JP6234908B2 - Method for producing eicosapentaenoic acid and / or docosahexaenoic acid-containing composition - Google Patents

Description

  The present invention relates to a method for producing an eicosapentaenoic acid and / or docosahexaenoic acid-containing composition.

  Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are highly unsaturated fatty acids, have recently been revealed in their pharmacological effects and are used as raw materials for pharmaceuticals and health foods. Polyunsaturated fatty acids have multiple double bonds and are not easily obtained by chemical synthesis. Accordingly, most of the highly unsaturated fatty acids used in the industry are produced by extraction or purification from raw materials derived from marine organisms rich in highly unsaturated fatty acids such as fish oil. However, since the raw material of biological origin is a mixture of various fatty acids having different numbers of carbon, the number and position of double bonds, and the composition ratio of stereoisomers, the content of desired EPA and DHA is not necessarily high. Absent.

  Therefore, conventionally, it has been required to selectively purify EPA and DHA from biological materials. In particular, when EPA or DHA is used as a raw material for foods and pharmaceuticals, high-purity EPA and DHA free from impurities are required. In addition, EPA and DHA purified from marine organism-derived raw materials emit a strong fishy odor when impurities are contained even in trace amounts. Therefore, foods and pharmaceuticals containing conventional EPA and DHA have EPA and DHA sealed in capsules. It was necessary to take measures against odors such as formulation with a masking agent. Therefore, there is a need for a method that can easily produce a composition containing EPA and DHA with high purity, which can be a raw material for pharmaceuticals and health foods.

  In Patent Documents 1 to 4, after an aqueous solution containing a silver salt is mixed with a raw material containing a highly unsaturated fatty acid such as EPA to form a complex of the highly unsaturated fatty acid and the silver salt, the aqueous phase is separated, Next, extraction with an organic solvent (Patent Document 1), heating to dissociate the complex (Patent Document 2), permeation through a liquid film (Patent Document 3), dilution stirring or complex dissociation agent, A method for obtaining highly unsaturated fatty acids and esters thereof by addition of a silver ion reducing agent (Patent Document 4) is described.

  Patent Document 5 discloses a method for producing EPA or an ester thereof having a concentration of 80% or more by stepwise distillation of a raw material containing a highly unsaturated fatty acid or an alkyl ester thereof using a distillation column having three or more stages. Have been described. Patent Document 6 discloses a high-purity eicosapentaene by subjecting a raw material containing a highly unsaturated fatty acid such as EPA to vacuum precision distillation, and mixing the obtained fraction containing EPA and its lower alcohol ester with an aqueous silver nitrate solution. A method for purifying an acid or its lower alcohol ester is described.

  However, in the methods described in Patent Documents 1 to 4, highly unsaturated fatty acids other than EPA and DHA simultaneously form a complex, and thus it is difficult to obtain high-purity EPA and DHA. In addition, arachidonic acid (AA), which has similar physical properties to EPA, by separation by distillation as described in Patent Document 5, and also by a combination of distillation and separation by silver nitrate aqueous solution as described in Patent Document 6. It is difficult to separate from other highly unsaturated fatty acids such as Eicosatetraenoic acid (ETA) and high purity EPA and DHA.

Japanese Patent No. 3001954 Japanese Patent No. 2786748 Japanese Patent No. 2935555 Japanese Patent No. 2895258 Japanese Patent No. 3005638 JP-A-7-242895

  The present invention relates to providing a method capable of more selectively purifying EPA and / or DHA from a raw material containing a highly unsaturated fatty acid.

  As a result of many studies, the present inventors have found that when an aqueous solution containing a complex of a highly unsaturated fatty acid such as EPA or DHA and a silver salt is brought into contact with an organic solvent under a specific temperature condition, a complex of EPA or DHA is obtained. Has been found to remain in the aqueous phase, while other undesired polyunsaturated fatty acids migrate to the organic solvent phase. Based on this finding, the present inventors mixed an alkyl ester of a highly unsaturated fatty acid with an aqueous solution containing a silver salt, and brought the mixture into contact with an organic solvent while maintaining a specific temperature. If the phase is discarded and the aqueous phase is recovered, the EPA and DHA complexes can be selectively separated from other highly unsaturated fatty acids, and if the separated EPA and DHA complexes are dissociated, It has been found that a composition containing EPA and DHA with high purity can be obtained.

