Composition containing highly unsaturated fatty acids or their alkyl esters and method for producing the same

Abstract

To provide a composition comprising highly enriched highly unsaturated fatty acid or alkyl ester thereof while containing fatty acid esters of 3-MCPD at adequately low concentrations, and to provide an efficient method for producing the composition.SOLUTION: A composition comprises fatty acids or fatty acid alkyl esters as its major component, the composition containing highly unsaturated fatty acid or alkyl ester thereof, wherein the proportion of the highly unsaturated fatty acid in the constituent fatty acids of the composition is 50 area% or more, and wherein the concentration of 3-MCPD, as found by analyzing the composition using American Oil Chemists' Society official method Cd 29b-13 assay A, is less than 1.80 ppm.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] 3-Chloropropane-1,2-diol (3-MCPD) is a compound suspected of being carcinogenic. Regulations have been established in the EU and other countries regarding the concentration of this substance in food. In fats and oils, it is known that 3-MCPD can be produced using diacylglycerol (DAG) and monoacylglycerol (MAG) as substrates (Non-Patent Literature 1). Fats and oils rich in diacylglycerol (DAG) Since the presence of 3-MCPD was pointed out, various methods have been attempted to reduce its content in oils and fats. For example, 3-MCPD is known to form at high temperatures, and Patent Documents 1 and 2 disclose that the concentration of substances that generate 3-MCPD in oils and fats can be reduced by adsorbent treatment, lowering the deodorization temperature, or shortening the treatment time. However, these are methods for reducing the concentration of substances that generate 3-MCPD in the production of triacylglycerols, and Patent Documents 1 and 2 do not describe methods for reducing the concentration of 3-MCPD fatty acid esters in the production of alkyl esters.

[0003] Polyunsaturated fatty acids (PUFAs) are known to possess various functionalities, and highly concentrated PUFAs are used in foods, supplements, pharmaceuticals, and cosmetics. When PUFAs are highly concentrated, increasing the proportion of the desired PUFA in the starting composition, they are converted from glycerides, primarily triacylglycerols, to alkyl esters with lower alcohols. Therefore, highly purified and concentrated PUFAs used in foods, supplements, pharmaceuticals, and cosmetics are often alkyl esters. To date, no method has been known to reduce the concentration of 3-MCPD fatty acid esters in such high-concentration PUFA alkyl esters.

[0004] Factors known to influence the formation of 3-MCPD fatty acid esters include the chlorine source, substrates such as MAG and DAG, and processing time at high temperatures (Non-Patent Documents 1 and 2). However, in oils used as raw materials for foods containing PUFAs, the content of chlorine sources and substrates such as MAG and DAG is usually small, and their impact on the purification of the target PUFA is extremely small. Therefore, the need to remove them is not generally recognized. Furthermore, the complete removal of chlorine sources and substrates such as MAG and DAG is not performed due to the technical difficulty and its impact on productivity, such as the recovery rate of PUFAs.

[0005] Rectification is a type of distillation that offers high separation capabilities, but it often requires heating at high temperatures of 150°C or above because it necessitates internal packing and reflux. Molecular distillation and short-path distillation are different. The heating temperature is 150°C or lower, and it can be carried out at a relatively low temperature compared to rectification, but repeated processing is required to sufficiently concentrate PUFA. Therefore, in the distillation of PUFA There is a risk of generating large amounts of 3-MCPD.

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

[0007] Therefore, purification processes involving heat treatment such as solvent removal and distillation carry a clear risk of generating 3-MCPD fatty acid esters, and methods such as urea addition and HPLC do not necessarily remove the diverse 3-MCPD fatty acid esters contained in the raw materials. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2011-147435 [Patent Document 2] Japanese 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) [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] Oils and fats containing PUFAs often contain 3-MCPD or its fatty acid esters derived from the raw materials or the oil extraction / refining process. Furthermore, when glycerides in oils and fats are alkylated, MAG and DAG remain or their concentrations increase, creating an environment conducive to the formation of 3-MCPD fatty acid esters. Moreover, the purification of alkyl esters often involves desolvation processes as well as distillation processes such as molecular distillation, short-path distillation, and rectification, and 3-MCPD fatty acid esters can be formed at each of these processes. Chromatography and urea addition do not involve heat, so the possibility of 3-MCPD fatty acid ester formation is low, but there is a risk that the concentration of already present 3-MCPD fatty acid esters cannot be sufficiently reduced. The present invention aims to provide a composition containing a highly concentrated PUFA or PUFA alkyl ester with a sufficiently low concentration of 3-MCPD fatty acid ester, and an efficient method for producing the same. [Means for solving the problem]

[0011] The inventors conducted extensive research to achieve the above objective and found that in distillation, the C20-C22 fatty acid alkyl ester fraction, in which PUFAs are concentrated, and the C14 fatty acid or C16 fatty acid fraction are concentrated. We found that the 3-MCPD mono fatty acid ester, when bonded to the parent compound, exhibits similar behavior. Furthermore, by reducing the content of monoacylglycerol (MAG) in the raw material, particularly MAGs to which C14 or C16 fatty acids are bonded, we were able to reduce the content of 3-MCPD mono in the C20-C22 fatty acid alkyl ester fraction. We found that it is possible to reduce the concentration of fatty acid esters. Furthermore, the inventors have found that the formation rate of 3-MCPD fatty acid esters changes significantly depending on the concentration of trace amounts of metal, such as iron, contained in the raw materials at a concentration of 1 ppm or less. It had been reported that iron affects the formation of 3-MCPD fatty acid esters from triacylglycerol (Non-Patent Literature 3). However, Non-Patent Literature 3 discloses extremely large amounts of Fe 2+ Or Fe 3+ This study investigated the formation of 3-MCPD fatty acid esters in the presence of iron, and it was previously unknown that trace amounts of iron, normally present in oils used as raw materials for PUFA alkyl ester production, would affect 3-MCPD fatty acid ester formation. Before concentration, PUFA alkyl esters produced from raw materials such as fish oil generally have a low degree of purification, so the iron concentration can vary depending on the iron content in the raw oil. Also, the iron concentration of PUFA alkyl esters before concentration can vary significantly depending on the quality of the extraction raw material, the extraction method, the purification method, etc. Therefore, when the iron concentration increases, the concentration of 3-MCPD fatty acid ester can 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 inventors further conducted 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, containing a highly unsaturated fatty acid or its alkyl ester, the proportion of the highly unsaturated fatty acid 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 being 1.80 ppm or less, said composition. 〔2〕The composition according to 〔1〕, wherein the proportion of the highly unsaturated fatty acid 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 value 〔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 feed 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 concentration purposes, The concentration of monoacylglycerol containing a fatty acid having 5 or more fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes as a constituent fatty acid is less than 10,000 ppm, and / or the iron concentration is less than 0.20 ppm, said composition. [8] The composition according to [7], wherein the concentration of monoacylglycerol containing a fatty acid having 6 or fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes as a constituent fatty acid 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 for concentration purposes 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 its alkyl ester, (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 the steps of (a) reducing the concentration of monoacylglycerol containing a fatty acid having 5 or more fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes in the composition of step (1) to less than 10,000 ppm, (b) reducing the iron concentration in the composition of step (1) to less than 0.20 ppm, and (c) reducing the chlorine concentration in the composition of step (1) to less than 10 ppm, and (3) A step of distilling the composition after step (2) and separating the main fraction, The method, including the method described above.

