Raw material composition and method for producing composition

Abstract

Provided are: a distillation raw material composition containing a highly unsaturated fatty acid alkyl ester, said composition having a sufficiently low concentration of a 3-MCPD fatty acid ester; and a method for efficiently producing a composition containing a highly unsaturated fatty acid or an alkyl ester thereof. A composition which is a distillation raw material composition containing a highly unsaturated fatty acid alkyl ester, the highly unsaturated fatty acid alkyl ester containing a highly unsaturated fatty acid alkyl ester as a concentration target, and having an iron concentration of less than 0.20 ppm.

Description

[0001] This application is a divisional application of the Chinese patent application whose original application was filed on December 12, 2019, with application number 201980081735.2 and invention name “Compositions containing highly unsaturated fatty acids or their alkyl esters and methods for producing the same”. Technical Field

[0002] The present invention relates to a composition containing highly unsaturated fatty acids or alkyl esters thereof and a method for producing the same. Background Art

[0003] 3-Chloropropane-1,2-diol (3-MCPD) is a compound suspected of being carcinogenic, and its concentration in food is regulated in various countries such as the EU. It is known that in oils and fats, diacylglycerols (DAG) and monoacylglycerols (MAG) are matrices and generate 3-MCPD (Non-Patent Document 1). After the presence of 3-MCPD in diacylglycerols (DAG) was pointed out for abundant oils and fats, attempts were made to reduce the content in oils and fats 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 generate 3-MCPD in oils and fats can be reduced by adsorbent treatment, lowering the deodorization temperature, or shortening the treatment time. However, these are methods for reducing the concentration of substances that generate 3-MCPD in the production of triacylglycerols, and Patent Documents 1 and 2 do not record methods for reducing the concentration of 3-MCPD fatty acid esters in the production of alkyl esters.

[0004] It is known that polyunsaturated fatty acids (PUFA) have various functions, and highly concentrated PUFAs are used in foods, supplements, pharmaceuticals, or cosmetics. Regarding PUFAs, when the proportion of desired PUFAs in the fatty acids of the starting composition is increased by high concentration, glycerides with triacylglycerol as the main component are converted into alkyl esters with lower alcohols. Therefore, in many cases, highly refined and concentrated PUFAs used in foods, supplements, pharmaceuticals, or cosmetics are alkyl esters. So far, there is no known method for reducing the concentration of 3-MCPD fatty acid esters in such high-concentration PUFA alkyl esters.

[0005] As factors that affect the generation of 3-MCPD fatty acid esters, chlorine sources, substrates such as MAG and DAG, and treatment time at high temperatures are known (Non-Patent Documents 1 and 2). However, for oils that are raw materials for foods containing PUFA, etc., usually, the content of substrates of 3-MCPD fatty acid esters such as chlorine sources and MAG and DAG is small, and the influence on the refining of PUFA as the target is extremely small, so it is generally believed that it is not necessary to remove them. In addition, considering its technical difficulty and the influence on productivity such as PUFA recovery rate, the complete removal of substrates such as chlorine sources and MAG and DAG is not performed.

[0006] Rectification is a distillation that can be expected to have high separation capabilities, but it requires internal fillings and reflux, so in many cases, high-temperature heating of 150°C or more is required. In molecular distillation and short-path distillation, the heating temperature is 150°C or less, and it can be carried out at a lower temperature than rectification, but in order to fully concentrate PUFA, the treatment must be repeated. Therefore, there is a risk of generating a large amount of 3-MCPD during the distillation of PUFA.

[0007] The urea addition method and the HPLC method are methods for separation using the structure of the fatty acids constituting the molecule (e.g., chain length, number of double bonds, etc.). However, when 3-MCPD in the raw material is present in the form of a di-fatty acid ester or a mono-fatty acid ester, it is difficult to separate it from the target alkyl ester using the type of fatty acid constituting it, and it is difficult to stably obtain fatty acid alkyl esters with reduced 3-MCPD fatty acid esters.

[0008] Therefore, there is a significant risk of generating 3-MCPD fatty acid esters in purification steps including desolvation, distillation, and other heat treatments. In addition, methods such as urea addition and HPLC are not necessarily able to remove various 3-MCPD fatty acid esters contained in the raw materials.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Laid-Open No. 2011-147435

[0012] Patent Document 2: Japanese Patent Application Laid-Open No. 2011-147436

[0013] Non-patent literature

[0014] Non-patent document 1: Eur. J. Lipid Sci. Technol. 114, 1268-1273 (2012)

[0015] Non-patent document 2: Eur. J. Lipid Sci. Technol. 115, 735-739 (2013)

[0016] Non-patent document 3: J. Agric. Food. Chem. 63 (6) 1839-48 (2015) Summary of the invention

[0017] Problems to be solved by the invention

[0018] Oils and fats containing PUFA often contain 3-MCPD or its fatty acid esters from raw materials, oil pressing (extraction) / refining processes. In addition, if the glycerides in the oils and fats are alkyl-esterified, MAG and DAG will remain, or their concentration will increase, forming an environment that is easy to generate 3-MCPD fatty acid esters. Furthermore, in many cases, the refining of alkyl esters includes a desolventizing 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. The temperature is not increased during chromatography and urea addition, so 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 fully reduced.

[0019] An object of the present invention is to provide a composition containing highly concentrated PUFA or PUFA alkyl esters with a sufficiently low concentration of 3-MCPD fatty acid esters, and a method for efficiently producing the composition.

[0020] Methods for solving problems

[0021] In order to achieve the above-mentioned purpose, the present inventors have repeatedly conducted in-depth research and found that, during distillation, the C20-C22 fatty acid alkyl ester component in which PUFA is concentrated exhibits similar behavior to the 3-MCPD mono-fatty acid ester combined with one molecule of C14 fatty acid or C16 fatty acid. It was further found that the concentration of 3-MCPD mono-fatty acid ester in the C20-C22 fatty acid alkyl ester component can be reduced by reducing the content of monoacylglycerol (MAG), especially MAG combined with C14 fatty acid or C16 fatty acid, in the raw material.

[0022] In addition, the present inventors have also found that the formation rate of 3-MCPD fatty acid esters is greatly changed by changes in the concentration of trace metals, such as iron, contained in the raw materials at a level of less than 1 ppm. It has been reported that iron has an effect on the formation of 3-MCPD fatty acid esters from triacylglycerols (Non-Patent Document 3). However, what is disclosed in Non-Patent Document 3 is the effect of extremely large amounts of Fe on the formation of 3-MCPD fatty acid esters. 2+ or Fe 3+ As a result of investigating the formation of 3-MCPD fatty acid esters in the presence of iron, it is unknown whether the extremely small amount of iron generally contained in oil, which is a raw material for producing PUFA alkyl esters, affects the formation of 3-MCPD fatty acid esters.

[0023] PUFA alkyl esters produced from raw materials such as fish oil are generally low in degree of refining before concentration, so the iron concentration varies depending on the iron content of the raw material oil. In addition, the iron concentration of PUFA alkyl esters before concentration may also vary greatly depending on the quality of the extracted raw materials, the extraction method, the refining method, etc. Therefore, when the iron concentration increases, the concentration of 3-MCPD fatty acid esters may increase unexpectedly 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 esters can be effectively suppressed.

[0024] Based on these findings, the present inventors have further conducted repeated studies and have completed the present invention.

[0025] That is, the present invention is as follows.

[0026] [1] A composition comprising fatty acids or fatty acid alkyl esters as main components, and containing highly unsaturated fatty acids or their alkyl esters, wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 50 area % or more, and when the composition is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association, the concentration of 3-MCPD produced is less than 1.80 ppm.

[0027] [2] The composition according to [1], wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 70 area % or more.

[0028] [3] The composition according to [1] or [2], wherein the concentration of 3-MCPD produced when the composition is analyzed using the official method Cd29b-13 analysis A of the American Oil Chemists Association is below the detection limit.

[0029] [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.

[0030] [5] The composition according to any one of [1] to [4], which is a distillate.

[0031] [6] The composition according to any one of [1] to [5], wherein the raw material is fish oil, microbial oil, plant oil or marine animal oil.

[0032] [7] A composition which is a distillation raw material composition containing a highly unsaturated fatty acid alkyl ester,

[0033] The highly unsaturated fatty acid alkyl ester contains the highly unsaturated fatty acid alkyl ester as the enrichment target,

[0034] The concentration of monoacylglycerol containing fatty acids having 5 or more fewer carbon atoms than the highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated is less than 10,000 ppm, and / or the iron concentration is less than 0.20 ppm.

[0035] [8] The composition according to [7], wherein the concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having six fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated is less than 10,000 ppm.

[0036] [9] The composition according to [7] or [8], wherein the chlorine concentration is less than 10 ppm.

[0037]

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

[0038]

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

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

[0039]

[12] A method for producing a composition containing highly unsaturated fatty acids or alkyl esters thereof, comprising:

[0040] (1) a step of alkyl-esterifying a raw material containing triglycerides containing highly unsaturated fatty acids as constituent fatty acids to prepare a composition containing highly unsaturated fatty acid alkyl esters,

[0041] (2) at least one selected from the following steps (a), (b) and (c):

[0042] (a) a step of reducing the concentration of monoacylglycerol containing fatty acids having a carbon number less than 5 or more than the carbon number of highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated, as constituent fatty acids, 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, (c) a step of reducing the chlorine concentration in the composition of step (1) to less than 10 ppm, and

[0043] (3) A step of distilling the composition after step (2) to separate a main fraction.

[0044]

[13] The method according to

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

[0045]

[14] The method according to

[12] or

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

[0046]

[15] The method according to any one of

[12] to

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

[0047]

[16] A method according to any one of

[12] to

[15] , wherein in step (2), the concentration of monoacylglycerol containing fatty acids having six fewer carbon atoms than the carbon atoms of highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated is reduced to less than 10,000 ppm.

[0048]

[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.

[0049]

[18] The method according to any one of

[12] to

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

[0050] In another embodiment, the present invention is as follows.

