Method for producing composition containing eicosapentaenoic acid alkyl ester

Abstract

The present invention provides a method for producing high-purity EPA. A method for producing a composition containing EPA alkyl esters, wherein the composition containing EPA alkyl esters contains 96% by mass or more of EPA alkyl esters in total fatty acids, and the ratio of trans isomers in the EPA alkyl esters is 1.5% by mass or less. The method comprises: (1) preparing a feedstock oil containing 50 to 92% by mass of EPA alkyl esters in total fatty acids, wherein the mass ratio of DHA acid alkyl esters to EPA alkyl esters contained in the feedstock oil is 3.3% by mass or less, and the ratio of trans isomers in the EPA alkyl esters contained in the feedstock oil is 2% by mass or less; (2) contacting the feedstock oil with an aqueous solution containing a silver salt; (3) recovering the aqueous solution containing the silver salt that has been in contact with the feedstock oil; (4) contacting the recovered aqueous solution containing the silver salt with an organic solvent; and (5) recovering the organic solvent that has been in contact with the aqueous solution containing the silver salt.

Description

Technical Field

[0001] The present invention relates to a method for producing a composition containing eicosapentaenoic acid alkyl ester. Background Art

[0002] As one of the highly unsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) has been used as a raw material for medicines or health foods because its pharmacological effects have been clarified in recent years. EPA used as a raw material for medicines or foods is required to be high-purity and free of impurities as much as possible. Despite this, since PUFAs such as EPA have multiple double bonds, it is not easy to obtain them by chemical synthesis. Most of the EPA used in industry is produced by extracting (extracting) or purifying raw materials from marine organisms rich in EPA, such as fish oil. However, since the oil raw materials from organisms are mixtures of various fatty acids with different carbon atoms, the number or position of double bonds, and the composition ratio of stereoisomers, the target EPA content may not be high. In addition, EPA purified from raw materials from marine organisms will emit a strong fishy smell even if it contains trace amounts of impurities. Therefore, if it is not encapsulated or masking agents are added to suppress the odor, it will cause discomfort when ingested. Therefore, it has been required to selectively purify EPA from oil raw materials from organisms.

[0003] PUFAs exist in cis and trans isomers. Most PUFAs in living organisms are cis isomers, but during the purification stage from biological raw materials, they are sometimes converted from cis isomers to trans isomers by heating, etc. Therefore, PUFAs with a thermal history that have been industrially purified from biological raw materials contain a certain amount of trans isomers. However, there are reports that trans fatty acids increase health risks, especially increase LDL cholesterol levels, and increase the risk of cardiovascular diseases. In EPA used as a raw material for medicines or foods, it is required to reduce the content of trans fatty acids as much as possible.

[0004] Patent Documents 1 to 4 describe methods in which a raw material containing PUFA such as EPA is mixed with an aqueous solution containing a silver salt to form a complex of PUFA and the silver salt, and then the aqueous layer containing the complex is subjected to organic solvent extraction (Patent Document 1), complex dissociation under heating (Patent Document 2), permeation into a liquid membrane (Patent Document 3), or dilution, or addition of a complex dissociating agent or a silver ion reducing agent (Patent Document 4), thereby obtaining the PUFA or its ester. Patent Document 5 describes that a mixed solution of a raw material containing PUFA such as EPA and / or DHA and an aqueous solution containing a silver salt is brought into contact with an organic solvent while being maintained at a specific temperature, and then the aqueous phase is recovered, and then an organic solvent is added to the aqueous phase, and then the organic solvent phase is recovered, thereby selectively purifying EPA and / or DHA. However, in the methods described in Patent Documents 1 to 5, PUFA other than EPA also forms a complex with the silver salt when the aqueous solution containing the silver salt is treated, so that it is difficult to selectively purify EPA.

[0005] Patent Document 6 describes that a raw material containing PUFAs such as EPA and / or DHA is mixed with an aqueous solution containing a silver salt, an organic solvent is added to the obtained aqueous phase, and the obtained organic solvent phase is subjected to vacuum distillation to produce a composition containing EPA with a high concentration and a low trans-isomer content. Patent Document 7 describes a method in which a raw material containing PUFAs such as EPA and / or DHA is subjected to vacuum precision distillation to obtain a fraction having a content of EPA or its lower alcohol ester of 50% by weight or more and a content of PUFA or its lower alcohol ester other than EPA of 10% by weight or less, and the fraction is then mixed with an aqueous silver nitrate solution, the impurity components in the upper layer are removed, and then the fraction is mixed with an organic solvent, and the organic solvent is removed from the obtained organic solvent phase to purify high-purity EPA or its lower alcohol ester.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent document 1: Japanese Patent No. 3001954;

[0009] Patent document 2: Japanese Patent No. 2786748;

[0010] Patent document 3: Japanese Patent No. 2935555;

[0011] Patent document 4: Japanese Patent No. 2895258;

[0012] Patent document 5: Japanese Patent No. 6234908;

[0013] Patent Document 6: International Publication No. 2014 / 054435;

[0014] Patent document 7: Japanese Patent Application Laid-Open No. 7-242895. Summary of the invention