That is, the present invention is a method for producing an eicosapentaenoic acid and / or docosahexaenoic acid-containing composition:
(1) contacting a raw material containing an alkyl ester of a highly unsaturated fatty acid containing eicosapentaenoic acid and / or docosahexaenoic acid with an aqueous solution containing a silver salt;
(2) contacting the organic solvent while maintaining the aqueous solution at 5-30 ° C., and then recovering the aqueous phase; and (3) recovering the organic solvent phase after adding the organic solvent to the aqueous phase. To do,
A method comprising:

  The present invention also provides a method for separating a complex of eicosapentaenoic acid and / or an alkyl ester of docosahexaenoic acid and silver from a complex of an alkyl ester of a highly unsaturated fatty acid containing eicosapentaenoic acid and / or docosahexaenoic acid and silver. A method wherein an aqueous solution comprising a complex of an alkyl ester of a highly unsaturated fatty acid containing eicosapentaenoic acid and / or docosahexaenoic acid and silver is contacted with an organic solvent while maintaining at 5-30 ° C. and then water A method is provided that includes recovering the phase.

  According to the method of the present invention, EPA and DHA can be selectively purified, and a highly pure EPA or DHA-containing composition having a low content of highly unsaturated fatty acids other than EPA and DHA can be obtained. . According to the method of the present invention, the desired EPA and DHA are separated from other highly unsaturated fatty acids, particularly arachidonic acid (AA) and eicosatetraenoic acid (ETA), which have been difficult to separate by conventional methods. Both can be easily separated. The method of the present invention is economical because the silver salt used for complex formation can be reused many times.

  In the production method of the eicosapentaenoic acid (EPA) and / or docosahexaenoic acid (DHA) -containing composition of the present invention, the raw material is mainly an oil-and-fat mixture derived from natural products, and other than EPA and DHA, together with EPA and DHA. Examples include those containing highly unsaturated fatty acids. Examples of highly unsaturated fatty acids other than EPA and DHA include arachidonic acid (AA), eicosatetraenoic acid (ETA), docosapentaenoic acid (DPA), and the like. Examples of such raw materials include marine animals such as fish, oils and fats derived from plankton, oils and fats derived from microorganisms such as algae, and among others, oils and fats derived from fishes such as sardines and yellowtails, and oils and fats derived from algae are preferable.

  The raw material is preferably an oil or fat containing 15% by mass or more, more preferably 40% by mass or more, of the target EPA and / or DHA with respect to the total amount of fatty acids. In addition, as the raw material, a raw material having a high content of EPA and / or DHA may be used, but from the viewpoint of cost and availability, the content of EPA and / or DHA is contained in the total fatty acid contained. , Preferably 60% by mass or less, more preferably 55% by mass or less, and still more preferably 50% by mass or less. EPA and / or DHA may be present in the form of free fatty acids in the raw material, or may be present in the form of fatty acid chains such as mono-, di- or triglycerides. In the production method of the present invention, by using a raw material having a target polyunsaturated fatty acid content of 15% by mass or more in the total contained fatty acid, the final EPA and / or DHA concentration is A composition of 70% by mass or more, preferably 75% by mass in the contained fatty acid can be obtained efficiently.

  In the production method of the present invention, the target EPA and / or DHA in the raw material is alkylesterified. Preferably, highly unsaturated fatty acids other than EPA and / or DHA are also alkylesterified. By alkylating the highly unsaturated fatty acid in the raw material, other highly unsaturated fatty acids can be efficiently removed from the target EPA and / or DHA in the production process of the composition of the present invention. .

  Examples of the alkyl group constituting the alkyl ester of the highly unsaturated fatty acid include a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a methyl group or an ethyl group, more preferably ethyl. It is a group. The highly unsaturated fatty acid alkyl ester can be produced by subjecting an oil containing highly unsaturated fatty acid and an acid having a desired alkyl group to an esterification reaction by a known method. For example, an alkyl esterified product of a highly unsaturated fatty acid can be easily obtained by saponifying an oil containing a highly unsaturated fatty acid triglyceride. The higher the degree of alkyl esterification, the better. The total amount of the desired polyunsaturated fatty acid (including free form) contained in the raw material is preferably 90% or more, more preferably 95% or more. It is good to be.