[0051] [A1] A composition comprising highly unsaturated fatty acids or their alkyl esters, wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 50 area % or more, and when the composition is analyzed using the official method Cd29b-13 analysis A of the American Oil Chemists Association, the concentration of 3-MCPD produced is less than 1.80 ppm.

[0052] [A2] The composition according to [A1], wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 70 area % or more.

[0053] [A3] A composition according to [A1] or [A2], wherein the concentration of 3-MCPD produced when the composition is analyzed using the official method Cd29b-13 analysis A of the American Oil Chemists Association is below the detection limit.

[0054] [A4] The composition according to any one of [A1] to [A3], wherein the highly unsaturated fatty acid is eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid, or a combination thereof.

[0055] [A5] The composition according to any one of [A1] to [A4], which is a distillate.

[0056] [A6] The composition according to any one of [A1] to [A5], wherein the raw material is derived from fish oil, microbial oil, plant oil or marine animal oil.

[0057] [A7] A composition which is a distillation raw material composition containing a highly unsaturated fatty acid alkyl ester,

[0058] The highly unsaturated fatty acid alkyl ester contains the highly unsaturated fatty acid alkyl ester as the enrichment target,

[0059] The concentration of monoacylglycerol containing fatty acids having 5 or more fewer carbon atoms than the highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated as constituent fatty acids is less than 10,000 ppm, or the iron concentration is less than 0.20 ppm.

[0060] [A8] The composition according to [A7], wherein the concentration of monoacylglycerol containing, as a constituent fatty acid, a fatty acid having 6 fewer carbon atoms than the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated is less than 10,000 ppm.

[0061] [A9] The composition according to [A7] or [A8], wherein the chlorine concentration is less than 10 ppm.

[0062] [A10] The composition according to any one of [A7] to [A9], wherein the highly unsaturated fatty acid alkyl ester to be concentrated is an alkyl ester of eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid or arachidonic acid, or a combination thereof.

[0063] [A11] The composition according to any one of [A7] to [A10], wherein the raw material is derived from fish oil, microbial oil, plant oil or marine animal oil.

[0064] [A12] A method for producing a composition containing highly unsaturated fatty acids or alkyl esters thereof, comprising:

[0065] (1) a step of alkyl-esterifying a raw material containing triglycerides containing highly unsaturated fatty acids as constituent fatty acids to prepare a composition containing highly unsaturated fatty acid alkyl esters,

[0066] (2) at least one selected from the following steps (a), (b) and (c):

[0067] (a) a step of reducing the concentration of monoacylglycerol containing fatty acids having a carbon number less than 5 or more than the carbon number of highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated, as constituent fatty acids, 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, (c) a step of reducing the chlorine concentration in the composition of step (1) to less than 10 ppm, and

[0068] (3) A step of distilling the composition after step (2) to separate a main fraction.

[0069] [A13] The method according to [A12], wherein the main fraction in step (3) is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association, and the concentration of 3-MCPD produced is less than 1.80 ppm.

[0070] [A14] The method according to [A12] or [A13], wherein step (2) (a) is carried out by silica gel chromatography.

[0071] [A15] The method according to any one of [A12] to [A14], wherein the distillation in step (3) is rectification.

[0072] [A16] The method according to any one of [A12] to [A15], wherein in step (2), the concentration of monoacylglycerol containing fatty acids having six fewer carbon atoms than the carbon atoms of highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated is reduced to less than 10,000 ppm.

[0073] [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.

[0074] [A18] The method according to any one of [A12] to [A17], wherein the raw material is derived from fish oil, microbial oil, plant oil or marine animal oil.

[0075] Effects of the Invention

[0076] According to the present invention, a composition having a low concentration of 3-MCPD fatty acid ester and containing a high concentration of PUFA or PUFA alkyl ester can be stably produced. DETAILED DESCRIPTION

[0077] The present invention is described in detail below.

[0078] In this specification, when labeling fatty acids, sometimes a numerical representation method that combines numbers and letters to simply represent the number of carbons, the number of double bonds, and the position of double bonds is used. For example, a saturated fatty acid with 20 carbon atoms can be labeled as "C20:0", a monounsaturated fatty acid with 18 carbon atoms can be labeled as "C18:1", and eicosapentaenoic acid can be labeled as "C20:5n-3", etc. "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 means that the bond between the 3rd carbon atom and the 4th carbon atom 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 determine the fatty acids labeled according to this method.

[0079] In the present specification, "highly unsaturated fatty acid" means a fatty acid having 18 or more carbon atoms and 3 or more double bonds. Highly unsaturated fatty acids may be, for example, fatty acids having 20 or more carbon atoms and 3 or more double bonds, or fatty acids having 20 or more carbon atoms and 5 or more double bonds. Examples of highly unsaturated fatty acids include α-linolenic acid (18:3n-3), γ-linolenic acid (18:3n-6), dihomo-γ-linolenic acid (20:3n-6), arachidonic acid (20:4n-6), eicosapentaenoic acid (20:5n-3), docosapentaenoic acid (22:5n-6), and docosahexaenoic acid (22:6n-3).

[0080] In this specification, "crude oil" means a mixture of lipids, and means oil in a state after extraction from a living organism. In this specification, "refined oil" means an oil that has been subjected to a crude oil refining treatment by removing substances other than target substances such as phospholipids and sterols by 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. A person skilled in the art can distinguish between refined oil and crude oil by conventional analysis.

[0081] In this specification, the composition containing highly unsaturated fatty acids or their alkyl esters means a fatty acid composition containing highly unsaturated fatty acids or a fatty acid alkyl ester composition containing the alkyl esters of highly unsaturated fatty acids. Here, the fatty acid composition is a composition with fatty acids as the main constituent, and the fatty acid alkyl ester composition is a composition with the alkyl esters of fatty acids as the main constituent.

[0082] In this specification, unless the context clearly indicates otherwise, a general term (e.g., highly unsaturated fatty acid, highly unsaturated fatty acid alkyl ester, etc.) does not exclude the possibility of multiple constituent elements. Therefore, a general term usually means the presence of at least one constituent element.

[0083] <Composition containing highly unsaturated fatty acid or its alkyl ester having reduced 3-MCPD fatty acid ester concentration>

[0084] The present invention provides a composition containing highly unsaturated fatty acids or their alkyl esters, wherein the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition is 50 area % or more, and when the composition is analyzed using the official method Cd29b-13 analysis A of the American Oil Chemists Association, the concentration of 3-MCPD generated is less than 1.80 ppm (sometimes referred to as the composition of the present invention in this specification).

[0085] In the present invention, the highly unsaturated fatty acid is not particularly limited as long as its alkyl ester is a substance obtained as a main distillate portion when concentrated 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.

[0086] The ratio of highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention is 50 area % or more, for example, it can be 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 addition, in one embodiment, the ratio of highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention is 99 area % or less, for example, it can be 98 area % or less, 95 area % or less, 90 area % or less, 85 area % or less, 80 area % or less, 75 area % or less, 70 area % or less, 65 area % or less, 60 area % or less, or 55 area % or less. The proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention may 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 %, 75 to 98 area %, 75 to 95 area %, 75 to 90 area %, 75 to 85 area % or 75 to 80 area %.

[0087] In one embodiment, the highly unsaturated fatty acid is eicosapentaenoic acid, and the ratio of the highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention is 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%.

[0088] In another embodiment, the highly unsaturated fatty acid is eicosapentaenoic acid, and the ratio of the highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention is 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%.

[0089] In still another embodiment, the highly unsaturated fatty acid may be eicosapentaenoic acid, and the proportion of highly unsaturated fatty acids in the constituent fatty acids of the composition of the present invention may be 60 to 80 area %, 65 to 80 area %, 70 to 80 area %, or 75 to 80 area %.

[0090] The composition of the present invention has a high ratio of highly unsaturated fatty acids and is therefore suitable as a raw material for pharmaceuticals and supplements containing highly unsaturated fatty acids as active ingredients.

[0091] In this specification, the "area %" representing the proportion of highly unsaturated fatty acids in the constituent fatty acids of a composition is: in a graph obtained by analyzing the composition using a gas chromatograph (GC-FID) with a hydrogen flame ionization detector (FID), the peaks of each component are identified, and the peak area of ​​each fatty acid is calculated using the Agilent ChemStation integration algorithm (version C.01.03

[37] , Agilent Technologies), and the content ratio of the peak component is expressed as the ratio of each peak area to the sum of the peak areas of the fatty acids. In the field of oil and fat chemistry, area % is used as a unit roughly synonymous with weight %. Reference is made to the Standard Oil and Fat Analysis Test Method 2013 Edition 2.4.2.1-2013 Fatty Acid Composition (FID Constant Temperature Gas Chromatography) established by the Japan Oil Chemicals Society (JOCS) and 2.4.2.2-2013 Fatty Acid Composition (FID Increasing Temperature Gas Chromatography) in the same document. The analysis conditions of the gas chromatography are as follows.

[0092] GC-FID measurement conditions

[0093] GC: 6890N (Agilent Technologies)

[0094] Column: DB-WAX (Agilent Technologies)

[0095] 30m×0.25mm ID, 0.25μm film thickness

[0096] Carrier gas: Helium, 1mL / min

[0097] Injection port: 250℃, 1μL, split (1:100)

[0098] Column temperature: 180℃→3℃ / min→230℃, hold for 15 minutes

[0099] Detector: FID, 250℃

[0100] Supplementary gas: nitrogen 45mL / min.

[0101] In the embodiment of the composition of the present invention being a fatty acid composition containing highly unsaturated fatty acids, the ratio of highly unsaturated fatty acids in the fatty acids is measured by the following method. That is, the fatty acid composition is subjected to GC under the aforementioned conditions on the basis of methyl esterification according to the content of AOCS official method Ce1b-89, and the area % of highly unsaturated fatty acids is calculated in the same manner.

