Composition containing highly unsaturated fatty acid or alkyl ester thereof 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 an alkyl ester thereof and a method for producing the same.

Background Art

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

[0003] Polyunsaturated fatty acids (PUFAs) are known to have various functionalities, and highly concentrated PUFAs are used in foods, supplements, pharmaceuticals, or cosmetics. When highly concentrated PUFAs increase the proportion of the desired PUFAs among the fatty acids of the starting composition, they are converted from glycerides mainly composed of triacylglycerol to alkyl esters with lower alcohols. Therefore, highly purified and concentrated PUFAs used in foods, supplements, pharmaceuticals, or cosmetics are often alkyl esters. A method for reducing the concentration of 3-MCPD fatty acid ester in such high-concentration PUFA alkyl esters has not been known so far.

[0004] As factors affecting the production of 3-MCPD fatty acid esters, a chlorine source, substrates such as MAG and DAG, and the treatment time at high temperatures are known (Non-Patent Documents 1 and 2). However, for oils used as raw materials for foods containing PUFA, etc., generally, the content of the chlorine source and substrates of 3-MCPD fatty acid esters such as MAG and DAG is small, and the influence on the purification of the target PUFA is extremely small. Therefore, the necessity of removing them has not been generally recognized. In addition, completely removing the chlorine source and substrates such as MAG and DAG has not been carried out due to its technical difficulty and the influence on productivity such as the recovery rate of PUFA.

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

[0006] The urea addition method and the HPLC method are methods of separating by the structure of the fatty acids constituting the molecule (for example, the chain length, the number of double bonds, etc.). However, when 3-MCPD exists as a di- or mono-fatty acid ester form in the raw material, it is difficult to separate it from the target alkyl ester depending on the type of fatty acid constituting it, and it is difficult to stably obtain a fatty acid alkyl ester with reduced 3-MCPD fatty acid ester.

[0007] Therefore, in the purification process including heat treatment such as desolventization and distillation, there is an obvious risk of generating 3-MCPD fatty acid esters, and methods such as urea addition and HPLC do not always remove various 3-MCPD fatty acid esters contained in the raw material.

Prior Art Documents

Patent Document

[0008]

Patent Document 1

Patent Document 2

Non-Patent Document

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0010] Oils and fats containing PUFA often contain 3-MCPD or its fatty acid esters derived from raw materials and oil extraction (extraction) and refining processes. Also, when the glycerides in oils and fats are alkyl esterified, MAG and DAG remain or rather their concentrations increase, creating an environment where 3-MCPD fatty acid esters are likely to be generated. Furthermore, the purification of alkyl esters often includes a solvent removal process and distillation processes such as molecular distillation, short-path distillation, and rectification, and 3-MCPD fatty acid esters can be generated in each process. In chromatography and urea addition, since no temperature is applied, the possibility of generating 3-MCPD fatty acid esters is low, but there is a risk that the concentration of 3-MCPD fatty acid esters already contained cannot be sufficiently reduced. The object of the present invention is to provide a composition containing 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 Problems

[0011] As a result of intensive studies to achieve the above object, the present inventors have found that in distillation, the C20-C22 fatty acid alkyl ester fraction in which PUFA is concentrated and the 3-MCPD mono-fatty acid ester in which a C14 fatty acid or a C16 fatty acid is bound by a single bond show similar behavior. Furthermore, by reducing the content of monoacylglycerol (MAG) in the raw material, particularly MAG bound with a C14 fatty acid or a C16 fatty acid, it has been found that the concentration of 3-MCPD mono-fatty acid ester in the C20-C22 fatty acid alkyl ester fraction can be reduced. In addition, the present inventors have found that the formation rate of 3-MCPD fatty acid ester changes significantly depending on the change in the concentration of trace metals of 1 ppm or less contained in the raw material, such as iron. It has been reported that iron affects the formation of 3-MCPD fatty acid ester from triacylglycerol (Non-Patent Document 3). However, what is disclosed in Non-Patent Document 3 is the result of examining the formation of 3-MCPD fatty acid ester in the presence of an extremely large amount of Fe or Fe and it has not been known that trace amounts of iron usually contained in oils used as raw materials for producing PUFA alkyl esters affect the formation of 3-MCPD fatty acid ester. 2+ or Fe 3+ ​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 becomes high, 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, wherein the proportion of the highly unsaturated fatty acid in the constituent fatty acids of the composition is 50 area% or more, and the concentration of 3-MCPD generated when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is 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 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, the composition according to 〔1〕 or 〔2〕. 〔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 feedstock composition containing a highly unsaturated fatty acid alkyl ester, The highly unsaturated fatty acid alkyl ester includes the 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) Step of distilling the composition after step (2) and collecting the main distillate fraction The method as described above, including

[13] When the main distillate fraction in step (3) is analyzed by the American Oil Chemists' Society official method Cd29b-13 as described in

[12] , the concentration of 3-MCPD generated when analyzed by assay 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 one of

[12] to

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

[16] In step (2), the concentration of monoacylglycerol containing a fatty acid having 6 or fewer carbon atoms than the number of carbon atoms of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes is reduced to less than 10,000 ppm. The method according to any one of

[12] to

[15] .

[17] The method according to any one of

[12] to

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

[18] The method according to any one of

[12] to

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

[0013] Also, in one aspect, the present invention is as follows. [A1] A composition containing a highly unsaturated fatty acid or an alkyl ester thereof, wherein the proportion of the highly unsaturated fatty acid in the constituent fatty acids of the composition is 50 area% or more, and when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the concentration of 3 -MCPD is less than 1.80 ppm. The above composition. [A2] The composition according to [A1], wherein the proportion of the highly unsaturated fatty acid in the constituent fatty acids of the composition is 70 area% or more. [A3] When the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A The composition according to [A1] or [A2], wherein the concentration of 3-MCPD generated upon analysis is less than the detection limit value. 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. The composition according to any one of [A1] to [A4], which is a distillate. The composition according to any one of [A1] to [A5], wherein the raw material is derived from fish oil, microbial oil, vegetable oil, or marine animal oil. The distillation raw material composition containing a highly unsaturated fatty acid alkyl ester, wherein the highly unsaturated fatty acid alkyl ester includes a highly unsaturated fatty acid alkyl ester for concentration purposes, the composition, wherein 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, or the iron concentration is less than 0.20 ppm. The composition according to [A7], wherein the concentration of monoacylglycerol containing a fatty acid having 6 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. The composition according to [A7] or [A8], wherein the chlorine concentration is less than 10 ppm. 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. The composition according to any one of [A7] to [A10], wherein the raw material is derived from fish oil, microbial oil, vegetable oil, or marine animal oil. A method for producing a composition containing a highly unsaturated fatty acid or its alkyl ester, (1) A step of preparing a composition containing a highly unsaturated fatty acid alkyl ester by alkyl esterifying a raw material containing a triglyceride having a highly unsaturated fatty acid as a constituent fatty acid , (2) (a) A step of reducing the concentration of a 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) a step of reducing the iron concentration in the composition of step (1) to less than 0.20 ppm, and (c) at least one selected from the steps of 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 collecting the main fraction The method as described above. [A13] The method according to [A12], wherein the concentration of 3-MCPD generated when the main fraction in step (3) is analyzed by the American Oil Chemists' Society official method Cd29b-13 a assay 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 one of [A12] to [A14], wherein the distillation in step (3) is fractional distillation. [A16] The method according to any one of [A12] to [A15], wherein in step (2), the concentration of a monoacylglycerol containing a fatty acid having 6 fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes is reduced to less than 10,000 ppm. [A17] The method according to any one of [A12] to [A16], wherein the highly unsaturated fatty acid is eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, or a combination thereof. [A18] The method according to any one of [A12] to [A17], wherein the raw material is derived from fish oil, microbial oil, vegetable oil, or marine animal oil. [Advantages of the Invention]