[13] The main fraction in step (3) is determined by the American Oil Chemists' Society official method Cd29b-13 as The method according to

[12] , wherein the concentration of 3-MCPD obtained when analyzed by say A is less than 1.80 ppm.

[14] The method according to

[12] or

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

[15] The method according to any of

[12] to

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

[16] The method according to any one of

[12] to

[15] , wherein in step (2), the concentration of monoacylglycerol containing a fatty acid with 6 fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration is reduced to less than 10,000 ppm.

[17] The method according to any one of

[12] to

[16] , wherein the polyunsaturated 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 embodiment, the present invention is as follows. [A1] A composition containing highly unsaturated fatty acids or their alkyl esters, 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, 3 - The composition wherein the concentration of MCPD 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 was divided according to the American Oil Chemists' Society official method Cd29b-13 assay A. The composition according to [A1] or [A2], wherein the concentration of 3-MCPD produced when analyzed is below 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] A composition described in any of [A1] to [A4], which is a distillate. [A6] A 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 contains highly unsaturated fatty acid alkyl ester for concentration purposes. The composition wherein the concentration of monoacylglycerol containing a fatty acid with a number of carbon atoms 5 or more less than the number of carbon atoms of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes is less than 10,000 ppm, or the iron concentration is less than 0.20 ppm. [A8] The composition according to [A7], wherein the concentration of monoacylglycerol containing a fatty acid with 6 fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes is less than 10,000 ppm. [A9] The composition according to [A7] or [A8], wherein the chlorine concentration is less than 10 ppm. [A10] The composition according to any one of [A7] to [A9], wherein the highly unsaturated fatty acid alkyl ester for concentration purposes is an alkyl ester of eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, or arachidonic acid, or a combination thereof. [A11] A 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 highly unsaturated fatty acids or their alkyl esters, (1) A process of alkylating a raw material containing a triglyceride that includes highly unsaturated fatty acids as constituent fatty acids, and preparing a composition containing highly unsaturated fatty acid alkyl ester. , (2) At least one selected from (a) a step of reducing the concentration of monoacylglycerol in the composition of step (1) to less than 10,000 ppm, which contains a fatty acid with a number of carbon atoms that is 5 or more fewer than the number of carbon atoms of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for the purpose of concentration, (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 after step (2) and separating the main fraction, The method, including the method described above. [A13] The main fraction in step (3) is determined by the American Oil Chemists' Society official method Cd29b-13 a The method according to [A12], wherein the concentration of 3-MCPD obtained when analyzed by ssay A is less than 1.80 ppm. [A14] The method according to [A12] or [A13], wherein step (2)(a) is performed by silica gel chromatography. [A15] The method according to any 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 a fatty acid with 6 fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration is reduced to less than 10,000 ppm. [A17] The method according to any 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 of [A12] to [A17], wherein the raw material is derived from fish oil, microbial oil, vegetable oil, or marine animal oil. [Effects of the Invention]

[0014] The present invention makes it possible to stably produce compositions containing high concentrations of PUFA or PUFA alkyl esters with low concentrations of 3-MCPD fatty acid esters. [Modes for carrying out the invention]

[0015] The present invention will be described in detail below. In this specification, when describing fatty acids, numerical representations using a combination of numbers and letters may be used to express the number of carbon atoms, the number of double bonds, and the position of the double bonds in a simplified manner. For example, a saturated fatty acid with 20 carbon atoms may be written as "C20:0", a monounsaturated fatty acid with 18 carbon atoms as "C18:1", and eicosapentaenoic acid 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 3rd and 4th 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 fatty acids described in this manner can be easily identified by those skilled in the art.

[0016] In this specification, "highly unsaturated fatty acid" means a fatty acid having 18 or more carbon atoms and 3 or more double bonds. Highly unsaturated fatty acids may be, for example, fatty acids with 20 or more carbon atoms and 3 or 4 or more double bonds, or fatty acids with 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, “crude oil” means a mixture of lipids, which is oil in the state extracted from living organisms. In this specification, “refined oil” means oil that has undergone a crude oil refining process in which crude oil is subjected to at least one oil refining process selected from the group consisting of a degumming process, a deacidifying process, a decolorizing process, and a deodorizing process, thereby removing substances other than the target product, such as phospholipids and sterols. Those skilled in the art can distinguish between refined oil and crude oil by ordinary analysis.

[0018] In this specification, a 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, a fatty acid composition is a composition whose main component is a fatty acid, and a fatty acid alkyl ester composition is a composition whose main component is an alkyl ester of a fatty acid.

[0019] In this specification, a general term (e.g., highly unsaturated fatty acid, highly unsaturated fatty acid alkyl ester, etc.) does not rule out the possibility of multiple components unless it is clearly indicated otherwise by the context. Therefore, a general term usually means that at least one component is present.

[0020] The present invention relates to a composition containing highly unsaturated fatty acids or their alkyl esters, wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 50 area% or more, and the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A. The present invention provides a composition having a concentration of 3-MCPD of less than 1.80 ppm (hereinafter referred to as the "composition of the present invention").

[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 distillate 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%, and 55 to 60% area, 60 to 99% area, 60 to 98% area, 60 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, It could be 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 may 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 may 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 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 may be 60 to 80 area%, 65 to 80 area%, 70 to 80 area%, or 75 to 80 area%.

[0026] Because the composition of the present invention has a high proportion of highly unsaturated fatty acids, it is suitable as a raw material for pharmaceuticals and supplements that contain highly unsaturated fatty acids as active ingredients.

[0027] In this specification, "area %" representing the proportion of highly unsaturated fatty acids in the constituent fatty acids of a composition is determined by identifying the peaks of each component in a chart obtained by analyzing the composition using gas chromatography-focal-ionization (GC-FID) with a flame ionization detector (FID), and using the Agilent ChemStation integration algorithm (revision C.01.03

[37] , Agilent Technologies). This method involves determining the peak area of ​​each fatty acid and expressing the content ratio of that peak component as the ratio of each peak area to the sum of the peak areas of all fatty acids. In the field of oil chemistry, area % is used almost synonymously with weight %. (References: 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 Method) and 2.4.2.2-2013 Fatty Acid Composition (FID Temperature-Boosting Gas Chromatography Method)) See the Rough Method. The analytical 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°C → 3°C / min → 230°C, held for 15 minutes. Detector: FID, 250℃ Makeup gas: Nitrogen 45 mL / min.

