fraction concentration, such as thin-film evaporators, single-evaporation devices, multi-effect evaporation devices, multi-chamber evaporation devices, natural circulation evaporators, forced circulation evaporators, falling film evaporators, rising film evaporators, and combinations thereof. In one aspect of the invention, examples of natural circulation evaporators (pages 13 / 23, CN 121443710 A) include external heating and calandria systems.

[0120] In one aspect of the present invention, at least one of the reservoirs of the evaporator used in the fraction concentration has an internal volume of 10L or more, 20L or more, 50L or more, 100L or more, 500L or more, or 1000L or more, and / or, less than 5000L, less than 2000L, less than 1000L, or less than 500L.

[0121] In one aspect of the present invention, at least one of the evaporators used in the fraction concentration blows an inert gas during the fraction concentration. Examples of inert gases include nitrogen, argon, carbon dioxide, or combinations thereof. In one aspect, nitrogen is an example. In one aspect of the present invention, an eluent evaporator is an example of an evaporator that blows an inert gas.

[0122] In this specification, "blowing an inert gas" means supplying an inert gas to an evaporator or similar device or container for the purpose of gas replacement. The amount and rate of supply of the inert gas are not particularly limited. Blowing nitrogen is one method of blowing an inert gas. It should be noted that burgeing refers to the process of blowing an inert gas until it is replaced by an inert gas.

[0123] In this specification, "residual elution dose" refers to the concentration of eluent contained in a fatty acid composition containing PUFA or its ester derivative obtained by fractional concentration. In one embodiment of the present invention, the fatty acid composition containing PUFA or its ester derivative obtained by fractional concentration 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 eluent.In another aspect of the invention, the fatty acid composition comprises an eluent of 100 ppm or less, 50 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, 0.5 ppm or less, or 0.16 ppm or less, and / or 0.001 ppm or more, 0.003 ppm or more, or 0.01 ppm or more.

[0124] In one aspect of the invention, the concentration of the fraction can be carried out at a heating temperature of 190°C or less, 160°C or less, 140°C or less, 135°C or less, 130°C or less, 120°C or less, or 100°C or less, and / or 40°C or more, 60°C or more, 80°C or more, 100°C or more, 120°C or more, 130°C or more, 135°C or more, 140°C or more, or 160°C or more.

[0125] In one aspect of the present invention, the concentration of the fraction can be carried out under conditions where the minimum pressure during decompression 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. In this specification, "minimum pressure during decompression" refers to the pressure at the highest vacuum level during decompression.

[0126] As one aspect of the present invention, a manufacturing method is provided, which includes: containing a fatty acid composition comprising PUFA or its ester derivative inside a container D under an inert gas atmosphere. In one aspect of the present invention, when containing the fatty acid composition in container D, the interior of container D is placed under an inert gas atmosphere. In one aspect of the present invention, the interior of container D may be placed under an inert gas atmosphere before, after, or simultaneously with containing the fatty acid composition. In one aspect of the invention, it is preferable to place the container D in an inert gas atmosphere before storing the fatty acid composition inside the container D.

[0127] In one aspect of the invention, the container D may be a container for storing a fatty acid composition containing PUFA or its ester derivatives, obtained by fractional concentration. In one aspect of the invention, the container for storing the fatty acid composition containing PUFA or its ester derivatives is sometimes referred to as a product container.

[0128] <Fatty Acid Composition> In this specification, "color b-value" refers to a value in a colorimetric system. In a colorimetric system, L represents lightness, and and represent hue and chroma. Here, represents the hue from blue to yellow, where 0 represents achromatic, the more positive the direction, the stronger the yellow, and the more negative the direction, the stronger the blue. The colorimetric system is also known as CIE LAB. The color b-value can be measured using a colorimeter by conventional methods.In this specification, the colorimeter is also referred to as a colorimeter.

[0129] In one aspect of the present invention, the color b value of the fatty acid composition containing PUFA or its ester derivative 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 another aspect 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 or more, 0.1 or more, or 0.3 or more. In one aspect of the present invention, the color b value of the fatty acid composition containing PUFA or its ester derivative can be 0 or more, and can be 0 to 3.2, 0 to 3, 0 to 2.8, 0 to 2.5, 0 to 2, 0 to 1.5, 0 to 1, or 0 to 0.7.