  Commercially available fats and oils may be used as the fats and oils containing the above highly unsaturated fatty acids and their alkyl esters, which are raw materials in the production method of the present invention. From the viewpoint of obtaining a high content of the highly unsaturated fatty acid and its alkyl ester, which are the objects of the present invention, commercially available oils and fats derived from fish oils in which the type and amount of the highly unsaturated fatty acid contained are standardized. It is preferable to use it.

  In each step of the method for producing the EPA and / or DHA-containing composition of the present invention shown below, the raw material is preferably applied in a liquid form. When the raw material is in a liquid form at the reaction temperature in each step, it can be applied to each step of the present invention as it is. In the case of a solid form at the reaction temperature in each step, the raw material can be appropriately dissolved or diluted in an organic solvent or other oil and applied. As the organic solvent, in order to perform the following step (1), an organic solvent separable from water is used, and examples thereof include ethyl acetate, chloroform, carbon tetrachloride, diethyl ether, hexane and the like. The organic solvent used for dissolving or diluting the raw material in the following step (1) may be the same organic solvent as the organic solvent used in the following step (2) or a different organic solvent.

The method for producing the EPA and / or DHA-containing composition of the present invention includes the following steps:
(1) contacting a raw material containing an alkyl ester of a highly unsaturated fatty acid containing EPA and / or DHA with an aqueous solution containing a silver salt;
(2) contacting the organic solvent while maintaining the aqueous solution at 5-30 ° C., and then recovering the aqueous phase; and (3) recovering the organic solvent phase after adding the organic solvent to the aqueous phase. To do.

  The production process (1) of the present invention is a purification technique that utilizes the fact that the solubility in an extraction solvent is changed by forming a complex of a silver salt at the double bond portion of a highly unsaturated fatty acid. This is a step of separating and purifying the fatty acid alkyl ester. More specifically, polyunsaturated fatty acids having 20 or more carbon atoms such as eicosapentaenoic acid (EPA), arachidonic acid (AA), eicosatetraenoic acid (ETA), docosahexaenoic acid (DHA), docosapentaene The alkyl ester of acid (DPA) can be separated and purified efficiently. This process can be performed according to the method described in patent documents 1-4 etc., for example.

  More specifically, an aqueous solution containing a silver salt is added to a raw material containing alkyl esters of EPA and / or DHA and other highly unsaturated fatty acids, and 5 minutes to 4 hours, preferably about 10 minutes to 2 hours. Stir. The upper limit of the reaction temperature at this time is the temperature at which the product of the step (1) becomes completely liquid, preferably about 80 ° C. or lower, and the lower limit is 5 ° C. or higher. Preferably, the reaction temperature is around room temperature (15-30 ° C.). By the above reaction, a complex of silver and a highly unsaturated fatty acid is generated in the aqueous solution. Since the complex is dissolved in the aqueous solution phase, the aqueous phase containing the complex may be recovered from the solution and used for the next step (2). When the aqueous phase is recovered, impurities such as fatty acids other than the highly unsaturated fatty acids that have not formed a complex can be easily removed. On the other hand, since the complex with the above silver salt is formed with the unsaturated bond of the unsaturated fatty acid, the highly unsaturated fatty acid other than the desired EPA and / or DHA is also complexed and recovered in the aqueous phase. The

  The silver salt is not particularly limited as long as it can form a complex with an unsaturated bond of a highly unsaturated fatty acid, but silver nitrate, silver perchlorate, silver tetrafluoroborate, silver acetate, etc. can be used. . Of these, silver nitrate is preferred. Examples of the solvent for the aqueous solution include water or a mixed medium of water and a compound having a hydroxyl group such as glycerin or ethylene glycol, and water is preferably used. The silver salt concentration in the aqueous solution may be 0.1 mol / L or more, but is preferably about 1 to 12 mol / L. The molar ratio of the highly unsaturated fatty acid and the silver salt is about 1: 100 to 100: 1, preferably about 1: 5 to 1: 1.

