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

Abstract

To provide a composition which includes a higher unsaturated fatty acid or a higher unsaturated fatty acid alkyl ester highly concentrated with sufficiently low concentration of 3-MCPD fatty acid ester and a method for efficient production thereof.SOLUTION: A composition includes a fatty acid or a fatty acid alkyl ester as a main component. The composition includes a higher unsaturated fatty acid or an alkyl ester thereof. The proportion of the higher unsaturated fatty acid in the constituent fatty acid of the composition is 50 area% or more. The composition has a 3-MCPD concentration of less than 1.80 ppm determined as analyzed according to the American Oil Chemists' Society's official method Cd29b-13 assay A.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition containing a highly unsaturated fatty acid or an alkyl ester thereof and a method for producing the same. [Background technology]

[0002] 3-Chloropropane-1,2-diol (3-MCPD) is a compound suspected of being carcinogenic, and its concentration in food is regulated in various countries, including the EU. It is also known that 3-MCPD is produced in fats and oils from diacylglycerol (DAG) or monoacylglycerol (MAG) as substrates (Non-Patent Document 1). Since the presence of 3-MCPD was pointed out in fats and oils rich in diacylglycerol (DAG), attempts have been made to reduce its content in fats and oils by various methods. For example, it is known that 3-MCPD is formed at high temperatures, and Patent Documents 1 and 2 disclose that the concentration of substances that produce 3-MCPD in fats and oils can be reduced by treating with an adsorbent, lowering the deodorization temperature, or shortening the treatment time. However, these are methods for reducing the concentration of substances that produce 3-MCPD in the production of triacylglycerol, and Patent Documents 1 and 2 do not disclose a method for reducing the concentration of 3-MCPD fatty acid esters in the production of alkyl esters.

[0003] Polyunsaturated fatty acids (PUFAs) are known to have various functionalities, and highly concentrated PUFAs are used in foods, supplements, medicines, or cosmetics. When the ratio of the desired PUFA among the fatty acids of the starting composition is increased by highly concentrating PUFAs, the PUFAs are converted from glycerides mainly composed of triacylglycerol to alkyl esters with lower alcohols. Therefore, the highly refined and concentrated PUFAs used in foods, supplements, medicines, or cosmetics are often alkyl esters. There has been no known method to reduce the concentration of 3-MCPD fatty acid esters for such high concentrations of PUFA alkyl esters.

[0004] Factors that affect the production of 3-MCPD fatty acid esters include chlorine sources, substrates such as MAG and DAG, and high-temperature treatment time (Non-Patent Documents 1 and 2). However, for oils that are raw materials for foods containing PUFA, the content of chlorine sources and substrates of 3-MCPD fatty acid esters such as MAG and DAG is usually small, and the impact on the purification of the target PUFA is extremely small, so the need to remove them is not generally recognized. In addition, complete removal of chlorine sources and substrates such as MAG and DAG has not been performed due to the technical difficulty and the impact on productivity such as the recovery rate of PUFA.

[0005] Rectification is a type of distillation that can be expected to have a high separation ability, but because it requires internal packing and reflux, it often requires heating at high temperatures of 150°C or higher. Molecular distillation and short-path distillation can be performed at relatively low temperatures, 150°C or lower, compared to rectification, but repeated treatment is required to sufficiently concentrate PUFA. Therefore, there is a risk of producing a large amount of 3-MCPD in the distillation of PUFA.

[0006] The urea addition method and the HPLC method are methods for separating fatty acids based on the structure (e.g., chain length, number of double bonds, etc.) of the fatty acids that make up the molecule. However, when 3-MCPD is present in the raw material as a di- or mono-fatty acid ester, it may be difficult to separate it from the target alkyl ester depending on the type of fatty acid that constitutes it, and it is difficult to stably obtain fatty acid alkyl esters with reduced 3-MCPD fatty acid esters.

[0007] Therefore, there is a clear risk of producing 3-MCPD fatty acid esters in purification processes that include heat treatment such as solvent removal and distillation, and methods such as urea addition and HPLC cannot always remove the various 3-MCPD fatty acid esters contained in the raw material. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent Publication 2011-147435 [Patent Document 2] Patent Publication No. 2011-147436 [Non-patent literature]

[0009] [Non-Patent Document 1] Eur. J. Lipid Sci. Technol. 114, 1268-1273 (2012) [Non-Patent Document 2] Eur. J. Lipid Sci. Technol. 115, 735-739 (2013) [Non-Patent Document 3] J. Agric. Food. Chem. 63(6) 1839-48 (2015) Summary of the Invention [Problem to be solved by the invention]

[0010] Oils and fats containing PUFA often contain 3-MCPD or its fatty acid esters derived from the raw materials and the oil extraction and refining processes. In addition, when the glycerides in oils and fats are alkylated, MAG and DAG remain or their concentrations increase, creating an environment that is favorable for the production of 3-MCPD fatty acid esters. Furthermore, the purification of alkyl esters often includes a desolvation process and distillation processes such as molecular distillation, short-path distillation, and rectification, and 3-MCPD fatty acid esters can be produced in each process. In chromatography and urea addition, the possibility of 3-MCPD fatty acid esters being produced is low because no heat is applied, but there is a risk that the concentration of 3-MCPD fatty acid esters already contained in the process cannot be sufficiently reduced. An object of the present invention is to provide a composition containing highly concentrated PUFA or PUFA alkyl esters, in which the concentration of 3-MCPD fatty acid esters is sufficiently low, and an efficient method for producing the same. [Means for solving the problem]

[0011] As a result of intensive research conducted by the present inventors to achieve the above object, they found that the C20-C22 fatty acid alkyl ester fraction in which PUFA is concentrated and the 3-MCPD mono fatty acid ester having one molecule of C14 or C16 fatty acid bonded thereto exhibit similar behavior during distillation. Furthermore, they found that the concentration of the 3-MCPD mono fatty acid ester in the C20-C22 fatty acid alkyl ester fraction can be reduced by reducing the content of monoacylglycerol (MAG) in the raw material, particularly the MAG having one molecule of C14 or C16 fatty acid bonded thereto. The present inventors also found that the rate of formation of 3-MCPD fatty acid esters changes significantly depending on the concentration of extremely small amounts of metals, such as iron, of 1 ppm or less contained in the raw material. It has been reported that iron affects the formation of 3-MCPD fatty acid esters from triacylglycerol (Non-Patent Document 3). However, Non-Patent Document 3 only discloses a method for the formation of 3-MCPD fatty acid esters using extremely large amounts of Fe 2+ or Fe 3+ This was the result of investigating the formation of 3-MCPD fatty acid esters in the presence of iron, and it was not known that the extremely small amounts of iron normally contained in the oil used as the raw material for producing PUFA alkyl esters affects the formation of 3-MCPD fatty acid esters. The PUFA alkyl ester before concentration produced from raw materials such as fish oil is generally low in refinement, so the iron concentration may vary depending on the iron content of the raw oil. The iron concentration of the PUFA alkyl ester before concentration may also vary greatly depending on the quality of the extracted raw material, the extraction method, the purification method, etc. Therefore, when the iron concentration is high, the concentration of 3-MCPD fatty acid ester may unexpectedly increase due to heat treatment such as distillation. By adjusting the iron concentration before heat treatment such as distillation to less than 0.20 ppm, such an unexpected increase in the concentration of 3-MCPD fatty acid ester can be effectively suppressed. Based on these findings, the present inventors conducted further research and completed the present invention. That is, the present invention is as follows.

[0012] [1] A composition containing a fatty acid or a fatty acid alkyl ester as a main component, the composition containing a highly unsaturated fatty acid or its alkyl ester, the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition being 50 area % or more, and the concentration of 3-MCPD generated when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is less than 1.80 ppm. [2] The composition according to [1], wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 70 area % or more. [3] The composition according to [1] or [2], wherein the concentration of 3-MCPD generated when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is less than the detection limit. [4] The composition according to any one of [1] to [3], wherein the highly unsaturated fatty acid is eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, or a combination thereof. [5] The composition according to any one of [1] to [4], which is a distillate. [6] The composition according to any one of [1] to [5], wherein the raw material is fish oil, microbial oil, vegetable oil, or marine animal oil. [7] A distillation raw material composition containing a highly unsaturated fatty acid alkyl ester, The highly unsaturated fatty acid alkyl ester includes a highly unsaturated fatty acid alkyl ester for the purpose of concentration, The composition has a concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number that is 5 or more less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated, of less than 10,000 ppm, and / or an iron concentration of less than 0.20 ppm. [8] The composition according to [7], wherein the concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number 6 less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated is less than 10,000 ppm. [9] The composition according to [7] or [8], wherein the chlorine concentration is less than 10 ppm.

[10] The composition according to any one of [7] to [9], wherein the highly unsaturated fatty acid alkyl ester to be concentrated is an alkyl ester of eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, or arachidonic acid, or a combination thereof.

[11] The composition according to any one of [7] to

[10] , wherein the raw material is fish oil, microbial oil, vegetable oil, or marine animal oil.

[12] A method for producing a composition containing a highly unsaturated fatty acid or an alkyl ester thereof, comprising the steps of: (1) a step of alkyl-esterifying a raw material containing a triglyceride containing a highly unsaturated fatty acid as a constituent fatty acid to prepare a composition containing a highly unsaturated fatty acid alkyl ester; (2) at least one selected from: (a) a step of reducing the concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number that is 5 or more less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated in the composition of step (1) to less than 10,000 ppm; (b) a step of reducing the iron concentration in the composition of step (1) to less than 0.20 ppm; and (c) a step of reducing the chlorine concentration in the composition of step (1) to less than 10 ppm; (3) distilling the composition obtained after step (2) and isolating a main fraction; The method comprising:

[13] The method according to

[12] , wherein the concentration of 3-MCPD produced in the main fraction in step (3) is less than 1.80 ppm when analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A.

[14] The method according to

[12] or

[13] , wherein step (2)(a) is carried out by silica gel chromatography.

[15] The method according to any one of

[12] to

[14] , wherein the distillation in the step (3) is rectification.

[16] The method according to any one of

[12] to

[15] , wherein in step (2), the concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number 6 less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated is reduced to less than 10,000 ppm.

[17] The method according to any one of

[12] to

[16] , wherein the highly unsaturated fatty acid is eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, or a combination thereof.

[18] The method according to any one of

[12] to

[17] , wherein the raw material is fish oil, microbial oil, vegetable oil, or marine animal oil.