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

Embodiments for Carrying Out the Invention

[0015] Hereinafter, the present invention will be described in detail. In this specification, when representing a fatty acid, a numerical expression that simply combines the number of carbon atoms, the number of double bonds, and the position of the double bond using numbers and alphabets may be used. For example, a saturated fatty acid with 20 carbon atoms is represented as "C20:0", a monounsaturated fatty acid with 18 carbon atoms is represented as "C18:1", and eicosapentaenoic acid can be represented as "C20:5 n-3" or the like. "n-" indicates the position of the double bond counted from the methyl end of the fatty acid. For example, if it is "n-3", it 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 those skilled in the art can easily identify the fatty acid represented according to this method.

[0016] In this specification, the "highly unsaturated fatty acid" means a fatty acid having 18 or more carbon atoms and 3 or more double bonds. The highly unsaturated fatty acid can be, for example, a fatty acid having 20 or more carbon atoms and 3 or more or 4 or more double bonds, or a fatty acid having 20 or more carbon atoms and 5 or more double bonds. Examples of the highly unsaturated fatty acid 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] As used herein, "crude oil" means a mixture of lipids and refers to oil in the state extracted from organisms. As used herein, "refined oil" means oil obtained by subjecting crude oil to at least one oil refining process selected from the group consisting of a degumming process, a deacidification process, a decolorization process, and a deodorization process to remove substances other than target substances such as phospholipids and sterols, i.e., crude oil refining treatment. Those skilled in the art can distinguish refined oil from crude oil by ordinary analysis.

[0018] As used herein, 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, the fatty acid composition is a composition having a fatty acid as a main constituent, and the fatty acid alkyl ester composition is a composition having an alkyl ester of a fatty acid as a main constituent.

[0019] As used herein, a generic term (such as highly unsaturated fatty acid, highly unsaturated fatty acid alkyl ester, etc.) does not exclude the possibility of the existence of a plurality of constituent elements unless it is clearly indicated otherwise from the context. Therefore, a generic term usually means that at least one constituent element exists.

[0020] <3-MCPD fatty acid ester concentration-reduced composition containing a highly unsaturated fatty acid or its alkyl ester> The present invention provides a composition containing a highly unsaturated fatty acid or its alkyl ester, wherein the proportion of the highly unsaturated fatty acid in the constituent fatty acids of the composition is 50 area% or more, and when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the concentration of 3-MCPD generated is less than 1.80 ppm (hereinafter sometimes 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 can be obtained as the main fraction upon concentration by distillation. The highly unsaturated fatty acid can be eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, or a combination thereof. In a preferred embodiment, the highly unsaturated fatty acid can be eicosapentaenoic acid, docosahexaenoic acid, or a combination thereof. In a more preferred embodiment, the highly unsaturated fatty acid can be eicosapentaenoic acid.

[0022] The proportion of the highly unsaturated fatty acid in the constituent fatty acids of the composition of the present invention can 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 another embodiment, the proportion of the highly unsaturated fatty acid in the constituent fatty acids of the composition of the present invention can 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 the highly unsaturated fatty acid in the constituent fatty acids of the composition of the present invention can be, for example, 50 to 99 area%, 50 to 98 area%, 50 to 95 area%, 50 to 90 area%, 50 to 85 area%, 50 to 80 area%, 50 to 75 area%, 50 to 70 area%, 50 to 65 area%, 50 to 60 area%, 55 to 99 area%, 55 to 98 area%, 55 to 95 area%, 55 to 90 area%, 55 to 85 area%, 55 to 80 area%, 55 to 75 area%, 55 to 70 area%, 55 to 65 area%, 55 to 60 area%, 60 to 99 area%, 60 to 98 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 can 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 aspect, the highly unsaturated fatty acid is eicosapentaenoic acid, and the proportion of the highly unsaturated fatty acid in the constituent fatty acids of the composition of the present invention can be 50 to 99 area%, 50 to 98 area%, 50 to 95 area%, 50 to 90 area%, 50 to 85 area%, 50 to 80 area%, 50 to 75 area%, 50 to 70 area%, 50 to 65 area%, 50 to 60 area%, 55 to 99 area%, 55 to 98 area%, 55 to 95 area%, 55 to 90 area%, 55 to 85 area%, 55 to 80 area%, 55 to 75 area%, 55 to 70 area%, 55 to 65 area%, or 55 to 60 area%.

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

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

[0026] Since the composition of the present invention has a high proportion of highly unsaturated fatty acids, it is suitable for pharmaceuticals and supplement raw materials containing highly unsaturated fatty acids as active ingredients.

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

[37] , Agilent Technologies), the peak area of each fatty acid is determined, and the content ratio of the component of that peak is indicated by the ratio of each peak area to the total peak area of the fatty acids. In the field of oil chemistry, area % is used almost synonymously with weight %. Refer to the Standard Oil Analysis Test Methods established by the Japan Oil Chemists' Society (JOCS) 2013 Edition 2.4.2.1-2013 Fatty Acid Composition (FID Isothermal Gas Chromatography Method) and 2.4.2.2-2013 Fatty Acid Composition (FID Temperature Programming Gas Chromatography Method). The analysis conditions for gas chromatography are as follows. GC-FID measurement conditions GC: 6890N (Agilent Technologies) Column: DB-WAX (Agilent Technologies) 30 m x 0.25 mm ID, 0.25 μm film thickness Carrier gas: Helium, 1 mL / min Injector: 250°C, 1 μL, Split (1:100) Column temperature: 180°C → 3°C / min → 230°C, held for 15 minutes Detector: FID, 250°C Makeup gas: Nitrogen 45 mL / min.