[0015] Problems to be solved by the invention

[0016] In the past, in the PUFA purification method using the formation of a complex between PUFA and a silver salt (so-called silver salt method) as described in Patent Documents 1 to 5, when the raw material oil and the aqueous solution containing the silver salt are mixed, PUFA other than EPA in the raw material oil also forms a complex at the same time, so it is difficult to obtain EPA with high purity. In addition, the PUFA purification method using distillation in combination with the silver salt method as described in Patent Documents 6 to 7 can produce a composition containing EPA at a higher concentration, but requires equipment for vacuum distillation, so the cost is high. Moreover, even in the methods described in Patent Documents 6 to 7, it is still difficult to separate EPA from other PUFAs such as arachidonic acid (AA) or eicosatetraenoic acid (ETA) having similar physical properties, and it is not easy to obtain a composition containing EPA at a high concentration and high purity. A method for producing a composition containing EPA at a high concentration and high purity that can be used as a raw material for medicines or health foods in a simpler and lower cost manner is required.

[0017] Means for solving problems

[0018] The present inventors have discovered that, in the method for purifying PUFA based on the formation of a complex between PUFA and a silver salt as described in Patent Document 1, a composition containing EPA at a high concentration and high purity can be obtained by using a fat raw material having a specific fatty acid composition in the raw oil treated with an aqueous solution containing a silver salt.

[0019] The present invention provides the following contents.

[0020] A method for producing a composition containing an eicosapentaenoic acid alkyl ester, the method comprising the following (1) to (5):

[0021] (1) preparing a feedstock oil containing 50 to 92% by mass of eicosapentaenoic acid alkyl ester in total fatty acids, wherein the mass ratio of docosahexaenoic acid alkyl ester to eicosapentaenoic acid alkyl ester contained in the feedstock oil is 3.3% by mass or less, and the ratio of trans isomers in the eicosapentaenoic acid alkyl ester contained in the feedstock oil is 2% by mass or less;

[0022] (2) contacting the raw oil with an aqueous solution containing a silver salt;

[0023] (3) recovering the aqueous solution containing the silver salt in contact with the crude oil;

[0024] (4) contacting the recovered aqueous solution containing the silver salt with an organic solvent; and

[0025] (5) by recovering the organic solvent in contact with the aqueous solution containing the silver salt, a composition containing an eicosapentaenoic acid alkyl ester is produced, wherein the composition contains 96% by mass or more of the eicosapentaenoic acid alkyl ester in the total fatty acids, and the ratio of the trans isomer in the eicosapentaenoic acid alkyl ester is 1.5% by mass or less.

[0026] [1] The method according to claim 1, wherein the content of the eicosapentaenoic acid alkyl ester in the feedstock oil is 60 to 85% by mass of the total fatty acids.

[0027] The method according to [1] or [2], wherein the content of docosahexaenoic acid alkyl ester in the feedstock oil is 2% by mass or less in the total fatty acids.

[0028] The method according to any one of [1] to [3], wherein the total content of arachidonic acid alkyl ester and eicosatetraenoic acid alkyl ester in the feedstock oil is 26% by mass or less relative to 100% by mass of eicosapentaenoic acid alkyl ester.

[0029] The method described in any one of [1] to [4] further comprises (3'): contacting the raw oil after the aqueous solution containing silver salt is recovered in the above (3) with a newly prepared aqueous solution containing silver salt, and then recovering the aqueous solution containing silver salt that has been in contact with the raw oil.

[0030] [5] The method described in claim 1, wherein the above (4) comprises: contacting the aqueous solution containing the silver salt recovered in the above (3) and (3') with an organic solvent.

[0031] The method according to any one of [1] to [6], wherein the temperature of the aqueous solution containing the silver salt when contacting the feedstock oil is 5 to 30°C.

[0032] The method according to any one of [1] to [7], wherein the temperature of the aqueous solution containing the silver salt when contacting with the organic solvent is 30 to 80°C.

[0033] Effects of the Invention

[0034] According to the method of the present invention, it is possible to obtain a composition which has a low content of PUFA other than EPA, a low ratio of trans isomers, and contains EPA at a high concentration and high purity. DETAILED DESCRIPTION

[0035] The present invention provides a method for producing a composition containing EPA alkyl esters. The method of the present invention comprises: (1) preparing a raw material oil containing EPA alkyl esters; (2) contacting the raw material oil with an aqueous solution containing a silver salt; (3) recovering the aqueous solution containing the silver salt that has been in contact with the raw material oil; (4) contacting the recovered aqueous solution containing the silver salt with an organic solvent; and (5) recovering the organic solvent that has been in contact with the aqueous solution containing the silver salt.

[0036] The raw material oil used in the method for producing the composition containing EPA alkyl ester of the present invention is an oil composition mainly containing EPA among the fatty acids contained. The raw material oil can be prepared from oils and fats derived from organisms containing EPA. Examples of the oils and fats derived from organisms include oils and fats derived from marine animals such as fish or plankton, oils and fats derived from microorganisms such as algae, and the like. Among them, preferred examples include oils and fats derived from fish such as sardines, mackerel, and tuna, and oils and fats derived from algae. The oils and fats derived from these organisms mainly contain fatty acids in the form of triglycerides in which three molecules of fatty acids are bonded to one molecule of glycerol. In addition, although the amount is small, diglycerides or monoglycerides, and free fatty acids may also be contained.