[0130] In this specification, "anisidine value" refers to the numerical value used in colorimetric quantification of carbonyl compounds using anisidine. It can be determined by measuring the yellow hue produced by the reaction of aldehydes, which are deterioration products of oils, with anisidine reagent in the presence of acetic acid, using an absorbance of 350 nm. This determination is performed using the method described as an official method (2.4, 26-81) in the standard oil analysis test method. For example, a UV-Vis spectrophotometer can be used for the determination.

[0131] In one aspect of the invention, the anisidine value of the fatty acid composition comprising PUFA or its ester derivative 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 another aspect of the present invention, the anisidine value of the fatty acid composition may be less than 15, less than 10, less than 5, less than 1, less than 0.7, less than 0.5, less than 0.3, or less than 0.25, and / or more than 0, more than 0.001, more than 0.003, or more than 0.01.

[0132] The fatty acid composition obtained by the manufacturing method in one aspect of the present invention may contain one highly unsaturated fatty acid or its ester derivative 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), octadecanoic acid, C18:3, C19:4, C20:4, C21:5, or a combination of two or more highly unsaturated fatty acids or their ester derivatives. Furthermore, its ester derivatives can be C1-C6 alkyl ester derivatives, such as methyl, ethyl, propyl, butyl, pentyl, or hexyl esters. In one embodiment of the invention, the fatty acid composition comprises EPA ethyl ester, DHA ethyl ester, or a combination thereof.In one embodiment of the invention, the fatty acid composition comprises EPA ethyl ester. In one embodiment of the present invention, the fatty acid composition may comprise a subset selected from crotonic acid, myristic acid, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, transoleic acid, 11-octadecenoic acid, codenoic acid, eicosaenoic acid, erucic acid, nervonic acid, linoleic acid, eicosadienoic acid, docosahexaenoic acid, linolenic acid, pinolenic acid, tungsten acid, 5,8,11-eicosatrienoic acid, dihomo-γ-linolenic acid, eicosatrienoic acid, octadecanoic acid, eicosatraenoic acid, adrenaline, 5Z,8Z,10E,12E,14Z-bosseopentaenoic acid, all-cis-4,7,10,13,16-octadecanoic acid, sardine acid, tetracosapentaenoic acid. docosahexaenoic acid (4,8,12,15,18,21-), herring acid, propionic acid, butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid, pechosaccharide, behenic acid, tridecanoic acid, lignoceric acid, pentadecanoic acid, ceric acid, carboceric acid, lignic acid, nonacosylic acid, bermudaolic acid, laccaeroic acid, psyllic acid, geddic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoic acid The above may be a combination of one highly unsaturated fatty acid or its ester derivative, namely, tetracontylic acid or tetracontylic acid, or a combination of two or more highly unsaturated fatty acids or their ester derivatives.

[0133] In one aspect of the invention, the fatty acid composition contains, relative to the total fatty acid composition, a highly unsaturated fatty acid or its ester derivative in proportions 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%.

[0134] In this specification, specific ranges are given using numerical values ​​prefixed with the term “about.” The term “about” is used in this specification to provide the exact number prefixed with it and to provide textual support for numbers close to or approximately equal to that number. In determining whether a number is close to or approximately equal to a specifically recorded number, an unrecorded number that is close to or close to the recorded number may be a number substantially equivalent to the specifically recorded number provided in the context in which it is presented. In one manner, approximately may refer to ±5%, ±2.5%, ±2%, or ±1% of the number mentioned.

[0135] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention pertains. All methods and materials similar to or equivalent to those described in this specification may also be used in the implementation or testing of the invention, and representative exemplified methods and materials are described herein.

[0136] It should be understood that, where a range of values ​​is provided, the values ​​between the upper and lower limits of that range, as well as any other recorded values ​​within its recorded range or values ​​within the recorded range, are included within the scope of the invention. Unless the context clearly indicates otherwise, up to one-tenth of the units of the values ​​between the upper and lower limits of that range are included within the scope of the invention. The upper and lower limits of their smaller ranges can be independently included in the smaller ranges, which are also included in the scope of the invention. In the case where the inclusion becomes one or both of the objects of all the specific exclusion limits, the range excluding any one or both of the limits they include is also included in the invention.