[0028] In embodiments of the present invention, where the composition 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. That is, the fatty acid composition is methyl esterified in accordance with the contents of AOCS official method Ce1b-89 and then subjected to GC under the above conditions, and the highly unsaturated fatty acids are measured in the same manner. Calculate the area percentage of the acid. In this case, the constituent fatty acids of the composition refer to the free fatty acids in the fatty acid composition.

[0029] In embodiments of the present invention, where the composition is a fatty acid alkyl ester composition containing an alkyl ester of a highly unsaturated fatty acid, the proportion of highly unsaturated fatty acids 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 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 acids of the composition refer to the fatty acids that make up the fatty acid alkyl ester of the fatty acid alkyl ester composition.

[0030] The alkyl group in highly unsaturated fatty acid alkyl esters is an alkyl group derived from a lower alcohol commonly used in the alkyl esterification of fatty acids, such as a C1 or C2 alkyl group (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 official assay Cd29b-13 assay. The concentration of 3-MCPD obtained when analyzed by A is reduced to less than 1.80 ppm (mg / kg), for example, 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 higher than 0 ppm. In one embodiment, the composition of the present invention is measured according to the American Oil Chemists' Society standard Cd29b-13 ass. The concentration of 3-MCPD obtained when analyzed by ay A may be 0.01 ppm or higher, for example, 0.02 ppm or higher. Furthermore, the composition of the present invention is such that when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the concentration of 3-MCPD obtained is 0.01 ppm or higher. The concentration is 0.01 ppm or greater, and may be 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. Furthermore, the composition of the present invention is described in the American Oil Chemists' Society official method Cd29b-13 The concentration of 3-MCPD obtained when analyzed by assay A is 0.02 ppm or less. Above, and possibly below 1.80 ppm, below 1.70 ppm, below 1.60 ppm, below 1.50 ppm, below 1.40 ppm, below 1.30 ppm, below 1.20 ppm, below 1.10 ppm, below 1.00 ppm, below 0.90 ppm, below 0.80 ppm, below 0.70 ppm, below 0.60 ppm, below 0.50 ppm, below 0.40 ppm, below 0.30 ppm, below 0.20 ppm, below 0.10 ppm, below 0.09 ppm, below 0.08 ppm, below 0.07 ppm, below 0.06 ppm, below 0.05 ppm, below 0.04 ppm, or below 0.03 ppm.

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

[0033] 100 mg of the sample was mixed with 3-MCPD-d5-dipalmitate standard solution (5 ppm). (Diluted with toluene to form 3-MCPD-d5) 100 μL, diethyl ether 600 Add μL and stir until completely dissolved, then cool at -22°C to -25°C for about 15 minutes. Then add 350 μL of sodium hydroxide-methanol solution (0.25 g sodium hydroxide dissolved in 100 mL methanol) and stir well, then react at -22°C to -25°C for 16 hours or more. At the same temperature, add acidic sodium bromide solution (600 ml sodium bromide) (g was dissolved in 1L of purified water, 3mL of 85% phosphoric acid was added, and 600μL was added to stop the reaction.) Next, the separated organic layer is concentrated by blowing nitrogen until it reaches approximately 100 μL. Then, 600 μL of hexane is added and the mixture is vigorously stirred, and the organic layer is allowed to stand for 5 to 10 minutes to remove it. This process is repeated twice. 600 μL of diethyl ether:ethyl acetate mixture (3:2, V / V) is added to the remaining aqueous layer and the mixture is vigorously stirred, and the organic layer is recovered. This process is repeated three times. The three recovered organic layers are combined and dehydrated with anhydrous sodium sulfate. The dehydrated organic layer is concentrated to 200 μL by blowing nitrogen, 20 μL of saturated phenylboronic acid-diethyl ether solution is added and the mixture is vigorously stirred for 10 seconds, and then the solvent is completely removed by blowing nitrogen. 200 μL of isooctane is added to this mixture and the mixture is vigorously stirred for 10 seconds, and the resulting solution is used as the GC-MS sample.

[0034] Calibration curves for 3-MCPD quantification are prepared by analyzing 3-MCPD standard solutions, which are prepared by dissolving 3-MCPD-dipalmitate in toluene at concentrations of 0 ppm, 0.5 ppm, 1 ppm, and 5 ppm, in the same manner as the samples described above.

[0035] The analytical conditions for GC-MS are 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 minute Column temperature: 85°C, hold for 0.5 minutes → 6°C / min → 150°C, hold for 5 minutes → 12°C / min → 180°C → 25°C / min → 280°C, hold for 7 minutes Ionization temperature: 200℃ Interface temperature: 200℃ Ionization method: EI, SIM. The m / z values ​​to be monitored are as follows. 3-MCPD-d5:m / z=149, 150, 201, 203 3-MCPD: m / z=146, 147, 196, 198 For quantitative analysis, m / z=150 for 3-MCPD-d5 and m / z=147 for 3-MCPD were used, while other values ​​were determined based on the target substance. Used for recognition.

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

[0037] In one embodiment of this specification, the composition of the present invention has a concentration of 3-MCPD when analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A. The concentration may be greater than or equal to 1.80 ppm and less than 1.80 ppm. In this case, the composition of the present invention may include a composition that does not contain 3-MCPD. In other embodiments, the composition of the present invention contains 3-MCPD (i.e., 3-MCPD or 3-MCPD fatty acid ester), and when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the resulting 3 - The concentration of MCPD may be less than 1.80 ppm.

[0038] The composition of the present invention is subjected to the American Oil Chemists' Society official assay Cd29b-13 assay. The concentration of 3-MCPD obtained when analyzed by A may be below the lower limit of quantification of the American Oil Chemists' Society official method Cd29b-13 assay A, and preferably below the detection limit. It could be full.

[0039] Highly unsaturated fatty acids such as eicosapentaenoic acid are known to be abundant in certain microbial oils, vegetable oils, or marine animal oils. Therefore, the compositions of the present invention may be made from these as raw materials. Specifically, the raw materials for the compositions of the present invention include fish oils such as sardine oil, tuna oil, skipjack tuna oil, menheiden 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, soybeans, rapeseed, etc.; yeasts such as those of the genus Yarrowia; and species of Mortierella, Penicillium, Aspergillus, and rosewood. Filamentous fungi belonging to genera such as Rhodotorula and Fusarium, Examples include oils derived from lipid-producing microorganisms such as algae of the genus Grenadine and stramenopiles. The composition of the present invention may also be an oil derived from a genetically modified microorganism into which a gene such as a genetically modified mutant Δ9 elongase gene has been introduced. Furthermore, oils derived from genetically modified plants such as Brassica species, sunflower, corn, cotton, flax, and safflower, into which a gene such as a mutant Δ9 elongase gene has been introduced by recombination technology, can also be used as a raw material oil. Examples of genetically modified vegetable oils and genetically modified microbial oils can be found in WO2012 / 027698, WO2010 / 033753, etc. In a preferred embodiment, the raw material for the composition of the present invention is a fish oil, microbial oil, vegetable oil, or marine animal oil, more preferably fish oil.