[0028] In an embodiment where the composition of the present invention is a fatty acid composition containing highly unsaturated fatty acids, the proportion of highly unsaturated fatty acids in the constituent fatty acids is measured by the following method. That is, the fatty acid composition is methyl esterified in accordance with the content of AOCS official method Ce1b-89 and then subjected to GC under the aforementioned conditions, and similarly for highly unsaturated fats Calculate the area percentage of the acid. In this case, the constituent fatty acids of the composition mean the free fatty acids in the fatty acid composition.

[0029] In an embodiment where the composition of the present invention is a fatty acid alkyl ester composition containing an alkyl ester of a highly unsaturated fatty acid, the ratio of the highly unsaturated fatty acid in the constituent fatty acids of the composition is measured by the following method. That is, the fatty acid alkyl ester composition is analyzed by gas chromatography under the above conditions, and the ratio (area %) of the peak area of the highly unsaturated fatty acid alkyl ester to the total peak area of the fatty acid alkyl esters is calculated. In this case, the constituent fatty acids of the composition mean the fatty acids constituting the fatty acid alkyl esters of the fatty acid alkyl ester composition.

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

[0031] The composition of the present invention is assayed by the American Oil Chemists' Society official method Cd29b-13 When analyzed by A, the concentration of 3-MCPD 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 the composition by the American Oil Chemists' Society official method Cd29b-13 ass ay A, the concentration of 3-MCPD that occurs when analyzed may be 0.01 ppm or more, for example 0.02 ppm or more. Further, the composition of the present invention is the 3-MCPD that occurs when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A concentration is 0.01 ppm or more 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. Further, the composition of the present invention is the composition by the American Oil Chemists' Society official method Cd29b-13 assay A, the concentration of 3-MCPD that occurs when analyzed is 0.02 ppm or more It may be above and less than 1.80 ppm, less than 1.70 ppm, less than 1.60 ppm, less than 1.50 ppm, less than 1.40 ppm, less than 1.30 ppm, less than 1.20 ppm, less than 1.10 ppm, less than 1.00 ppm, less than 0.90 ppm, less than 0.80 ppm, less than 0.70 ppm, less than 0.60 ppm, less than 0.50 ppm, less than 0.40 ppm, less than 0.30 ppm, less than 0.20 ppm, less than 0.10 ppm, less than 0.09 ppm, less than 0.08 ppm, less than 0.07 ppm, less than 0.06 ppm, less than 0.05 ppm, less than 0.04 ppm, or less than 0.03 ppm.

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

[0033] 100 mg of the sample is added with 100 μL of a 3-MCPD-d5-dipalmitic acid ester standard solution (diluted with toluene to 5 ppm to be 3-MCPD-d5), 600 μL of diethyl ether, stirred and mixed until completely dissolved, and then cooled at -22°C to -25°C for about 15 minutes. Then, 350 μL of a sodium hydroxide-methanol solution (dissolving 0.25 g of sodium hydroxide in 100 mL of methanol) is added and stirred well, and then reacted at -22°C to -25°C for 16 hours or more. At the same temperature, 600 μL of an acidic sodium bromide solution (dissolving 600 g of sodium bromide in 1 L of purified water and adding 3 mL of 85% phosphoric acid) is added to stop the reaction Subsequently, nitrogen is blown to concentrate until the separated organic layer becomes about 100 μL. Then, 600 μL of hexane is added, stirred vigorously, and allowed to stand for 5 to 10 minutes to remove the organic layer twice. To the remaining aqueous layer, 600 μL of a diethyl ether:ethyl acetate mixed solution (3:2, V / V) is added, stirred vigorously, and the organic layer is recovered three times in succession. 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 a saturated phenylboronic acid-diethyl ether solution is added, stirred vigorously for 10 seconds, and then nitrogen is blown to completely remove the solvent. 200 μL of isooctane is added here, stirred vigorously for 10 seconds, and the resulting solution is used as a GC-MS sample.

[0034] The calibration curve for 3-MCPD quantification is prepared by analyzing 3-MCPD standard solutions in which 3-MCPD-dipalmitate is dissolved in toluene so that the 3-MCPD concentration becomes 0 ppm, 0.5 ppm, 1 ppm, and 5 ppm in the same manner as the above samples.

[0035] The analysis conditions for GC-MS are as follows. GC-MS conditions GC: GC-2010 and GCMS-QP2010 (Shimadzu Corporation) Column: DB-5ms (Agilent Technologies) 30 m x 0.25 mm ID, 0.25 μm film thickness Carrier gas: Helium, 1.2 mL / min Inlet: 250 °C, 1 μL, splitless injection, sampling time 1 minute Column temperature: Hold at 85 °C for 0.5 minute → 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 °C Interface temperature: 200 °C Ionization method: EI, m / z for SIM monitoring is as follows 3-MCPD-d5: m / z = 149, 150, 201, 203 3-MCPD: m / z = 146, 147, 196, 198 For quantification, m / z = 150 of 3-MCPD-d5 and m / z = 147 of 3-MCPD are used, and the others are used for the confirmation of the target substance.

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

[0037] In one aspect of the present specification, when the composition of the present invention is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the concentration of 3-MCPD generated may be 0 ppm or more and less than 1.80 ppm. In this case, the composition of the present invention may include a composition containing no 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 concentration of 3 -MCPD generated may be less than 1.80 ppm.

[0038] When the composition of the present invention is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the concentration of 3-MCPD generated may be less than the lower limit of quantification of the method of the American Oil Chemists' Society official method Cd29b-13 assay A, preferably less than the detection limit.

[0039] ​​Highly unsaturated fatty acids such as eicosapentaenoic acid are known to be abundantly contained in certain microbial oils, vegetable oils, or marine animal oils. Therefore, the composition of the present invention can be made from them. Specifically, as raw materials for the composition of the present invention, there are fish oils such as sardine oil, tuna oil, bonito oil, menhaden oil, cod liver oil, herring oil, capelin oil, and salmon oil, marine animal oils derived from crustaceans such as krill, vegetable oils derived from perilla, flax, soybean, rapeseed, etc., yeasts such as the genus Yarrowia, filamentous fungi belonging to the genus Mortierella, the genus Penicillium, the genus Aspergillus, the genus Rhodotorula, the genus Fusarium, etc., algae such as the genus Euglena, and oils derived from lipid-producing microorganisms such as stramenopiles. The composition of the present invention may be an oil derived from a genetically modified recombinant microorganism into which a gene such as a mutated Δ9 elongase gene has been introduced. Also, an oil derived from a genetically modified plant of oilseed plants such as Brassica species, sunflower, corn, cotton, flax, safflower, etc., into which a gene such as a mutated Δ9 elongase gene has been introduced by recombinant technology can also be used as a raw material oil. For genetically modified vegetable oils and genetically modified microbial oils, etc., those described in, for example, WO2012 / 027698, WO2010 / 033753, etc. can be exemplified. In a preferred embodiment, the raw material of the composition of the present invention is a marine animal oil such as fish oil, microbial oil, vegetable oil, or marine animal oil, and more preferably fish oil. Filamentous fungi belonging to the genus Rhodotorula, the genus Fusarium, etc. Algae such as the genus Euglena, and oils derived from lipid-producing microorganisms such as stramenopiles are exemplified. The composition of the present invention may be an oil derived from a genetically modified recombinant microorganism into which a gene such as a mutated Δ9 elongase gene has been introduced. Also, an oil derived from a genetically modified plant of oilseed plants such as Brassica species, sunflower, corn, cotton, flax, safflower, etc., into which a gene such as a mutated Δ9 elongase gene has been introduced by recombinant technology can also be used as a raw material oil. For genetically modified vegetable oils and genetically modified microbial oils, etc., those described in, for example, WO2012 / 027698, WO2010 / 033753, etc. can be exemplified. In a preferred embodiment, the raw material of the composition of the present invention is a marine animal oil such as fish oil, microbial oil, vegetable oil, or marine animal oil, and more preferably fish oil.