[0102] In this case, the constituent fatty acids of the composition mean the free fatty acids in the fatty acid composition.

[0103] In an embodiment of the composition of the present invention, which is a fatty acid alkyl ester composition containing an alkyl ester of a highly unsaturated fatty acid, the ratio of the highly unsaturated fatty acids in the constituent fatty acids of the composition is measured by the following method. That is, the fatty acid alkyl ester composition is analyzed by gas chromatography under the above-mentioned conditions, and the ratio (area %) of the peak area of ​​the highly unsaturated fatty acid alkyl ester to the sum of the peak areas of the fatty acid alkyl esters is calculated.

[0104] In this case, the constituent fatty acid of the composition means the fatty acid constituting the fatty acid alkyl ester of the fatty acid alkyl ester composition.

[0105] The alkyl group in the highly unsaturated fatty acid alkyl ester is an alkyl group derived from a lower alcohol commonly used in the alkyl esterification of fatty acids, and examples thereof include an alkyl group having 1 or 2 carbon atoms (i.e., a methyl group or an ethyl group). In a preferred embodiment, the highly unsaturated fatty acid alkyl ester may be a highly unsaturated fatty acid ethyl ester.

[0106] In the composition of the present invention, the concentration of 3-MCPD produced when the composition is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association is reduced to less than 1.80ppm (mg / kg), for example, less than 1.70ppm, less than 1.60ppm, less than 1.50ppm, less than 1.40ppm, less than 1.30ppm, less than 1.20ppm, less than 1.10ppm, less than 1.00ppm, less than 0.90ppm, less than 0.80ppm, less than 0.70ppm, less than 0.60ppm, less than 0.50ppm, less than 0.40ppm, less than 0.30ppm, less than 0.20ppm, less than 0.10ppm, less than 0.09ppm, less than 0.08ppm, less than 0.07ppm, less than 0.06ppm, less than 0.05ppm, less than 0.04ppm, less than 0.03ppm, less than 0.02ppm or less than 0.01ppm. In some embodiments, the concentration of 3-MCPD is greater than 0ppm. In one embodiment, in the composition of the present invention, the concentration of 3-MCPD generated when the composition is analyzed by the American Oil Chemists Association official method Cd29b-13 analysis A is 0.01 ppm or more, for example, 0.02 ppm or more. In addition, in the composition of the present invention, the concentration of 3-MCPD generated when the composition is analyzed using the official method Cd29b-13 analysis A of the American Oil Chemists Association 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.In addition, in the composition of the present invention, the concentration of 3-MCPD generated when the composition is analyzed using the official method Cd29b-13 analysis A of the American Oil Chemists Association 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.

[0107] In this specification, the analysis by the American Oil Chemists Society official method (AOCS official method) Cd29b-13 analysis A is performed by the following steps well known to those skilled in the art.

[0108] Add 100 μL of 3-MCPD-d5-dipalmitate standard solution (diluted with toluene to form 5 ppm 3-MCPD-d5) and 600 μL of ether to 100 mg of sample, stir and mix until completely dissolved, and then cool at -22°C to -25°C for about 15 minutes. Then, add 350 μL of sodium hydroxide-methanol solution (0.25 g of sodium hydroxide dissolved in 100 mL of methanol) and stir thoroughly, and then react at -22°C to -25°C for more than 16 hours. At the same temperature, add 600 μL of acidic sodium bromide solution (600 g of sodium bromide dissolved in 1 L of purified water, and add 3 mL of 85% phosphoric acid) to stop the reaction, and then blow nitrogen to concentrate until the organic layer separated into two layers becomes about 100 μL. Then, add 600 μL of hexane, stir vigorously, and let stand for 5 to 10 minutes to remove the organic layer. This operation is repeated twice. Add 600 μL of ether:ethyl acetate mixture (3:2, V / V) to the remaining water layer, stir vigorously, recover the organic layer, and repeat this operation three times. Combine the organic layers recovered three times and dehydrate with anhydrous sodium sulfate. Blow nitrogen gas to concentrate the dehydrated organic layer to 200 μL, add 20 μL of saturated phenylboronic acid-ether solution, stir vigorously for 10 seconds, and blow nitrogen gas to completely remove the solvent. Add 200 μL of isooctane, stir vigorously for 10 seconds, and use the resulting solution as a GC-MS sample.

[0109] A calibration curve for quantification of 3-MCPD was prepared by analyzing a 3-MCPD standard solution prepared by dissolving 3-MCPD-dipalmitate in toluene so that the 3-MCPD concentration was 0 ppm, 0.5 ppm, 1 ppm, and 5 ppm in the same manner as the above sample.

[0110] The analysis conditions of GC-MS are as follows.

[0111] GC-MS conditions

[0112] GC: GC-2010 and GCMS-QP2010 (Shimadzu Corporation)

[0113] Column: DB-5ms (Agilent Technologies)

[0114] 30m×0.25mm ID, 0.25μm film thickness

[0115] Carrier gas: Helium, 1.2mL / min

[0116] Injection port: 250℃, 1μL, splitless sampling time 1 minute

[0117] Column temperature: 85℃ for 0.5 minutes → 6℃ / minute → 150℃ for 5 minutes → 12℃ / minute → 180℃ → 25℃ / minute → 280℃ for 7 minutes

[0118] Ionization temperature: 200°C

[0119] Interface temperature: 200℃

[0120] Ionization method: EI, SIM detection m / z is as follows

[0121] 3-MCPD-d5: m / z=149, 150, 201, 203

[0122] 3-MCPD: m / z=146, 147, 196, 198

[0123] Note that m / z = 150 of 3-MCPD-d5 and m / z = 147 of 3-MCPD were used for quantification, and the others were used for confirmation of target substances.

[0124] In the above analysis method, both the free form and the ester form of 3-MCPD in the sample are detected as 3-MCPD without distinction. 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.

[0125] In one embodiment of the present specification, in the composition of the present invention, the concentration of 3-MCPD produced when the composition is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association can be above 0 ppm and below 1.80 ppm. In this case, the composition of the present invention may include a composition that does not contain 3-MCPD. In other embodiments, the composition of the present invention contains 3-MCPD (i.e., 3-MCPD or 3-MCPD fatty acid ester), and the concentration of 3-MCPD produced when the composition is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association can be below 1.80 ppm.

[0126] In the composition of the present invention, when the composition is analyzed using the official method Cd29b-13 analysis A of the American Oil Chemists Association, the concentration of 3-MCPD produced can be lower than the quantitative lower limit of the method of the official method Cd29b-13 analysis A of the American Oil Chemists Association, preferably lower than the detection limit.

[0127] It is known that certain types of microbial oils, vegetable oils, or marine oils contain a large amount of highly unsaturated fatty acids such as eicosapentaenoic acid. Therefore, the composition of the present invention can be made of them. As the raw material of the composition of the present invention, specifically, sardine oil, tuna oil, bonito oil, menhaden oil, cod liver oil, herring oil, capelin oil and salmon oil, fish oils such as krill, marine oils from crustacean sources, vegetable oils from sources such as perilla, flax, soybean, rapeseed, yeasts such as Yarrowia, filamentous fungi belonging to Mortierella, Penicillium, Aspergillus, Rhodotorula, Fusarium, algae such as Euglena, and oils from microorganisms such as unequal flagella that produce lipids can be exemplified. The composition of the present invention may also be an oil derived from a genetically recombinant microorganism into which a gene such as a mutant Δ9 elongase gene with a gene modification has been introduced. In addition, an oil derived from a genetically recombinant plant obtained from an oilseed plant such as Brassica species, sunflower, corn, cotton, flax, safflower, etc. into which a gene such as a mutant Δ9 elongase gene has been introduced by recombinant technology may also be used as a raw material oil. Regarding genetically recombinant plant oils and genetically recombinant microbial oils, for example, substances described in WO2012 / 027698, WO2010 / 033753, etc. may be exemplified. In a preferred embodiment, the raw material of the composition of the present invention is aquatic animal oil such as fish oil, microbial oil, vegetable oil, or marine animal oil, more preferably fish oil.

[0128] In the raw materials of the composition of the present invention, highly unsaturated fatty acids are mainly contained in the form of glycerides. For example, fish oil contains a variety of fatty acids with carbon numbers of 14 to 22 and double bonds of 0 to 6 in the form of glycerides. After the glycerides are reacted with lower alcohols such as ethanol in the presence of a catalyst or an enzyme to cause the fatty acids contained in the glycerides to undergo alkyl esterification, the fatty acid alkyl esters other than the target highly unsaturated fatty acid alkyl esters (such as EPA alkyl esters) are removed, thereby producing highly unsaturated fatty acid alkyl esters (such as EPA alkyl esters) of high purity. In one embodiment, the removal of fatty acid alkyl esters other than the target highly unsaturated fatty acid alkyl esters (such as EPA alkyl esters) can be carried out by distillation. The inventors of the present invention have found that during distillation, a large amount of 3-MCPD fatty acid esters from monoacylglycerol or diacylglycerol with a larger molecular weight are mixed in the main distillate residue. Therefore, in a preferred embodiment, the composition of the present invention can be a distillate (fraction).

[0129] It is known that a trans isomer is generated as a thermally modified product from a highly unsaturated fatty acid alkyl ester by a heat treatment during distillation (e.g. European Journal of Lipid Science and Technology, 108 (2006) 589-597. "Geometrical isomerization of eicosapentaenoic anddocosahexaenoic acid at high temperatures"; JAOCS, 66 (1989) 1822-1830. "Eicosapentaenoic acid geometricalisomer artifacts in heated fish oil esters"). Therefore, in one embodiment, the composition of the present invention may further contain a trans isomer of a highly unsaturated fatty acid alkyl ester. The concentration of the trans isomer in the composition of the present invention 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. In addition, the concentration of the trans isomer in the composition of the present invention is 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.

[0130] In this specification, the concentration of the trans isomer is a value measured by GC analysis. Specifically, it is measured by the following procedure.

[0131] 10 mg of the sample was dissolved in 1 mL of hexane and subjected to GC analysis under the following conditions.