[0040] The raw materials for the composition of the present invention mainly contain highly unsaturated fatty acids in the form of glycerides. For example, fish oil contains many types 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, the glycerides are reacted with a lower alcohol such as ethanol to alkylate the fatty acids contained in the glycerides, and then the desired highly unsaturated fatty acid alkyl ester (e.g., EPA alkyl ester) is obtained. By removing the external fatty acid alkyl ester, a highly pure, highly unsaturated fatty acid alkyl ester (e.g., EPA alkyl ester) can be produced. In one embodiment, Other than the target highly unsaturated fatty acid alkyl ester (e.g., EPA alkyl ester) The removal of fatty acid alkyl esters can be carried out by distillation. The inventors have found that the main residue obtained from distillation contains a large amount of 3-MCPD fatty acid esters derived from mono or diacylglycerols with relatively high molecular weights. Therefore, in a preferred embodiment, the composition of the present invention may be a distillate (fraction).

[0041] It is known that heat treatment during distillation generates trans isomers, which are heat-denatured products, from highly unsaturated fatty acid alkyl esters (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”). Therefore, in one embodiment, the composition of the present invention is a highly unsaturated fatty acid alkyl ester. The composition of the present invention may further contain trans isomers of ster. The concentration of the trans isomer in the composition of the present invention 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. The concentration of the trans isomer in the composition of the present invention may also 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. It may be 0.05% or more of the area.

[0042] In this specification, the concentrations of trans isomers are measured by GC analysis. Specifically, they are measured by the following procedure. Dissolve 10 mg of the sample in 1 mL of hexane and subject it 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°C → 3°C / min → 230°C, held for 15 minutes. Detector: FID, 250℃ Makeup gas: Nitrogen 45 mL / min.

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

[0044] For example, for the trans isomer of dihomo-γ-linolenate ethyl ester (DGLA-E), the sum of the areas of the relative retention time peaks is measured by the GC analysis described above, the ratio of the trans isomer to DGLA-E is determined, and the isomer concentration in the sample is calculated from the DGLA-E concentration in the sample. Isomer A: Relative retention time 1.001~1.009 Isomer B: Relative retention time 1.01~1.03 (Assume the retention time of DGLA is 1.)

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

[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. Furthermore, 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, when the composition of the present invention is measured using gas chromatography under the above analytical conditions, the concentration of the trans isomer of EPA ethyl 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. Alternatively, when the composition of the present invention is measured using gas chromatography under the above analytical conditions, the concentration of the trans isomer of EPA ethyl 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.

[0048] 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 DGLA 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. Furthermore, 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 DGLA 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.

[0049] In a preferred embodiment, when the composition of the present invention is measured using gas chromatography under the above analytical conditions, the concentration of the trans isomer of DGLA ethyl 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. Alternatively, when the composition of the present invention is measured using gas chromatography under the above analytical conditions, the concentration of the trans isomer of DGLA ethyl 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.

[0050] Fish oils and microbial oils containing highly unsaturated fatty acids also contain cholesterol in addition to triglycerides. Highly unsaturated fatty acid concentrates prepared from these raw materials also contain cholesterol (WO2012 / 118173). Furthermore, alkali esterification or urea addition to cholesterol-containing oils does not completely remove the cholesterol. Therefore, in certain embodiments of this specification, the compositions of the present invention contain 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. Alternatively, 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 This compound has a steroid skeleton represented by O, and exists in natural products as either a free or esterified form. The esterified form is acylcholesterol, in which a fatty acid is bonded to the hydroxyl (OH) group. In this invention, cholesterol content refers to the total content of both the free and esterified forms. The cholesterol content is measured by the following method.

[0052] To approximately 0.1 g of the sample, add 1 mL of 0.1 g / L 5α-cholestane as an internal standard, then add 1 mL of 2 mol / L potassium hydroxide / aqueous ethanol solution, and incubate at 100°C for 10 minutes. Heat the mixture. After cooling, add 3 mL of petroleum ether and 3 mL of saturated ammonium sulfate, stir, and after standing, collect the upper layer and measure it by gas chromatography under the following measurement conditions. To determine the relative sensitivity between 5α-cholestane and free cholesterol, 5α-cholestane and The total cholesterol content is calculated by measuring hexane solutions containing 25 mg of cholesterol each using gas chromatography. Gas chromatography analysis conditions Model: Agilent 6890 GC system (Agilent Corporation) 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 (39.3 kPa, constant pressure)

[0053] In one embodiment, the composition of the present invention may contain saturated fatty acids having 18 or fewer carbon atoms or their alkyl esters as impurities. In this case, the proportion of saturated fatty acids having 18 or fewer carbon atoms in the constituent fatty acids of the composition of the present invention is 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 3 area%. The product may be less than %.

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

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

[0056] <Method for producing a composition containing highly unsaturated fatty acids or their alkyl esters> The present invention provides a method for producing the above-mentioned composition of the present invention. The method is as follows: (1) A step of alkylating a raw material containing a triglyceride that includes highly unsaturated fatty acids as constituent fatty acids to prepare a composition containing highly unsaturated fatty acid alkyl ester, (2) At least one step selected from (a) reducing the concentration of monoacylglycerol in the composition of step (1) to less than 10,000 ppm, which contains a fatty acid with a number of carbon atoms that is 5 or more fewer than the number of carbon atoms of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for the purpose of concentration, to less than 10,000 ppm, (b) reducing the iron concentration in the composition of step (1) to less than 0.20 ppm, and (c) reducing the chlorine concentration in the composition of step (1) to less than 10 ppm, (3) Distillation of the composition after step (2) to separate the main fraction. This includes (hereinafter also referred to as the method of the present invention).

[0057] By the method of the present invention, a composition mainly containing a fatty acid or a fatty acid alkyl ester can be produced, wherein the composition contains 95% or more by weight, 96% or more by weight, 97% or more by weight, 98% or more by weight, 99% or more by weight, 99.5% or more by weight, or 99.9% or more by weight of the fatty acid or fatty acid alkyl ester.

[0058] In this specification, step (1) may be referred to as the alkyl esterification step, and step (2)(a The first step is sometimes called the monoacylglycerol removal step, the second step (2)(b) is sometimes called the iron removal step, the third step (2)(c) is sometimes called the chlorine removal step, and the third step (3) is sometimes called the distillation step.

[0059] Examples of raw materials in the method of the present invention include the oils described in the above-mentioned composition of the present invention, specifically fish oils such as sardine oil, tuna oil, skipjack tuna oil, menheiden 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, soybeans, rapeseed, etc.; oils derived from lipid-producing microorganisms such as yeasts of the genus Yarrowia, filamentous fungi belonging to the genera Mortierella, Penicillium, Aspergillus, Rhodotorula, Fusarium, etc.; algae such as Euglena, etc.; and stramenopiles. The raw materials in the method of the present invention may also be oils derived from genetically modified microorganisms into which genes such as the genetically modified mutant Δ9 elongase gene have been introduced. Furthermore, genetically modified plant-derived oils from oilseed plants such as Brassica species, sunflower, corn, cotton, flax, and safflower, into which genes such as the mutated Δ9 elongase gene have been introduced by recombination technology, can also be used as raw material oils. Examples of genetically modified vegetable oils and genetically modified microbial oils can be found in WO2012 / 027698, WO2010 / 033753, etc. In a preferred embodiment, the raw material in the method of the present invention is fish oil, microbial oil, vegetable oil, or marine animal oil, and more preferably fish oil.