[0013] In another aspect, the present invention is as follows. [A1] A composition containing a highly unsaturated fatty acid or an alkyl ester thereof, wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 50 area % or more, and when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the concentration of 3-MCPD generated is less than 1.80 ppm. [A2] The composition according to [A1], wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 70 area % or more. [A3] The composition described in [A1] or [A2], wherein the concentration of 3-MCPD generated when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is less than the detection limit. [A4] The composition according to any one of [A1] to [A3], wherein the highly unsaturated fatty acid is eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, or a combination thereof. [A5] The composition according to any one of [A1] to [A4], which is a distillate. [A6] The composition according to any one of [A1] to [A5], wherein the raw material is derived from fish oil, microbial oil, vegetable oil, or marine animal oil. [A7] A distillation raw material composition containing a highly unsaturated fatty acid alkyl ester, The highly unsaturated fatty acid alkyl ester includes a highly unsaturated fatty acid alkyl ester for the purpose of concentration, The composition has a concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number that is 5 or more less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated, of less than 10,000 ppm, or an iron concentration of less than 0.20 ppm. [A8] The composition according to [A7], in which the concentration of monoacylglycerol containing as a constituent fatty acid a fatty acid having 6 fewer carbon atoms than the number of carbon atoms of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated is less than 10,000 ppm. [A9] The composition according to [A7] or [A8], having a chlorine concentration of less than 10 ppm. [A10] The composition according to any one of [A7] to [A9], wherein the highly unsaturated fatty acid alkyl ester to be concentrated is an alkyl ester of eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, or arachidonic acid, or a combination thereof. [A11] The composition according to any one of [A7] to [A10], wherein the raw material is derived from fish oil, microbial oil, vegetable oil, or marine animal oil. [A12] A method for producing a composition containing a highly unsaturated fatty acid or an alkyl ester thereof, comprising the steps of: (1) a step of alkyl-esterifying a raw material containing a triglyceride containing a highly unsaturated fatty acid as a constituent fatty acid to prepare a composition containing a highly unsaturated fatty acid alkyl ester; (2) at least one selected from: (a) a step of reducing the concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number that is 5 or more less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated in the composition of step (1) to less than 10,000 ppm; (b) a step of reducing the iron concentration in the composition of step (1) to less than 0.20 ppm; and (c) a step of reducing the chlorine concentration in the composition of step (1) to less than 10 ppm; (3) distilling the composition obtained after step (2) and isolating a main fraction; The method comprising: [A13] The method according to [A12], wherein the concentration of 3-MCPD produced when the main fraction in step (3) is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is less than 1.80 ppm. [A14] The method according to [A12] or [A13], wherein step (2)(a) is carried out by silica gel chromatography. [A15] The method according to any one of [A12] to [A14], wherein the distillation in step (3) is rectification. [A16] The method according to any one of [A12] to [A15], wherein in step (2), the concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number 6 less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated is reduced to less than 10,000 ppm. [A17] The method according to any one of [A12] to [A16], wherein the highly unsaturated fatty acid is eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, or a combination thereof. [A18] The method according to any one of [A12] to [A17], wherein the raw material is derived from fish oil, microbial oil, vegetable oil, or marine animal oil. Effect of the Invention

[0014] According to the present invention, a composition containing a high concentration of PUFA or PUFA alkyl ester with a low concentration of 3-MCPD fatty acid ester can be stably produced. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in detail below. In this specification, when describing fatty acids, a numerical expression may be used that expresses the number of carbon atoms, the number of double bonds, and the position of the double bonds in a simplified manner by combining numbers and letters. For example, a saturated fatty acid with 20 carbon atoms may be described as "C20:0", a monounsaturated fatty acid with 18 carbon atoms may be described as "C18:1", eicosapentaenoic acid may be described as "C20:5 n-3", etc. "n-" indicates the position of the double bond counted from the methyl end of the fatty acid, for example, "n-3" indicates that the bond between the third and fourth carbon atoms counted from the methyl end of the fatty acid is a double bond. This method is well known to those skilled in the art, and those skilled in the art can easily identify fatty acids described according to this method.

[0016] As used herein, "highly unsaturated fatty acid" refers to a fatty acid having 18 or more carbon atoms and 3 or more double bonds. The highly unsaturated fatty acid may be, for example, a fatty acid having 20 or more carbon atoms and 3 or more or 4 or more double bonds, or a fatty acid having 20 or more carbon atoms and 5 or more double bonds. Examples of highly unsaturated fatty acids include α-linolenic acid (18:3 n-3), γ-linolenic acid (18:3 n-6), dihomo-γ-linolenic acid (20:3 n-6), arachidonic acid (20:4 n-6), eicosapentaenoic acid (20:5 n-3), docosapentaenoic acid (22:5 n-6), and docosahexaenoic acid (22:6 n-3).

[0017] In this specification, the term "crude oil" refers to a mixture of lipids and oil extracted from an organism. In this specification, the term "refined oil" refers to oil that has been subjected to a crude oil refining process in which the crude oil is subjected to at least one oil and fat refining process selected from the group consisting of a degumming process, a deacidification process, a bleaching process, and a deodorizing process, and substances other than the target substance such as phospholipids and sterols are removed. Those skilled in the art can distinguish between refined oil and crude oil by ordinary analysis.

[0018] In this specification, the composition containing a highly unsaturated fatty acid or its alkyl ester means a fatty acid composition containing a highly unsaturated fatty acid or a fatty acid alkyl ester composition containing an alkyl ester of a highly unsaturated fatty acid. Here, the fatty acid composition is a composition whose main component is a fatty acid, and the fatty acid alkyl ester composition is a composition whose main component is an alkyl ester of a fatty acid.

[0019] In this specification, a generic term (e.g., polyunsaturated fatty acid, polyunsaturated fatty acid alkyl ester, etc.) does not exclude the possibility that multiple components are present, unless the context clearly indicates otherwise. Thus, a generic term usually means that at least one component is present.

[0020] <Composition containing highly unsaturated fatty acid or its alkyl ester with reduced concentration of 3-MCPD fatty acid ester> The present invention provides a composition containing a highly unsaturated fatty acid or an alkyl ester thereof, wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 50 area % or more, and when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the concentration of 3-MCPD produced is less than 1.80 ppm (sometimes referred to as the composition of the present invention in this specification).

[0021] In the present invention, the highly unsaturated fatty acid is not particularly limited as long as its alkyl ester is obtained as the main fraction when concentrated by distillation.The highly unsaturated fatty acid may be eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, or a combination thereof.In a preferred embodiment, the highly unsaturated fatty acid may be eicosapentaenoic acid, docosahexaenoic acid, or a combination thereof.In a more preferred embodiment, the highly unsaturated fatty acid may be eicosapentaenoic acid.

[0022] The proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention may be 50 area% or more, for example, 55 area% or more, 60 area% or more, 65 area% or more, 70 area% or more, 75 area% or more, 80 area% or more, 85 area% or more, 90 area% or more, 95 area% or more, or 96 area% or more. In one embodiment, the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention may be 99 area% or less, for example, 98 area% or less, 95 area% or less, 90 area% or less, 85 area% or less, 80 area% or less, 75 area% or less, 70 area% or less, 65 area% or less, 60 area% or less, or 55 area% or less. The proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention is, for example, 50 to 99 area%, 50 to 98 area%, 50 to 95 area%, 50 to 90 area%, 50 to 85 area%, 50 to 80 area%, 50 to 75 area%, 50 to 70 area%, 50 to 65 area%, 50 to 60 area%, 55 to 99 area%, 55 to 98 area%, 55 to 95 area%, 55 to 90 area%, 55 to 85 area%, 55 to 80 area%, 55 to 75 area%, 55 to 70 area%, 55 to 65 area%, 55 to 60 area%, 60 to 99 area%, 60 to 98 ... to 95 area%, 60 to 90 area%, 60 to 85 area%, 60 to 80 area%, 60 to 75 area%, 65 to 99 area%, 65 to 98 area%, 65 to 95 area%, 65 to 90 area%, 65 to 85 area%, 65 to 80 area%, 65 to 75 area%, 70 to 99 area%, 70 to 98 area%, 70 to 95 area%, 70 to 90 area%, 70 to 85 area%, 70 to 80 area%, 70 to 75 area%, 75 to 99 area%, 75 to 98 area%, 75 to 95 area%, 75 to 90 area%, 75 to 85 area%, or 75 to 80 area%.

[0023] In one embodiment, the highly unsaturated fatty acid is eicosapentaenoic acid, and the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention can be 50 to 99 area%, 50 to 98 area%, 50 to 95 area%, 50 to 90 area%, 50 to 85 area%, 50 to 80 area%, 50 to 75 area%, 50 to 70 area%, 50 to 65 area%, 50 to 60 area%, 55 to 99 area%, 55 to 98 area%, 55 to 95 area%, 55 to 90 area%, 55 to 85 area%, 55 to 80 area%, 55 to 75 area%, 55 to 70 area%, 55 to 65 area%, or 55 to 60 area%.

[0024] In another embodiment, the highly unsaturated fatty acid is eicosapentaenoic acid, and the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention can be 60 to 99 area%, 60 to 98 area%, 60 to 95 area%, 60 to 90 area%, 60 to 85 area%, 65 to 99 area%, 65 to 98 area%, 65 to 95 area%, 65 to 90 area%, 65 to 85 area%, 70 to 99 area%, 70 to 98 area%, 70 to 95 area%, 70 to 90 area%, 70 to 85 area%, 75 to 99 area%, 75 to 98 area%, 75 to 95 area%, 75 to 90 area%, or 75 to 85 area%.

[0025] In yet another aspect, the polyunsaturated fatty acid is eicosapentaenoic acid, and the proportion of polyunsaturated fatty acids in the constituent fatty acids of the composition of the present invention may be 60 to 80 area%, 65 to 80 area%, 70 to 80 area%, or 75 to 80 area%.

[0026] The composition of the present invention has a high proportion of polyunsaturated fatty acids and is therefore suitable as a raw material for pharmaceuticals and supplements that contain polyunsaturated fatty acids as active ingredients.