[0132] [GC analysis conditions]

[0133] GC: 6890N (Agilent Technologies)

[0134] Column: DB-WAX (Agilent Technologies)

[0135] 30m×0.25mm ID, 0.25μm film thickness

[0136] Carrier gas: Helium, 1mL / min

[0137] Injection port: 250℃, 1μL, split (1:100)

[0138] Column temperature: 180℃→3℃ / min→230℃ for 15 minutes

[0139] Detector: FID, 250℃

[0140] Supplementary gas: nitrogen 45mL / min.

[0141] For example, for the trans isomers (five types, A to E) of EPA ethyl ester (EPA-E), the concentration is calculated as follows.

[0142] In the sample from which saturated fatty acids or monounsaturated fatty acids with a C21 or higher content have been removed by distillation, in the trans isomer of EPA ethyl ester (EPA-E), when the retention time of EPA-E is taken as 1, the relative retention time is 0.98 to 0.99 for isomer A, 1.01 to 1.02 for isomer B, 1.02 to 1.03 for isomer C, and 1.04 to 1.05 for isomers D and E, which form overlapping peaks. Among the peaks of the five isomers, the peaks of isomers D and E overlap, so four peaks are detected. The total area of ​​the peaks with these relative retention times is taken as the peak area of ​​the trans isomer of EPA-E. The ratio of the trans isomer to EPA-E is obtained, and the isomer concentration in the sample is calculated from the EPA-E concentration in the sample.

[0143] For samples containing saturated fatty acids with C21 or more or monounsaturated fatty acids, separation was performed using a silver nitrate column with Discovery Ag-ION 750 mg / 6 mL (SUPELCO) to remove saturated fatty acids with C21 or more and monounsaturated fatty acids, and then analysis was performed as described above.

[0144] For example, for the trans isomer of dihomo-γ-linolenic acid ethyl ester (DGLA-E), the total area of ​​the peaks with the following relative retention times is measured by the above-mentioned GC analysis to determine the ratio of the trans isomer to DGLA-E, and the isomer concentration in the sample is calculated from the DGLA-E concentration in the sample.

[0145] Isomer A: Relative retention time 1.001~1.009

[0146] Isomer B: Relative retention time 1.01~1.03

[0147] (The holding time of DGLA is set to 1.)

[0148] The content of trans isomers of other highly unsaturated fatty acid alkyl esters can also be determined by conventional methods.

[0149] When the composition of the present invention is measured using gas chromatography under the above analysis conditions, the concentration of the trans isomer of EPA alkyl 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. In addition, when the composition of the present invention is measured using gas chromatography under the above analysis conditions, the concentration of the trans isomer of EPA alkyl ester is 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.

[0150] In a preferred embodiment, when the composition of the present invention is measured using gas chromatography under the above-mentioned analysis conditions, the concentration of the trans isomer of EPA ethyl ester is 2.5 area % or less, for example, it can be 2.3 area % or less, 2.0 area % or less, 1.8 area % or less, 1.6 area % or less, 1.4 area % or less, 1.2 area % or less, 1.0 area % or less, 0.9 area % or less, 0.8 area % or less, 0.7 area % or less, 0.6 area % or less, or 0.5 area % or less. In addition, when the composition of the present invention is measured using gas chromatography under the above-mentioned analysis conditions, the concentration of the trans isomer of EPA ethyl ester is 0.01 area % or more, for example, it can be 0.02 area % or more, 0.03 area % or more, 0.04 area % or more, or 0.05 area % or more.

[0151] When the composition of the present invention is measured using gas chromatography under the above-mentioned analysis conditions, the concentration of the trans isomer of DGLA alkyl 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. In addition, when the composition of the present invention is measured using gas chromatography under the above-mentioned analysis conditions, the concentration of the trans isomer of DGLA alkyl ester is 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.

[0152] In a preferred embodiment, when the composition of the present invention is measured using gas chromatography under the above-mentioned analysis conditions, the concentration of the trans isomer of DGLA ethyl ester is 2.5 area % or less, for example, it can be 2.3 area % or less, 2.0 area % or less, 1.8 area % or less, 1.6 area % or less, 1.4 area % or less, 1.2 area % or less, 1.0 area % or less, 0.9 area % or less, 0.8 area % or less, 0.7 area % or less, 0.6 area % or less, or 0.5 area % or less. In addition, when the composition of the present invention is measured using gas chromatography under the above-mentioned analysis conditions, the concentration of the trans isomer of DGLA ethyl ester is 0.01 area % or more, for example, it can be 0.02 area % or more, 0.03 area % or more, 0.04 area % or more, or 0.05 area % or more.

[0153] Fish oil and microbial oil containing highly unsaturated fatty acids contain cholesterol in addition to triglycerides. The highly unsaturated fatty acid concentrated oil prepared from these stock oils also contains cholesterol (WO2012 / 118173). In addition, even if the oil containing cholesterol is subjected to alkali esterification or urea addition, cholesterol will not be completely removed. Therefore, in the specific embodiment of this specification, the composition of the present invention contains cholesterol. The cholesterol content can be, for example, less than 1.5% by weight, less than 0.3% by weight or less than 0.2% by weight. In addition, the cholesterol content can be, for example, more than 0.01% by weight or more than 0.02% by weight.

[0154] Cholesterol is a substance with the molecular formula C 27 H 46 The compound of the steroid skeleton represented by O exists as a free body or an ester body in natural substances. The ester body is an acyl cholesterol with a fatty acid bonded to the hydroxyl (OH) group. The cholesterol content in the present invention means the total content of the free body and the ester body. The cholesterol content is measured by the following method.

[0155] 1 mL of 0.1 g / L 5α-cholestane as an internal standard was added to about 0.1 g of the sample, and 1 mL of 2 mol / L potassium hydroxide / aqueous ethanol solution was added, followed by heating at 100°C for 10 minutes. After cooling, 3 mL of petroleum ether and 3 mL of saturated ammonium sulfate were added, stirred, and the upper layer was recovered after standing, and measured by gas chromatography under the following measurement conditions. In order to determine the relative sensitivity of 5α-cholestane and free cholesterol, hexane solutions each containing 25 mg of 5α-cholestane and cholesterol were measured by gas chromatography to calculate the total cholesterol content.

[0156] Gas chromatography analysis conditions

[0157] Model: Agilent 6890GC system (Agilent Technologies, Inc.)

[0158] Column: DB-1J&W 123-1012

[0159] Column temperature: 270°C

[0160] Injection temperature: 300℃

[0161] Injection method: split flow

[0162] Split ratio: 50:1

[0163] Detector temperature: 300°C

[0164] Detector: FID

[0165] Carrier gas: Helium (39.3kPa, constant pressure)

[0166] In one embodiment, the composition of the present invention may contain a saturated fatty acid having a carbon number of 18 or less or an alkyl ester thereof as an impurity. In this case, the proportion of the saturated fatty acid having a carbon number of 18 or less in the constituent fatty acids of the composition of the present invention may be 0.1 area % or more, for example, 0.2 area % or more or 0.3 area % or more, and less than 10 area %, for example, less than 5 area %, less than 4 area % or less than 3 area %.

[0167] The highly unsaturated fatty acid can be obtained by hydrolyzing the highly unsaturated fatty acid alkyl ester produced by the above method.

[0168] Composition of the present invention is a composition containing fatty acid or fatty acid alkyl ester as a main component, usually containing more than 50% by weight, more than 55% by weight, more than 60% by weight, more than 65% by weight, more than 70% by weight, more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight or more than 99.9% by weight of fatty acid or fatty acid alkyl ester. The content of fatty acid or fatty acid alkyl ester in the composition of the present invention can be confirmed by known methods such as TLC / FID.

[0169] <Method for producing a composition containing a highly unsaturated fatty acid or an alkyl ester thereof>

[0170] The present invention provides a method for producing the above-mentioned composition of the present invention. The method comprises:

[0171] (1) a step of alkyl-esterifying a raw material containing triglycerides containing highly unsaturated fatty acids as constituent fatty acids to prepare a composition containing highly unsaturated fatty acid alkyl esters,

[0172] (2) at least one step selected from the following steps (a), (b) and (c):

[0173] (a) a step of reducing the concentration of monoacylglycerol containing fatty acids having a carbon number less than 5 or more than the carbon number of highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated, as constituent fatty acids, 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, (c) a step of reducing the chlorine concentration in the composition of step (1) to less than 10 ppm, and

[0174] (3) A step of distilling the composition after step (2) to separate a main fraction (hereinafter also referred to as the method of the present invention).

[0175] The method of the present invention can produce a composition containing fatty acids or fatty acid alkyl esters as main components, 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 fatty acids or fatty acid alkyl esters.

[0176] 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.

[0177] As the raw material in the method of the present invention, the oils described above in connection with the composition of the present invention can be cited, specifically, fish oils such as sardine oil, tuna oil, bonito oil, menhaden oil, cod liver oil, herring oil, capelin oil, and salmon oil, marine oils derived from crustaceans such as krill, plant oils derived from perilla, flax, soybeans, and rapeseed, yeasts such as Yarrowia, filamentous fungi belonging to the genera Mortierella, Penicillium, Aspergillus, Rhodotorula, and Fusarium, algae such as Euglena, and oils derived from lipid-producing microorganisms such as anisoflagellates can be cited. The raw material in the method of the present invention may also be oils derived from genetically modified microorganisms into which genes such as a mutant Δ9 elongase gene with a modified gene have been introduced. In addition, genetically modified plant-derived oils obtained from oilseed plants such as Brassica species, sunflower, corn, cotton, flax, safflower, etc., into which genes such as mutant Δ9 elongase genes are introduced by recombinant technology, can also be used as raw material oils. Regarding genetically modified plant oils and genetically modified microbial oils, for example, substances described in WO2012 / 027698, WO2010 / 033753, etc. can be exemplified. In a preferred embodiment, the raw material in the method of the present invention is fish oil, microbial oil, plant oil, or aquatic animal oil such as marine animal oil, more preferably fish oil.