[0060] Crude oil refining process The raw material oil used in the alkyl esterification in step (1) may be crude oil or refined oil. For example, any method is acceptable for obtaining crude oil from fish or other marine products, but in the case of fish oil, it is usually extracted by the following method: The whole fish or the processing residue such as the head, skin, bones, and internal organs of the fish generated from seafood processing is crushed and steamed, then pressed to separate it into a broth (stick water) and pressed meal. The oil obtained together with the broth is separated from the broth by centrifugation to obtain crude fish oil. Generally, crude fish oil is refined into refined fish oil through a crude oil refining process that removes substances other than the target product, such as phospholipids and sterols, by performing processes such as degumming, deacidification, decolorization using activated clay or activated carbon, washing, and deodorization by steam distillation, depending on the raw material. In the embodiments of the present invention, this refined fish oil can also be used as a raw material.

[0061] Step (1) (Alkyl esterification step) The raw material oil, or fat, is broken down into lower alcohol esters by alcohol decomposition using lower alcohols. Examples of lower alcohols commonly used for the alkyl esterification of fatty acids include those with one or two carbon atoms. Alcohol decomposition involves adding a lower alcohol, such as ethanol, to the oil and reacting it with a catalyst or enzyme to produce alkyl esters from fatty acids bonded to glycerol. Catalysts such as alkaline catalysts and acid catalysts are used. Lipase is used as the enzyme.

[0062] The reaction efficiency of alcohol decomposition of fatty acids has been empirically demonstrated to be high, and after alcohol decomposition, compositions mainly containing these fatty acids in their alkyl ester forms are obtained. However, this does not completely exclude the presence of fatty acids in forms other than alkyl esters.

[0063] Process (2) Step (2) is a pre-treatment step for distillation and is at least one step selected from (a) to (c) below. That is, step (2) is any one of (a) to (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b), and It could be (c).

[0064] (a) Monoacylglycerol removal process In step (2)(a), the composition containing the fatty acid alkyl ester prepared in step (1) is reduced in monoacylglycerol content, which is a substrate for 3-MCPD fatty acid ester, before being concentrated by distillation. This reduces the amount of 3-MCPD fatty acid ester that is generated by the heat treatment during distillation and mixed into the main fraction.

[0065] Existing techniques such as repeated alkyl esterification and adsorbent treatment can be used to remove monoacylglycerol from the fatty acid alkyl ester composition prepared in step (1).

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

[0067] Examples of adsorbent treatments 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 treatment with silica gel (e.g., Microsphere D75-60A), fatty acid alkyl esters are applied to a silica gel-packed column in order to adsorb them onto the silica gel. Then, ethyl acetate / hexane (1:50) is passed through the column, the eluent is fractionated, and the fraction from which MAG and DAG have been removed from the fatty acid alkyl esters is recovered. This fraction is desolvented to obtain fatty acid alkyl esters. Activated clay treatment can be performed, for example, by adding 5% activated clay relative to the oil, stirring under reduced pressure at 120°C 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 a constituent fatty acid with a carbon number 5 or less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration. Here, the highly unsaturated fatty acid alkyl ester for concentration 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 of step (3), the conditions are set so that the highly unsaturated fatty acid alkyl ester for concentration is separated as the main fraction. Therefore, the highly unsaturated fatty acid alkyl ester separated as the main fraction and concentrated in the distillation of step (3) is the highly unsaturated fatty acid alkyl ester for concentration.

[0069] When there are two or more highly unsaturated fatty acid alkyl esters for concentration purposes (for example, when attempting to concentrate a combination of alkyl esters of two or more highly unsaturated fatty acids selected from eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, and arachidonic acid), the concentration of monoacylglycerol containing a fatty acid with a number of carbon atoms at least 5 less than the one with the most carbon atoms is reduced.

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

[0071] In step (2)(a), the concentration of monoacylglycerol containing a fatty acid with 5 or more fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes is set to less than 10,000 ppm, less than 9,000 ppm, and 8,000 ppm. The concentration is reduced to less than 0 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 monoglycerol before reduction is 10,000 ppm or higher, or above the above upper limit. For example, if the highly unsaturated fatty acid alkyl ester for concentration is eicosapentaenoic acid (20:5 n-3) alkyl ester, dihomo-γ-linolenic acid (20:3 n-6) alkyl ester, or arachidonic acid (20:4 n-6) alkyl ester, or a combination thereof, the concentration of monoacylglycerol containing a fatty acid with 15 or fewer carbon atoms (preferably a fatty acid with 14 carbon atoms) as a constituent fatty acid is reduced to the above concentration. If the highly unsaturated fatty acid alkyl ester for concentration is docosahexaenoic acid (22:6 n-3) alkyl ester, the concentration of monoacylglycerol containing a fatty acid with 17 or fewer carbon atoms (preferably a fatty acid with 16 carbon atoms) as a constituent fatty acid is reduced to the above concentration. Furthermore, if the highly unsaturated fatty acid alkyl ester for concentration purposes is a combination of one or more selected from docosahexaenoic acid (22:6 n-3) alkyl ester, 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 a fatty acid with 17 or fewer carbon atoms (preferably a fatty acid with 14 carbon atoms and a fatty acid with 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 may be produced from monoacylglycerol containing a fatty acid with 5 or more fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration may be included in the main fraction together with the highly unsaturated fatty acid alkyl ester. Therefore, when obtaining the highly unsaturated fatty acid alkyl ester for concentration as the main fraction in the distillation of step (3), by pre-reducing the concentration of monoacylglycerol containing a fatty acid with 5 or more fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration, a composition containing a highly unsaturated fatty acid alkyl ester with a reduced concentration of 3-MCPD fatty acid ester can be obtained.

[0073] The monoacylglycerols removed in step (2)(a) may be monoacylglycerols containing fatty acids with 5-10, 5-9, 5-8, 5-7, 5-6, or 6 fewer carbon atoms than the highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl ester for concentration purposes. In step (2)(a), the total concentration of these monoacylglycerols should be reduced to the above-mentioned concentration.

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

[0075] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester for concentration purposes is 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 for concentration purposes is docosahexaenoic acid (22:6 n-3) alkyl ester and eicosapentaenoic acid (20:5 The combination is one or more selected from n-3) alkyl esters, dihomo-γ-linolenic acid (20:3 n-6) alkyl esters, and arachidonic acid (20:4 n-6) alkyl esters, and the monoacylglycerol removed in step (2)(a) may be monomyristate glycerol and monopalmitate glycerol.