[0137] In this specification, unless otherwise specified, "%" means "weight %".

[0138] The specific manner described in this specification is of course subject to variation, and therefore the invention is not limited to such specific manner. It should be understood that the scope of the invention is considered to be defined only by the appended claims, and therefore the terminology used in this specification is only for describing specific manners and not for limitation.

[0139] It should be understood that, unless otherwise specified, the singular form of a statement in this specification also includes its plural form. Therefore, it should be understood that, unless otherwise specified, articles in the singular form (e.g., "a", "an", "the", etc. in the English context) also include their plural forms.

[0140] A UV / Vis absorbance detector comprising a scanning spectrophotometer with a grating optical system can be used. A deuterium lamp can be used as a light source in the ultraviolet range of 190–360 nm. A tungsten lamp can be used as a light source in the visible range of 360–800 nm. The use of deuterium lamps and / or tungsten lamps, either alone or in combination, provides a simple means of detecting the absorbing material as it leaves the column.

[0141] A photodiode array (hereinafter also referred to as PDA) detector is an ultraviolet / visible absorbance detector capable of very rapidly collecting data within a selected spectral range. Absorbance spectral data associated with each chromatographic peak can be collected and stored. The stored data can be compared with pure standard spectra from a library. Because the characteristic spectra associated with the unseparated components are highly likely to differ, the spectra obtained by the PDA detector are useful in the identification of components with overlapping peaks and difficult separation.

[0142] Fluorescence detectors are useful in the detection of components exhibiting chemiluminescent properties such as fluorescence or phosphorescence. They are at least one order of magnitude more sensitive than UV absorbance detectors. Fluorescence is typically observed by detecting emission radiation separated by a grating at an angle of 90 degrees relative to the excitation beam. The amount of fluorescent substances can be enhanced by post-column derivatization of the dissolved compound using special reagents or by pre-column derivatization of the sample itself. Specification 16 / 23 pages 20 CN 121443710 A

[0143] Refractive index detectors respond to almost all solutes. Refractive index detectors are also known as RI detectors. The difference in refractive index between the reference mobile phase and the column effluent results in the detection of separated components as peaks on the chromatogram. Due to its extremely high sensitivity to the mobile phase, this detector cannot be used within an LC pump without sufficient pulse decay, and is also unsuitable for gradient applications due to varying mobile phase compositions. The detection limit is typically lower than that observed by an absorbance detector.

[0144] The conductivity detector provides high-sensitivity detection of all chargeable components. This detector can easily and reliably detect anions, cations, metals, organic acids, and surfactants down to the ppb level, and therefore can be used with LC systems. The addition of a chemical inhibitor between the column and the conductivity detector reduces the conductivity of the eluent, enabling the use of gradient dissolution and determination of ppb levels with minimal baseline drift.To typically identify low-level anions, the eluent is converted to a low-conductivity acid with weak ionization, thereby reducing background noise. For example, Na2CO3 is converted to carbonic acid. Simultaneously, the anions of the components are converted to their corresponding high-conductivity acids, relatively increasing the signal of the components. For example, NaCl is converted to HCl.

[0145] In this specification, the "area %" indicating the proportion of each fatty acid in the composition refers to the percentage of a component's content expressed as the ratio of the peak area of ​​each fatty acid to the sum of the peak areas of the fatty acids in a gas chromatography (hereinafter also referred to as GC-FID) analysis of the composition, where the peaks of each component are identified, and the peak area of ​​each fatty acid is calculated using the Agilent ChemStation integral algorithm. In the field of oleochemistry, area % is used with approximately the same meaning as weight %. The analysis conditions are based on the 2013 versions of the Standard Oil and Fat Analysis Test Method 2.4.2.1–2013 Fatty Acid Composition (FID Isothermal Gas Chromatography) and 2.4.2.2–2013 Fatty Acid Composition (FID Heated Gas Chromatography) established by the Japan Oil Chemistry Society (JOCS). The analytical conditions for gas chromatography (hereinafter, GC) are as follows.