[0027] In this specification, the "area %" which represents the proportion of highly unsaturated fatty acids in the constituent fatty acids of a composition, is a percentage of the content of the component of a peak, which is determined by identifying the peak of each component in a chart obtained by analyzing the composition using a gas chromatography with a flame ionization detector (GC-FID), and calculating the peak area of ​​each fatty acid using the Agilent ChemStation integration algorithm (Revision C.01.03

[37] , Agilent Technologies). In the field of oil chemistry, area % is used as almost synonymous with weight %. See the Japan Oil Chemists' Society (JOCS) Standard Methods for Analysis of Fats and Oils, 2013 Edition, 2.4.2.1-2013 Fatty Acid Composition (FID Constant Temperature Gas Chromatography) and 2.4.2.2-2013 Fatty Acid Composition (FID Temperature Programmed Gas Chromatography). The analysis conditions for gas chromatography are as follows: GC-FID measurement conditions GC: 6890N (Agilent Technologies) Column: DB-WAX (Agilent Technologies) 30mx 0.25mm ID, 0.25μm film thickness Carrier gas: Helium, 1 mL / min Inlet: 250℃, 1 μL, Split (1:100) Column temperature: 180℃ → 3℃ / min → 230℃, hold for 15 minutes Detector: FID, 250℃ Make-up gas: Nitrogen 45 mL / min.

[0028] In an embodiment in which the composition of the present invention is a fatty acid composition containing highly unsaturated fatty acids, the proportion of highly unsaturated fatty acids in the constituent fatty acids is measured by the following method: the fatty acid composition is methyl-esterified in accordance with the content of AOCS official method Ce1b-89, and then subjected to GC under the above-mentioned conditions, and the area percentage of highly unsaturated fatty acids is calculated in the same manner. In this case, the constituent fatty acids of the composition refer to the free fatty acids in the fatty acid composition.

[0029] In an embodiment in which the composition of the present invention is a fatty acid alkyl ester composition containing an alkyl ester of a highly unsaturated fatty acid, the proportion of the highly unsaturated fatty acid in the constituent fatty acids of the composition is measured by the following method: That is, the fatty acid alkyl ester composition is analyzed by gas chromatography under the above-mentioned conditions, and the ratio (area %) of the peak area of ​​the highly unsaturated fatty acid alkyl ester to the sum of the peak areas of the fatty acid alkyl esters is calculated. In this case, the constituent fatty acid of the composition means a fatty acid that constitutes the fatty acid alkyl ester of the fatty acid alkyl ester composition.

[0030] The alkyl group in the highly unsaturated fatty acid alkyl ester is an alkyl group derived from a lower alcohol that is generally used in the alkyl esterification of fatty acids, and examples of such alkyl groups include alkyl groups having 1 or 2 carbon atoms (i.e., a methyl group or an ethyl group). In a preferred embodiment, the highly unsaturated fatty acid alkyl ester may be a highly unsaturated fatty acid ethyl ester.

[0031] The composition of the present invention is subjected to the American Oil Chemists' Society Cd29b-13 assay. The concentration of 3-MCPD resulting from the extraction of 3-MCPD from the feedstock is reduced to less than 1.80 ppm (mg / kg), e.g., less than 1.70 ppm, less than 1.60 ppm, less than 1.50 ppm, less than 1.40 ppm, less than 1.30 ppm, less than 1.20 ppm, less than 1.10 ppm, less than 1.00 ppm, less than 0.90 ppm, less than 0.80 ppm, less than 0.70 ppm, less than 0.60 ppm, less than 0.50 ppm, less than 0.40 ppm, less than 0.30 ppm, less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, less than 0.05 ppm, less than 0.04 ppm, less than 0.03 ppm, less than 0.02 ppm, or less than 0.01 ppm. In some embodiments, the concentration of 3-MCPD is greater than 0 ppm. In one embodiment, the composition of the present invention may have a concentration of 3-MCPD of 0.01 ppm or more, for example 0.02 ppm or more, when the composition is analyzed by the American Oil Chemists' Society Cd29b-13 assay A. In addition, the composition of the present invention may have a concentration of 3-MCPD of 0.01 ppm or more, for example 0.02 ppm or more, when the composition is analyzed by the American Oil Chemists' Society Cd29b-13 assay A. The resulting concentration of 3-MCPD when analyzed by HPLC-MS / MS / MS-MS can be greater than or equal to 0.01 ppm and less than 1.80 ppm, less than 1.70 ppm, less than 1.60 ppm, less than 1.50 ppm, less than 1.40 ppm, less than 1.30 ppm, less than 1.20 ppm, less than 1.10 ppm, less than 1.00 ppm, less than 0.90 ppm, less than 0.80 ppm, less than 0.70 ppm, less than 0.60 ppm, less than 0.50 ppm, less than 0.40 ppm, less than 0.30 ppm, less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, less than 0.05 ppm, less than 0.04 ppm, less than 0.03 ppm, or less than 0.02 ppm.In addition, the composition of the present invention is a composition that is used in the Cd29b-13 assay according to the American Oil Chemists' Society official method. The resulting concentration of 3-MCPD when analyzed by HPLC-MS / MS / MS-MS can be greater than or equal to 0.02 ppm and less than 1.80 ppm, less than 1.70 ppm, less than 1.60 ppm, less than 1.50 ppm, less than 1.40 ppm, less than 1.30 ppm, less than 1.20 ppm, less than 1.10 ppm, less than 1.00 ppm, less than 0.90 ppm, less than 0.80 ppm, less than 0.70 ppm, less than 0.60 ppm, less than 0.50 ppm, less than 0.40 ppm, less than 0.30 ppm, less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, less than 0.05 ppm, less than 0.04 ppm, or less than 0.03 ppm.

[0032] In this specification, the analysis by the American Oil Chemists' Society official method (AOCS official method) Cd29b-13 assay A is performed according to the following procedure, which is well known to those skilled in the art.

[0033] Add 100μL of 3-MCPD-d5-dipalmitic acid ester standard solution (diluted with toluene to 5ppm 3-MCPD-d5) and 600μL of diethyl ether to 100mg of sample, stir and mix until completely dissolved, then cool at -22℃ to -25℃ for about 15 minutes. Then add 350μL of sodium hydroxide-methanol solution (0.25g of sodium hydroxide dissolved in 100mL of methanol), stir well, and react at -22℃ to -25℃ for 16 hours or more. At the same temperature, add 600μL of acidic sodium bromide solution (600g of sodium bromide dissolved in 1L of purified water, add 3mL of 85% phosphoric acid) to stop the reaction, then concentrate by blowing nitrogen until the organic layer separated into two layers is about 100μL. Then add 600μL of hexane, stir vigorously, leave to stand for 5 to 10 minutes, and remove the organic layer twice. Add 600μL of a diethyl ether:ethyl acetate mixture (3:2, V / V) to the remaining aqueous layer and stir vigorously. Repeat this process three times to recover the organic layer. Combine the three recovered organic layers and dehydrate them over anhydrous sodium sulfate. After dehydration, concentrate the organic layer to 200μL by blowing in nitrogen. Add 20μL of a saturated phenylboronic acid-diethyl ether solution and stir vigorously for 10 seconds, then blow in nitrogen to completely remove the solvent. Add 200μL of isooctane and stir vigorously for 10 seconds. Use the resulting solution as a GC-MS sample.

[0034] A calibration curve for quantification of 3-MCPD is prepared by dissolving 3-MCPD dipalmitate in toluene to give 3-MCPD concentrations of 0 ppm, 0.5 ppm, 1 ppm, and 5 ppm, and analyzing the 3-MCPD standard solutions in the same manner as for the above samples.

[0035] The GC-MS analysis conditions were as follows: GC-MS conditions GC: GC-2010 and GCMS-QP2010 (Shimadzu Corporation) Column: DB-5ms (Agilent Technologies) 30mx 0.25mm ID, 0.25μm film thickness Carrier gas: Helium, 1.2 mL / min Injection port: 250℃, 1 μL, splitless, sampling time 1 min Column temperature: 85°C, hold for 0.5 min → 6°C / min → 150°C, hold for 5 min → 12°C / min → 180°C → 25°C / min → 280°C, hold for 7 min Ionization temperature: 200℃ Interface temperature: 200℃ Ionization method: EI, SIM The m / z monitored are as follows 3-MCPD-d5:m / z=149, 150, 201, 203 3-MCPD: m / z=146, 147, 196, 198 For quantification, m / z = 150 for 3-MCPD-d5 and m / z = 147 for 3-MCPD are used, and the others are used to confirm the target substances.

[0036] In the above analytical method, both the free form and the ester form of 3-MCPD in a sample are detected without being distinguished as 3-MCPD. Therefore, the measured value in the above analytical method represents the total content (ppm (mg / kg)) of the free form of 3-MCPD originally present in the sample and the free form of 3-MCPD that can be formed from the ester form.

[0037] In one embodiment herein, the composition of the present invention may have a 3-MCPD concentration of 0 ppm or more and less than 1.80 ppm when the composition is analyzed by the American Oil Chemists' Society Official Method Cd29b-13 assay A. In this case, the composition of the present invention may include a composition that does not contain 3-MCPD. In another embodiment, the composition of the present invention may contain 3-MCPD (i.e., 3-MCPD or 3-MCPD fatty acid esters), and may have a 3-MCPD concentration of less than 1.80 ppm when the composition is analyzed by the American Oil Chemists' Society Official Method Cd29b-13 assay A.

[0038] The composition of the present invention may have a concentration of 3-MCPD that is less than the lower limit of quantification, and preferably less than the detection limit, of the American Oil Chemist's Society Official Method Cd29b-13 Assay A, when the composition is analyzed by the American Oil Chemist's Society Official Method Cd29b-13 Assay A.

[0039] It is known that highly unsaturated fatty acids such as eicosapentaenoic acid are contained in large amounts in certain microbial oils, vegetable oils, or marine animal oils. Therefore, the composition of the present invention can be made from them. Specific examples of the raw materials for the composition of the present invention include fish oils such as sardine oil, tuna oil, bonito oil, menhaden oil, cod liver oil, herring oil, capelin oil, and salmon oil, marine animal oils derived from crustaceans such as krill, vegetable oils derived from perilla, flax, soybean, rapeseed, etc., yeasts such as the genus Yarrowia, filamentous fungi such as those belonging to the genus Mortierella, the genus Penicillium, the genus Aspergillus, the genus Rhodotorula, and the genus Fusarium, algae such as the genus Euglena, and oils derived from microorganisms that produce lipids, such as stramenopiles. The composition of the present invention may be oil derived from a genetically modified microorganism into which a gene such as a genetically modified mutant Δ9 elongase gene has been introduced. In addition, oil derived from a genetically modified plant of an oilseed plant such as Brassica spp., sunflower, corn, cotton, flax, safflower, etc., into which a gene such as a mutant Δ9 elongase gene has been introduced by recombinant technology, can also be used as a raw material oil. Examples of genetically modified vegetable oils and genetically modified microbial oils include those described in WO2012 / 027698, WO2010 / 033753, etc. In a preferred embodiment, the raw material of the composition of the present invention is a fish oil, a microbial oil, a vegetable oil, or an aquatic animal oil such as a marine animal oil, more preferably a fish oil.