[0037] Methods for preparing the above-mentioned raw oil from organisms containing EPA include: a method of heating and pressing the organisms containing EPA to extract oil; and a method of further subjecting the extracted oil to urea treatment, chromatography, distillation, supercritical treatment, etc. to separate the oil components.

[0038] The PUFA in the feedstock oil is converted into alkyl esters. By converting the PUFA in the feedstock oil into alkyl esters, the target EPA can be efficiently separated from other PUFAs other than EPA in the production method of the present invention. As the alkyl group constituting the alkyl ester of the PUFA, there can be listed: a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a methyl group or an ethyl group, and more preferably an ethyl group. The alkyl ester of the PUFA can be produced by subjecting a fat containing PUFA and an alcohol having a desired alkyl group to an esterification reaction using a known method. For example, the alkyl ester of PUFA can be obtained by reacting PUFA triglycerides contained in the fat with a lower alcohol in the presence of a catalyst or an enzyme to form an alkyl ester.

[0039] In the total fatty acids of the feedstock oil, the content of EPA alkyl esters in the feedstock oil is 50 to 92 mass%, preferably 50 to 85 mass%, more preferably 50 to 75 mass%, further preferably 60 to 85 mass%, further preferably 70 to 85 mass%, further preferably 75 to 85 mass%. By using a feedstock oil having a content of EPA alkyl esters of 50 mass% or more in the total fatty acids, a composition containing EPA at a high concentration (96 mass% or more in the total fatty acids) can be efficiently obtained. On the other hand, from the perspective of the cost of the feedstock oil and the perspective of keeping the trans isomer ratio in the feedstock oil at a low level as described later, the content of EPA alkyl esters in the feedstock oil can be as low as 92 mass% or less, and more preferably 85 mass% or less.

[0040] The stock oil mainly contains EPA, and may also contain other PUFAs other than EPA. As the other PUFA, docosahexaenoic acid (DHA), arachidonic acid (AA), eicosatetraenoic acid (ETA), etc. are listed. Preferably, the content of PUFA alkyl esters other than EPA, such as DHA, AA, and ETA in the stock oil is low. In more detail, the mass ratio of DHA alkyl esters to EPA alkyl esters in the stock oil relative to 100% by mass of EPA alkyl esters is as long as it is 3.3% by mass or less, preferably 2.7% by mass or less, more preferably 2.0% by mass or less, and further preferably 1.0% by mass or less. Preferably, the content of DHA alkyl esters in the stock oil is less than 2% by mass of the total fatty acids in the stock oil, and more preferably 1.5% by mass or less. In addition, it is preferred that the ratio of the total content of AA alkyl esters and ETA alkyl esters in the stock oil to the content of EPA alkyl esters is less than 26% by mass, more preferably less than 15% by mass, and further preferably less than 11% by mass, relative to 100% by mass of EPA alkyl esters.

[0041] The above-mentioned raw material oil containing high concentration of EPA can be prepared by heating the above-mentioned organisms containing EPA, such as marine animals such as fish, and squeezing them, and then further separating the oil components from the obtained oil-containing product by urea treatment, chromatography treatment, distillation treatment, supercritical treatment, etc. Preferably, the raw material oil used in the present invention is a raw material oil prepared by a method including heat treatment such as distillation.

[0042] The heat treatment during the stock oil modulation process as described above sometimes generates unpreferable trans isomers in the resulting stock oil. The method of the present invention is effective for removing trans isomers generated during the modulation process of such stock oil. The stock oil used in the method of the present invention may contain trans isomers of EPA alkyl esters. The ratio of trans isomers in the EPA alkyl esters contained in the stock oil is preferably 2% by mass or less, more preferably 1.5% by mass or less, and further preferably 1.0% by mass or less. By using a stock oil in which the trans isomer ratio of EPA is 2% by mass or less, a composition containing EPA at a high concentration (more than 96% by mass of the total fatty acids contained) and in which the ratio of trans isomers in EPA is reduced compared to the stock oil and is 1.5% by mass or less can be efficiently obtained.

[0043] In the feedstock oil used in the production method of the present invention, commercially available fats and oils can be used as long as the content of EPA alkyl esters, the content of other PUFA alkyl esters, or the content of trans isomers are within the above ranges. From the viewpoint of cost and stabilization of the target EPA yield, it is preferred to use commercially available fish oil-derived fats and oils in which the types or amounts of PUFAs contained are standardized.

[0044] From the viewpoint of maintaining the quality of the produced EPA alkyl ester-containing composition and preventing the deterioration of the aqueous solution containing the silver salt, the feedstock oil used in the production method of the present invention preferably has a low oxidation index. The oxidation index of lipids can be represented by peroxide value (POV), acid value (AV), etc. The POV (mEq / kg) of the feedstock oil used in the present invention is preferably 10 or less, more preferably 5 or less, or the AV (mg / g) is preferably 0.3 or less, more preferably 0.2 or less. It is further preferred that the POV of the feedstock oil is 10 or less and the AV is 0.3 or less, and it should be noted that the POV is preferably 5 or less and the AV is 0.2 or less. POV can be measured by iodine titration (ISO 3960: 2007) or the like. AV can be measured by potassium hydroxide titration (ISO 660: 2009) or the like.