[0146] GC-FID Measurement Conditions GC: 7890B or 8890 (Agilent Technologies) Detector: FID Column: 30m × 0.25mm, 0.25μm film thickness (equivalent to a capillary column of USP PHASE G16) Column Temperature: Constant temperature around 200°C Detector Temperature: Constant temperature around 300°C Sample Introgression Temperature: Constant temperature around 300°C Carrier Gas: Helium Flow Rate: Ethyl eicosate retention time adjusted to approximately 30 minutes Split Ratio: 1:100 Make-up Gas: Nitrogen 30mL / min Area Measurement Range: From the peak of the solvent to approximately 2.5 times the retention time of ethyl eicosate Analyte Injection Volume: 1.0μL Analysis Time: Approximately 75 minutes In one embodiment of the invention, an internal standard can be used when performing analysis using gas chromatography. To determine the relative retention time of the analyte with respect to the internal standard or to aid in the quantification of the analyte, the internal standard can be added to the sample as a reference marker. The internal standard can be suitably selected by those skilled in the art as a compound that is very similar to but not identical to the target analyte, such as a deuterated derivative of the target analyte. For quantification purposes, the internal standard can be calibrated by plotting the ratio of the analyte's signal to the internal standard's signal as a function of the standard analyte concentration. Here, the standard is a sample of known concentration prepared by those skilled in the art for reference in quantifying unknown analyte samples (pages 17 / 23, CN 121443710 A).

[0147] Examples The present invention will be described in more detail below through examples, but the present invention is not limited to these examples. It should be noted that in the following examples, unless otherwise specified, "%" means "weight %".

[0148] In the examples of this application, the measuring devices used are as follows.

[0149] • Oxygen concentration in the headspace: measured using a residual oxygen meter "Pack Keeper" RO-103KS (manufactured by Iijima Electronics Co., Ltd.) according to the instructions of the device.

[0150] • Dissolved oxygen concentration in methanol: measured using a dissolved oxygen meter: FOR-21 (manufactured by Automatic System Research Co., Ltd.) according to the instructions of the device.

[0151] • Color b value: measured using a color and turbidity simultaneous measuring instrument: TZ-6000 (manufactured by Nippon Denshoku Kogyo Co., Ltd.), C light source, 2-degree field of view, 20mm test tube, according to the instructions of the device. In this specification, the color b value represents the value in the colorimetric system / CIE LAB.

[0152] • Anisidine value: Determined using a UV-Vis spectrophotometer: V-730 (DS type) (manufactured by Nippon Spectrophotometer Co., Ltd.), following the procedure described in USP401 for anisidine value.

[0153] • Acid value: Determined using an automatic titration apparatus: 876 Dosimat (manufactured by Metrohm), following the procedure described in USP401 for acid value.

[0154] • Fatty acid composition and isomer analysis: Determined using GC-FID. The analytical conditions for gas chromatography are described separately.

[0155] • Methanol residue: Methanol residue was analyzed using headspace gas chromatography (hereinafter also referred to as HS-GC), which combines headspace and gas chromatography. The analytical conditions for headspace and GC are described below.

[0156] Headspace device conditions Headspace sampler: G4557A (Agilent Technologies) Equilibrium temperature in vial: constant temperature around 100°C Equilibrium time in vial: 20 minutes Injection line temperature: 150°C Carrier gas: Helium Pressurization pressure: 103 kPa Pressurization time: 0.01 minutes Sample injection volume: 1.0 mL GC‑FID measurement conditions GC: 7890B or 6850A (Agilent Technologies) Detector: FID Column: 30 m × 0.25 mm, 0.50 μm film thickness (capillary column equivalent to USP PHASE G16) Column temperature: hold at about 40°C for 5 minutes → heat up at 30°C / minute → hold at about 240°C for 5 minutes Sample introduction part temperature: constant temperature around 250°C Detector temperature: constant temperature around 280°C Carrier gas: Helium Flow rate: the retention time of methanol is adjusted to about 4.3 minutes Description Page 18 / 23 22 CN 121443710 A Split ratio: 1:10 Make-up gas: Nitrogen 25 mL / min. Vial volume: 10 mL Vial stirring level: 5 Analysis time: about 16.7 minutes <Preparation method of main fraction> Fish oil ethyl ester with EPA-E content of 16 area% or more in fatty acid ethyl esters is prepared by ethylating fish oil through a conventional method. Using a multi-stage rectification apparatus, the fish oil ethyl ester is subjected to continuous rectification under the conditions that the pressure at the top of the column is 26.7 Pa or less and the temperature is 190°C or less, and fractionated into an initial fraction, a main fraction, and a residual fraction, thereby obtaining the main fraction. Herein, the initial fraction is a fraction with higher volatility than the main fraction, and takes ethyl esters of fatty acids with less than C20 as the main component. The main fraction takes ethyl esters of C20 fatty acids as the main component, and the content of EPA-E in the fatty acid ethyl esters is 70 area% or more. The residual fraction is a fraction with lower volatility than the main fraction, and takes ethyl esters of fatty acids with C21 or more as the main component.