[0178] Crude oil refining process

[0179] The raw material oil used in the alkyl esterification in step (1) may be a crude oil or a refined oil. For example, any method may be used to obtain crude oil from aquatic raw materials such as fish or seafood. In the case of fish oil, it is usually collected by the following method. The whole fish or the processing residues such as the head, skin, middle bones, and internal organs of the fish produced in the processing of aquatic products are crushed and boiled, and then pressed to separate the cooking juice (pressed liquid) and the pressed crude powder. The fat obtained together with the cooking juice is separated from the cooking juice by centrifugal separation to obtain crude fish oil.

[0180] Generally, crude fish oil is subjected to a degumming process, a deacidification process, a decolorization process using activated clay or activated carbon, a water washing process, a deodorization process using steam distillation, etc., depending on the raw material, and then undergoes a crude oil refining process to remove substances other than target substances such as phospholipids and sterols to produce refined fish oil. In the embodiment of the present invention, the refined fish oil can also be used as a raw material.

[0181] Step (1) (Alkyl esterification step)

[0182] By alcoholysis using a lower alcohol, fats and oils as feedstock oils are decomposed into lower alcohol esters. Examples of lower alcohols include substances commonly used in alkyl esterification of fatty acids, such as lower alcohols with 1 or 2 carbon atoms. Alcoholysis is a reaction in which a lower alcohol such as ethanol is added to fats and oils and reacted with a catalyst or an enzyme to generate alkyl esters from fatty acids bound to glycerol. As catalysts, alkaline catalysts, acid catalysts, etc. are used. As enzymes, lipase is used.

[0183] It is empirically determined that alcoholysis of fatty acids is highly efficient, and after alcoholysis, a composition containing fatty acids in the form of their alkyl esters is obtained. However, it is not entirely excluded that fatty acids in forms other than the alkyl ester form are contained.

[0184] Process (2)

[0185] Step (2) is a pre-treatment step for distillation, and is at least one step selected from the following steps (a) to (c). That is, step (2) may be any one of (a) to (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c).

[0186] (a) Monoacylglycerol Removal Step

[0187] In step (2) (a), the content of monoacylglycerol, which is a substrate for 3-MCPD fatty acid esters, is reduced before the fatty acid alkyl ester-containing composition prepared in step (1) is concentrated by distillation. This can reduce the amount of 3-MCPD fatty acid esters that are mixed into the main fraction due to the heat treatment during distillation.

[0188] When removing monoacylglycerol from the fatty acid alkyl ester-containing composition prepared in step (1), conventional techniques such as repeated alkyl esterification treatment and adsorbent treatment can be used.

[0189] Esterification is an equilibrium reaction, and the amount of residual glycerides depends on the ratio of alcohol to glycerol as a byproduct. By subjecting the ester component obtained after alkyl esterification to alkyl esterification again, the ratio of alcohol to glycerol can be tilted significantly toward the alcohol side, thereby reducing glycerides.

[0190] As adsorbent treatment, for example, silica gel chromatography, activated clay treatment, acidic clay treatment, activated carbon treatment, silica gel treatment, etc. can be cited. Silica gel chromatography can be performed, for example, according to the following steps. In the treatment using silica gel (such as Microsphere D75-60A), in order to adsorb fatty acid alkyl esters on silica gel, fatty acid alkyl esters are loaded onto a silica gel packed column. Then, ethyl acetate / hexane (1:50) is passed through the column, the eluent is separated, and the components after removing MAG and DAG from the fatty acid alkyl esters are recovered. The component is desolventized to obtain fatty acid alkyl esters. In addition, activated clay treatment can be performed, for example, by the following method: adding activated clay in an amount of 5% relative to the oil, stirring at 120°C under reduced pressure for 2 hours, and filtering. For other adsorbent treatments, conventional methods can also be used.

[0191] The monoacylglycerol removed in (a) of step (2) contains fatty acids having a carbon number less than 5 or more than the carbon number of the highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters as the concentration target as constituent fatty acids. Here, the highly unsaturated fatty acid alkyl esters as the concentration target refer to the highly unsaturated fatty acid alkyl esters that are intended to be concentrated in the method of the present invention. That is, in the distillation of step (3), the conditions are set in such a way that the highly unsaturated fatty acid alkyl esters as the concentration target are separated as the main fraction. Therefore, in the distillation of step (3), the highly unsaturated fatty acid alkyl esters separated and concentrated as the main fraction are the highly unsaturated fatty acid alkyl esters as the concentration target.

[0192] When there are two or more highly unsaturated fatty acid alkyl esters as the concentration target (for example, when one wants 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 fatty acids having 5 or more fewer carbon atoms than the substance with the largest carbon number as constituent fatty acids is reduced.

[0193] The constituent fatty acid of the monoacylglycerol removed in (a) of step (2) may be either saturated fatty acid or unsaturated fatty acid, but it is preferred to remove monoacylglycerol of saturated fatty acid.

[0194] In step (2) (a), the concentration of monoacylglycerol containing fatty acids having 5 or more fewer carbon numbers than the carbon numbers of highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated is reduced to less than 10,000 ppm, less than 9,000 ppm, less than 8,000 ppm, less than 7,000 ppm, less than 6,000 ppm, less than 5,000 ppm, less than 4,000 ppm, less than 3,000 ppm, less than 2,000 ppm, less than 1,000 ppm, less than 900 ppm, less than 800 ppm, less than 700 ppm, less than 600 ppm, or less than 500 ppm. In some embodiments, the concentration of the monoacylglycerol is higher than 0 ppm. In some embodiments, the concentration of the monoglycerol before reduction is 10,000 ppm or more or more than the above upper limit. For example, when the highly unsaturated fatty acid alkyl ester as the concentration target is eicosapentaenoic acid (20:5n-3) alkyl ester, dihomo-γ-linolenic acid (20:3n-6) alkyl ester, or arachidonic acid (20:4n-6) alkyl ester, or a combination thereof, the concentration of monoacylglycerol containing fatty acids with a carbon number of 15 or less (preferably fatty acids with a carbon number of 14) as constituent fatty acids is reduced to the above concentration. When the highly unsaturated fatty acid alkyl ester as the concentration target is docosahexaenoic acid (22:6n-3) alkyl ester, the concentration of monoacylglycerol containing fatty acids with a carbon number of 17 or less (preferably fatty acids with a carbon number of 16) as constituent fatty acids is reduced to the above concentration. Furthermore, when the highly unsaturated fatty acid alkyl ester used as the concentration target is a combination of docosahexaenoic acid (22:6n-3) alkyl ester and one or more selected from eicosapentaenoic acid (20:5n-3) alkyl ester, dihomo-γ-linolenic acid (20:3n-6) alkyl ester and arachidonic acid (20:4n-6) alkyl ester, the concentration of monoacylglycerol containing fatty acids with a carbon number of 17 or less (preferably fatty acids with a carbon number of 14 and fatty acids with a carbon number of 16) as constituent fatty acids is reduced to the above-mentioned concentration.

[0195] In the distillation of step (3), 3-MCPD fatty acid esters that can be produced from monoacylglycerol containing fatty acids as constituent fatty acids whose carbon number is 5 or more less than that of highly unsaturated fatty acids constituting highly unsaturated fatty acid alkyl esters constituting the concentration target can be contained in the main fraction together with the highly unsaturated fatty acid alkyl esters. Therefore, when highly unsaturated fatty acid alkyl esters as the concentration target are obtained as the main fraction in the distillation of step (3), the concentration of monoacylglycerol containing fatty acids as constituent fatty acids whose carbon number is 5 or more less than that of highly unsaturated fatty acids constituting the concentration target highly unsaturated fatty acid alkyl esters is reduced in advance, thereby obtaining a composition containing highly unsaturated fatty acid alkyl esters with a reduced concentration of 3-MCPD fatty acid esters.

[0196] The monoacylglycerol removed in (a) of step (2) may be a monoacylglycerol containing, as a constituent fatty acid, a fatty acid having a carbon number that is 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, or 6 less than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester that is the concentration target. In (a) of step (2), the total concentration of these monoacylglycerols may be reduced to the above concentration.

[0197] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester to be concentrated may be eicosapentaenoic acid (20:5n-3) alkyl ester, dihomo-γ-linolenic acid (20:3n-6) alkyl ester or arachidonic acid (20:4n-6) alkyl ester, or a combination thereof, and the monoacylglycerol removed in (a) of step (2) may be monomyristic acid glycerol.

[0198] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester to be concentrated may be docosahexaenoic acid (22:6n-3) alkyl ester, and the monoacylglycerol removed in step (2) (a) may be monopalmitin glycerol.

[0199] In a preferred embodiment, the highly unsaturated fatty acid alkyl esters to be concentrated may be a combination of docosahexaenoic acid (22:6n-3) alkyl esters and one or more selected from eicosapentaenoic acid (20:5n-3) alkyl esters, dihomo-γ-linolenic acid (20:3n-6) alkyl esters and arachidonic acid (20:4n-6) alkyl esters, and the monoacylglycerols removed in (a) of step (2) may be monomyristic acid glyceryl and monopalmitic acid glyceryl.

[0200] In the present specification, the concentrations of monoacylglycerol (MAG) and diacylglycerol (DAG) in the composition are values ​​(ppm (mg / kg)) calculated from the measured values ​​obtained by the following method.

[0201] Collect and weigh 100 μL of the composition, dissolve it in 400 μL of hexane, and subject 150 μL of the resulting solution to thin layer chromatography (TLC) under the following TLC conditions to separate MAG and DAG. Scrape all the MAG and DAG bands confirmed by UV 254nm, 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. Add 1 mL of 0.1 mg / mL C23:0FAME (tricosanoic acid methyl ester) hexane solution and 5 mL of saturated saline, stir well, use the obtained hexane layer as a sample, perform GC-FID analysis under the following conditions, and calculate the concentration of each fatty acid using the following calculation formula.