[0040] The raw materials of 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 an enzyme, glyceride is reacted with a lower alcohol such as ethanol to alkyl-esterify the fatty acids contained in the glyceride, and then the target highly unsaturated fatty acid alkyl ester (for example, EPA alkyl ester) By removing other fatty acid alkyl esters, highly pure highly unsaturated fatty acid alkyl esters (for example, EPA alkyl esters) can be produced. In one embodiment , the removal of fatty acid alkyl esters other than the target highly unsaturated fatty acid alkyl ester (for example, EPA alkyl ester) can be carried out by distillation. The present inventors have found that a large amount of 3-MCPD fatty acid ester derived from relatively high molecular weight mono- or diacylglycerol is mixed into the main residue in distillation. Therefore, in a preferred embodiment, the composition of the present invention can be a distillate (fraction).

[0041] It is known that heat treatment during distillation generates trans isomers, which are heat-denatured products, from highly unsaturated fatty acid alkyl esters (for example, 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 aspect, the composition of the present invention may further contain trans isomers of highly unsaturated fatty acid alkyl esters. The concentration of trans isomers in the composition of the present invention can be 2.5 area% or less, for example, 2.3 area% or less, 2.0 area% or less, 1.8 area% or less, 1.6 area% or less, 1.4 area% or less, 1.2 area% or less, 1.0 area% or less, 0.9 area% or less, 0.8 area% or less, 0.7 area% or less, 0.6 area% or less, or 0.5 area% or less. Also, the concentration of trans isomers in the composition of the present invention can be 0.01 area% or more, for example, 0.02 area% or more, 0.03 area% or more, 0.04 area% or more, or 0.05 area% or more.

[0042] In this specification, the concentration of the trans isomer is a measured value by GC analysis. Specifically, it is 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) 30 m x 0.25 mm ID, 0.25 μm film thickness Carrier gas: Helium, 1 mL / min Inlet: 250°C, 1 μL, Split (1:100) Column temperature: 180°C → 3°C / min → 230°C, held for 15 minutes Detector: FID, 250°C Make-up gas: Nitrogen 45 mL / min.

[0043] For example, for the trans isomers (five types of A to E) of EPA ethyl ester (EPA-E), the concentration is calculated as follows. In a sample from which saturated fatty acids or monounsaturated fatty acids with 21 or more carbon atoms have been removed by distillation, for the trans isomers of EPA ethyl ester (EPA-E), when the retention time of EPA-E is set to 1, the relative retention times are: isomer A is 0.98 - 0.99, isomer B is 1.01 - 1.02, isomer C is 1.02 - 1.03, and isomers D and E have overlapping peaks and are both 1.04 - 1.05. Among the peaks of the five types of isomers, the peaks of isomers D and E overlap, so four peaks are detected. The sum of the areas of these peaks with relative retention times is taken as the peak area of the EPA-E trans isomers. The ratio of the trans isomers to EPA-E is determined, and the concentration of the isomers in the sample is calculated from the concentration of EPA-E in the sample. For samples containing saturated fatty acids or monounsaturated fatty acids with 21 or more carbon atoms, remove the saturated fatty acids and monounsaturated fatty acids with 21 or more carbon atoms by silver nitrate column fractionation using Discovery Ag-ION 750 mg / 6 mL (Supelco), and then analyze as described above.

[0044] For example, for the trans isomers of dihomo-γ-linolenic acid ethyl ester (DGLA-E), measure the total area of the peaks with the following relative retention times by the above GC analysis, determine the ratio of the trans isomers to DGLA-E, and calculate the isomer concentration in the sample 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 (Take the retention time of DGLA as 1.)

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

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

[0047] In a preferred embodiment, when the composition of the present invention is measured under the above analysis conditions using gas chromatography, 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. Further, when the composition of the present invention is measured under the above analysis conditions using gas chromatography, 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 under the above analysis conditions using gas chromatography, the concentration of the trans isomer of 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. Further, when the composition of the present invention is measured under the above analysis conditions using gas chromatography, the concentration of the trans isomer of 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 under the above analysis conditions using gas chromatography, the concentration of the trans isomer of DGLA ethyl ester is 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. Further, when the composition of the present invention is measured under the above analysis conditions using gas chromatography, the concentration of the trans isomer of DGLA ethyl ester can be 0.01 area% or more, for example, 0.02 area% or more, 0.03 area% or more, 0.04 area% or more, or 0.05 area% or more.

[0050] Fish oils and microbial oils containing highly unsaturated fatty acids contain cholesterol in addition to triglycerides. Cholesterol is also contained in highly unsaturated fatty acid concentrated oils prepared from these raw material oils (WO2012 / 118173). Further, even when alkali esterification or urea addition is performed on an oil containing cholesterol, cholesterol is not completely removed. Therefore, in a specific embodiment herein, the composition of the present invention contains cholesterol. The cholesterol content can be, for example, 1.5% by weight or less, 0.3% by weight or less, or 0.2% by weight or less. Also, the cholesterol content can be, for example, 0.01% by weight or more, or 0.02% by weight or more.

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

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

[0053] In one aspect, the composition of the present invention may contain a saturated fatty acid having 18 or fewer carbon atoms or an alkyl ester thereof as an impurity. In this case, the proportion of the saturated fatty acid 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 less than 3 area %.

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

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

[0056] <Method for producing a composition containing a highly unsaturated fatty acid or an alkyl ester thereof> The present invention provides a method for producing the composition of the present invention. The method includes (1) A step of alkyl esterifying a raw material containing a triglyceride containing a highly unsaturated fatty acid as a constituent fatty acid to prepare a composition containing a highly unsaturated fatty acid alkyl ester, (2) At least one step selected from the group consisting of: (a) a step of reducing the concentration of a monoacylglycerol containing a fatty acid having 5 or less carbon atoms less than the carbon number of 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) 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, and (3) A step of distilling the composition after step (2) and collecting the main fraction (hereinafter, also referred to as the method of the present invention).