[0045] The mass of each constituent fatty acid in the total fatty acids in the fats and oils can be measured by gas chromatography (for example, the method described in Reference Example 1 described later).

[0046] In the method for producing a composition containing EPA alkyl esters of the present invention, the above-mentioned raw material oil is preferably used in liquid form (liquid state). When the raw material oil is in liquid form at the reaction temperature of each step, it can be directly used in each step of the method of the present invention. When the raw material oil is in solid form (solid state) at the reaction temperature in each step, the raw material oil can be appropriately dissolved or diluted in an organic solvent or other oil for use. As the organic solvent, an organic solvent that can be separated from water is used in order to smoothly carry out the following steps (2) to (3), for example, ethyl acetate, chloroform, carbon tetrachloride, diethyl ether, hexane, cyclohexane, etc.

[0047] In the method for producing a composition containing EPA alkyl esters of the present invention, first, (1) a feedstock oil containing EPA alkyl esters and other PUFA alkyl esters in specific amounts as described above is prepared. Preferably, the feedstock oil of such a composition is a feedstock oil having a thermal history prepared by the method including heat treatment as described above. Then, the feedstock oil prepared in (1) is subjected to the following steps (2) and (3):

[0048] (2) contacting the feedstock oil with an aqueous solution containing a silver salt (hereinafter also referred to as a silver salt solution);

[0049] (3) Recovering the silver salt solution in contact with the crude oil.

[0050] Steps (2) and (3) are steps for separating EPA alkyl esters from the feedstock oil by utilizing the change in the solubility of PUFA alkyl esters containing EPA in the extraction solvent by forming a complex with the carbon-carbon double bond of PUFA by a silver salt. This step can be carried out, for example, according to the methods described in Patent Documents 1 to 6.

[0051] The silver salt is not particularly limited as long as it can form a complex with the unsaturated bond of PUFA, and examples thereof include silver nitrate, silver perchlorate, silver tetrafluoroborate, silver acetate, etc. Among them, silver nitrate is preferred. As the solvent of the silver salt solution, water or a mixed medium of water and a compound having a hydroxyl group such as glycerol or ethylene glycol can be mentioned, and water is preferably used. The concentration of the silver salt in the silver salt solution can be 0.1 mol / L or more, preferably about 1 to 20 mol / L. The molar ratio of PUFA to silver salt in the feedstock oil can be 1:100 to 100:1, preferably about 1:5 to 1:1. The number of moles of PUFA in the feedstock oil can be calculated by dividing the total mass of PUFA in the feedstock oil by the average molecular weight of PUFA. The average molecular weight of PUFA can be calculated from the molecular weight of each PUFA and the composition ratio in the feedstock oil. The total mass and composition ratio of PUFA in the feedstock oil can be calculated by gas chromatography (for example, under the conditions described in Reference Example 1 described later).

[0052] In the above (2), there is no particular limitation on the method for contacting the stock oil with the silver salt solution. For example, the stock oil and the silver salt solution may be put into a reaction tank separately and contacted in the reaction tank, or the pre-mixed stock oil and the silver salt solution may be put into the reaction tank and contacted in the reaction tank. Alternatively, according to the method described in WO2017 / 191821, droplets of the silver salt solution are passed through the stock oil filling the reaction tank, so that the stock oil and the silver salt solution can be contacted.

[0053] In the above (2), the temperature of the silver salt solution when in contact with the stock oil (the reaction temperature when the stock oil and the silver salt solution are in contact) is preferably about 80°C or less and 5°C or more, more preferably 5 to 30°C, and even more preferably 15 to 30°C. As a method for maintaining the reaction temperature in the above range, there can be mentioned: a method of heating or cooling the stock oil and / or the silver salt solution to the above range before bringing them into contact; a method of maintaining the temperature of the reaction tank for bringing the stock oil and the silver salt solution into contact in the above range; and a combination of these methods. In the above (2), the contact time between the stock oil and the silver salt solution is preferably 5 minutes to 4 hours, and more preferably 10 minutes to 2 hours.

[0054] By contacting the raw material oil with the silver salt solution, a complex of PUFA and silver (also referred to as PUFA-silver complex in this specification) is generated in the silver salt solution. The formed complex moves to the water layer, i.e., the silver salt solution. Then, in the above (3), by recovering the silver salt solution in contact with the raw material oil, a solution containing the PUFA-silver complex can be obtained.

[0055] The contact between the stock oil and the silver salt solution in (2) and (3) above, and the recovery of the silver salt solution after the contact can be carried out in an intermittent manner or in a continuous manner. For example, in an intermittent manner, the silver salt solution can be added to the stock oil containing EPA alkyl ester, and the stock oil and the silver salt solution can be brought into contact while stirring, and then the mixed solution can be left to stand to separate the water layer and recover it. In a continuous manner, for example, the silver salt solution can be continuously added to the reaction tank while allowing droplets of the silver salt solution to pass through the stock oil filling the reaction tank, and on the other hand, the silver salt solution after passing through can be continuously recovered, or accumulated and recovered. In another example of the continuous method, a flow path type agitator is used as the reaction tank. The stock oil and the silver salt solution can be added from one port of the agitator, and they can be transferred to another port while stirring and transported to a separation tank, and the water layer can be separated in the separation tank and recovered.