  In the step (2) of the production method of the present invention, the aqueous solution containing the complex of the polyunsaturated fatty acid and the silver salt produced in the step (1) is brought into contact with an organic solvent, and then the aqueous solution and the organic solvent are mixed. This is a step of recovering the aqueous phase from the mixed solution. During contact with the organic solvent, the aqueous solution is maintained at a temperature at which the desired EPA and / or DHA complex does not dissociate. Specifically, the temperature of the aqueous solution in step (2) is preferably maintained at 5 to 30 ° C, more preferably 10 to 25 ° C. More preferably, in addition to the temperature of the aqueous solution in the step (2) being within the above temperature range, the reaction temperature in the step (1), that is, the silver-polyunsaturated fatty acid complex is converted in the step (1). It is in the range of +20 to −15 ° C., preferably in the range of +5 to −10 ° C., more preferably lower than the temperature of step (1) and within −10 ° C. with respect to the generated temperature. .

  In the step (2), the organic solvent added to the aqueous solution containing the complex of the highly unsaturated fatty acid alkyl ester is preferably hexane or cyclohexane, more preferably cyclohexane. The amount of the aqueous solution and the organic solvent is preferably 100: 3-300, more preferably 100: 3-200, as a volume ratio. Preferably, in the step (2), the aqueous solution and the organic solvent are mixed so as to have the volume ratio under the temperature condition, and the mixture is mixed for 5 minutes to 4 hours, preferably 10 minutes to 2 Stir for about an hour, more preferably for about 30 minutes to 2 hours. When the mixed solution is allowed to stand after completion of the stirring, the aqueous phase and the organic solvent phase are separated, so the organic solvent phase is removed and the aqueous phase is recovered. Furthermore, a new organic solvent may be added to the recovered aqueous phase and step (2) may be repeated.

  The aqueous solution containing the complex of the highly unsaturated fatty acid and silver salt obtained in the step (1) includes an EPA and / or DHA alkyl ester complex and an alkyl of a highly unsaturated fatty acid other than EPA and / or DHA. Mixed with ester complexes. When this aqueous solution is contacted with an organic solvent under the specific temperature conditions described above, the other polyunsaturated fatty acid complex dissociates and the fatty acid migrates into the organic solvent phase, whereas EPA and / or DHA is maintained in the aqueous phase as a complex. Therefore, in the step (2), the EPA and / or DHA can be separated from other highly unsaturated fatty acids if the aqueous phase is recovered after contacting the aqueous solution and the organic solvent under the temperature conditions. it can. As described above, in the method of the present invention, an organic solvent may be used in step (1) in order to dissolve or dilute the raw material. However, in step (1), after the raw material is dissolved or diluted in an organic solvent, an aqueous solution containing a silver salt is added thereto, and then the aqueous phase is recovered, as in step (2). Other polyunsaturated fatty acids cannot be removed effectively.

  Accordingly, one embodiment of the present invention is an EPA and / or DHA alkyl ester from a mixture of various polyunsaturated fatty acid alkyl esters and silver complexes comprising EPA and / or DHA obtained in step (1). The step (2) may be performed with the intention of separating the complex of silver and silver. Since the undesired polyunsaturated fatty acid is removed from the aqueous phase recovered in the step (2), if this is subjected to the step (3) described later, high-purity EPA and / or DHA alkyl ester. Can be recovered.

  Step (3) of the production method of the present invention is a step of adding an organic solvent to the aqueous phase recovered in the above step (2) and then recovering the organic solvent phase. In step (3), an organic solvent is added to dissociate the EPA and / or DHA alkyl ester complex in the aqueous phase, and the dissociated EPA and / or DHA alkyl ester is extracted into the organic solvent phase. If the organic solvent phase is then recovered, the desired EPA and / or DHA alkyl ester can be recovered. The said process can be performed according to the method described in patent documents 1-4 etc., for example.

  In the step (3), as an organic solvent to be added to the aqueous phase, solubility of alkyl esters of EPA and / or DHA such as hexane, ether, ethyl acetate, butyl acetate, chloroform, cyclohexane, benzene, toluene, xylene, etc. And a solvent which is high and can be separated from water.