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

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

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

[0060] Crude oil purification process The raw material oil used in the alkyl esterification of process (1) may be crude oil or refined oil. For example, any method may be used to obtain crude oil from fishery raw materials such as fish or seafood. In the case of fish oil, it is usually obtained by the following method. The whole fish or processing residues such as fish heads, skins, backbones, and internal organs generated from fishery processing are pulverized and steamed, and then pressed to separate into cooking juice (stick water) and pressed meal. The oil and fat obtained together with the cooking juice is separated from the cooking juice by centrifugation to obtain crude fish oil. Generally, crude fish oil is refined through a crude oil refining process that removes substances other than target substances such as phospholipids and sterols by performing a degumming process, a deacidification process, a decolorization process using activated clay or activated carbon, a water washing process, a deodorization process by steam distillation, etc., according to the raw materials, to obtain refined fish oil. In the embodiments of the present invention, this refined fish oil can also be used as a raw material.

[0061] Process (1) (Alkyl esterification process) The oil and fat, which is the raw material oil, is decomposed into lower alcohol esters by alcoholysis using a lower alcohol. Examples of the lower alcohol include those commonly used in the alkyl esterification of fatty acids, such as lower alcohols having 1 or 2 carbon atoms. Alcoholysis involves adding a lower alcohol, such as ethanol, and a catalyst or enzyme to the oil and fat and reacting them to generate alkyl esters from the fatty acids bound to glycerin. As the catalyst, an alkali catalyst, an acid catalyst, etc. are used. As the enzyme, lipase is used.

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

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

[0064] (a) Monoacylglycerol removal process In step (2)(a), before concentrating the composition containing the fatty acid alkyl ester prepared in step (1) by distillation, the content of monoacylglycerol serving as a substrate for 3-MCPD fatty acid ester is reduced. Thereby, the amount of 3-MCPD fatty acid ester generated by the heat treatment during distillation and mixed into the main fraction can be reduced.

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

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

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

[0068] The monoacylglycerol removed in step (2)(a) contains, as the constituent fatty acid, a fatty acid having 5 or more fewer carbon atoms than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes. Here, the highly unsaturated fatty acid alkyl ester for concentration purposes 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 purposes is fractionated as the main fraction. Therefore, in the distillation of step (3), the highly unsaturated fatty acid alkyl ester fractionated as the main fraction and concentrated is the highly unsaturated fatty acid alkyl ester for concentration purposes.

[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, as the constituent fatty acid, a fatty acid having 5 or more fewer carbon atoms than the carbon number of the one with the most carbon atoms is reduced.

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

[0071] In step (2)(a), the concentration of the monoacylglycerol containing, as a constituent fatty acid, 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 is less than 10,000 ppm, less than 9,000 ppm, 8,00 Reduce it 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 more or the above upper limit or more. For example, when 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, reduce the concentration of the monoacylglycerol containing a fatty acid having 15 or less carbon atoms (preferably a fatty acid having 14 carbon atoms) as a constituent fatty acid to the above concentration. When the highly unsaturated fatty acid alkyl ester for concentration purposes is docosahexaenoic acid (22:6 n-3) alkyl ester, reduce the concentration of the monoacylglycerol containing a fatty acid having 17 or less carbon atoms (preferably a fatty acid having 16 carbon atoms) as a constituent fatty acid to the above concentration. Also, when the highly unsaturated fatty acid alkyl ester for concentration purposes is a combination of docosahexaenoic acid (22:6 n-3) alkyl ester and one or more selected from eicosapentaenoic acid (20:5 n-3) alkyl ester, dihomo-γ-linolenic acid (20:3 n-6) alkyl ester, and arachidonic acid (20:4 n-6) alkyl ester, reduce the concentration of the monoacylglycerol containing a fatty acid having 17 or less carbon atoms (preferably a fatty acid having 14 carbon atoms and a fatty acid having 16 carbon atoms) as a constituent fatty acid to the above concentration.

[0072] In the distillation of step (3), the 3-MCPD fatty acid ester that can be generated from a monoacylglycerol containing a fatty acid having 5 or fewer carbon atoms less than the number of carbon atoms of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes 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 purposes as the main fraction in the distillation of step (3), by previously reducing the concentration of the monoacylglycerol containing a fatty acid having 5 or fewer carbon atoms less than the number of carbon atoms of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes as the constituent fatty acid, 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 monoacylglycerol removed in step (2)(a) may be a monoacylglycerol containing a fatty acid having 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, or 6 fewer carbon atoms than the number of carbon atoms of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester for concentration purposes as the constituent fatty acid. In step (2)(a), the concentration of these monoacylglycerols may be reduced to the above-mentioned concentration in total.

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

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

[0076] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester 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, and the monoacylglycerol removed in step (2)(a) can be glycerol monomyristate and glycerol monopalmitate.

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

[0078] Collect 100 μL of the composition and weigh it. Subject 150 μL of the solution dissolved in 400 μL of hexane to thin-layer chromatography (TLC) under the following TLC conditions to separate MAG and DAG. Scrape off all the bands of MAG and DAG confirmed at UV254 nm, add 1 mL of 1N sodium methoxide / methanol solution, stir well, and heat for 5 minutes. Then, cool to room temperature, add 1 mL of 1N hydrochloric acid, and stir well. 0.1 mg / mL 1 mL of a C23:0 FAME (methyl tricosanoate) hexane solution and saturated Add 5 mL of brine, stir well, and use the obtained hexane layer as a sample for GC-FID analysis under the following conditions, and calculate the concentration of each fatty acid using the following calculation formula. Fatty acid concentration [mg / kg] = (peak area of fatty acid / peak area of C23:0) × (10 5 / sample amount [mg] subjected to TLC) TLC conditions TLC plate: PLC Silica gel 60F 254 0.5 mm, 10 cm × 10 cm 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) 30 m x 0.25 mm ID, 0.25 μm film thickness Carrier gas: Helium, 1 mL / min Inlet: 250°C, 1 μL, Split (1:100) Column temperature: 180°C → 3°C / min → 230°C, held for 15 minutes Detector: FID, 250°C Make-up gas: Nitrogen 45 mL / min.

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

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

[0081] Perform ICP-MS analysis on the sample solution and calibration curve samples under the following analysis conditions, create a calibration curve by automatic calculation using the software attached to the instrument, and quantify the iron content in the sample solution.