[0056] Preferably, the production method of the present invention further comprises the following step (3') after the above step (3): contacting the stock oil after the silver salt solution is recovered in the above step (3) with a new silver salt solution, and then recovering the silver salt solution in contact with the stock oil. The stock oil separated from the silver salt solution in the above step (3) may still contain EPA that has not formed a complex. Therefore, by contacting the separated stock oil with a new silver salt solution, the PUFA remaining in the stock oil can be converted into a PUFA-silver complex, so that the recovery rate of EPA (the ratio of the amount of EPA recovered to the amount of EPA in the stock oil) is improved. In the above step (3'), as the new silver salt solution in contact with the stock oil, a newly prepared silver salt solution can be used, or the silver salt solution after PUFA is recovered in the step (5) described later can be washed and used as needed. The conditions (temperature, etc.) when the stock oil is in contact with the silver salt solution can be appropriately set within the range of the conditions described in the above step (2). The above step (3') can be repeated two or more times. In this case, the raw oil separated from the silver salt solution in the first step (3') is supplied to the second step (3'), and this operation is repeated as needed. The raw oil used in the step (3') preferably contains 5% by mass or more of EPA alkyl esters in the total fatty acids. The number of times of the step (3') can be appropriately determined according to the content of EPA alkyl esters in the raw oil. The silver salt solution recovered in the step (3') can be combined with the silver salt solution containing PUFA-silver complex recovered separately and supplied to the extraction step of PUFA alkyl esters described later (the following (4) and (5)), or it can be supplied to the extraction step alone.

[0057] The silver salt solution containing the PUFA-silver complex recovered above is used in the extraction step of PUFA alkyl ester ((4) and (5) described below). If necessary, the silver salt solution containing the PUFA-silver complex recovered may be washed with an organic solvent before being used in the extraction step. In the washing, the recovered silver salt solution may be brought into contact with an organic solvent, preferably stirred, and then the aqueous layer may be separated and recovered. The organic solvent used for the washing may be any organic solvent that can be used to dissolve the feedstock oil. The washing conditions (temperature, etc.) may be appropriately set within the range of the conditions described in relation to (2) above.

[0058] In the method for producing a composition containing EPA alkyl esters of the present invention, after recovering the silver salt solution containing the PUFA-silver complex, the following (4) and (5) are performed:

[0059] (4) contacting the recovered silver salt solution with an organic solvent;

[0060] (5) Recovering the organic solvent in contact with the silver salt solution.

[0061] Steps (4) and (5) are steps of adding an organic solvent to the silver salt solution containing the PUFA-silver complex recovered in step (3), extracting the EPA alkyl ester in the solution into the organic solvent, and then recovering the organic solvent containing the EPA alkyl ester. This step can be carried out, for example, according to the methods described in Patent Documents 1 and 5.

[0062] Examples of the organic solvent used in (4) above include hexane, diethyl ether, ethyl acetate, butyl acetate, chloroform, cyclohexane, benzene, toluene, xylene, and other solvents that have high solubility in PUFA and can be separated from water. Among them, hexane or cyclohexane is preferred. The amount of the silver salt solution and the organic solvent is preferably 100:3 to 300, more preferably 100:3 to 200 in volume ratio.

[0063] The method for contacting the organic solvent with the silver salt solution is not particularly limited. For example, the silver salt solution recovered from the reaction tank is transported to an extraction tank for contacting the silver salt solution with the organic solvent, where it is contacted with the organic solvent. By contacting the organic solvent with the silver salt solution, the PUFA alkyl ester in the silver salt solution is transferred to the organic solvent. Then, the organic solvent contacted with the silver salt solution is recovered in the above (5), thereby obtaining an organic solvent containing the target EPA alkyl ester.

[0064] The temperature of the silver salt solution when in contact with the organic solvent (the reaction temperature when the organic solvent and the silver salt solution are in contact) is preferably higher than the reaction temperature in (2), i.e., the formation temperature of the PUFA-silver complex. It is more preferred that the temperature be 20°C or higher than the reaction temperature in (2), i.e., the formation temperature of the complex. For example, the reaction temperature in step (4) is preferably 30 to 80°C, more preferably 50 to 70°C. As methods for maintaining the reaction temperature in the above range, there can be cited: a method of heating the silver salt solution and / or the organic solvent to the above range and then bringing them into contact, a method of maintaining the temperature of the extraction tank in the above range, and a combination of these methods. In (4), the contact time of the organic solvent and the silver salt solution is preferably 5 minutes to 4 hours, more preferably 10 minutes to 2 hours, and even more preferably 30 minutes to 2 hours.