[0040] The raw material of the composition of the present invention contains polyunsaturated fatty acids mainly in the form of glycerides. For example, fish oil contains many kinds of fatty acids with 14 to 22 carbon atoms and 0 to 6 double bonds in the form of glycerides. In the presence of a catalyst or enzyme, glyceride is reacted with a lower alcohol such as ethanol to convert the fatty acids contained in the glyceride into alkyl esters, and then fatty acid alkyl esters other than the desired polyunsaturated fatty acid alkyl ester (e.g., EPA alkyl ester) are removed, whereby a high-purity polyunsaturated fatty acid alkyl ester (e.g., EPA alkyl ester) can be produced. In one embodiment, the removal of fatty acid alkyl esters other than the desired polyunsaturated fatty acid alkyl ester (e.g., EPA alkyl ester) can be carried out by distillation. The present inventors have found that the main fraction of the distillation is contaminated with a large amount of 3-MCPD fatty acid esters derived from mono- or diacylglycerols with relatively large molecular weights. Therefore, in a preferred embodiment, the composition of the present invention can be a distillate (fraction).

[0041] It is known that heat treatment during distillation produces a trans isomer, which is a thermally denatured product, from the highly unsaturated fatty acid alkyl ester (e.g., European Journal of Lipid Science and Technology, 108 (2006) 589-597. "Geometrical isomerization of eicosapentaenoic and docosahexaenoic acid at high temperatures"; JAOCS, 66 (1989) 1822-1830. "Eicosapentaenoic acid geometrical isomer artifacts in heated fish oil esters"). Thus, in one embodiment, the composition of the present invention may further contain a trans isomer of the highly unsaturated fatty acid alkyl ester. The concentration of the trans isomer in the composition of the present invention can be 2.5 area% or less, for example, 2.3 area% or less, 2.0 area% or less, 1.8 area% or less, 1.6 area% or less, 1.4 area% or less, 1.2 area% or less, 1.0 area% or less, 0.9 area% or less, 0.8 area% or less, 0.7 area% or less, 0.6 area% or less, or 0.5 area% or less. The concentration of the trans isomer in the composition of the present invention can be 0.01 area% or more, for example, 0.02 area% or more, 0.03 area% or more, 0.04 area% or more, or 0.05 area% or more.

[0042] In this specification, the concentration of the trans isomer is a value measured by GC analysis. Specifically, the concentration is measured by the following procedure. Dissolve 10 mg of sample in 1 mL of hexane and subject to GC analysis under the following conditions. [GC analysis conditions] GC: 6890N (Agilent Technologies) Column: DB-WAX (Agilent Technologies) 30mx 0.25mm ID, 0.25μm film thickness Carrier gas: Helium, 1 mL / min Inlet: 250℃, 1 μL, Split (1:100) Column temperature: 180℃ → 3℃ / min → 230℃, hold for 15 minutes Detector: FID, 250℃ Make-up gas: Nitrogen 45 mL / min.

[0043] For example, for the trans isomers (five types, A to E) of EPA ethyl ester (EPA-E), the concentration is calculated as follows: In a sample in which saturated fatty acids or monounsaturated fatty acids of C21 or more have been removed by distillation, the relative retention times of the trans isomers of EPA ethyl ester (EPA-E) are 0.98-0.99 for isomer A, 1.01-1.02 for isomer B, 1.02-1.03 for isomer C, and 1.04-1.05 for isomers D and E, which overlap when the retention time of EPA-E is taken as 1. Of the peaks of the five isomers, the peaks of isomers D and E overlap, so four peaks are detected. The sum of the areas of the peaks at these relative retention times is the peak area of ​​the EPA-E trans isomer. The ratio of the trans isomer to EPA-E is calculated, and the concentration of the isomer in the sample is calculated from the concentration of EPA-E in the sample. For samples containing saturated or monounsaturated fatty acids of C21 or higher, the saturated or monounsaturated fatty acids of C21 or higher are removed by silver nitrate column fractionation using Discovery Ag-ION 750 mg / 6 mL (Sperco) before analysis as described above.

[0044] For example, for the trans isomer of dihomo-gamma-linolenic acid ethyl ester (DGLA-E), the sum of the areas of the peaks at the following relative retention times is measured by the above-mentioned GC analysis, the ratio of the trans isomer to DGLA-E is determined, and the concentration of the isomer in the sample is calculated from the concentration of DGLA-E in the sample. Isomer A: Relative retention time 1.001-1.009 Isomer B: Relative retention time 1.01-1.03 (The retention time of DGLA is 1.)

[0045] The content of trans isomers in other highly unsaturated fatty acid alkyl esters can also be measured by a standard method.

[0046] When the composition of the present invention is measured using gas chromatography under the above analytical conditions, the concentration of the trans isomer of the EPA alkyl ester may be 2.5 area% or less, for example, 2.3 area% or less, 2.0 area% or less, 1.8 area% or less, 1.6 area% or less, 1.4 area% or less, 1.2 area% or less, 1.0 area% or less, 0.9 area% or less, 0.8 area% or less, 0.7 area% or less, 0.6 area% or less, or 0.5 area% or less. When the composition of the present invention is measured using gas chromatography under the above analytical conditions, the concentration of the trans isomer of the EPA alkyl ester may be 0.01 area% or more, for example, 0.02 area% or more, 0.03 area% or more, 0.04 area% or more, or 0.05 area% or more.

[0047] In a preferred embodiment, the composition of the present invention, when measured using gas chromatography under the above analytical conditions, may have a concentration of trans isomer of EPA ethyl ester of 2.5 area% or less, for example, 2.3 area% or less, 2.0 area% or less, 1.8 area% or less, 1.6 area% or less, 1.4 area% or less, 1.2 area% or less, 1.0 area% or less, 0.9 area% or less, 0.8 area% or less, 0.7 area% or less, 0.6 area% or less, or 0.5 area% or less. Also, the composition of the present invention, when measured using gas chromatography under the above analytical conditions, may have a concentration of trans isomer of EPA ethyl ester of 0.01 area% or more, for example, 0.02 area% or more, 0.03 area% or more, 0.04 area% or more, or 0.05 area% or more.

[0048] When the composition of the present invention is measured by gas chromatography under the above-mentioned analytical conditions, the concentration of the trans isomer of DGLA alkyl ester can be 2.5 area% or less, for example, 2.3 area% or less, 2.0 area% or less, 1.8 area% or less, 1.6 area% or less, 1.4 area% or less, 1.2 area% or less, 1.0 area% or less, 0.9 area% or less, 0.8 area% or less, 0.7 area% or less, 0.6 area% or less, or 0.5 area% or less.In addition, when the composition of the present invention is measured by gas chromatography under the above-mentioned analytical conditions, the concentration of the trans isomer of DGLA alkyl ester can be 0.01 area% or more, for example, 0.02 area% or more, 0.03 area% or more, 0.04 area% or more, or 0.05 area% or more.

[0049] In a preferred embodiment, when the composition of the present invention is measured by gas chromatography under the above-mentioned analytical conditions, the concentration of the trans isomer of DGLA ethyl ester can be 2.5 area% or less, for example, 2.3 area% or less, 2.0 area% or less, 1.8 area% or less, 1.6 area% or less, 1.4 area% or less, 1.2 area% or less, 1.0 area% or less, 0.9 area% or less, 0.8 area% or less, 0.7 area% or less, 0.6 area% or less, or 0.5 area% or less.In addition, when the composition of the present invention is measured by gas chromatography under the above-mentioned analytical conditions, the concentration of the trans isomer of DGLA ethyl ester can be 0.01 area% or more, for example, 0.02 area% or more, 0.03 area% or more, 0.04 area% or more, or 0.05 area% or more.

[0050] In addition to triglycerides, fish oils and microbial oils containing highly unsaturated fatty acids also contain cholesterol. Highly unsaturated fatty acid concentrated oils prepared from these raw oils also contain cholesterol (WO2012 / 118173). Furthermore, even if cholesterol-containing oils are subjected to alkali esterification or urea addition, cholesterol is not completely removed. Therefore, in certain embodiments herein, the composition of the present invention contains cholesterol. The cholesterol content may be, for example, 1.5% by weight or less, 0.3% by weight or less, or 0.2% by weight or less. Furthermore, the cholesterol content may be, for example, 0.01% by weight or more, or 0.02% by weight or more.

[0051] Cholesterol has the molecular formula C 27 H 46 Cholesterol is a compound having a steroid skeleton represented by O, and exists in natural products as a free form or an ester form. The ester form is an acyl cholesterol in which a fatty acid is bound to the hydroxyl group (OH group). The cholesterol content in the present invention means the total content of the free form and the ester form. The cholesterol content is measured by the following method.

[0052] Add 1 mL of 0.1 g / L 5α-cholestane as an internal standard to approximately 0.1 g of sample, add 1 mL of 2 mol / L potassium hydroxide / aqueous ethanol solution, and heat at 100°C for 10 minutes. After cooling, add 3 mL of petroleum ether and 3 mL of saturated ammonium sulfate, stir, and allow to stand, then collect the upper layer and measure it by gas chromatography under the following measurement conditions. To determine the relative sensitivity of 5α-cholestane and free cholesterol, measure a hexane solution in which 25 mg each of 5α-cholestane and cholesterol are dissolved by gas chromatography, and calculate the total cholesterol amount. Gas chromatographic analysis conditions Model: Agilent 6890 GC system (Agilent) Column: DB-1 J&W 123-1012 Column temperature: 270℃ Injection temperature: 300℃ Injection method: Split Split ratio: 50:1 Detector temperature: 300℃ Detector: FID Carrier gas: Helium (39.3kPa, constant pressure)

[0053] In one embodiment, the composition of the present invention may contain saturated fatty acids having 18 or less carbon atoms or alkyl esters thereof as impurities. In this case, the ratio of saturated fatty acids having 18 or less carbon atoms in the constituent fatty acids of the composition of the present invention may be 0.1 area% or more, for example, 0.2 area% or more, or 0.3 area% or more, and less than 10 area%, for example, less than 5 area%, less than 4 area%, or less than 3 area%.

[0054] The highly unsaturated fatty acid can be obtained by hydrolyzing the highly unsaturated fatty acid alkyl ester produced by the above method.

[0055] The composition of the present invention is a composition containing fatty acid or fatty acid alkyl ester as a main component, and usually contains fatty acid or fatty acid alkyl ester at 50% by weight or more, 55% by weight or more, 60% by weight or more, 65% by weight or more, 70% by weight or more, 75% by weight or more, 80% by weight or more, 85% by weight or more, 90% by weight or more, 95% by weight or more, 96% by weight or more, 97% by weight or more, 98% by weight or more, 99% by weight or more, 99.5% by weight or more, or 99.9% by weight or more. The content of fatty acid or fatty acid alkyl ester in the composition of the present invention can be confirmed by a known method such as TLC / FID.