[0157] The preparation of the main fraction can be carried out, for example, by referring to the methods described in Japanese Patent Application Laid-open No. Hei 04-041457, Japanese Patent Application Laid-open No. Hei 04-128250, and Japanese Patent Application Laid-open No. Hei 05-222392.

[0158] <HPLC purification method> According to the process flow diagram shown in Figure 1, purified EPA-E is produced from the main fraction. That is, the main fraction in the main fraction tank is purified by HPLC using an eluent from an eluent tank, and the obtained fractions are stored in a fraction tank. The intermediate EPA-E obtained by concentrating the fractions by an eluent evaporator is stored in a buffer tank. The purified EPA-E obtained by further concentrating the intermediate EPA-E by a thin film evaporator is stored in a product tank.The EPA-E in the fatty acid ethyl esters of the main fraction is 70% or more by area, and the EPA-E in the fatty acid ethyl esters of purified EPA-E is 96% or more by area.

[0159] The apparatus shown in this process diagram is a closed system except for the eluent tank. The main fraction tank, fraction tank, and product tank, excluding the eluent evaporator, buffer tank, and thin film evaporator after depressurization, are purged with nitrogen using a gas sealing unit and a vent valve.

[0160] In this specification, the main fraction is contained in a fatty acid mixture containing PUFA or its ester derivatives. The intermediate EPA-E and purified EPA-E are contained in a fatty acid composition containing PUFA or its ester derivatives.

[0161] The temperature of the eluent tank is 38°C to 42°C, and the volume ratio of methanol used as eluent to the headspace is 11:89 to 13:87. Here, the volume ratio is "eluent: headspace". For the eluent tank, the vent pipe (V) is opened, and the top space is filled with air, or nitrogen is blown from the vent pipe (V) to purge the top space with nitrogen.

[0162] The temperature of the main fraction tank is 39.5℃~40.5℃, and the volume ratio of the main fraction to the top space is 5:95~90:10. Here, the volume ratio is "main fraction: top space". The main fraction tank is purged with nitrogen. The gas in the top space is released when the main fraction is received through the equipped gas sealing unit and the vent valve (G), and nitrogen is blown when the main fraction is transferred to the HPLC to purge the top space of the main fraction tank with nitrogen.

[0163] In the HPLC, at a temperature of 38℃~42℃, methanol (b) from the eluent tank is used as the eluent, and ODS is used as the stationary phase. The main fraction (a) transferred from the main fraction tank is purified, and the fraction (c) containing EPA-E 96% or more in fatty acid ethyl esters is stored in the fraction tank.

[0164] The fractionation tank is not temperature-regulated, and the temperature is gradually reduced from approximately 40°C. The volume ratio of the fraction to the headspace is 5:95 to 65:35. Here, the volume ratio is "fraction: headspace". The fractionation tank is purged with nitrogen. Nitrogen is released from the headspace when receiving the fraction from the HPLC through the equipped gas sealing unit and vent valve (G), and nitrogen is purged when the fraction is transferred to the eluent evaporator to purge the headspace of the fractionation tank. Specification 19 / 23 pages 23 CN 121443710 A

[0165] DL-α-tocopherol in an amount of 0.05% relative to EPA-E is added to the stored fraction (d), and then methanol removal is performed using the eluent evaporator. In the eluent evaporator, methanol removal is carried out by blowing a small amount of nitrogen while maintaining a vacuum of 35.8 kPa to 36.2 kPa using an absolute pressure gauge. The temperature gradually increases from 41°C to 42°C and finally reaches 130°C to 133°C. The intermediate EPA-E(e), with a residual methanol content of about 500 ppm, is stored in a buffer tank.On the other hand, the methanol (f) liquefied after vacuum distillation is recovered into the eluent tank.