[0202] Fatty acid concentration [mg / kg] = (fatty acid peak area / C23:0 peak area) × (10 5 / sample amount for TLC [mg])

[0203] TLC conditions

[0204] TLC plate: PLC silica gel 60F 254 0.5mm, 10cm×10cm

[0205] Developing solvent: hexane: ether: acetic acid (7:3:0.1, vol / vol / vol)

[0206] GC-FID measurement conditions

[0207] GC: 6890N (Agilent Technologies)

[0208] Column: DB-WAX (Agilent Technologies)

[0209] 30m×0.25mm ID, 0.25μm film thickness

[0210] Carrier gas: Helium, 1mL / min

[0211] Injection port: 250℃, 1μL, split (1:100)

[0212] Column temperature: 180℃→3℃ / min→230℃ for 15 minutes

[0213] Detector: FID, 250℃

[0214] Supplementary gas: nitrogen 45mL / min.

[0215] (b) Iron Removal Step

[0216] In step (2) (b), the iron concentration is reduced to less than 0.20ppm, less than 0.10ppm, less than 0.09ppm, less than 0.08ppm, less than 0.07ppm, less than 0.06ppm or less than 0.05ppm. In some embodiments, the iron concentration is higher than 0ppm. In some embodiments, the iron concentration before reduction is greater than 0.20ppm or greater than the above upper limit. By reducing the iron concentration, the generation of 3-MCPD fatty acid esters can be suppressed.

[0217] In this specification, the iron concentration (or iron content) is a value (ppm (mg / kg)) calculated from the measured value obtained by ICP-MS. Specifically, it is calculated by the following steps.

[0218] 1 g of the test composition was weighed and the volume was made up to 10 mL with butyl acetate (for atomic absorption analysis, Wako Pure Chemical Industries, Ltd.) to prepare a sample solution.

[0219] Conostan S-21 (10 ppm (Wt.)) was used as a standard sample. The standard sample was 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).

[0220] The sample solution and the calibration curve sample were subjected to ICP-MS analysis under the following analysis conditions, and a calibration curve was prepared by automatic calculation using software attached to the device to quantify the iron content of the sample solution.

[0221] Measurement device: Agilent 7700 series ICP-MS (Agilent Technologies)

[0222] RF power: 1550W

[0223] Sampling position: 10mm

[0224] Carrier gas: 0.45L / min

[0225] Optional gas: 20%

[0226] Supplementary gas: 0.20L / min

[0227] Spray chamber temperature: -5°C

[0228] Sample introduction: negative pressure suction

[0229] Measurement mode: He mode

[0230] He cell gas flow rate: 4.3mL / min

[0231] Determination of elements: 56 Fe

[0232] The iron content in the sample composition was calculated from the iron content of the quantitative sample solution by the following formula.

[0233] Iron content in sample composition [ppm] = C / (W×100)

[0234] C: Iron content of the sample solution measured by ICP-MS (μg / L)

[0235] W: The amount of sample composition collected (g)

[0236] The iron concentration can 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 different from the monoacylglycerol removal process. In some embodiments, the iron concentration is higher than 0 ppm. As a technique for reducing the iron concentration, in addition to the techniques that can be used in the monoacylglycerol removal process described above, examples include acid washing and ion exchange. These removal techniques can be carried out by conventional methods.

[0237] (c) Chlorine Removal Step

[0238] 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 greater than 10 ppm or greater than the above upper limit. By reducing the chlorine concentration, the formation of 3-MCPD fatty acid esters can be suppressed.

[0239] In this specification, the chlorine concentration (or chlorine content) is a value (ppm (mg / kg)) calculated from a measured value obtained by ICP-MS. Specifically, it is calculated by the following procedure.

[0240] 1 g of the test composition was weighed and the volume was made up to 10 mL with butyl acetate (for atomic absorption analysis, Wako Pure Chemical Industries, Ltd.) to prepare a sample solution.

[0241] Conostan Cl Std. (1000 ppm (Wt.)) was used as a standard sample. The standard sample was 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).

[0242] The sample solution and the calibration curve sample were subjected to ICP-MS analysis under the following analysis conditions, and a calibration curve was prepared by automatic calculation using software attached to the device to quantify the chlorine content of the sample solution.

[0243] Measurement device: Agilent 7700 series ICP-MS (Agilent Technologies)

[0244] RF power: 1550W

[0245] Sampling position: 10mm

[0246] Carrier gas: 0.45L / min

[0247] Optional gas: 20%

[0248] Supplementary gas: 0.20L / min

[0249] Spray chamber temperature: -5°C

[0250] Sample introduction: negative pressure suction

[0251] Measurement mode: He mode

[0252] He cell gas flow rate: 4.3mL / min

[0253] Determination of elements: 35 Cl

[0254] The chlorine content in the sample composition was calculated from the chlorine content of the quantitative sample solution by the following formula.

[0255] Chlorine content in sample composition [ppm] = C / (W×100)

[0256] C: Chlorine content of the sample solution measured by ICP-MS (μg / L)

[0257] W: The amount of sample composition collected (g)

[0258] The chlorine concentration can 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 different from the monoacylglycerol removal process. In some embodiments, the chlorine concentration is higher than 0 ppm. As a technique for reducing the chlorine concentration, in addition to the techniques that can be used in the monoacylglycerol removal process described above, there can be cited techniques such as degumming and deacidification that are commonly used in the oil and fat refining process. These removal techniques can be carried out by conventional methods.

[0259] Step (3) (Distillation step)

[0260] The composition in which the monoacylglycerol concentration is reduced in step (2) is distilled to separate the main fraction. Even if 3-MCPD fatty acid esters are generated due to the heat treatment during distillation, by setting the conditions so that the main fraction contains highly unsaturated fatty acid alkyl esters as the concentration target, it is possible to obtain a composition containing highly unsaturated fatty acid alkyl esters, and the concentration of 3-MCPD generated when the composition is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association can be less than 1.80 ppm. Such distillation conditions can be appropriately set according to the highly unsaturated fatty acid alkyl esters as the concentration target.

[0261] The distillation process can be carried out, for example, by rectification (precision distillation), molecular distillation or short-path distillation. They can be carried out by conventional methods, for example, the methods described in Japanese Patent Laid-Open No. 4-128250, Japanese Patent Laid-Open No. 5-222392, Japanese Patent Laid-Open No. 4-41457, Japanese Patent Laid-Open No. 6-33088 can be used.

[0262] The distillation is carried out under high vacuum, and the highly unsaturated fatty acid alkyl ester is used as the main fraction, and the more volatile initial fraction and the less volatile residual fraction are separated and obtained. The distillation conditions can be set in a manner that the highly unsaturated fatty acid alkyl ester as the concentration target is concentrated as the main fraction, for example, the temperature can be 150°C to 200°C, for example, 160°C to 200°C, 170°C to 200°C, and the pressure can be 1 to 300 Pa, for example, 1 to 200 Pa, 1 to 100 Pa, 1 to 50 Pa. Preferably, the main fraction is obtained at 170 to 200°C under a vacuum degree of 1 to 50 Pa.

[0263] Examples of conditions for molecular distillation or short-path distillation include temperatures of 80 to 150° C., for example, 80 to 130° C., 80 to 120° C., and a temperature of less than 10×10 -1 Pa, for example, less than 10×10 -2 Pa, less than 10×10 -3 Pa's pressure.

[0264] The concentration of 3-MCPD produced when the main fraction in step (3) is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association is less than 1.80ppm, for example, less than 1.70ppm, less than 1.60ppm, less than 1.50ppm, less than 1.40ppm, less than 1.30ppm, less than 1.20ppm, less than 1.10ppm, less than 1.00ppm, less than 0.90ppm, less than 0.80ppm, less than 0.70ppm, less than 0.60ppm, less than 0.50ppm, less than 0.40ppm, less than 0.30ppm, less than 0.20ppm, less than 0.10ppm, less than 0.09ppm, less than 0.08ppm, less than 0.07ppm, less than 0.06ppm, less than 0.05ppm, less than 0.04ppm, less than 0.03ppm, less than 0.02ppm or less than 0.01ppm. In some embodiments, the concentration of 3-MCPD is 0ppm or more. In particular, the concentration of 3-MCPD generated when the main fraction in step (3) is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association can be 0.01 ppm or more, for example, 0.02 ppm or more. In addition, when the main fraction in step (3) is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association, the concentration of 3-MCPD produced may 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.In addition, when the main fraction in step (3) is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association, the concentration of 3-MCPD produced may 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.

[0265] Chromatographic process

[0266] The method of the present invention may further include a purification step by chromatography such as high performance liquid chromatography (HPLC) after step (3).

[0267] The chromatography step performed by HPLC or the like after the distillation step is a step for reducing the content of the non-target components by removing the non-target components in the composition obtained in the distillation step, and further concentrating the highly unsaturated fatty acid alkyl esters in the distilled composition. The chromatography step can be performed according to a conventionally known method, such as the method described in Japanese Patent Laid-Open No. 5-222392. As the chromatogram used in the concentration treatment, for example, reverse phase column chromatography can be cited. As examples of stationary phases (adsorbents), polymer beads, polystyrene preferably networked with DVB (divinylbenzene), and silica gel can be cited. Preferably, reverse phase bonded silica gel containing C8 or C18 alkanes is cited, and C18 bonded reverse phase silica gel is particularly preferred. The adsorbent used in the chromatogram after the distillation of the present invention is preferably non-polar. As long as it is an adsorbent of a reverse phase distribution system, it can be used without particular restrictions. For example, octadecylsilyl (ODS) silica gel can be cited, which can be used as an ODS column.

[0268] The size of the column used in the device is not particularly limited, and depends on the amount of the sample to be purified. Those skilled in the art can easily determine the column of the appropriate size that should 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.

[0269] The temperature of the mobile phase and column is not particularly limited and depends on the solubility of the separation target substance in the mobile phase. A person skilled in the art can easily determine the appropriate mobile phase and column temperature to be used. The temperature of the column is typically 0 to 70°C, preferably 20 to 40°C.