[0056] <Method of producing a composition containing highly unsaturated fatty acid or its alkyl ester> The present invention provides a method for producing the composition of the present invention, comprising the steps of: (1) a step of alkyl-esterifying a raw material containing a triglyceride containing a highly unsaturated fatty acid as a constituent fatty acid to prepare a composition containing a highly unsaturated fatty acid alkyl ester; (2) at least one step selected from (a) a step of reducing the concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number that is 5 or more less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated in the composition of step (1) to less than 10,000 ppm, (b) a step of reducing the iron concentration in the composition of step (1) to less than 0.20 ppm, and (c) a step of reducing the chlorine concentration in the composition of step (1) to less than 10 ppm; (3) A step of distilling the composition obtained after step (2) and isolating a main fraction. (hereinafter, also referred to as the method of the present invention).

[0057] The method of the present invention can produce a composition containing a fatty acid or a fatty acid alkyl ester as a main component, the composition containing fatty acid or fatty acid alkyl ester at 95% by weight or more, 96% by weight or more, 97% by weight or more, 98% by weight or more, 99% by weight or more, 99.5% by weight or more, or 99.9% by weight or more.

[0058] In this specification, step (1) may be referred to as an alkyl esterification step, step (2)(a) may be referred to as a monoacylglycerol removal step, step (2)(b) may be referred to as an iron removal step, step (2)(c) may be referred to as a chlorine removal step, and step (3) may be referred to as a distillation step.

[0059] Examples of the raw material in the method of the present invention include the oils described above for the composition of the present invention, specifically, fish oils such as sardine oil, tuna oil, bonito oil, menhaden oil, cod liver oil, herring oil, capelin oil, and salmon oil, marine animal oils derived from crustaceans such as krill, vegetable oils derived from perilla, flax, soybean, rapeseed, etc., yeasts such as the genus Yarrowia, filamentous fungi belonging to the genus Mortierella, Penicillium, Aspergillus, Rhodotorula, and Fusarium, algae such as the genus Euglena, and oils derived from microorganisms that produce lipids such as stramenopiles. The raw material in the method of the present invention may be oils derived from genetically modified microorganisms into which a gene such as a genetically modified mutant Δ9 elongase gene has been introduced. In addition, oils derived from genetically modified plants such as Brassica species, sunflower, corn, cotton, flax, safflower, etc., into which genes such as a mutant Δ9 elongase gene have been introduced by recombinant technology, can also be used as the raw oil. Examples of genetically modified vegetable oils and genetically modified microbial oils include those described in WO2012 / 027698, WO2010 / 033753, etc. In a preferred embodiment, the raw material in the method of the present invention is a fish oil, a microbial oil, a vegetable oil, or an aquatic animal oil such as a marine animal oil, more preferably a fish oil.

[0060] Crude oil refining process The raw oil used in the alkyl esterification in step (1) may be crude oil or refined oil. For example, any method may be used to obtain crude oil from fish or marine product raw materials, but in the case of fish oil, it is usually extracted by the following method. Whole fish or processing residues such as fish heads, skin, backbone, and internal organs generated during seafood processing are crushed and steamed, and then pressed to separate into broth (stick water) and pressed meal. The fats and oils obtained together with the broth are separated from the broth by centrifugation to obtain crude fish oil. In general, crude fish oil is refined into refined fish oil through a crude oil refining process in which substances other than the target substance such as phospholipids and sterols are removed by carrying out a degumming process, a deacidification process, a decolorization process using activated clay or activated carbon, a water washing process, a deodorization process by steam distillation, etc. according to the raw material. In an embodiment of the present invention, this refined fish oil can also be used as the raw material.

[0061] Step (1) (Alkyl esterification step) The raw material oil, fats and oils, are decomposed into lower alcohol esters by alcoholysis using a lower alcohol. Examples of lower alcohols include those commonly used in alkyl esterification of fatty acids, such as lower alcohols with one or two carbon atoms. Alcohololysis involves adding a lower alcohol, such as ethanol, and a catalyst or enzyme to fats and oils to cause a reaction, thereby producing alkyl esters from fatty acids bound to glycerin. Examples of catalysts that can be used include alkali catalysts and acid catalysts. Examples of enzymes that can be used include lipase.

[0062] It has been empirically proven that the reaction efficiency of alcoholysis of fatty acids is high, and after alcoholysis, a composition containing fatty acids mainly in their alkyl ester form is obtained, although this does not completely exclude the inclusion of fatty acids in forms other than the alkyl ester form.

[0063] Process (2) Step (2) is a pretreatment step for distillation and is at least one step selected from the following steps (a) to (c): That is, step (2) may be any one of steps (a) to (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b), and (c).

[0064] (a) Monoacylglycerol removal step In step (2)(a), the content of monoacylglycerol, which is a substrate for 3-MCPD fatty acid esters, is reduced before the composition containing fatty acid alkyl esters prepared in step (1) is concentrated by distillation, thereby reducing the amount of 3-MCPD fatty acid esters that are generated by heat treatment during distillation and contaminate the main fraction.

[0065] For removing monoacylglycerol from the composition containing the fatty acid alkyl ester prepared in step (1), existing techniques such as repeated alkyl esterification treatment or treatment with an adsorbent can be used.

[0066] Esterification is an equilibrium reaction, and the amount of remaining glyceride depends on the ratio of alcohol to the by-product glycerin. By subjecting the ester fraction obtained after alkyl esterification to alkyl esterification again, the ratio of alcohol to glycerin can be shifted significantly toward the alcohol side, thereby reducing the amount of glyceride.

[0067] Examples of the adsorbent treatment include silica gel chromatography, activated clay treatment, acid clay treatment, activated carbon treatment, and silica gel treatment. Silica gel chromatography can be performed, for example, by the following procedure. In the treatment with silica gel (e.g., Microsphere D75-60A), the fatty acid alkyl ester is applied to a silica gel-packed column to adsorb the fatty acid alkyl ester to the silica gel. Then, ethyl acetate / hexane (1:50) is passed through the column, the eluate is fractionated, and a fraction in which MAG and DAG have been removed from the fatty acid alkyl ester is recovered. The fraction is desolvated to obtain the fatty acid alkyl ester. The activated clay treatment can be performed, for example, by adding activated clay in an amount of 5% relative to the oil, stirring at 120° C. under reduced pressure for 2 hours, and then filtering. Other adsorbent treatments can also be performed by standard methods.

[0068] The monoacylglycerol removed in step (2)(a) contains, as a constituent fatty acid, a fatty acid having a carbon number that is 5 or more fewer than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated. Here, the highly unsaturated fatty acid alkyl ester to be concentrated refers to the highly unsaturated fatty acid alkyl ester intended to be concentrated in the method of the present invention. That is, in the distillation in step (3), the conditions are set so that the highly unsaturated fatty acid alkyl ester to be concentrated is separated as a main fraction. Therefore, in the distillation in step (3), the highly unsaturated fatty acid alkyl ester separated as a main fraction and concentrated is the highly unsaturated fatty acid alkyl ester to be concentrated.

[0069] When there are two or more types of highly unsaturated fatty acid alkyl esters to be concentrated (for example, when concentrating a combination of alkyl esters of two or more types of highly unsaturated fatty acids selected from eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, and arachidonic acid), the concentration of monoacylglycerol containing as a constituent fatty acid a fatty acid with a carbon number that is 5 or more fewer than the carbon number of the fatty acid with the greatest carbon number is reduced.

[0070] The constituent fatty acid of the monoacylglycerol removed in step (2)(a) may be either a saturated fatty acid or an unsaturated fatty acid, but it is preferable to remove monoacylglycerol of a saturated fatty acid.

[0071] In step (2)(a), the concentration of monoacylglycerol containing as a constituent fatty acid a fatty acid having a carbon number of 5 or more less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated is reduced to less than 10,000 ppm, less than 9,000 ppm, less than 8,000 ppm, less than 7,000 ppm, less than 6,000 ppm, less than 5,000 ppm, less than 4,000 ppm, less than 3,000 ppm, less than 2,000 ppm, less than 1,000 ppm, less than 900 ppm, less than 800 ppm, less than 700 ppm, less than 600 ppm, or less than 500 ppm. In some embodiments, the concentration of the monoacylglycerol is higher than 0 ppm. In some embodiments, the concentration of the monoacylglycerol before reduction is 10,000 ppm or more or the upper limit value above. For example, when the highly unsaturated fatty acid alkyl ester to be concentrated is an eicosapentaenoic acid (20:5 n-3) alkyl ester, a dihomo-γ-linolenic acid (20:3 n-6) alkyl ester, an arachidonic acid (20:4 n-6) alkyl ester, or a combination thereof, the concentration of monoacylglycerol containing a fatty acid having 15 or less carbon atoms (preferably a fatty acid having 14 carbon atoms) as a constituent fatty acid is reduced to the above concentration. When the highly unsaturated fatty acid alkyl ester to be concentrated is a docosahexaenoic acid (22:6 n-3) alkyl ester, the concentration of monoacylglycerol containing a fatty acid having 17 or less carbon atoms (preferably a fatty acid having 16 carbon atoms) as a constituent fatty acid is reduced to the above concentration. Furthermore, when the highly unsaturated fatty acid alkyl ester to be concentrated is a combination of docosahexaenoic acid (22:6 n-3) alkyl ester and one or more selected from eicosapentaenoic acid (20:5 n-3) alkyl ester, dihomo-γ-linolenic acid (20:3 n-6) alkyl ester, and arachidonic acid (20:4 n-6) alkyl ester, the concentration of monoacylglycerol containing fatty acids having 17 or less carbon atoms (preferably fatty acids having 14 carbon atoms and fatty acids having 16 carbon atoms) as constituent fatty acids is reduced to the above concentration.

[0072] In the distillation of step (3), 3-MCPD fatty acid esters that can be generated from monoacylglycerols containing, as constituent fatty acids, fatty acids with a carbon number that is 5 or more less than the carbon number of the highly unsaturated fatty acid that constitutes the highly unsaturated fatty acid alkyl ester to be concentrated can be contained in the main fraction together with the highly unsaturated fatty acid alkyl esters. Therefore, when obtaining the highly unsaturated fatty acid alkyl ester to be concentrated as the main fraction in the distillation of step (3), the concentration of monoacylglycerols containing, as constituent fatty acids, fatty acids with a carbon number that is 5 or more less than the carbon number of the highly unsaturated fatty acid that constitutes the highly unsaturated fatty acid alkyl ester to be concentrated can be reduced in advance to obtain a composition containing highly unsaturated fatty acid alkyl esters with a reduced concentration of 3-MCPD fatty acid esters.