[0166] The vacuum degree of the buffer tank is 35.8 kPa to 36.2 kPa with an absolute pressure gauge, and the temperature is 130°C to 133°C. The volume ratio of intermediate EPA-E to headspace is 6:94 to 9:91. Here, the volume ratio is "intermediate EPA-E: headspace". On the other hand, the methanol (h) liquefied after vacuum distillation is recovered into the eluent tank.

[0167] The stored intermediate EPA-E (g) is further de-methanoled using a thin-film evaporator. The further de-methanoling in the thin-film evaporator is carried out at a vacuum degree of 1 to 20 Pa with an absolute pressure gauge and a temperature of 110°C to 127°C, with a residual methanol content of less than 5 ppm. DL-α-tocopherol is added in such a way that it is 0.2% of the total amount of EPA-E, and the purified EPA-E (i) is stored in the product tank. On the other hand, a small amount of methanol (j) liquefied after vacuum distillation is extracted as waste solvent to the eluent storage tank.

[0168] The temperature of the product tank is 20°C to 22°C, and the volume ratio of purified EPA-E to headspace is 0:100 to 81:19. Here, the volume ratio is "purified EPA-E: headspace". The product tank is purged with nitrogen. When receiving purified EPA-E, the gas in the headspace is released through the equipped gas sealing unit and the vent valve (G). When transferring purified EPA-E, nitrogen is purged to purge the headspace of the product tank with nitrogen.

[0169] Example 1 Effect of oxygen concentration in the eluent tank on dissolved oxygen in the eluent In Example 1a, methanol obtained by vacuum distillation and liquefaction was added dropwise to the eluent tank in which the oxygen concentration in the headspace was 0.00% by volume due to purging with nitrogen. The oxygen concentration in the headspace and the dissolved oxygen in the methanol as the eluent were measured at this time. In Example 1b, the oxygen concentration in the headspace was 12.50% by volume, and other procedures were the same as in Example 1a.

[0170] In this example, the temperature for measuring the dissolved oxygen in methanol was 23°C, and the measurement time from when the sensor of the dissolved oxygen meter was placed in methanol and the measurement began until the value of the dissolved oxygen meter was read was 15 minutes. The results are shown in Table 1.

[0171] According to this example, the reduction of the oxygen concentration in the headspace of the eluent tank by nitrogen purging of the eluent tank is related to the reduction of the dissolved oxygen in the eluent.

[0172] [Table 1]

[0173] Example 2 Oxygen concentration in the fractionation tank with or without nitrogen purging of the eluent tank Example 2a Eluent tank with nitrogen purging Methanol obtained by vacuum distillation was added dropwise to the eluent tank, which was purged by blowing nitrogen into the eluent tank, and stored as the eluent. The methanol was passed through HPLC and collected in the fractionation tank that had been purged with nitrogen. Before storage, the volume ratio of methanol in the fractionated tank to the top space was 5:95, and after storage, the volume ratio was 65:35.Then, while purging nitrogen, methanol as a fraction was transferred to restore the volume ratio to 5:95 before storage. Here, their volume ratio is "fraction: headspace" as specified on page 24 of CN 121443710 A, 20 / 23.

[0174] The oxygen concentration shift in the fraction tank was measured when the cycle of storing the fraction and transferring it while purging nitrogen was repeated 10 times. The results are shown in Table 2.

[0175] Example 2b Nitrogen-free purging of the eluent tank Methanol obtained by vacuum distillation was added dropwise to the eluent tank, which was filled with air without purging nitrogen, and stored as the eluent. The methanol was passed through HPLC and stored in a fraction tank that had been purged with nitrogen. In this example, the conditions other than nitrogen purging of the eluent tank were the same as in Example 2a. The results are shown in Table 2.

[0176] According to this example, nitrogen purging of the eluent tank can reduce the oxygen concentration in the headspace of the fraction tank. It can be seen that without nitrogen purging, oxygen enters together with methanol during storage. If the storage and transfer cycle is repeated more than 5 times, the oxygen concentration shift is approximately constant.