[0065] The contact of the organic solvent with the silver salt solution in (4) and (5) above, and the recovery of the organic solvent after the contact can be carried out in an intermittent manner or in a continuous manner. For example, in an intermittent manner, the silver salt solution can be added to the organic solvent, the organic solvent and the silver salt solution can be brought into contact while stirring, and then the mixed solution can be allowed to stand to separate the organic solvent layer and recover it. In an example of a continuous method, a flow-type stirrer is used as an extraction tank. The organic solvent and the silver salt solution can be added from one port of the stirrer, transferred to another port while stirring and transported to a separation tank, and the organic solvent layer can be separated in the separation tank and recovered.

[0066] The silver salt solution separated from the organic solvent in the above step may be contacted with the organic solvent again as needed. For example, the silver salt solution separated from the organic solvent in the above step and the additional silver salt solution recovered in the above step (3) as needed are added to the organic solvent. The organic solvent used here may be the organic solvent recovered in the above step, or a part or all of the new organic solvent may be used. The conditions (temperature, etc.) during the contact may be the same as those in the above step (4). This operation may be further repeated. By contacting the organic solvent with the silver salt solution again, PUFA alkyl esters are further extracted, so that more target EPA alkyl esters can be recovered.

[0067] Furthermore, the recovered organic solvent may be passed through an adsorbent such as silica gel, activated carbon, or silicon dioxide, thereby further removing residual silver ions.

[0068] It should be noted that the silver salt solution separated from the organic solvent in the above step may be contacted with the stock oil again. In the stock oil used here, as long as the content of EPA alkyl esters, the content of other PUFA alkyl esters or the content of trans isomers are within the above ranges, the stock oil separated from the silver salt solution in the above step may be reused, or a part or all of the new stock oil may be used. The conditions (temperature, etc.) during the contact may be the same as those in (2) above. This operation may be further repeated. By contacting the stock oil with the silver salt solution again, a large amount of silver salt solution containing PUFA-silver complex can be recovered.

[0069] The steps of the above production method of the present invention are preferably carried out under low oxygen conditions. Low oxygen conditions can be achieved, for example, by making the system of the method of the present invention (e.g., reaction tank, extraction tank, separation tank, their connecting paths, etc.) a closed system isolated from the outside air, keeping the inside of the system under a nitrogen environment, and filling the inside of the system with liquid (raw oil, silver salt solution, or organic solvent). It is preferred that the system be a closed system isolated from the outside air and filled with raw oil or silver salt solution. In the case where the process in the reaction tank and the extraction tank is carried out in a continuous manner, if the system is temporarily filled with liquid, the low oxygen state can be maintained afterwards. It is preferred that the low oxygen conditions in this specification refer to conditions where the oxygen concentration is less than 0.4%, more preferably less than 0.1%. In addition, it is preferred that the method of the present invention is carried out under light protection. By carrying out the method of the present invention under low oxygen conditions and light protection, the pH drop of the silver salt solution can be suppressed, or the oxidation of fats and oils in the raw oil, silver salt solution and organic solvent can be suppressed, and the deterioration of the silver salt solution or the deterioration of the purified PUFA-containing composition can be prevented.

[0070] In the method for producing a composition containing EPA alkyl esters of the present invention, the organic solvent recovered in the above (5) contains EPA alkyl esters separated from the feedstock oil. Therefore, the recovered organic solvent can be obtained in the form of a composition containing EPA alkyl esters. If necessary, the recovered organic solvent can be further purified by concentration, chromatography, distillation, etc. to prepare a composition containing EPA alkyl esters at a higher concentration. It should be noted that the organic solvent after the EPA alkyl esters are separated by purification can be reused for the extraction of EPA alkyl esters in the above (4).

[0071] The composition containing EPA alkyl ester obtained by the production method of the present invention contains 96% by mass or more of EPA alkyl esters in the total fatty acids contained, and the ratio of trans isomers in the EPA alkyl esters is 1.5% by mass or less, preferably 1% by mass or less. In addition, according to the production method of the present invention, an EPA recovery rate of 90% or more, preferably 95% or more, can be achieved. Therefore, according to the production method of the present invention, a composition containing high-purity EPA with a trans isomer ratio of 1.5% or less at a high concentration of 96% by mass or more can be produced with a high yield. Example

[0072] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0073] (Reference Example 1) Analysis of fatty acid composition ratio

[0074] 12.5 mg of the sample was diluted in 1 mL of n-hexane and analyzed for the content ratio of each fatty acid in the total fatty acids using a gas chromatograph (Type 7890GC; manufactured by Agilent Technologies) under the following conditions. The results are expressed as mass % converted from the area of ​​the chromatogram.

[0075] <Injection port conditions>

[0076] Inlet temperature: 250°C, split ratio: 10.

[0077] <Column conditions>

[0078] Column: DB-WAX 0.25 mm × 30 m manufactured by J&W;

[0079] Column temperature: 210°C;

[0080] He flow rate: 1.0 mL / min, He pressure: 20 PSI.

[0081] <Testing conditions>

[0082] H2 flow rate: 40mL / min, air flow rate: 450mL / min;

[0083] He flow rate: 1.00 mL / min, DET temperature: 260°C.