[0270] As the solvent used in the mobile phase, for example, short-chain alcohols can be cited. Short-chain alcohols typically have 1 to 6 carbon atoms. As preferred examples of short-chain alcohols, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol and tert-butanol can be cited. The solvent used in the mobile phase is preferably methanol or ethanol, more preferably methanol. In order to shorten the elution time, it is preferred not to deliberately add water to the short-chain alcohol.

[0271] <Distillation Raw Material Composition>

[0272] The present invention provides a distillation raw material composition for obtaining the above-mentioned composition of the present invention, and the use of the distillation raw material composition as a distillation raw material in the method for producing the above-mentioned composition of the present invention. The distillation raw material composition contains highly unsaturated fatty acid alkyl esters, and the content of monoacylglycerol is reduced, or the iron concentration is reduced. The highly unsaturated fatty acid alkyl esters contain highly unsaturated fatty acid alkyl esters as a concentration target. By distilling the distillation raw material composition to obtain highly unsaturated fatty acid alkyl esters as a fraction, the content of 3-MCPD fatty acid esters can be reduced while concentrating the highly unsaturated fatty acid alkyl esters.

[0273] The distillation raw material composition of the present invention can be produced by alkyl esterifying the oil listed as the raw material of the composition of the present invention, reducing the content of monoacylglycerol or reducing the iron concentration. In one embodiment, the distillation raw material composition of the present invention is produced from raw materials such as fish oil, microbial oil, vegetable oil or marine animal oil or obtained from the raw material by the above-mentioned method for producing the composition of the present invention. In a preferred embodiment, the raw material of the distillation raw material composition of the present invention is fish oil. Alkyl esterification and reduction of the content of monoacylglycerol or iron concentration can be carried out by the method described in the above-mentioned method of the present invention. In a preferred embodiment, the highly unsaturated fatty acid alkyl ester contained in the distillation raw material composition of the present invention may be a highly unsaturated fatty acid ethyl ester. In addition, the highly unsaturated fatty acid alkyl ester as the concentration target may be an alkyl ester of eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, or arachidonic acid, or a combination thereof. In a preferred embodiment, the highly unsaturated fatty acid alkyl ester as the concentration target may be an alkyl ester of eicosapentaenoic acid or docosahexaenoic acid, or a combination thereof. In a more preferred embodiment, the highly unsaturated fatty acid alkyl ester as the concentration target may be an alkyl ester of eicosapentaenoic acid or docosahexaenoic acid.

[0274] The distillation raw material composition of the present invention is a composition containing 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 fatty acid alkyl ester.

[0275] The proportion of highly unsaturated fatty acids relative to the total fatty acids in the distillation raw material composition of the present invention is 5 area % or more, for example, 10 area % or more, 15 area % or more, or 20 area % or more. In addition, the proportion of highly unsaturated fatty acids relative to the total fatty acids in the distillation raw material composition of the present invention is less than 70 area %, for example, less than 65 area %, less than 60 area % or less than 55 area %.

[0276] The distillation raw material composition of the present invention may contain a saturated fatty acid having 18 or less carbon atoms or an alkyl ester thereof as an impurity. In this case, the proportion of the saturated fatty acid having 18 or less carbon atoms in the constituent fatty acids of the distillation raw material composition of the present invention may be 0.1 area % or more, for example, 0.2 area % or more, 0.3 area % or more, 0.4 area % or more, or 0.5 area % or more, and less than 50 area %, for example, less than 40 area % or less than 30 area %.

[0277] In a preferred embodiment, the concentration of monoacylglycerol containing fatty acids having 5 or more fewer carbon numbers than the carbon numbers of highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated as constituent fatty acids in the distillation raw material composition of the present invention is less than 10,000 ppm, less than 9,000 ppm, less than 8,000 ppm, less than 7,000 ppm, less than 6,000 ppm, less than 5,000 ppm, less than 4,000 ppm, less than 3,000 ppm, less than 2,000 ppm, less than 1,000 ppm, less than 900 ppm, less than 800 ppm, less than 700 ppm, less than 600 ppm, or less than 500 ppm. In some embodiments, the concentration of such monoacylglycerol is higher than 0 ppm.

[0278] The monoacylglycerol to which the above upper limit is applied may be a monoacylglycerol containing, as a constituent fatty acid, 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 to be concentrated.

[0279] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester used as the concentration target may be eicosapentaenoic acid (20:5n-3) alkyl ester, dihomo-γ-linolenic acid (20:3n-6) alkyl ester or arachidonic acid (20:4n-6) alkyl ester, or a combination thereof, and the monoacylglycerol may be monomyristic acid glyceride.

[0280] In a preferred embodiment, the highly unsaturated fatty acid alkyl ester to be concentrated may be docosahexaenoic acid (22:6n-3) alkyl ester, and the monoacylglycerol may be monopalmitin glycerol.

[0281] In a preferred embodiment, the highly unsaturated fatty acid alkyl esters used as the concentration target may be a combination of docosahexaenoic acid (22:6n-3) alkyl ester and one or more selected from eicosapentaenoic acid (20:5n-3) alkyl esters, dihomo-γ-linolenic acid (20:3n-6) alkyl esters and arachidonic acid (20:4n-6) alkyl esters, and the monoacylglycerols may be monomyristic acid glyceryl and monopalmitic acid glyceryl.

[0282] In a preferred embodiment, 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 greater than 0 ppm.

[0283] In a preferred embodiment, the chlorine concentration in the distillation feed composition of the present invention is less than 10 ppm, such as less than 9 ppm, less than 8 ppm or less than 7 ppm. In some embodiments, the chlorine concentration is greater than 0 ppm.

[0284] <Use form>

[0285] The utilization form of the composition of the present invention is not particularly limited, and is preferably an oral dosage form. Typically, it can be made into the form of oral preparations such as granules, tablets, capsules, and liquids. The uses of the composition of the present invention include, for example, food and beverages (health foods, nutritional supplements, special health foods, supplements, dairy products, refreshing drinks, pet food and beverages, livestock feeds, etc.), pharmaceuticals, quasi-drugs, etc., and are particularly preferably supplements and pharmaceuticals. In addition to food materials or food, it can also be used as an additive for animal feed. Therefore, the composition of the present invention can be used as a material or active ingredient for these food and beverages, pharmaceuticals, and quasi-drugs, and can be preferably used in their manufacture.

[0286] Examples of the present invention are described below, but the present invention is not limited to these examples.

[0287] It should be noted that, in the examples, unless otherwise stated, all % means weight %. Furthermore, unless otherwise stated, ppm means weight ppm (i.e., mg / kg).

[0288] In the examples, the 3-MCPD concentration means the measured value obtained by the AOCS official method Cd29b-13 analysis A. In addition, a 3-MCPD concentration of 0.00 ppm means that 3-MCPD was not detected in the above analysis method (i.e., below the detection limit).

[0289] In the examples, the iron concentration of 0.00 ppm means that no iron was detected in the above-mentioned measurement using ICP-MS (ie, below the detection limit).

[0290] Example

[0291] [Experiment 1] Effect of iron content on the formation of 3-MCPD

[0292] The oil obtained by deacidifying sardine crude oil by short-path distillation was ethyl esterified with an alkaline catalyst and then subjected to silica gel purification to separate the ethyl ester component. For silica gel purification, a glass hollow column filled with Microsphere gel D-75-60A (AGC Si-Tech Co., Ltd.) 5 times the amount of the sample was used, and hexane / ethyl acetate (50:1) was used as the eluent.

[0293] Regarding the ethyl ester fraction (fish oil ethyl ester (EE)), it was confirmed that no bands of DAG and MAG were detected by thin layer chromatography (TLC). In addition, as a result of measuring the iron concentration, no iron was detected in the ethyl ester fraction.

[0294] To the fish oil EE, 1000 ppm of monomyristyl glyceride (Wako Pure Chemical Industries, Ltd., product code 321-32412) was added. Furthermore, an aqueous solution of iron (II) sulfate 7 hydrate was added so that the iron content was 0.10 ppm (Example 2) or 1.00 ppm (Comparative Example 1), or no addition (Example 1), and then ethanol was added and homogenized. Then, the solvent was completely removed by evaporation and vacuum.

[0295] Each test area was stirred in an oil bath at 210° C. under a nitrogen flow, and samples were taken over time to measure the 3-MCPD concentration. Table 1 shows the change in 3-MCPD concentration during heating of each test area.

[0296] [Table 1]

[0297] Table 1

[0298]

[0299] In Comparative Example 1, to which 1.00 ppm of iron was added, 0.61 ppm of 3-MCPD was generated after heating for 4 hours. On the other hand, in Examples 1 and 2, in which the iron concentrations were adjusted to 0.00 ppm and 0.10 ppm, respectively, the 3-MCPD contents were 0.08 ppm and 0.15 ppm even after heating for 4 hours, which were very low values ​​compared to 0.61 ppm in Comparative Example 1.

[0300] [Experiment 2] Effect of MAG content on the formation of 3-MCPD

[0301] Fish oil EE prepared in the same manner as in Experiment 1 (iron concentration adjusted to 10 ppm) was directly heated at 120°C for 1 hour under a nitrogen flow, or monomyristyl glyceryl (Wako Pure Chemical Industries, Ltd., product code 321-32412) was added at a concentration of 1 to 10% and heated at 120°C for 1 hour under a nitrogen flow.

[0302] The 3-MCPD concentration after heating is shown in Table 2. It can be seen that, in general, even at a relatively low temperature of 120° C. used in the distillation removal of fatty acid ethyl esters by molecular distillation, as the MAG concentration increases, the 3-MCPD concentration increases.