[0073] The monoacylglycerol removed in step (2)(a) may be a monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number that is 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, or 6 fewer than the carbon number of the highly unsaturated fatty acid that constitutes the highly unsaturated fatty acid alkyl ester to be concentrated. In step (2)(a), the concentration of these monoacylglycerols may be reduced to the above-mentioned concentration in total.

[0074] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester to be enriched is an eicosapentaenoic acid (20:5 n-3) alkyl ester, a dihomo-γ-linolenic acid (20:3 n-6) alkyl ester, or an arachidonic acid (20:4 n-6) alkyl ester, or a combination thereof, and the monoacylglycerol removed in step (2)(a) may be glycerol monomyristate.

[0075] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester to be enriched is a docosahexaenoic acid (22:6 n-3) alkyl ester, and the monoacylglycerol removed in step (2)(a) may be glycerol monopalmitate.

[0076] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester to be concentrated is a combination of docosahexaenoic acid (22:6 n-3) alkyl ester and one or more selected from eicosapentaenoic acid (20:5 n-3) alkyl ester, dihomo-γ-linolenic acid (20:3 n-6) alkyl ester, and arachidonic acid (20:4 n-6) alkyl ester, and the monoacylglycerol removed in step (2)(a) may be glycerol monomyristate and glycerol monopalmitate.

[0077] In this specification, the concentrations of monoacylglycerol (MAG) and diacylglycerol (DAG) in the composition are values ​​(ppm (mg / kg)) calculated from values ​​measured by the following method.

[0078] 100μL of the composition is collected and weighed, and 150μL of the solution is dissolved in 400μL of hexane. The resulting solution is subjected to thin layer chromatography (TLC) under the following TLC conditions to separate MAG and DAG. All MAG and DAG bands confirmed at UV254nm are scraped off, 1mL of 1N sodium methoxide / methanol solution is added, and the mixture is stirred well and heated for 5 minutes. Then, the mixture is cooled to room temperature, and 1mL of 1N hydrochloric acid is added and stirred well. The hexane layer obtained by adding 1mL of 0.1mg / mL C23:0 FAME (tricosanoic acid methyl ester) hexane solution and 5mL of saturated saline and stirring well is used as a sample, and GC-FID analysis is performed under the following conditions, and the concentration of each fatty acid is calculated using the following formula. Fatty acid concentration [mg / kg] = (fatty acid peak area / C23:0 peak area) × (10 5 / Amount of sample subjected to TLC [mg] TLC conditions TLC plate: PLC Silica gel 60F 254 0.5mm, 10cm x 10cm Developing solvent: hexane:diethyl ether:acetic acid (7:3:0.1, vol / vol / vol) GC-FID measurement conditions GC: 6890N (Agilent Technologies) Column: DB-WAX (Agilent Technologies) 30mx 0.25mm ID, 0.25μm film thickness Carrier gas: Helium, 1 mL / min Inlet: 250℃, 1 μL, Split (1:100) Column temperature: 180℃ → 3℃ / min → 230℃, hold for 15 minutes Detector: FID, 250℃ Make-up gas: Nitrogen 45 mL / min.

[0079] (b) Iron removal process In step (2)(b), the iron concentration is reduced to less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, or less than 0.05 ppm. In some embodiments, the iron concentration is greater than 0 ppm. In some embodiments, the iron concentration before reduction is greater than or equal to 0.20 ppm or the upper limit value. By reducing the iron concentration, the production of 3-MCPD fatty acid esters can be suppressed.

[0080] In this specification, the iron concentration (or iron content) is a value (ppm (mg / kg)) calculated from a value measured by ICP-MS. Specifically, it is calculated by the following procedure. After weighing out 1 g of the test composition, the solution is diluted to 10 mL with butyl acetate (for atomic absorption spectrometry, Wako Pure Chemical Industries, Ltd.) to prepare a sample solution. Conostan S-21 (10 ppm (Wt.)) is used as a standard sample. This standard sample is diluted with butyl acetate to prepare calibration curve samples (0 μg / L, 0.1 μg / L, 0.5 μg / L, 1 μg / L, 5 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L).

[0081] The sample liquid and the calibration curve sample are subjected to ICP-MS analysis under the analytical conditions below, a calibration curve is created by automatic calculation using the software provided with the device, and the iron content of the sample liquid is quantified.

[0082] Measurement equipment: Agilent 7700 series ICP-MS (Agilent Technologies) RF Power: 1550W Sampling position: 10mm Carrier gas: 0.45L / min Optional gas: 20% Make-up gas: 0.20L / min Spray chamber temperature: -5℃ Sample introduction: Negative pressure suction Measurement mode: He mode He cell gas flow rate: 4.3mL / min Measuring element: 56 Fe

[0083] From the measured iron content of the sample liquid, the iron content in the sample composition is calculated according to the following formula. Iron content in sample composition [ppm] = C / (W x 100) C: Iron content of the sample solution measured by ICP-MS (μg / L) W: Amount of sample composition collected (g)

[0084] The iron concentration may be reduced to less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, or less than 0.05 ppm by a process other than the monoacylglycerol removal process. In some embodiments, the iron concentration is greater than 0 ppm. Techniques for reducing the iron concentration include, in addition to the techniques that can be used in the monoacylglycerol removal process described above, for example, acid washing and ion exchange. These removal techniques can be carried out by conventional methods.

[0085] (c) Chlorine removal process In step (2)(c), the chlorine concentration is reduced to less than 10 ppm, for example, less than 9 ppm, less than 8 ppm, or less than 7 ppm. In some embodiments, the chlorine concentration is higher than 0 ppm. In some embodiments, the chlorine concentration before reduction is 10 ppm or more or the upper limit value or more. By reducing the chlorine concentration, the production of 3-MCPD fatty acid esters can be suppressed.

[0086] In this specification, the chlorine concentration (or chlorine content) is a value (ppm (mg / kg)) calculated from a value measured by ICP-MS. Specifically, it is calculated by the following procedure. After weighing out 1 g of the test composition, the solution is diluted to 10 mL with butyl acetate (for atomic absorption spectrometry, Wako Pure Chemical Industries, Ltd.) to prepare a sample solution. Conostan Cl Std. (1000 ppm (Wt.)) is used as the standard sample. This standard sample is diluted with butyl acetate to prepare calibration curve samples (0 μg / L, 0.1 μg / L, 0.5 μg / L, 1 μg / L, 5 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L).

[0087] The sample liquid and the calibration curve sample are subjected to ICP-MS analysis under the analytical conditions below, a calibration curve is created by automatic calculation using the software attached to the device, and the chlorine content of the sample liquid is quantified.

[0088] Measurement equipment: Agilent 7700 series ICP-MS (Agilent Technologies) RF Power: 1550W Sampling position: 10mm Carrier gas: 0.45L / min Optional gas: 20% Make-up gas: 0.20L / min Spray chamber temperature: -5℃ Sample introduction: Negative pressure suction Measurement mode: He mode He cell gas flow rate: 4.3mL / min Measuring element: 35 Cl

[0089] From the measured chlorine content of the sample solution, the chlorine content in the sample composition is calculated according to the following formula. Chlorine content in sample composition [ppm] = C / (W x 100) C: Chlorine content of the sample solution measured by ICP-MS (μg / L) W: Amount of sample composition collected (g)

[0090] The chlorine concentration may be reduced to less than 10 ppm, for example, less than 9 ppm, less than 8 ppm, or less than 7 ppm, by a process other than the monoacylglycerol removal process. In some embodiments, the chlorine concentration is higher than 0 ppm. Techniques for reducing the chlorine concentration include, in addition to the techniques that can be used in the monoacylglycerol removal process described above, techniques such as degumming and deacidification that are commonly used in the refining process of oils and fats. These removal techniques can be carried out by conventional methods.

[0091] Step (3) (distillation step) The composition in which the concentration of monoacylglycerol has been reduced in step (2) is distilled to separate the main fraction. Even if 3-MCPD fatty acid esters are generated by the heat treatment during distillation, by setting conditions under which the main fraction contains the highly unsaturated fatty acid alkyl esters to be concentrated, it is possible to obtain a composition containing highly unsaturated fatty acid alkyl esters, in which the concentration of 3-MCPD generated is less than 1.80 ppm when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A. Such distillation conditions can be appropriately set depending on the highly unsaturated fatty acid alkyl esters to be concentrated.

[0092] The distillation step can be carried out by, for example, rectification (precise distillation), molecular distillation, or short-path distillation. These can be carried out by a conventional method, for example, the methods described in JP-A-4-128250, JP-A-5-222392, JP-A-4-41457, JP-A-6-33088, etc. can be used.

[0093] The rectification is carried out under high vacuum, and the highly unsaturated fatty acid alkyl ester is separated as the main fraction from the more volatile initial fraction and the less volatile residue. The conditions of the rectification may be set so that the highly unsaturated fatty acid alkyl ester to be concentrated is concentrated as the main fraction, and for example, the temperature may be 150°C to 200°C, for example, 160°C to 200°C, or 170°C to 200°C, and the pressure may be 1 to 300 Pa, for example, 1 to 200 Pa, 1 to 100 Pa, or 1 to 50 Pa. It is preferable to obtain the main fraction at a vacuum degree of 1 to 50 Pa and 170 to 200°C.

[0094] Examples of conditions for molecular distillation or short path distillation include temperatures of 80 to 150°C, e.g., 80 to 130°C, 80 to 120°C, and 10 x 10 -1 Less than Pa, e.g. 10 x 10 -2 Less than 10 x 10 Pa -3 Pressures of less than 1 Pa can be mentioned.