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

[0083] From the iron content in the quantified sample solution, calculate the iron content in the sample composition using the following formula. Iron content in the sample composition [ppm] = C / (W × 100) C: Iron content in the sample solution measured by ICP-MS (μg / L) W: Sampling amount of the sample composition (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. Technologies for reducing the iron concentration include, in addition to the technologies that can be used in the aforementioned monoacylglycerol removal process, for example, pickling and ion exchange. These removal technologies can be performed by established 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 the above upper limit or higher. By reducing the chlorine concentration, the formation of 3-MCPD fatty acid esters can be suppressed.

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

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

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

[0089] From the chlorine content of the quantified sample solution, calculate the chlorine content in the sample composition by the following formula. Chlorine content [ppm] in the sample composition = C / (W × 100) C: Chlorine content (μg / L) of the sample solution measured by ICP-MS W: Sampling amount (g) of the sample composition

[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 step. In some embodiments, the chlorine concentration is higher than 0 ppm. As techniques for reducing the chlorine concentration, in addition to the techniques that can be used in the aforementioned monoacylglycerol removal step, for example, techniques such as degumming and deacidification generally used in the refining process of fats and oils can be mentioned. These removal techniques can be carried out by established methods.

[0091] Process (3) (Distillation process) Distill the composition with reduced monoacylglycerol concentration in step (2), and collect the main fraction. Even if 3-MCPD fatty acid ester is generated by the heat treatment during distillation, by setting conditions such that the main fraction contains highly unsaturated fatty acid alkyl ester for concentration purposes, a composition containing highly unsaturated fatty acid alkyl ester, when the composition is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the concentration of 3-M A composition with a CPD concentration of less than 1.80 ppm can be obtained. Such distillation conditions can be appropriately set according to the highly unsaturated fatty acid alkyl ester for concentration purposes.

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

[0093] Fine distillation is carried out under high vacuum, and highly unsaturated fatty acid alkyl esters are taken as the main distillate, which can be obtained by separating more volatile initial distillate and less volatile residue. The conditions for fine distillation may be set such that the highly unsaturated fatty acid alkyl esters for concentration purposes are concentrated as the main distillate. 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 distillate at 170 - 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 distillate 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 distillate fraction in step (3) is analyzed by the American Oil Chemists' Society Official Method Cd29b - 13 assay A, the 3 - The concentration of MCPD can be 0.01 ppm or more, for example, 0.02 ppm or more. Also, 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 that occurs can be 0.01 ppm or more 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. Also, 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 that occurs can be 0.02 ppm or more 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 purification step by chromatography such as high performance liquid column chromatography (HPLC) after step (3).

[0097] The chromatography process such as HPLC following the distillation process is a process of further concentrating highly unsaturated fatty acid alkyl esters in the composition after distillation by reducing the content of unwanted components, such as by removing the unwanted components in the composition obtained in the distillation process. The chromatography process can be carried out according to a conventionally known method, for example, the method described in JP-A-5-222392. Examples of the chromatography used for the concentration treatment include, for example, reverse-phase column chromatography. Examples of the stationary phase (adsorbent) include polymer beads, preferably polystyrene reticulated with DVB (divinylbenzene), and silica gel, preferably reverse-phase bonded silica gel containing C8 or C18 alkane, and C18-bonded reverse-phase silica gel is particularly preferred. The adsorbent used in the chromatography after distillation of the present invention is preferably nonpolar. Any adsorbent of the reverse-phase partition system can be used without particular limitation, and examples thereof include 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 the sample to be purified. A person skilled in the art can easily determine an appropriate size of the column to be used. The diameter of each column is typically 10 to 800 mm, preferably 50 to 800 mm, more preferably 300 to 800 mm, and most preferably 600 to 800 mm. The length of each column is typically 10 to 200 cm, preferably 25 to 150 cm. The mobile phase and the temperature of the column are not particularly limited, but depend on the solubility of the substance to be separated in the mobile phase. A person skilled in the art can easily determine an appropriate mobile phase and the temperature of the column to be used. The temperature of the column is typically 0 to 70 ° C, preferably 20 ° C to 40 ° C.

[0099]

[0100] ​Examples of solvents used in the mobile phase include short-chain alcohols. Short-chain alcohols typically have 1 to 6 carbon atoms. Examples of suitable short-chain alcohols include methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, s-butanol, and t-butanol. The solvent used in the mobile phase is preferably methanol or ethanol, more preferably methanol. For short-chain alcohols, it is preferable not to add water intentionally in order to shorten the elution time.

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

[0102] The distillation feedstock composition of the present invention can be produced by alkyl esterifying the oil listed as the raw material of the composition of the present invention to reduce the content of monoacylglycerol or reduce the iron content. In one embodiment, the distillation feedstock 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, for example, by the method for producing the composition of the present invention described above. In a preferred embodiment, the raw material of the distillation feedstock composition of the present invention is fish oil. The alkyl esterification and the reduction of the content of monoacylglycerol or the iron content can be carried out by the methods described in the above method of the present invention. In a preferred embodiment, the highly unsaturated fatty acid alkyl ester contained in the distillation feedstock composition of the present invention can be a highly unsaturated fatty acid ethyl ester. Also, the highly unsaturated fatty acid alkyl ester for concentration purposes can be an alkyl ester of eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, or arachidonic acid, or a combination thereof. In a preferred embodiment, the highly unsaturated fatty acid alkyl ester for concentration purposes can be an alkyl ester of eicosapentaenoic acid or docosahexaenoic acid, or a combination thereof. In a more preferred embodiment, the highly unsaturated fatty acid alkyl ester for concentration purposes can be an alkyl ester of eicosapentaenoic acid.

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

[0104] The ratio of the highly unsaturated fatty acid to the total fatty acids in the distillation feedstock composition of the present invention is 5 area% or more and, for example, can be 10 area% or more, 15 area% or more, or 20 area% or more. Also, the ratio of the highly unsaturated fatty acid to the total fatty acids in the distillation feedstock composition of the present invention can be less than 70 area%, for example, less than 65 area%, less than 60 area%, or less than 55 area%.

[0105] The distillation feedstock composition of the present invention may contain a saturated fatty acid having 18 or fewer carbon atoms or an alkyl ester thereof as an impurity. In this case, the proportion of the saturated fatty acid having 18 or fewer carbon atoms in the constituent fatty acids of the distillation feedstock 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 less than 40 area%, or less than 30 area%.

[0106] In a preferred embodiment, for a 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, its concentration in the distillation feedstock 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 monoacylglycerol to which the above upper limit applies may be a monoacylglycerol containing a fatty acid having 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, or 6 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.

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

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

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

[0111] In a preferred aspect, the iron concentration in the distillation feed 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 higher than 0 ppm.

[0112] In a preferred aspect, the chlorine concentration in the distillation feed 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 higher than 0 ppm.