[0084] The fatty acids analyzed are as follows: EPA-E: eicosapentaenoic acid ethyl ester, DHA-E: docosahexaenoic acid ethyl ester, AA-E: arachidonic acid ethyl ester, ETA-E: eicosatetraenoic acid ethyl ester.

[0085] The EPA-E recovery rate was calculated by the following formula.

[0086] EPA-E recovery rate (%) = (mass of EPA-E in product (%) × mass of product) / (mass of EPA-E in feedstock oil (%) × mass of feedstock) × 100

[0087] (Crude Oil)

[0088] Feedstock oils 1 to 13 having the fatty acid compositions shown in Table 1 were prepared by distillation purification.

[0089]

[0090] (Example 1)

[0091] 30 g of the raw material oil 1 and 14 mL of cyclohexane were stirred and mixed thoroughly to dissolve. The obtained 41 g of solution and 150 g of silver salt solution (50% by mass silver nitrate aqueous solution) were added to a flask and stirred at 300 rpm for 20 minutes at 20°C under a nitrogen environment (oxygen concentration 0.4%). The stirred liquid was allowed to stand at 20°C for 15 minutes, and the separated organic layer and water layer (silver salt solution containing PUFA-silver complex) were recovered respectively. 150 g of the newly prepared silver salt solution was added to the recovered organic layer, and stirred at 300 rpm for 20 minutes at 20°C under a nitrogen environment (oxygen concentration 0.4%), and the stirred liquid was allowed to stand at 20°C for 15 minutes. The separated water layer was recovered, 15 mL of cyclohexane was added thereto, and stirred at 300 rpm for 20 minutes at 20°C. The stirred liquid was allowed to stand at 20°C for 15 minutes, and the separated water layer (silver salt solution containing PUFA-silver complex) was recovered. The two aqueous layers were mixed, 280 mL of cyclohexane was added, and the mixture was stirred at 300 rpm for 20 minutes at 60° C. to extract the PUFA ethyl ester in the aqueous layer into the cyclohexane. The liquid after stirring was allowed to stand, and the separated organic layer was recovered and then concentrated to obtain a composition containing PUFA ethyl ester.

[0092] (Example 2)

[0093] A composition containing PUFA ethyl ester was obtained by the same procedure as in Example 1 except that the raw material oil 2 was used.

[0094] (Example 3)

[0095] 30 g of the raw material oil 3 and 35 mL of cyclohexane were stirred and mixed thoroughly to dissolve. The obtained 57 g of solution and 380 g of silver salt solution (50% by mass silver nitrate aqueous solution) were added to a flask and stirred at 300 rpm for 20 minutes at 20°C under a nitrogen environment (oxygen concentration 0.4%). The stirred liquid was allowed to stand at 20°C for 15 minutes. The separated water layer was recovered, 15 mL of cyclohexane was added thereto, and the mixture was stirred at 300 rpm for 20 minutes at 20°C. The stirred liquid was allowed to stand at 20°C for 15 minutes, and the separated water layer (silver salt solution containing PUFA-silver complex) was recovered. The obtained water layer was heated to 60°C, 350 mL of cyclohexane was added, and the mixture was stirred at 300 rpm for 20 minutes at 60°C to extract the PUFA ethyl ester in the water layer into cyclohexane. The stirred liquid was allowed to stand, the separated organic layer was recovered, and then concentrated to obtain a composition containing PUFA ethyl ester.

[0096] (Example 4)

[0097] A composition containing PUFA ethyl ester was obtained by the same procedure as in Example 1 except that raw material oil 4 was used.

[0098] (Example 5)

[0099] 30 g of the raw material oil 11 and 14 mL of cyclohexane were stirred and mixed thoroughly to dissolve. The obtained 41 g of solution and 120 g of silver salt solution (50% by mass silver nitrate aqueous solution) were added to a flask and stirred at 300 rpm for 20 minutes at 20°C under a nitrogen environment (oxygen concentration 0.4%). The stirred liquid was allowed to stand at 20°C for 15 minutes, and the separated organic layer and water layer (silver salt solution containing PUFA-silver complex) were recovered respectively. 120 g of the newly prepared silver salt solution was added to the recovered organic layer, and stirred at 300 rpm for 20 minutes at 20°C under a nitrogen environment (oxygen concentration 0.4%), and the stirred liquid was allowed to stand at 20°C for 15 minutes. The separated water layer was recovered, 60 mL of cyclohexane was added thereto, and stirred at 300 rpm for 20 minutes at 20°C. The stirred liquid was allowed to stand at 20°C for 15 minutes, and the separated water layer (silver salt solution containing PUFA-silver complex) was recovered. The two aqueous layers were mixed, 450 mL of cyclohexane was added, and the mixture was stirred at 300 rpm for 20 minutes at 60° C. to extract the PUFA ethyl ester in the aqueous layer into the cyclohexane. The stirred liquid was allowed to stand, and the separated organic layer was recovered and then concentrated to obtain a composition containing PUFA ethyl ester.

[0100] (Example 6)

[0101] A composition containing PUFA ethyl ester was obtained by the same procedure as in Example 5 except that the raw material oil 12 was used.