[0095] The main fraction in step (3) was subjected to the American Oil Chemists' Society official Cd29b-13 assay. The resulting concentration of 3-MCPD when analyzed by A is less than 1.80 ppm, e.g., less than 1.70 ppm, less than 1.60 ppm, less than 1.50 ppm, less than 1.40 ppm, less than 1.30 ppm, less than 1.20 ppm, less than 1.10 ppm, less than 1.00 ppm, less than 0.90 ppm, less than 0.80 ppm, less than 0.70 ppm, less than 0.60 ppm, less than 0.50 ppm, less than 0.40 ppm, less than 0.30 ppm, less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, less than 0.05 ppm, less than 0.04 ppm, less than 0.03 ppm, less than 0.02 ppm, or less than 0.01 ppm. In some embodiments, the concentration of 3-MCPD is 0 ppm or more. In particular, when the main fraction in step (3) is analyzed by the American Oil Chemists' Society Official Cd29b-13 Assay A, the concentration of 3-MCPD generated may be 0.01 ppm or more, for example, 0.02 ppm or more. In addition, when the main fraction in step (3) is analyzed by the American Oil Chemists' Society Official Cd29b-13 Assay A, the concentration of 3-MCPD generated may be 0.01 ppm or more, for example, 0.02 ppm or more. The resulting concentration of 3-MCPD when analyzed by A can be greater than or equal to 0.01 ppm and less than 1.80 ppm, less than 1.70 ppm, less than 1.60 ppm, less than 1.50 ppm, less than 1.40 ppm, less than 1.30 ppm, less than 1.20 ppm, less than 1.10 ppm, less than 1.00 ppm, less than 0.90 ppm, less than 0.80 ppm, less than 0.70 ppm, less than 0.60 ppm, less than 0.50 ppm, less than 0.40 ppm, less than 0.30 ppm, less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, less than 0.05 ppm, less than 0.04 ppm, less than 0.03 ppm, or less than 0.02 ppm.In addition, the main fraction in step (3) was analyzed by the American Oil Chemists' Society official Cd29b-13 assay. The resulting concentration of 3-MCPD when analyzed by A can be greater than or equal to 0.02 ppm and less than 1.80 ppm, less than 1.70 ppm, less than 1.60 ppm, less than 1.50 ppm, less than 1.40 ppm, less than 1.30 ppm, less than 1.20 ppm, less than 1.10 ppm, less than 1.00 ppm, less than 0.90 ppm, less than 0.80 ppm, less than 0.70 ppm, less than 0.60 ppm, less than 0.50 ppm, less than 0.40 ppm, less than 0.30 ppm, less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, less than 0.05 ppm, less than 0.04 ppm, or less than 0.03 ppm.

[0096] Chromatography Steps The method of the present invention may further comprise, after step (3), a purification step by chromatography, such as high performance liquid column chromatography (HPLC).

[0097] The chromatography step by HPLC or the like following the distillation step is a step of further concentrating the highly unsaturated fatty acid alkyl ester in the composition after distillation by removing undesired components in the composition obtained in the distillation step, thereby reducing the content of undesired components. The chromatography step can be carried out according to a conventionally known method, for example, the method described in JP-A-5-222392. Examples of the chromatography used in the concentration treatment include reversed-phase column chromatography. Examples of the stationary phase (adsorbent) include polymer beads, preferably polystyrene reticulated with DVB (divinylbenzene), and silica gel, preferably reversed-phase bonded silica gel containing C8 or C18 alkanes, and C18 bonded reversed-phase silica gel is particularly preferred. The adsorbent used in the chromatography after distillation of the present invention is preferably non-polar. Any adsorbent of the reversed-phase distribution system can be used without particular limitation, for example, octadecylsilyl (ODS) silica gel can be used as an ODS column.

[0098] The dimensions of the columns used in the device are not particularly limited, but depend on the amount of sample to be purified. Those skilled in the art can easily determine the appropriate size of column to be used. The diameter of each column is typically 10 to 800 mm, preferably 50 to 800 mm, more preferably 300 to 800 mm, and most preferably 600 to 800 mm. The length of each column is typically 10 to 200 cm, preferably 25 to 150 cm.

[0099] The temperature of the mobile phase and the column is not particularly limited, but depends on the solubility of the substance to be separated in the mobile phase. A person skilled in the art can easily determine the appropriate mobile phase and column temperature to be used. The column temperature is typically 0 to 70°C, preferably 20 to 40°C.

[0100] Solvents used in the mobile phase include, for example, short-chain alcohols. Short-chain alcohols typically have 1 to 6 carbon atoms. Examples of suitable short-chain alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, and t-butanol. The solvent used in the mobile phase is preferably methanol or ethanol, more preferably methanol. It is preferable that no water is intentionally added to the short-chain alcohol in order to shorten the elution time.

[0101] <Distillation raw material composition> The present invention provides a distillation raw material composition for obtaining the composition of the present invention, and the use of the distillation raw material composition as a distillation raw material in the method for producing the composition of the present invention. The distillation raw material composition contains a highly unsaturated fatty acid alkyl ester, and the content of monoacylglycerol is reduced or the iron concentration is reduced. The highly unsaturated fatty acid alkyl ester includes a highly unsaturated fatty acid alkyl ester for the purpose of concentration. By distilling the distillation raw material composition and obtaining the highly unsaturated fatty acid alkyl ester as a fraction, the highly unsaturated fatty acid alkyl ester can be concentrated and the content of 3-MCPD fatty acid ester can be reduced.

[0102] The distillation raw material composition of the present invention can be produced by alkyl esterifying the oil listed as the raw material of the composition of the present invention, and reducing the monoacylglycerol content or reducing the iron concentration. In one embodiment, the distillation raw material composition of the present invention is produced or obtained from a raw material such as fish oil, microbial oil, vegetable oil, or marine animal oil, for example, by the above-mentioned method for producing the composition of the present invention. In a preferred embodiment, the raw material of the distillation raw material composition of the present invention is fish oil. The alkyl esterification and reduction of the monoacylglycerol content or iron concentration can be performed by the method described in the above-mentioned method of the present invention. In a preferred embodiment, the highly unsaturated fatty acid alkyl ester contained in the distillation raw material composition of the present invention can be a highly unsaturated fatty acid ethyl ester. The highly unsaturated fatty acid alkyl ester for the purpose of concentration can be an alkyl ester of eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, or arachidonic acid, or a combination thereof. In a preferred embodiment, the highly unsaturated fatty acid alkyl ester for the purpose of concentration can be an alkyl ester of eicosapentaenoic acid or docosahexaenoic acid, or a combination thereof. In a more preferred embodiment, the highly unsaturated fatty acid alkyl ester for the purpose of concentration can be an alkyl ester of eicosapentaenoic acid.

[0103] The distillation raw material composition of the present invention is a composition containing a fatty acid alkyl ester as a main component, and contains fatty acid alkyl ester in an amount of 95% by weight or more, 96% by weight or more, 97% by weight or more, 98% by weight or more, 99% by weight or more, or 99.5% by weight or more.

[0104] The ratio of polyunsaturated fatty acids to total fatty acids in the distillation raw material composition of the present invention may be 5 area% or more, for example, 10 area% or more, 15 area% or more, or 20 area% or more. The ratio of polyunsaturated fatty acids to total fatty acids in the distillation raw material composition of the present invention may be less than 70 area%, for example, less than 65 area%, less than 60 area%, or less than 55 area%.

[0105] The distillation raw material composition of the present invention may contain saturated fatty acids having 18 or less carbon atoms or alkyl esters thereof as impurities. In this case, the ratio of saturated fatty acids having 18 or less carbon atoms in the constituent fatty acids of the distillation raw material composition of the present invention may be 0.1 area% or more, for example, 0.2 area% or more, 0.3 area% or more, 0.4 area% or more, or 0.5 area% or more, and less than 50 area%, for example, less than 40 area% or less than 30 area%.

[0106] In a preferred embodiment, the concentration of monoacylglycerols containing as constituent fatty acids a fatty acid having a carbon number 5 or more less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated in the distillation raw material composition of the present invention is less than 10,000 ppm, less than 9,000 ppm, less than 8,000 ppm, less than 7,000 ppm, less than 6,000 ppm, less than 5,000 ppm, less than 4,000 ppm, less than 3,000 ppm, less than 2,000 ppm, less than 1,000 ppm, less than 900 ppm, less than 800 ppm, less than 700 ppm, less than 600 ppm, or less than 500 ppm. In some embodiments, the concentration of such monoacylglycerols is higher than 0 ppm.

[0107] The monoacylglycerol to which the above upper limit applies may be a monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number that is 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, or 6 less than the carbon number of the highly unsaturated fatty acid that constitutes the highly unsaturated fatty acid alkyl ester to be concentrated.

[0108] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester for enrichment purposes is an eicosapentaenoic acid (20:5 n-3) alkyl ester, a dihomo-γ-linolenic acid (20:3 n-6) alkyl ester, or an arachidonic acid (20:4 n-6) alkyl ester, or a combination thereof, and the monoacylglycerol can be glycerol monomyristate.

[0109] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester for enrichment purposes can be a docosahexaenoic acid (22:6 n-3) alkyl ester, and the monoacylglycerol can be glycerol monopalmitate.

[0110] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester for the purpose of enrichment is a combination of docosahexaenoic acid (22:6 n-3) alkyl ester and one or more selected from eicosapentaenoic acid (20:5 n-3) alkyl ester, dihomo-γ-linolenic acid (20:3 n-6) alkyl ester, and arachidonic acid (20:4 n-6) alkyl ester, and the monoacylglycerol may be glycerol monomyristate and glycerol monopalmitate.

[0111] In preferred aspects, the iron concentration in the distillation feed compositions of the invention is less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, or less than 0.05 ppm. In some embodiments, the iron concentration is greater than 0 ppm.

[0112] In preferred embodiments, the chlorine concentration in the distillation feed composition of the present invention is less than 10 ppm, such as less than 9 ppm, less than 8 ppm, or less than 7 ppm. In some embodiments, the chlorine concentration is greater than 0 ppm.

[0113] <Usage form> The form of use of the composition of the present invention is not particularly limited, but is preferably an oral dosage form, and can typically be in the form of oral preparations such as granules, tablets, capsules, and liquids. The uses of the composition of the present invention include, for example, food and drink (health foods, nutritional supplements, foods for specified health uses, supplements, dairy products, soft drinks, food and drink for pets, livestock feed, etc.), medicines, and quasi-drugs, and are particularly preferably supplements and medicines. In addition to food ingredients or foods, the composition may be used as an additive component for animal feed. Therefore, the composition of the present invention can be used as a material or active ingredient for these food and drink, medicine, and quasi-drugs, and can be preferably used in the production of these.