[0113] <Usage form> The usage form of the composition of the present invention is not particularly limited, but it is preferably an oral dosage form, and typically can be in the form of oral preparations such as granules, tablets, capsules, solutions, etc. The uses of the composition of the present invention include, for example, foods and drinks (health foods, dietary supplements, foods for specified health uses, supplements, dairy products, soft drinks, pet foods, livestock feeds, etc.), pharmaceuticals, quasi-drugs, etc. In particular, it is preferably a supplement and a pharmaceutical. In addition to food materials or foods, it may also be used as an additive component for animal feeds. Therefore, the composition of the present invention can be used as a material or an active ingredient of these foods and drinks, pharmaceuticals, and quasi-drugs, and can be preferably used in the production of these.

[0114] Examples of the present invention are described below, but the present invention is not limited thereto at all. In the examples, those indicated by % are % by weight unless otherwise specified. Also, ppm is ppm by weight (i.e., mg / kg) unless otherwise specified. In the examples, the 3-MCPD concentration means the measured value by the American Oil Chemists' Society official method (AOCS official method) Cd29b-13 assay A. Also, a 3-MCPD concentration of 0.00 ppm means that 3-MCPD was not detected in the above analysis method (i.e., it was below the detection limit). In the examples, an iron concentration of 0.00 ppm means that iron was not detected in the measurement by the above ICP-MS (i.e., it was below the detection limit).

Examples

[0115] [Test 1] Influence of iron content on the production of 3-MCPD The oil obtained by deacidifying sardine crude oil by short-path distillation was esterified with an ethyl ester using an alkali catalyst, and then subjected to silica gel purification to separate the ethyl ester fraction. In the silica gel purification, a glass open column filled with 5 times the amount of Microsphere gel D-75-60A (AGC SI TECH Co., Ltd.) of the sample was used, and hexane / ethyl acetate (50:1) was used as the eluent. It was confirmed by thin layer chromatography (TLC) that no bands of DAG or MAG were detected in this ethyl ester fraction (fish oil ethyl ester (EE)). As a result of the measurement, 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). In addition, an aqueous solution of iron(II) sulfate heptahydrate was added to the sample with an iron content of 0.10 ppm (actual The solvent was added to the mixture so that the concentration was 1.00 ppm (Example 2) or 1.00 ppm (Comparative Example 1), or no solvent was added (Example 1), and then ethanol was added and homogenized.Then, the solvent was completely removed using an evaporator and vacuum drawing.

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

[0118] [Table 1]

[0119] In Comparative Example 1, in which 1.00 ppm of iron was added, 0.61 ppm of 3-MCPD was produced after heating for 4 hours. On the other hand, in Examples 1 and 2, in which the iron concentrations were adjusted to 0.00 ppm and 0.10 ppm, respectively, 4.0 ppm of 3-MCPD was produced. Even after heating for 2 h, the 3-MCPD content was 0.08 ppm and 0.15 ppm, which is significantly lower than the 0.61 ppm in Comparative Example 1. It was always low.

[0120] [Test 2] Effect of MAG content on the formation of 3-MCPD The fish oil EE (iron concentration adjusted to 10 ppm) prepared in the same manner as in Test 1 was used as it was, or glycerol monomyristate (Wako Pure Chemical Industries, product code 321-32412) was added at a concentration of 1-10% and heated at 120 °C for 1 hour under a nitrogen stream. After heating, the 3-MCPD concentration is shown in Table 2. It was found that even at a relatively low temperature of 120 °C, which is generally adopted for molecular distillation of fatty acid ethyl esters, the 3-MCPD concentration increases as the MAG concentration increases.

[0121] As shown in Table 2. It was found that even at a relatively low temperature of 120 °C, which is generally adopted for molecular distillation of fatty acid ethyl esters, the 3-MCPD concentration increases as the MAG concentration increases. After heating, the 3-MCPD concentration is shown in Table 2. It was found that even at a relatively low temperature of 120 °C, which is generally adopted for molecular distillation of fatty acid ethyl esters, the 3-MCPD concentration increases as the MAG concentration increases.

[0122]

Table 2

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

[0124]

Table 3

[0125] Subsequently, fish oil ethyl ester 2 from which MAG and DAG were removed from fish oil ethyl ester 1, fish oil ethyl ester 3 obtained by adding glycerol monomyristate to fish oil ethyl ester 2, and fish oil ethyl ester 4 obtained by adding glycerol monopalmitate to fish oil ethyl ester 2 were prepared. Fish oil ethyl ester 2 was prepared by the following method. Fish oil ethyl ester 2 was prepared by the following method. A mixed solution obtained by mixing 600 g of fish oil ethyl ester 1 with 2400 mL of hexane was passed through a column packed with 1200 g of silica gel (Microsphere D75-60A) in a hexane slurry to adsorb the fish oil ethyl ester onto the silica gel. Then, ethyl acetate / hexane (1:50) was passed through the column, and the eluate was fractionated to recover the fraction in which MAG and DAG were removed from the fish oil ethyl ester. The solvent was removed from the recovered fraction by an evaporator and vacuum pumping to obtain 585 g of fish oil ethyl ester free of MAG and DAG. The fish oil ethyl ester 2 thus obtained contained no MAG and DAG and had an iron concentration of 0.05 ppm and a chlorine concentration of 7 ppm. 0.1 g of glycerol monomyristate was mixed with 100 g of fish oil ethyl ester 2 and completely dissolved and homogenized to prepare fish oil ethyl ester 3. Also, 0.1 g of glycerol monopalmitate (Tokyo Chemical Industry, product code G0083) was mixed with 100 g of fish oil ethyl ester 2 and completely dissolved and homogenized to prepare fish oil ethyl ester 4. Fish oil ethyl ester 3 or 4 was used as a sample (raw material composition for distillation) and subjected to precision distillation including the following first precision distillation step and second precision distillation step. The first precision distillation step is a step of removing fractions C18 and below. A fractionating tube with a vacuum jacket (φ25 mm, Kiriyama Glass) was used for the fractionating tube, and 5 pieces of Sulzer Lab Packing EX (25 mm × 50 mm, Sulzer Chemtech) were used for the internal packing. The liquid temperature at the bottom of the column (bottom temperature) was set to 185°C or lower, the vapor temperature at the top of the column (top temperature) was set to 135°C or lower, and the pressure before the vacuum pump (top pressure, i.e., degree of vacuum) was set to 30 Pa or lower, and precision distillation was performed for a heating time of 4.0 hours. In this first precision distillation step, the fraction C18 and below was taken as the initial distillate.

[0126] 100 g of fish oil ethyl ester 2 was mixed with 0.1 g of glycerol monomyristate and completely dissolved and homogenized to prepare fish oil ethyl ester 3. Also, 0.1 g of glycerol monopalmitate (Tokyo Chemical Industry, product code G0083) was mixed with 100 g of fish oil ethyl ester 2 and completely dissolved and homogenized to prepare fish oil ethyl ester 4. Fish oil ethyl ester 3 or 4 was used as a sample (raw material composition for distillation) and subjected to precision distillation including the following first precision distillation step and second precision distillation step.