[0303] [Table 2]

[0304] Table 2

[0305]

[0306] [Experiment 3] Effect of MAG content on the formation of 3-MCPD in fish oil ethyl ester distillate

[0307] Fish oil containing 20 area % EPA was ethyl esterified using an alkaline catalyst according to a conventional method to prepare fish oil ethyl ester 1. In fish oil ethyl ester 1, the ratio of EPA in the fatty acid composition was 20 area %, DAG and MAG containing C14:0 as constituent fatty acids were contained at the concentrations shown in Table 3, the iron concentration was 0.2 ppm, and the chlorine concentration was 17 ppm.

[0308] [Table 3]

[0309] Table 3 DAG and MAG content in fish oil ethyl ester 1 [mg / kg]

[0310]

[0311] Next, fish oil ethyl ester 2 obtained by removing MAG and DAG from fish oil ethyl ester 1, fish oil ethyl ester 3 obtained by adding monomyristyl glyceryl to fish oil ethyl ester 2, and fish oil ethyl ester 4 obtained by adding monopalmityl glyceryl to fish oil ethyl ester 2 were prepared.

[0312] Fish oil ethyl ester 2 was prepared by the following method.

[0313] 600 g of fish oil ethyl ester 1 was mixed with 2400 mL of hexane, and the resulting mixture was passed through a column obtained by filling 1200 g of silica gel (Microsphere D75-60A) with a slurry of hexane, so that the fish oil ethyl ester was adsorbed on the silica gel. Then, ethyl acetate / hexane (1:50) was passed through the column, and the eluent was separated to recover the component after MAG and DAG were removed from the fish oil ethyl ester. The solvent was removed from the recovered component by using an evaporator and vacuuming to obtain 585 g of fish oil ethyl ester free of MAG and DAG. The fish oil ethyl ester 2 obtained by the above operation was completely free of MAG and DAG, and the iron concentration was 0.05 ppm and the chlorine concentration was 7 ppm.

[0314] 0.1 g of monomyristic acid glyceryl was mixed with 100 g of fish oil ethyl ester 2, and the mixture was completely dissolved and homogenized to prepare fish oil ethyl ester 3. Furthermore, 0.1 g of monopalmitic acid glyceryl (Tokyo Chemical Industry, product code G0083) was mixed with 100 g of fish oil ethyl ester 2, and the mixture was completely dissolved and homogenized to prepare fish oil ethyl ester 4.

[0315] 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.

[0316] The first precision distillation step is a step to remove components below C18. The fractionation tube uses a fractionation tube with a vacuum jacket ( Kiriyama Glass), 5 Sulzer Lab packing EX (25mm×50mm, Sulzer Chemtech) were used as internal fillers. The liquid temperature in the bottom kettle (tower bottom temperature) was set to below 185°C, the vapor temperature at the top of the tower (tower top temperature) was set to below 135°C, the pressure before the vacuum pump (tower top pressure, i.e., vacuum degree) was set to below 30Pa, and the heating time was 4.0 hours for precision distillation. Through this first precision distillation step, the components below C18 were removed as the primary distillation part, and the primary distillation removal residue in which EPA was concentrated was obtained.

[0317] Then, in the second precision distillation step, the residual fraction obtained in the first precision distillation step is removed and the following precision distillation is performed. The fractionation tube uses a fractionation tube with a vacuum jacket ( Kiriyama Glass), and five Sulzer Lab Packing EX (25 mm × 50 mm, SulzerChemtech) were used as internal fillers. The liquid temperature in the bottom kettle (tower bottom temperature) was set to 195°C, the vapor temperature at the top of the tower (tower top temperature) was set to 150°C, the pressure before the vacuum pump (tower top pressure, i.e., vacuum degree) was set to 30 Pa, and the heating time was 3.5 hours for precision distillation. Through this second precision distillation step, the components above C22 were removed as the residue (residue fraction) to obtain the main distillation fraction.

[0318] Using 80g of fish oil ethyl ester 3 as raw material, 26g of EPA was concentrated in the primary distillation residue through the first precision distillation step. Using 25g of the obtained primary distillation residue as raw material, 11g of EPA was concentrated in the main distillation part through the second precision distillation step. In the main distillation part, EPA was concentrated, 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, the isomer formation caused by heating during distillation was also confirmed, and 0.8 area% of the trans isomer of EPA ethyl ester was confirmed in the main distillate (the total value of 5 trans isomers. Not shown in Table 4. The same applies below.).

[0319] The 3-MCPD concentration was 0.00 ppm in the fish oil ethyl ester 3 as the raw material, but increased to 0.01 ppm in the main fraction after distillation.

[0320] Using 77.8 g of fish oil ethyl ester 4 as a raw material, a primary distillation residue of 29.5 g of EPA was obtained by the first precision distillation step. Using 26.4 g of the primary distillation residue as a raw material, a main distillation fraction of 12.6 g of EPA was obtained by the second precision distillation step. In the main distillation fraction, EPA was concentrated, 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 caused by heating during distillation was also confirmed, and 1.6 area % of the trans isomer of EPA ethyl ester was confirmed in the main distillation.

[0321] The 3-MCPD concentration in the main fraction was 0.00 ppm, and no increase in concentration was observed compared with the fish oil ethyl ester 4 used as the raw material.

[0322] [Table 4]

[0323]

[0324] The distribution of 3-MCPD in each fraction of fish oil ethyl ester 3 and 4 was investigated. Table 5 shows the material balance of 3-MCPD contained in the main fraction and the main fraction residue.

[0325] [Table 5]

[0326]

[0327] It can be seen that the 3-MCPD concentration in the main distillate is strongly affected by the specific MAG in the raw material. In particular, in the main distillate from which EPA, i.e., C20 fatty acid components, is separated, when MAG containing C14:0, i.e., C14 fatty acid as a constituent fatty acid is added to fish oil ethyl ester 3, 28.0% of the total 3-MCPD is contained, and when MAG containing C16:0, i.e., C16 fatty acid as a constituent fatty acid is added to fish oil ethyl ester 4, all 3-MCPD is contained in the main distillate residue. According to this result, it is suggested that when C20 fatty acid ethyl ester containing EPA ethyl ester is recovered as the main distillate, 3-MCPD fatty acid ester formed by MAG containing C14 fatty acid as a constituent fatty acid will be mixed into the main distillate, but 3-MCPD fatty acid ester formed by MAG containing C16 fatty acid as a constituent fatty acid will hardly be mixed into the main distillate. That is, it can be considered that when the highly unsaturated fatty acid as the refining target is C20-PUFA such as EPA, C14 saturated fatty acid strongly affects the 3-MCPD concentration in the distillate.

[0328] It should be noted that in each test, the primary fraction did not contain 3-MCPD and 3-MCPD fatty acid esters.

[0329] Industrial Availability

[0330] According to the present invention, a composition having a low concentration of 3-MCPD fatty acid ester and containing a high concentration of PUFA alkyl ester can be stably produced.

[0331] With respect to all publications, patent applications, patents and other documents mentioned in this specification, if each publication, patent application, patent or other document is incorporated by reference in its entirety into this specification, it is incorporated by reference into this specification in the manner specifically and individually indicated. With respect to definitions contained in articles incorporated by reference, they are excluded only to the extent that they conflict with definitions in this disclosure.

[0332] Other embodiments are described in the appended claims.

Claims

1. A composition which is a distillation raw material composition containing highly unsaturated fatty acid alkyl esters, The highly unsaturated fatty acid alkyl ester contains the highly unsaturated fatty acid alkyl ester as the concentration target, and the iron concentration is less than 0.20 ppm.

2. The composition according to claim 1, wherein the concentration of monoacylglycerol containing fatty acids having a carbon number less than 5 or more than the carbon number of the highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated as constituent fatty acids is less than 10,000 ppm.

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

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

5. The composition according to claim 1, wherein the highly unsaturated fatty acid alkyl ester to be concentrated is an alkyl ester of eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid or arachidonic acid, or a combination thereof.

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

7. A method for producing a composition containing highly unsaturated fatty acids or alkyl esters thereof, comprising: (1) a step of alkyl-esterifying a raw material containing triglycerides containing highly unsaturated fatty acids as constituent fatty acids to prepare a composition containing highly unsaturated fatty acid alkyl esters, (2) a step (b) of reducing the iron concentration in the composition of step (1) to less than 0.20 ppm, and (3) A step of distilling the composition after step (2) to separate a main fraction.

8. The method according to claim 7, wherein the step (2) further comprises at least one of the following steps (a) and (c): (a) a step of reducing the concentration of monoacylglycerol containing, as a constituent fatty acid, fatty acids having a carbon number less than 5 or more than the carbon number of the highly unsaturated fatty acid constituting the highly unsaturated fatty acid alkyl ester to be concentrated, in the composition of step (1) to less than 10,000 ppm; (c) A step of reducing the chlorine concentration in the composition of step (1) to less than 10 ppm.

9. The method according to claim 7, wherein the concentration of 3-MCPD produced when the main fraction in step (3) is analyzed by the official method Cd29b-13 analysis A of the American Oil Chemists Association is less than 1.80 ppm.

10. The method according to claim 8, wherein step (2) (a) is performed by silica gel chromatography.

11. The method according to claim 7, wherein the distillation in step (3) is rectification.

12. The method according to claim 8, wherein in step (2), the concentration of monoacylglycerol containing fatty acids having six fewer carbon atoms than the carbon atoms of highly unsaturated fatty acids constituting the highly unsaturated fatty acid alkyl esters to be concentrated is reduced to less than 10,000 ppm.

13. The method according to claim 7, wherein the highly unsaturated fatty acid is eicosapentaenoic acid, docosahexaenoic acid, dihomo-γ-linolenic acid, arachidonic acid or a combination thereof.

14. The method according to any one of claims 7 to 13, wherein the raw material is fish oil, microbial oil, vegetable oil or marine animal oil.

15. The method according to claim 7, wherein the ratio of highly unsaturated fatty acids in the constituent fatty acids of the composition is 70 area % or more.

16. The method of claim 7 or 15, wherein the composition, when analyzed using the American Oil Chemists Association Official Method Cd29b-13 Analysis A, produces a 3-MCPD concentration below the detection limit.

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