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

[0078] 100 μL of the composition was taken and weighed, and 150 μL of the solution, prepared by dissolving it in 400 μL of hexane, was subjected to thin-layer chromatography (TLC) under the following TLC conditions to separate MAG and DAG. All MAG and DAG bands confirmed at UV 254 nm were scraped off, 1 mL of 1N sodium methoxide / methanol solution was added, and the mixture was stirred well and heated for 5 minutes. After that, it was cooled to room temperature, 1 mL of 1N hydrochloric acid was added, and the mixture was stirred well. 0.1 mg / mL C23:0 FAME (methyl tricosanoate) hexane solution 1 mL and saturated Add 5 mL of saline solution and stir well to obtain a hexane layer. This hexane layer is used as the sample, and GC-FID analysis is performed under the following conditions. The concentrations of each fatty acid are then calculated using the following formula. Fatty acid concentration [mg / kg] = (peak area of ​​fatty acids / peak area of ​​C23:0) × (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°C → 3°C / min → 230°C, held for 15 minutes. Detector: FID, 250℃ Makeup 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 higher than 0 ppm. In some embodiments, the iron concentration before reduction is 0.20 ppm or higher or above the above upper limit. By reducing the iron concentration, the formation of 3-MCPD fatty acid esters can be suppressed.

[0080] In this specification, iron concentration (or iron content) is a value calculated from measurements taken by ICP-MS (ppm (mg / kg)). Specifically, it is calculated by the following procedure. After weighing 1 g of the test composition, it is diluted to 10 mL using butyl acetate (for atomic absorption spectrometry, Wako Pure Chemical Industries, Ltd.) to prepare the sample solution. Conostan S-21 (10 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, 100 μg / L).

[0081] ICP-MS analysis is performed on the sample solution and calibration curve sample under the following analytical conditions. A calibration curve is created by automatic calculation using the software attached to the instrument, and the iron content of the sample solution is quantified.

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

[0083] The iron content in the sample composition is calculated from the quantified iron content of the sample solution using the following formula. Iron content in the sample composition [ppm] = C / (W × 100) C: Iron content of the sample solution measured by ICP-MS (μg / L) W: Amount of sample composition taken (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 separate from the monoacylglycerol removal process. In some embodiments, the iron concentration is higher 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, pickling and ion exchange, for example. 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 higher, or above the above upper limit. By reducing the chlorine concentration, the formation of 3-MCPD fatty acid esters can be suppressed.

[0086] In this specification, chlorine concentration (or chlorine content) is a value calculated from measurements taken by ICP-MS (ppm (mg / kg)). Specifically, it is calculated by the following procedure. After weighing 1 g of the test composition, it is diluted to 10 mL using butyl acetate (for atomic absorption spectrometry, Wako Pure Chemical Industries, Ltd.) to prepare the sample solution. Conostan Cl Std. (1000 ppm (Wt.)) was 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, 100 μg / L).

[0087] ICP-MS analysis is performed on the sample solution and calibration curve sample under the following analytical conditions. A calibration curve is created by automatic calculation using the software attached to the instrument, and the chlorine content of the sample solution is quantified.

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

[0089] The chlorine content in the sample composition is calculated from the quantified chlorine content of the sample solution using the following formula. Chlorine content in the sample composition [ppm] = C / (W × 100) C: Chlorine content of the sample solution measured by ICP-MS (μg / L) W: Amount of sample composition taken (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 separate from 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, which are commonly used in the refining process of oils and fats. These removal techniques can be carried out by conventional methods.

[0091] Process (3) (Distillation Process) In step (2), the composition with reduced monoacylglycerol concentration is distilled, and the main fraction is separated. Even if 3-MCPD fatty acid esters are produced by the heat treatment during distillation, by setting conditions that the main fraction contains the highly unsaturated fatty acid alkyl esters intended for concentration, a composition containing highly unsaturated fatty acid alkyl esters is obtained, and when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the 3-MCPD fatty acid esters produced are obtained. A composition with a CPD concentration of less than 1.80 ppm can be obtained. The distillation conditions for such a composition can be appropriately set depending on the highly unsaturated fatty acid alkyl ester to be concentrated.

[0092] The distillation process can be carried out, for example, by rectification (precise distillation), molecular distillation, or short-stroke distillation. These can be carried out by standard methods, and for example, methods described in Japanese Patent Publication Nos. 4-128250, 5-222392, 4-41457, and 6-33088 can be used.

[0093] Fine distillation is carried out under high vacuum, and highly unsaturated fatty acid alkyl esters are taken as the main fraction, and can be obtained by separating more volatile initial fraction and less volatile residue. The conditions for fine distillation may be set so that the highly unsaturated fatty acid alkyl esters for the purpose of concentration are concentrated as the main fraction. For example, the temperature is 150°C to 200°C, such as 160 - 200°C, 170 - 200°C, and the pressure can be 1 - 300 Pa, such as 1 - 200 Pa, 1 - 100 Pa, 1 - 50 Pa. It is preferable to obtain the main fraction at 170°C to 200°C under a vacuum degree of 1 - 50 Pa.

[0094] Examples of the conditions for molecular distillation or short-path distillation include a temperature of 80 - 150°C, such as 80 - 130°C, 80 - 120°C, and a pressure of less than 10×10 -1 Pa, such as less than 10×10 -2 Pa, less than 10×10 -3 Pa.

[0095] When the main fraction in step (3) 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, such as 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 method Cd29b - 13 assay A, the 3 - The concentration of MCPD may be 0.01 ppm or higher, for example, 0.02 ppm or higher. Furthermore, the main fraction in step (3) is subjected to the American Oil Chemists' Society official Cd29b-13 assay. The concentration of 3-MCPD obtained when analyzed by A may be 0.01 ppm or greater, 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. Furthermore, when the main fraction in step (3) was analyzed using the American Oil Chemists' Society official method Cd29b-13 assay A, The concentration of 3-MCPD may be 0.02 ppm or greater, 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 process The method of the present invention may further include a chromatographic purification step, such as high-performance liquid column chromatography (HPLC), after step (3).

[0097] The chromatography step, such as HPLC, that follows the distillation step is a step to further concentrate the highly unsaturated fatty acid alkyl ester in the distilled composition by removing unwanted components from the composition obtained in the distillation step, thereby reducing the content of unwanted components. The chromatography step can be carried out according to conventionally known methods, for example, the method described in Japanese Patent Application Publication No. 5-222392. Examples of chromatography used in the concentration process include reversed-phase column chromatography. Examples of stationary phases (adsorbents) include polymer beads, preferably DVB (divinylbenzene) and a network of polystyrene, and Examples include silica gel, preferably reverse-phase linked silica gel containing a C8 or C18 alkane, with C18 linked reverse-phase linked silica gel being particularly preferred. The adsorbent used in the post-distillation chromatography of the present invention is preferably nonpolar. Any reverse-phase partition adsorbent can be used without particular limitation, for example, octadecylsilyl (ODS) silica gel, which can be used as an ODS column.