[0127] The first precision distillation step is a step of removing fractions C18 and below. A fractionating tube with a vacuum jacket (φ25 mm, Kiriyama Glass) was used for the fractionating tube, and 5 pieces of Sulzer Lab Packing EX (25 mm × 50 mm, Sulzer Chemtech) were used for the internal packing. The liquid temperature at the bottom of the column (bottom temperature) was set to 185°C or lower, the vapor temperature at the top of the column (top temperature) was set to 135°C or lower, and the pressure before the vacuum pump (top pressure, i.e., degree of vacuum) was set to 30 Pa or lower, and precision distillation was performed for a heating time of 4.0 hours. In this first precision distillation step, the fraction C18 and below was taken as the initial distillate. The second precision distillation step is a step of removing fractions C20 and above. A fractionating tube with a vacuum jacket (φ25 mm, Kiriyama Glass) was used for the fractionating tube, and 5 pieces of Sulzer Lab Packing EX (25 mm × 50 mm, Sulzer Chemtech) were used for the internal packing. The liquid temperature at the bottom of the column (bottom temperature) was set to 220°C or lower, the vapor temperature at the top of the column (top temperature) was set to 170°C or lower, and the pressure before the vacuum pump (top pressure, i.e., degree of vacuum) was set to 30 Pa or lower, and precision distillation was performed for a heating time of 4.0 hours. In this second precision distillation step, the fraction C20 and above was taken as the residue.

[0128] The second precision distillation step is a step of removing fractions C20 and above. A fractionating tube with a vacuum jacket (φ25 mm, Kiriyama Glass) was used for the fractionating tube, and 5 pieces of Sulzer Lab Packing EX (25 mm × 50 mm, Sulzer Chemtech) were used for the internal packing. The liquid temperature at the bottom of the column (bottom temperature) was set to 220°C or lower, the vapor temperature at the top of the column (top temperature) was set to 170°C or lower, and the pressure before the vacuum pump (top pressure, i.e., degree of vacuum) was set to 30 Pa or lower, and precision distillation was performed for a heating time of 4.0 hours. In this second precision distillation step, the fraction C20 and above was taken as the residue. The second precision distillation step is a step of removing fractions C20 and above. A fractionating tube with a vacuum jacket (φ25 mm, Kiriyama Glass) was used for the fractionating tube, and 5 pieces of Sulzer Lab Packing EX (25 mm × 50 mm, Sulzer Chemtech) were used for the internal packing. The liquid temperature at the bottom of the column (bottom temperature) was set to 220°C or lower, the vapor temperature at the top of the column (top temperature) was set to 170°C or lower, and the pressure before the vacuum pump (top pressure, i.e., degree of vacuum) was set to 30 Pa or lower, and precision distillation was performed for a heating time of 4.0 hours. In this second precision distillation step, the fraction C20 and above was taken as the residue. Removed to obtain a residue without the first fraction in which EPA was concentrated.

[0129] Subsequently, in the second precision distillation step, the following precision distillation was performed on the residue without the first fraction obtained in the first precision distillation step. A fractionating column with a vacuum jacket (φ25 mm, Kiriyama Glass) was used as the fractionating column, and five Sulzer Lab Packing EX (25 mm × 50 mm, Sulzer Chemtech) were used as the internal packing. The liquid temperature at the bottom of the column (bottom temperature) was 195 °C, the vapor temperature at the top of the column (top temperature) was 150 °C, and the pressure before the vacuum pump (top pressure, i.e., degree of vacuum) was 30 Pa. Precision distillation was performed for 3.5 hours of heating time. In this second precision distillation step, fractions of C22 or higher were removed as the residue, and the main fraction was obtained.

[0130] Using 80 g of fish oil ethyl ester 3 as a raw material, 26 g of a residue without the first fraction in which EPA was concentrated was obtained in the first precision distillation step. Using 25 g of the obtained residue without the first fraction as a raw material, 11 g of the main fraction in which EPA was concentrated was obtained in the second precision distillation step. The main fraction was concentrated with EPA, and as shown in Table 4 the proportion of EPA in the fatty acid composition increased from 20.9% to 73.1%. On the other hand, isomer formation due to heating during distillation was also observed, and 0.8 area% of trans isomers of EPA ethyl ester (total value of five types of trans isomers. Not shown in Table 4. The same applies hereinafter.) was observed in the main fraction. Also, the 3-MCPD concentration was 0.00 ppm in the fish oil ethyl ester 3 as the raw material, and increased to 0.01 ppm in the main fraction after distillation.

[0131]

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

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

[0134]

Table 4

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

[0136]

Table 5

[0137] It was found that the concentration of 3-MCPD in the main distillate is strongly affected by specific MAGs in the raw material. In the main distillate obtained by specifically separating EPA, that is, the C20 fatty acid component, when MAG containing C14:0, that is, C14 fatty acid as a constituent fatty acid, was added to ethyl ester of fish oil 3, 28.0% of the total 3-MCPD was contained, whereas when MAG containing C16:0, that is, C16 fatty acid as a constituent fatty acid, was added to ethyl ester of fish oil 4, all of the 3-MCPD was contained in the residue of the main distillate. From this fact, ​​​When recovering C20 fatty acid ethyl esters containing EPA ethyl ester as the main fraction, C14 3-MCPD fatty acid esters formed from MAG containing C14 fatty acids as constituent fatty acids are mixed into the main fraction However, it was shown that 3-MCPD fatty acid esters formed from MAG containing C16 fatty acids as constituent fatty acids hardly mix into the main fraction. That is, when the highly unsaturated fatty acid to be purified is a C20-PUFA such as EPA, it can be said that C14 saturated fatty acids strongly affect the 3-MCPD concentration in the distillate. In each test, the initial fraction did not contain any 3-MCPD or 3-MCPD fatty acid esters.

Industrial Applicability

[0138] According to the present invention, a composition containing a high-concentration PUFA alkyl ester with a low 3-MCPD fatty acid ester concentration can be stably produced.

[0139] All publications, patent applications, patents, and other documents mentioned in this specification are incorporated herein by reference as if each publication, patent application, patent, or other document was specifically and individually indicated as being incorporated by reference in its entirety. Definitions contained in the incorporated text 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 a fatty acid or a fatty acid alkyl ester as a main component, the composition containing a highly unsaturated fatty acid or an alkyl ester thereof, 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 is less than 1.80 ppm, (1) The concentration of trans isomers of highly unsaturated fatty acid alkyl esters is 0.01 area % or more, or (2) The composition having a cholesterol content of 0.01% by weight or more.

2. 2. The composition according to claim 1, wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of said composition is 70 area % or more.

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

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

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. The 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 a fish oil, a microbial oil, a vegetable oil, or a marine animal oil.

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