[0177] [Table 2]

[0178] Example 3 Oxygen concentration in the top space of the eluent tank and color b value of intermediate EPA-E in the buffer tank Specification 21 / 23 pages 25 CN 121443710 A The color b value of intermediate EPA-E in the buffer tank after nitrogen purging of the eluent tank, HPLC purification and eluent evaporation was confirmed.

[0179] Methanol stored in the eluent tank was used as the eluent. The main fraction stored in the nitrogen-purged main fraction tank was purified by HPLC. The fraction with an eicosapentaenoic acid ethyl ester ratio of 96% or more in the fatty acid composition was stored in the nitrogen-purged fraction tank. DL-α-tocopherol was added to the preserved fraction at a rate of 0.05% relative to EPA-E, and then methanol was removed using an eluent evaporator, resulting in intermediate EPA-E stored in a tank with a residual methanol content of approximately 500 ppm.

[0180] In this embodiment, the volume ratio of the fraction to the headspace was 5:95 to 65:35, the same as in Example 2, and the cycle of receiving and transferring was repeated. Here, the volume ratio is "fraction: headspace".

[0181] The oxygen concentration in the headspace of the eluent tank and the color b value of the intermediate EPA-E in the buffer tank were measured after five cycles of receiving and transferring the fraction tank. The results are shown in Table 3.

[0182] According to this embodiment, by using the eluent in the eluent tank that has been nitrogen-purged, the color difference (color b value) of the intermediate EPA-E can be reduced.

[0183] [Table 3]

[0184] Example 4 Nitrogen purging of eluent tank and confirmation of the physical properties of purified EPA-E The effect of the presence or absence of nitrogen purging of eluent tank on the color b value, anisidine value, and acid value of purified EPA-E in the product tank after HPLC purification, eluent evaporation, and thin film evaporation.

[0185] The intermediate EPA-E was stored in a buffer tank using the same steps as in Example 3. After further methanol removal from the intermediate EPA-E using a thin film evaporator, DL-α-tocopherol was added to adjust the amount to 0.2% of the total amount of EPA-E, and the purified EPA-E was stored in the product tank after nitrogen purging.

[0186] The results of measuring the physical properties and fatty acid composition of purified EPA-E based on the presence or absence of nitrogen purging of eluent tank are shown in Table 4.

[0187] According to this example, it can be seen that by using an eluent that has undergone nitrogen purging, the color b value and anisidine value of purified EPA-E can be reduced. On the other hand, there is almost no difference in acid value, methanol residue, and isomer ratio.

[0188] [Table 4] Specification 22 / 23 pages 26 CN 121443710 A

[0189] Explanation of reference numerals V: Vent pipe G: Gas sealing unit and vent 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: Small amount of methanol liquefied after vacuum distillation. Specification 23 / 23 pages 27 CN 121443710 A Figure 1 Specification Figure 1 / 1 page 28 CN 121443710 A.

Claims

1. A method for producing a fatty acid composition, characterized by, The fatty acid composition contains highly unsaturated fatty acids, i.e., PUFAs, or ester derivatives thereof, The production method includes: (1) a solvent is housed in a container A, and an eluent is obtained in an atmosphere of an inert gas inside the container A; (2) purification of a fatty acid mixture containing PUFAs or ester derivatives thereof is performed by chromatography using the eluent as a mobile phase, and a fraction containing PUFAs or ester derivatives thereof is obtained; and (3) concentration of the fraction containing PUFAs or ester derivatives thereof is performed, and a fatty acid composition containing PUFAs or ester derivatives thereof is obtained.

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

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

4. The production method according to any one of claims 1 to 3, wherein The solvent contains a solvent recovered from the fraction containing PUFAs or ester derivatives thereof by the concentration of the fraction.

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

6. The production method according to any one of claims 1 to 5, wherein The oxygen concentration of the headspace of the 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 production method according to any one of claims 1 to 6, wherein The fraction containing PUFAs or ester derivatives thereof is housed inside a container C in an atmosphere of an inert gas.

8. The production method according to any one of claims 1 to 7, wherein The fraction containing PUFAs or ester derivatives thereof is housed inside a container C in an atmosphere of an inert gas, and the oxygen concentration of the headspace of the container C after the fraction is housed 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 production method according to any one of claims 1 to 8, wherein The fatty acid mixture containing PUFAs or ester derivatives thereof is housed inside a container B in an atmosphere of an inert gas before the purification of the fatty acid mixture.