  In step (3), in order to facilitate the dissociation of the EPA and / or DHA complex, an organic solvent is preferably added to the aqueous phase, and the resulting mixture is reacted in the above step (1). Heating is performed so that the temperature is higher than the temperature at which the silver-polyunsaturated fatty acid complex is formed in step (1). More preferably, the temperature is higher than 15 ° C, more preferably higher than 20 ° C with respect to the reaction temperature in the above step (1). The aqueous phase is then removed and the organic solvent phase containing the EPA and / or DHA alkyl ester is recovered. Alternatively, remaining silver ions may be further removed by passing the recovered organic solvent phase through an adsorbent such as silica gel, activated carbon, or silicon dioxide.

  The aqueous phase removed in the step (3) is an aqueous solution containing the silver salt used in the production of the silver-polyunsaturated fatty acid complex in the step (1). Therefore, the aqueous phase removed in step (3) can be recovered and used again as the silver salt aqueous solution in step (1) of the method of the present invention. Also in the conventional method, the silver salt aqueous solution remaining after dissociating the highly unsaturated fatty acid from the complex was recovered and reused for purification of the highly unsaturated fatty acid. However, since this silver salt aqueous solution gradually deteriorates as the content of impurities increases with repeated use, repeated use of the silver salt aqueous solution may lead to a reduction in the quality of the highly unsaturated fatty acid to be purified. On the other hand, since the aqueous silver nitrate solution used in the method of the present invention has a lower impurity content than the conventional method, the quality of the EPA or DHA-containing composition purified even after reuse is lowered. There are few things.

  Therefore, one embodiment of the present invention uses a silver salt aqueous solution containing a silver salt contained in the aqueous phase remaining after recovering the organic solvent phase in the above step (3), to produce new EPA and / or DHA. It can be a method for producing an EPA and / or DHA-containing composition comprising repeating the above steps (1) to (3) based on a raw material containing an alkyl ester of a highly unsaturated fatty acid containing. In this method, the number of repetitions of the step may be 2 or more, but is preferably 10 times or more, more preferably 20 times or more, still more preferably 50 times or more, still more preferably 10 to 70 times. . In this method, the aqueous phase removed in the step (3) may be reused as it is in the step (1), but may be reused after appropriately adjusting the concentration of the silver salt.

  The EPA and / or DHA alkyl ester-containing composition produced by the production method of the present invention comprises 70% by mass or more, preferably 75% by mass, of the target EPA and / or DHA alkyl ester in the total fatty acids contained. Contains above. The desired EPA and / or DHA alkyl ester contained in the composition is preferably an EPA alkyl ester. The alkyl ester-containing composition of EPA and / or DHA of the present invention has a boiling point close to that of EPA and DHA, and has a low content of highly unsaturated fatty acids such as AA and ETA that are difficult to separate in a purification operation by distillation. It is also suitable as a raw material for preparing a EPA and / or DHA-containing composition with higher purity by an operation such as distillation. Therefore, by using the alkyl ester-containing composition of EPA and / or DHA of the present invention, high-purity EPA and / or DHA can be more easily produced. Further, according to the present invention, the silver salt used for the production of the silver-polyunsaturated fatty acid complex can be reused more times, so that the cost required for the polyunsaturated fatty acid purification can be reduced. it can.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited only to these Examples.

In the following examples, the composition analysis method for polyunsaturated fatty acids is as follows.
10 mg of the measurement sample was diluted in 10 mL of n-hexane, and the content ratio of each fatty acid in the total fatty acids was analyzed using a gas chromatography analyzer (Type 6890 GC; manufactured by Agilent Technologies) under the following conditions. The result was expressed as mass% converted from the area of the chromatogram.
<Column conditions>
Column: J-W DB-WAX 0.25 mm × 30 m, column temperature: 210 ° C.
He flow rate: 1.0 ml / min, He pressure: 20 PSI
<Detection conditions>
H ₂ flow rate: 40 ml / min, Air flow rate: 450 ml / min
He flow rate: 1.00 ml / min, DET temperature: 260 ° C

  Moreover, in the following Examples, the calculation method of the recovery rate of EPA alkyl ester (EPA-E) and DHA alkyl ester (DHA-E) is as follows.