[0102] (Example 7)

[0103] A composition containing PUFA ethyl ester was obtained by the same procedure as in Example 1 except that the raw material oil 13 was used.

[0104] (Comparative Example 1)

[0105] A composition containing PUFA ethyl ester was obtained by the same procedure as in Example 1 except that raw material oil 5 was used.

[0106] (Comparative Example 2)

[0107] A composition containing PUFA ethyl ester was obtained by the same procedure as in Example 3 except that raw material oil 6 was used.

[0108] (Comparative Example 3)

[0109] A composition containing PUFA ethyl ester was obtained by the same procedure as in Example 1 except that raw material oil 7 was used.

[0110] (Comparative Example 4)

[0111] 30 g of the raw material oil 8 and 70 mL of cyclohexane were stirred and mixed thoroughly to dissolve. The obtained 84 g of solution and 380 g of silver salt solution (50% by mass silver nitrate aqueous solution) were added to a flask and stirred at 300 rpm for 20 minutes at 20°C under a nitrogen environment (oxygen concentration 0.4%). The stirred liquid was allowed to stand at 20°C for 15 minutes. The separated water layer was recovered, 38 mL of cyclohexane was added thereto, and the mixture was stirred at 300 rpm for 20 minutes at 20°C. The stirred liquid was allowed to stand at 20°C for 15 minutes, and the separated water layer (silver salt solution containing PUFA-silver complex) was recovered. The obtained water layer was heated to 60°C, 350 mL of cyclohexane was added, and the mixture was stirred at 300 rpm for 20 minutes at 60°C to extract the PUFA ethyl ester in the water layer into cyclohexane. The stirred liquid was allowed to stand, the separated organic layer was recovered, and then concentrated to obtain a composition containing PUFA ethyl ester.

[0112] (Comparative Examples 5 to 6)

[0113] A composition containing PUFA ethyl ester was obtained by the same procedure as in Comparative Example 4 except that raw material oils 9 and 10 were used.

[0114] (Test 1)

[0115] The fatty acid composition of the compositions obtained in Examples 1 to 7 and Comparative Examples 1 to 6 was determined according to the procedure of Reference Example 1. The results are shown in Table 2. As shown in Table 2, when the EPA-E content in the feedstock oil is high, and the DHA-E / EPA-E ratio and the amount of EPA-E trans isomers are both low, a composition containing more than 96% by mass of EPA-E in the total fatty acids and the ratio of trans isomers in EPA-E being less than 1.5% by mass can be obtained. If the amount of EPA-E in the feedstock oil is too low, a product with a sufficiently high amount of EPA-E cannot be obtained. In addition, if the DHA-E / EPA-E ratio or the amount of EPA-E trans isomers increases, there is a tendency for the EPA-E recovery rate to decrease.

[0116]

Claims

1. A method for producing a composition containing an eicosapentaenoic acid alkyl ester, the method comprising the following (1) to (5): (1) A raw material oil containing 50 to 92% by mass of eicosapentaenoic acid alkyl ester in total fatty acids is prepared, wherein: The mass ratio of docosahexaenoic acid alkyl ester to eicosapentaenoic acid alkyl ester contained in the feed oil is 3.3 mass % or less, and the ratio of trans isomers in the eicosapentaenoic acid alkyl ester contained in the feed oil is 2 mass % or less; (2) contacting the feedstock oil with an aqueous solution containing a silver salt; (3) recovering the aqueous solution containing the silver salt that has been in contact with the crude oil; (4) contacting the recovered aqueous solution containing the silver salt with an organic solvent; and (5) recovering the organic solvent in contact with the aqueous solution containing the silver salt, thereby producing a composition containing an eicosapentaenoic acid alkyl ester, wherein the composition contains 96% by mass or more of the eicosapentaenoic acid alkyl ester in the total fatty acids, and the ratio of the trans isomer in the eicosapentaenoic acid alkyl ester is 1.5% by mass or less, The steps of the above production method are performed under low oxygen conditions.

2. The method of claim 1, wherein: The content of the eicosapentaenoic acid alkyl ester in the feedstock oil is 60 to 85% by mass of the total fatty acids.

3. The method of claim 1, wherein: The content of docosahexaenoic acid alkyl ester in the feedstock oil is 2% by mass or less in the total fatty acids.

4. The method of claim 1, wherein: The total content of arachidonic acid alkyl ester and eicosatetraenoic acid alkyl ester in the feedstock oil is 26% by mass or less relative to 100% by mass of eicosapentaenoic acid alkyl ester.

5. The method according to claim 1, further comprising (3'): contacting the raw oil after the aqueous solution containing silver salt is recovered in the above (3) with a newly prepared aqueous solution containing silver salt, and then recovering the aqueous solution containing silver salt that has been in contact with the raw oil.

6. The method of claim 5, wherein: The above (4) comprises: contacting the aqueous solution containing the silver salt recovered in the above (3) and (3') with an organic solvent.

7. The method of claim 1, wherein: The temperature of the aqueous solution containing the silver salt when it comes into contact with the feedstock oil is 5 to 30°C.

8. The method of claim 1, wherein: The temperature of the aqueous solution containing the silver salt when it comes into contact with the organic solvent is 30 to 80°C.

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