[0114] Examples of the present invention will be described below, but the present invention is not limited to these in any way. In the examples, all percentages are by weight unless otherwise specified, and ppm is by weight (i.e., mg / kg) unless otherwise specified. In the examples, the 3-MCPD concentration refers to the value measured by the American Oil Chemists' Society official method, Cd29b-13 assay A. In addition, a 3-MCPD concentration of 0.00 ppm means that 3-MCPD was not detected by the above analytical method (i.e., it was below the detection limit). In the examples, an iron concentration of 0.00 ppm means that no iron was detected in the above-mentioned ICP-MS measurement (i.e., it was below the detection limit). EXAMPLES

[0115] [Test 1] Effect of iron content on the formation of 3-MCPD Sardine crude oil was deacidified by short-path distillation, and the oil was ethyl-esterified with an alkali catalyst, and then subjected to silica gel purification to separate the ethyl ester fraction. For silica gel purification, a glass open column packed with Microsphere gel D-75-60A (AGC Si-Tech Co., Ltd.) in an amount five times the amount of the sample was used, and hexane / ethyl acetate (50:1) was used as the eluent. It was confirmed by thin layer chromatography (TLC) that no bands of DAG or MAG were detected in this ethyl ester fraction (fish oil ethyl ester (EE)).In addition, the iron concentration was measured and no iron was detected in the ethyl ester fraction.

[0116] To this fish oil EE, 1000 ppm of glycerol monomyristate (Wako Pure Chemical Industries, product code 321-32412) was added. Furthermore, an aqueous solution of iron(II) sulfate heptahydrate was added so that the iron content became 0.10 ppm (Example 2) or 1.00 ppm (Comparative Example 1), or no iron was added (Example 1), and then ethanol was added to homogenize. After that, the solvent was completely removed using an evaporator and vacuum drawing.

[0117] For each test group, the mixture was stirred in an oil bath at 210°C under a nitrogen stream, and samples were taken over time to measure the 3-MCPD concentration. The changes in 3-MCPD concentration during heating for each test group are shown in Table 1.

[0118] [Table 1]

[0119] In Comparative Example 1, in which 1.00 ppm of iron was added, 0.61 ppm of 3-MCPD was produced after heating for 4 hours. On the other hand, in Examples 1 and 2, in which the iron concentrations were adjusted to 0.00 ppm and 0.10 ppm, respectively, the 3-MCPD contents were 0.08 ppm and 0.15 ppm even after heating for 4 hours, which were significantly lower than the 0.61 ppm in Comparative Example 1.

[0120] [Test 2] Effect of MAG content on the formation of 3-MCPD Fish oil EE (iron concentration adjusted to 10 ppm) prepared in the same manner as in Test 1 was used as is, or glycerol monomyristate (Wako Pure Chemical Industries, product code 321-32412) was added at a concentration of 1 to 10%, and heated at 120°C for 1 hour under a nitrogen stream.

[0121] The 3-MCPD concentration after heating is shown in Table 2. It was found that even at a relatively low temperature of 120°C, which is generally used for the removal of fatty acid ethyl esters by molecular distillation, the 3-MCPD concentration increases with increasing MAG concentration.

[0122] [Table 2]

[0123] [Test 3] Effect of MAG content on the formation of 3-MCPD in fish oil ethyl ester distillates Fish oil containing 20 area % EPA was ethyl-esterified with an alkali catalyst according to a conventional method to prepare fish oil ethyl ester 1. Fish oil ethyl ester 1 had a fatty acid composition in which the proportion of EPA was 20 area %, and contained DAG and MAG containing C14:0 as constituent fatty acids at the concentrations shown in Table 3, an iron concentration of 0.2 ppm, and a chlorine concentration of 17 ppm.

[0124] [Table 3]

[0125] Next, fish oil ethyl ester 2 was prepared by removing MAG and DAG from fish oil ethyl ester 1, fish oil ethyl ester 3 was prepared by adding glycerol monomyristate to fish oil ethyl ester 2, and fish oil ethyl ester 4 was prepared by adding glycerol monopalmitate to fish oil ethyl ester 2. Fish oil ethyl ester 2 was prepared by the following method. The mixture obtained by mixing 600 g of fish oil ethyl ester 1 with 2400 mL of hexane was passed through a column packed with 1200 g of silica gel (Microsphere D75-60A) and a slurry of hexane, and the fish oil ethyl ester was adsorbed on the silica gel. Then, ethyl acetate / hexane (1:50) was passed through the column to fractionate the eluent, and a fraction in which MAG and DAG were removed from the fish oil ethyl ester was collected. The collected fraction was desolvated by an evaporator and vacuum drawing to obtain 585 g of fish oil ethyl ester free of MAG and DAG. The fish oil ethyl ester 2 thus obtained contained no MAG or DAG, and had an iron concentration of 0.05 ppm and a chlorine concentration of 7 ppm.

[0126] 100 g of fish oil ethyl ester 2 was mixed with 0.1 g of glycerol monomyristate, completely dissolved, and homogenized to prepare fish oil ethyl ester 3. Also, 100 g of fish oil ethyl ester 2 was mixed with 0.1 g of glycerol monopalmitate (Tokyo Chemical Industry, product code G0083), completely dissolved, and homogenized to prepare fish oil ethyl ester 4.

[0127] The fish oil ethyl ester 3 or 4 was used as a sample (raw material composition for distillation) and subjected to precision distillation including the following first precision distillation step and second precision distillation step.

[0128] The first precision distillation step is a step to remove the C18 and below fractions. A vacuum jacketed fractionating column (φ25mm, Kiriyama Glass) was used as the fractionating column, and five Sulzer Lab Packing EX (25mm x 50mm, Sulzer Chemtech) were used as the internal packing. Precision distillation was performed with the liquid temperature in the bottom pot (column bottom temperature) set to 185°C or less, the top vapor temperature (column top temperature) set to 135°C or less, and the pressure before the vacuum pump (column top pressure, i.e., vacuum degree) set to 30 Pa or less, with a heating time of 4.0 hours. In this first precision distillation step, the C18 and below fractions were removed as the first distillation, and a first distillation residue in which EPA was concentrated was obtained.

[0129] Then, in the second precision distillation process, the following precision distillation was performed on the initial distillation residue obtained in the first precision distillation process. A vacuum jacketed fractionating column (φ25mm, Kiriyama Glass) was used as the fractionating column, and five Sulzer Labpacking EX (25mm x 50mm, Sulzer Chemtech) were used as the internal packing. Precision distillation was performed for 3.5 hours with the liquid temperature in the bottom pot (bottom temperature) at 195°C, the top vapor temperature (top temperature) at 150°C, and the pressure before the vacuum pump (top pressure, i.e., vacuum degree) at 30 Pa. In this second precision distillation process, the C22 and higher fractions were removed as the residue (residue), and the main fraction was obtained.

[0130] Using 80 g of fish oil ethyl ester 3 as a raw material, 26 g of initial distillation residue in which EPA was concentrated was obtained in the first precision distillation step. Using 25 g of the initial distillation residue obtained as a raw material, 11 g of main fraction in which EPA was concentrated was obtained in the second precision distillation step. EPA was concentrated in the main fraction, and as shown in Table 4, the proportion of EPA in the fatty acid composition increased from 20.9% to 73.1%. On the other hand, isomer formation due to heating during distillation was also observed, and 0.8 area % of the trans isomer of EPA ethyl ester (total value of five types of trans isomers; not shown in Table 4; the same applies below) was found in the main fraction.

[0131] The 3-MCPD concentration was 0.00 ppm in the raw material fish oil ethyl ester 3, but increased to 0.01 ppm in the main fraction after distillation.

[0132] Using 77.8 g of fish oil ethyl ester 4 as a raw material, 29.5 g of initial distillation residue in which EPA was concentrated was obtained in the first precision distillation step. Using 26.4 g of the initial distillation residue obtained as a raw material, 12.6 g of main fraction in which EPA was concentrated was obtained in the second precision distillation step. EPA was concentrated in the main fraction, and as shown in Table 4, the proportion of EPA in the fatty acid composition increased from 20.9% to 77.4%. Meanwhile, isomer formation due to heating during distillation was also observed, and 1.6 area % of the trans isomer of EPA ethyl ester was observed in the main fraction.

[0133] The 3-MCPD concentration in the main fraction was 0.00 ppm, and no increase was observed from the concentration in the raw material, fish oil ethyl ester 4.

[0134] [Table 4]

[0135] The distribution of 3-MCPD in each fraction of fish oil ethyl ester 3 and 4 was investigated. The material balance of 3-MCPD contained in the main fraction and the main fraction residue is shown in Table 5.

[0136] [Table 5]

[0137] It was found that the 3-MCPD concentration in the main fraction was strongly influenced by the specific MAG in the feedstock. In the main fraction, which was specifically fractionated to extract EPA, i.e., C20 fatty acid components, 28.0% of the total 3-MCPD was contained in the case of fish oil ethyl ester 3, in which MAG containing C14:0, i.e., C14 fatty acid as a constituent fatty acid, was added, whereas in the case of fish oil ethyl ester 4, in which MAG containing C16:0, i.e., C16 fatty acid as a constituent fatty acid, was added, all of the 3-MCPD was contained in the main fraction. This shows that when C20 fatty acid ethyl esters including EPA ethyl esters are recovered as the main fraction, 3-MCPD fatty acid esters formed from MAG containing C14 fatty acid as a constituent fatty acid are mixed into the main fraction, but 3-MCPD fatty acid esters formed from MAG containing C16 fatty acid as a constituent fatty acid are hardly mixed into the main fraction. In other words, when the polyunsaturated fatty acid to be refined is a C20-PUFA such as EPA, it can be said that the C14 saturated fatty acid has a strong influence on the 3-MCPD concentration in the distillate. In each test, the initial fraction contained absolutely no 3-MCPD or 3-MCPD fatty acid esters. [Industrial Applicability]

[0138] According to the present invention, compositions containing a high concentration of PUFA alkyl esters and a low concentration of 3-MCPD fatty acid esters can be stably produced.

[0139] All publications, patent applications, patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in any text incorporated by reference are excluded to the extent that they conflict with definitions in this disclosure.

[0140] Other embodiments are within the scope of the following claims.

Claims

1. A distillation raw material composition containing a highly unsaturated fatty acid alkyl ester, The highly unsaturated fatty acid alkyl ester includes a highly unsaturated fatty acid alkyl ester for the purpose of concentration, The composition has a concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number that is 5 or more less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated, of less than 10,000 ppm, and / or an iron concentration of less than 0.20 ppm.

2. The composition according to claim 1, wherein the concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number 6 less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated is less than 10,000 ppm.

3. 3. The composition of claim 1 or 2, wherein the chlorine concentration is less than 10 ppm.

4. The composition according to any one of claims 1 to 3, wherein the highly unsaturated fatty acid alkyl ester for the purpose of concentration is an alkyl ester of eicosapentaenoic acid, docosahexaenoic acid, dihomo-gamma-linolenic acid, or arachidonic acid, or a combination thereof.

5. The composition according to any one of claims 1 to 4, wherein the raw material is a fish oil, a microbial oil, a vegetable oil, or a marine animal oil.

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