[0098] The dimensions of the column used in the apparatus 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 use. 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 temperatures of the mobile phase and column are not particularly limited, but depend on the solubility of the substance to be separated in the mobile phase. Those skilled in the art can easily determine the appropriate mobile phase and column temperatures to be used. The column temperature is typically 0 to 70°C, preferably 20 to 40°C.

[0100] Examples of solvents used in the mobile phase include short-chain alcohols. Short-chain alcohols typically have 1 to 6 carbon atoms. Suitable examples of 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, and more preferably methanol. It is preferable not to intentionally add water 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 described above, and the use of the distillation raw material composition as a distillation raw material in a method for producing the composition of the present invention described above. The distillation raw material composition contains highly unsaturated fatty acid alkyl esters, and has a reduced monoacylglycerol content or reduced iron content. The highly unsaturated fatty acid alkyl esters include highly unsaturated fatty acid alkyl esters for concentration purposes. By distilling the distillation raw material composition and obtaining highly fatty acid alkyl esters as a fraction, the highly fatty acid alkyl esters can be concentrated and the content of 3-MCPD fatty acid esters can be reduced.

[0102] The distillation raw material composition of the present invention can be produced by alkylating the oil listed as a raw material for the composition of the present invention to reduce the monoacylglycerol content or the iron content. In one embodiment, the distillation raw material composition of the present invention is produced from or obtained from raw materials such as fish oil, microbial oil, vegetable oil, or marine animal oil by the above-described method for producing the composition of the present invention. In a preferred embodiment, the raw material for the distillation raw material composition of the present invention is fish oil. Alkyl esterification and reduction of monoacylglycerol content or iron content can be carried out by the method described in the above-described 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 may be a highly unsaturated fatty acid ethyl ester. Furthermore, the highly unsaturated fatty acid alkyl ester for concentration purposes may 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 concentration purposes may 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 concentration purposes may be an alkyl ester of eicosapentaenoic acid.

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

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

[0105] The distillation raw material composition of the present invention may contain saturated fatty acids having 18 or fewer carbon atoms or their alkyl esters as impurities. In this case, the proportion of saturated fatty acids having 18 or fewer carbon atoms in the constituent fatty acids of the distillation raw material composition of the present invention is 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. It may be less than 40% of the area, or less than 30% of the area.

[0106] In preferred embodiments, the concentration of monoacylglycerol 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 monoacylglycerol is higher than 0 ppm.

[0107] The monoacylglycerols to which the above upper limits apply may be monoacylglycerols that contain as constituent fatty acids fatty acids with 5-10, 5-9, 5-8, 5-7, 5-6, or 6 fewer carbon atoms than the highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl ester for concentration purposes.

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

[0109] In preferred embodiments, the highly unsaturated fatty acid alkyl ester for concentration purposes is docosahexaenoic acid (22:6 n-3) alkyl ester, and the monoacylglycerol may be glycerol monopalmitate.

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

[0111] In preferred embodiments, the iron concentration in the distillation raw material composition of the present 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 a preferred embodiment, the chlorine concentration in the distillation raw material composition of the present invention is 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 greater than 0 ppm.

[0113] <Usage form> The form in which the composition of the present invention is used is not particularly limited, but it is preferably in the form of an oral dosage form, and typically it can be in the form of an oral formulation such as granules, tablets, capsules, or liquids. The composition of the present invention can be used in, for example, foods and beverages (health foods, nutritional supplements, foods for specified health uses, supplements, dairy products, soft drinks, pet foods and beverages, livestock feed, etc.), pharmaceuticals, quasi-drugs, etc., and is particularly preferably used in supplements and pharmaceuticals. In addition to food materials or foods, it may also be used as an additive component for animal feed. Accordingly, the composition of the present invention can be used as a material or active ingredient in these foods and beverages, pharmaceuticals, and quasi-drugs, and can be preferably used in their manufacture.

[0114] Examples of the present invention are described below, but the present invention is not limited to these examples. In the examples, percentages (%) are weight percentages unless otherwise specified. Also, ppm (ppm) is weight ppm (i.e., mg / kg) unless otherwise specified. In the examples, the 3-MCPD concentration refers to the measurement value obtained by the American Oil Chemists' Society (AOCS) official method Cd29b-13 assay A. 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 measurement by ICP-MS (i.e., it was below the detection limit). [Examples]

[0115] [Experiment 1] Effect of iron content on the formation of 3-MCPD Sardine crude oil was deoxidized by short-step distillation, and the resulting oil was ethyl esterified using an alkaline catalyst. The oil was then subjected to silica gel purification, and the ethyl ester fraction was separated. For silica gel purification, a glass open column packed with five times the volume of Microsphere gel D-75-60A (AGC SI-TEC Co., Ltd.) was used, and hexane / ethyl acetate (50:1) was used as the eluent. Thin-layer chromatography (TLC) confirmed that no DAG and MAG bands were detected in this ethyl ester fraction (fish oil ethyl ester (EE)). Furthermore, the iron content was also confirmed. The measurement results showed that no iron was detected in the ethyl ester fraction.

[0116] This fish oil EE contains glycerol monomyristate (Wako Pure Chemical Industries, product code 321-32412). A 1000 ppm amount was added. Furthermore, an aqueous solution of iron(II) sulfate heptahydrate was added with an iron content of 0.10 ppm (actual The solution was added to a concentration of 1.00 ppm (Example 2) or 1.00 ppm (Comparative Example 1), or not added at all (Example 1), and then ethanol was added to homogenize it. The solvent was then completely removed by evaporating and vacuuming.

[0117] Each test section was stirred in an oil bath at 210°C under a nitrogen stream, and the sump was monitored over time. The 3-MCPD concentration was measured. Table 1 shows the change in 3-MCPD concentration during heating for each test group.

[0118] [Table 1]

[0119] In Comparative Example 1, where 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, where the iron concentration was adjusted to 0.00 ppm and 0.10 ppm, respectively, 4 Even after heating for a period of time, the 3-MCPD content was 0.08 ppm and 0.15 ppm, which is lower than the 0.61 ppm in Comparative Example 1. It was always a low value.

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

[0121] The 3-MCPD concentration after heating is shown in Table 2. Generally, this is obtained by molecular distillation of fatty acid ethyl esters. It was found that even at the relatively low temperature of 120°C used in the previous study, the 3-MCPD concentration increased as the MAG concentration increased.

[0122] [Table 2]

[0123] [Experiment 3] Effect of MAG content on the formation of 3-MCPD in fish oil ethyl ester distillate Fish oil containing 20% ​​EPA was ethyl esterified with an alkaline catalyst according to a conventional method to prepare fish oil ethyl ester 1. Fish oil ethyl ester 1 has a fatty acid composition in which EPA is present in 20% area. The product percentage contains DAG and MAG, which include C14:0 as a constituent fatty acid, at the concentrations shown in Table 3, and iron The concentration was 0.2 ppm, and the chlorine concentration was 17 ppm.