10. The manufacturing method according to claim 9, wherein, The oxygen concentration of the headspace of the 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 production method according to any one of claims 1 to 10, wherein The fatty acid composition containing PUFAs or ester derivatives thereof is housed inside a container D in an atmosphere of an inert gas.

12. The manufacturing method according to claim 11, wherein, The oxygen concentration of the headspace of the 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 production method according to any one of claims 1 to 12, wherein The inert gas inside the container A, the container B, the container C, and the container D is each independently nitrogen, argon, or carbon dioxide.

14. The production method according to any one of claims 1 to 13, wherein The inert gas inside the container A, the container B, the container C, and the container D is nitrogen.

15. The production method according to any one of claims 1 to 14, wherein The proportion of PUFAs or ester derivatives thereof in all fatty acids or ester derivatives thereof in the fatty acid composition containing PUFAs or ester derivatives thereof is 97% or more, 96% or more, 90% or more, 80% or more, 70% or more, or 50% or more.

16. The production method according to any one of claims 1 to 15, wherein The color b value of the fatty acid composition containing the PUFA or the 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 production method according to any one of claims 1 to 16, wherein The anisidine value of the fatty acid composition containing the PUFA or the 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 production method according to any one of claims 1 to 17, wherein The residual eluent amount of the fatty acid composition containing the PUFA or the 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 production method according to any one of claims 1 to 18, wherein The maximum temperature in the concentration of the fraction is 190°C or less, 160°C or less, 140°C or less, 135°C or less, 130°C or less, 120°C or less, or 100°C or less, and / or, 40°C or more, 60°C or more, 80°C or more, 100°C or more, 120°C or more, 130°C or more, 135°C or more, 140°C or more, or 160°C or more.

20. The production method according to any one of claims 1 to 19, wherein The minimum pressure in the concentration of the fraction under reduced pressure 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 production method according to any one of claims 1 to 20, wherein The solvent and the eluent are a mixture of one or two or more selected from methanol, ethanol, 2-propanol, acetonitrile, hexane, or a mixture of one or more of them with water.

22. The production method according to any one of claims 1 to 21, wherein The purification of the fatty acid mixture uses a silica gel-based adsorbent or a polymer-based adsorbent as a stationary phase.

23. The production method according to any one of claims 1 to 22, wherein The purification of the fatty acid mixture uses a reverse phase-based adsorbent as a stationary phase.

24. The production method according to any one of claims 1 to 23, wherein The concentration of the fraction uses one or more evaporators, and the inner volume of at least one of the liquid reservoirs of the evaporators is 10 L or more, 20 L or more, 50 L or more, 100 L or more, 500 L or more, or 1000 L or more, and / or, 5000 L or less, 2000 L or less, 1000 L or less, or 500 L or less.

25. The production method according to any one of claims 1 to 24, wherein The concentration of the fraction uses one or more evaporators, and an inert gas is blown into at least one of the evaporators during the concentration of the fraction.

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

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

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

29. A fatty acid composition, characterized in that, A fatty acid mixture containing a PUFA or an ester derivative thereof produced by the production method described in any one of claims 1 to 28.

30. A fatty acid composition, characterized in that, A fatty acid mixture containing a highly unsaturated fatty acid, that is, a PUFA, or an ester derivative thereof, The proportion of the PUFA or ester derivative thereof in the total fatty acids or ester derivatives thereof 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 of claim 30, wherein, The anisidine value of the fatty acid composition containing the PUFA or 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 of claim 30 or 31, wherein, The residual eluent amount of the fatty acid composition containing the PUFA or 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 or a combination of two or more selected from the group consisting of EPA, DHA, n-3 DPA, DGLA, and ARA.

34. The fatty acid composition according to any one of claims 30 to 33, wherein, The ester derivative of the PUFA 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 ester derivative thereof is an ethyl ester of EPA or an ethyl ester of DHA. The PUFA is one or a combination of two or more selected from the group consisting of EPA, DHA, n-3 DPA, DGLA, and ARA. The ester derivative of the PUFA is a C1 to C6 alkyl ester derivative of the PUFA. The PUFA or ester derivative thereof is an ethyl ester of EPA or an ethyl ester of DHA.

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