[0124] [Table 3]

[0125] Next, fish oil ethyl ester 2 is obtained by removing MAG and DAG from fish oil ethyl ester 1, fish oil ethyl ester 3 is obtained by adding monomyristate glycerol to fish oil ethyl ester 2, and fish oil ethyl ester 2 is obtained by adding monopalmitate glycerol. Steal 4 was prepared. Fish oil ethyl ester 2 was prepared by the following method. Mixing 600 g of fish oil ethyl ester 1 with 2400 mL of hexane yielded a mixture, which was then used to obtain silica. 1200 g of gel (Microsphere D75-60A) was passed through a column packed with hexane slurry to adsorb fish oil ethyl ester onto silica gel. Subsequently, ethyl acetate / hexane (1:50) was passed through the column to fractionate the eluent, and the fraction from which MAG and DAG were removed from the fish oil ethyl ester was recovered. The recovered fraction was desolvented by evaporator and vacuum. 585 g of fish oil ethyl ester free from MAG and DAG was obtained. Ruester 2 contained no MAG or DAG, and had an iron concentration of 0.05 ppm and a chlorine concentration of 7 ppm.

[0126] Mix 0.1 g of glycerol monomyristate with 100 g of ethyl fish oil 2, and completely Fish oil ethyl ester 3 was prepared by dissolving and homogenizing the two components. Furthermore, fish oil ethyl ester 4 was prepared by mixing 0.1 g of glycerol monopalmitate (Tokyo Chemical Industries, product code G0083) with 100 g of fish oil ethyl ester 2, and then completely dissolving and homogenizing the two components.

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

[0128] The first precision distillation step is the process of removing fractions below C18. A vacuum-jacketed fractionation tube (φ25mm, Kiriyama Glass) was used, and five Sulzer Lab Packing EX (25mm x 50mm, Sulzer Chemtec) were used as internal packing. The liquid temperature in the bottom vessel (bottom temperature) was kept below 185°C, and the top vapor temperature (top temperature) was kept below 135°C. Below, precision distillation was performed with the pressure before the vacuum pump (top pressure, i.e., vacuum level) set to 30 Pa or less, and the heating time 4.0 hours. In this first precision distillation step, the fraction below C18 was used as the initial distillate. After removal, the initial distillate residue with concentrated EPA was obtained.

[0129] Subsequently, in the second precision distillation process, the following precision distillation was performed on the initial residue obtained in the first precision distillation process. A vacuum-jacketed fractionation tube (φ25mm, Kiriyama Glass) was used as the fractionation tube, and five Sulzer Lab Packing EX (25mm x 50mm, Sulzer Chemtec) were used as the internal packing material. The liquid temperature in the bottoming vessel (bottoming temperature) was set to Precision distillation was performed at 195°C, with a top vapor temperature (top temperature) of 150°C and a pressure before the vacuum pump (top pressure, i.e., vacuum level) of 30 Pa, for a heating time of 3.5 hours. During the distillation process, fractions with a C22 or higher content were removed as residue to obtain the main distillate.

[0130] Using 80 g of fish oil ethyl ester 3 as the raw material, a first precision distillation step yielded 26 g of a residue after the initial distillate, which was concentrated with EPA. Using the obtained 25 g of residue after the initial distillate as the raw material, a second precision distillation step yielded 11 g of a main distillate, which was concentrated with EPA. The main distillate was concentrated with EPA, 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, heating during distillation Isomer formation was also observed, with 0.8 area% of the trans isomer of EPA ethyl ester in the main fraction. (Total value for 5 trans isomers. Not shown in Table 4. The same applies below.) was observed.

[0131] Furthermore, the 3-MCPD concentration in the raw material, fish oil ethyl ester 3, is 0.00 ppm, and after distillation... In the main fraction, the concentration increased to 0.01 ppm.

[0132] Using 77.8 g of fish oil ethyl ester 4 as the raw material, a first precision distillation step yielded 29.5 g of the initial distillate residue, which was concentrated with EPA. Using the obtained initial distillate residue of 26.4 g as the raw material, a second precision distillation step yielded 12.6 g of the main distillate, which was concentrated with EPA. The main distillate was concentrated with EPA, as shown in Table 4. As shown above, the proportion of EPA in the fatty acid composition increased from 20.9% to 77.4%. On the other hand, during distillation... Isomer formation was also observed upon heating, with 1.6 area% of EPA ethyl ester present in the main fraction. A lance isomer was observed.

[0133] Furthermore, the concentration of 3-MCPD in the main distillate was 0.00 ppm, and in the raw material, fish oil ethyl ester 4... No increase from the concentration was observed.

[0134] [Table 4]

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

[0136] [Table 5]

[0137] It was found that the concentration of 3-MCPD in the main fraction was strongly influenced by specific MAGs in the raw materials. In the main fraction from which EPA, i.e., C20 fatty acid components, was specifically separated, fish oil ethyl ester 3 was C14:0, In other words, when MAG containing C14 fatty acids as constituent fatty acids was added, 28.0% of the total 3-MCPD was contained in it, whereas when MAG containing fish oil ethyl ester 4 (C16:0, i.e., C16 fatty acids as constituent fatty acids) was added, all of the 3-MCPD was contained in the main residue. Furthermore, when recovering C20 fatty acid ethyl esters containing EPA ethyl ester as the main deposit, C14 The 3-MCPD fatty acid ester, formed from MAG containing the fatty acid of the above as a constituent fatty acid, is mixed into the main deposit. However, it was shown that 3-MCPD fatty acid esters formed from MAG containing C16 fatty acids as constituent fatty acids hardly contaminate the main distillate. In other words, when the highly unsaturated fatty acid targeted for purification is a C20-PUFA such as EPA, C14 saturated fatty acids strongly influence the 3-MCPD concentration in the distillate. Furthermore, in each test, the initial distillate contained no 3-MCPD or 3-MCPD fatty acid esters at all. [Industrial applicability]

[0138] The present invention makes it possible to stably produce compositions containing high concentrations of PUFA alkyl esters with low concentrations of 3-MCPD fatty acid esters.

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

[0140] Other embodiments are described in the following claims.

Claims

1. A composition containing fatty acids or fatty acid alkyl esters as the main component, wherein it contains highly unsaturated fatty acids or their alkyl esters, the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 50 area % or more, and the concentration of 3-MCPD obtained when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is less than 1.80 ppm, and (1) The concentration of the trans isomer of the highly unsaturated fatty acid alkyl ester is 0.01 area percent or more, (2) The composition having a cholesterol content of 0.01% by weight or more.

2. The composition according to claim 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 claim 1 or 2, wherein the concentration of 3-MCPD obtained when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is below the detection limit.

4. The composition according to claim 1 or 2, wherein the concentration of 3-MCPD obtained when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is 0.01 ppm or higher.

5. The composition according to any one of claims 1 to 4, wherein the highly unsaturated fatty acid is eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, or a combination thereof.

6. A composition according to any one of claims 1 to 5, which is a distillate.

7. The composition according to any one of claims 1 to 6, wherein the raw material is fish oil, microbial oil, vegetable oil, or marine animal oil.

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