The impurity ratio of the composition is determined based on the content of the desired EPA and DHA in the composition (arachidonic acid alkyl ester (AA-E) and eicosatetra, which are difficult to separate by conventional methods such as chromatography). The content of enoic acid alkyl ester (ETA-E)) was calculated according to the following formula.
Impurity ratio (%)
= (Total content of AA-E and ETA-E / Total content of EPA-E and DHA-E) × 100

  In the evaluation of the composition, the composition score and the recovery score of each composition were obtained based on the impurity ratio and the recovery rate of EPA-E and DHA-E according to the criteria in Table 1 below. Furthermore, comprehensive evaluation of the composition was performed based on those total scores.

(Reference Example 1) Raw material A
To 1 kg of sardine oil, 1000 mL of absolute ethanol in which 50 g of sodium hydroxide was dissolved was added, and after mixing and stirring at 70 to 80 ° C. for 1 hour, 500 mL of water was further added and mixed well, and allowed to stand for 1 hour. The separated aqueous layer was removed, and the oil layer was washed with water several times to neutralize the washing liquid, to obtain 820 g of ethyl esterified sardine oil (hereinafter referred to as “raw material A”).
As shown in Table 2, the raw material A contains 2.14% by mass of arachidonic acid (AA), 1.70% by mass of eicosatetraenoic acid (ETA), and 42.96 eicosapentaenoic acid (EPA) in the total fatty acids. Mass%, docosahexaenoic acid (DHA) 7.71 mass% was contained.

(Reference Example 2) Raw material B
To 1 kg of the raw material A prepared above, 600 mL of n-hexane was added, and well stirred and dissolved. To this, 2500 mL of an aqueous solution containing 50% by mass of silver nitrate was added and stirred at 20 ° C. for 1 hour. After standing, the aqueous phase was separated, and the remaining organic layer was concentrated to obtain 550 g of ethyl esterified sardine oil (hereinafter referred to as “raw material B”).
As shown in Table 2, the raw material B contained AA 3.72% by mass, ETA 2.73% by mass, EPA 18.65% by mass, and DHA 0.91% by mass in the total fatty acids.

(Reference Example 3) Raw material C
In the same manner as in Reference Example 2, 550 g of ethyl esterified sardine oil (hereinafter referred to as “raw material C”) was obtained.
As shown in Table 2, the raw material C contained AA 3.81% by mass, ETA 2.75% by mass, EPA 20.46% by mass, and DHA 1.23% by mass in the total fatty acids.

Example 1
Step (1): 5 mL of cyclohexane was added to 30 g of the raw material A, and the mixture was thoroughly stirred and dissolved. 80 mL of an aqueous solution containing 50% by mass of silver nitrate was added thereto, and the mixture was stirred at 20 ° C. for 1 hour. After allowing the solution to stand, the separated organic phase was removed, and the aqueous phase was recovered.
Step (2): While maintaining the aqueous phase obtained in Step (1) at 20 ° C., 5 mL of cyclohexane was added and stirred for 30 minutes. After the mixture was allowed to stand, the separated organic phase was removed, and the aqueous phase was recovered.
Step (3): The aqueous phase obtained in Step (2) is brought to 60 ° C., 150 mL of cyclohexane is added, and the mixture is stirred for 30 minutes at 60 ° C. to extract the fatty acid ethyl ester in the aqueous phase into the organic phase. . After the mixture was allowed to stand, the separated organic phase was collected and concentrated to obtain a fatty acid ethyl ester-containing composition.

(Example 2)
Step (1): 15 mL of n-hexane was added to 30 g of the raw material B, and the mixture was thoroughly stirred and dissolved. 60 mL of an aqueous solution containing 50% by mass of silver nitrate was added thereto, and the mixture was stirred at 25 ° C. for 1 hour. After allowing the solution to stand, the separated organic phase was removed, and the aqueous phase was recovered.
Step (2): While maintaining the aqueous phase obtained in Step (1) at 25 ° C., 50 mL of n-hexane was added and stirred for 30 minutes. After the mixture was allowed to stand, the separated organic phase was removed, and the aqueous phase was recovered.
Step (3): The aqueous phase obtained in Step (2) is brought to 60 ° C., 150 mL of n-hexane is added, and the mixture is stirred for 30 minutes at 60 ° C. to convert the fatty acid ethyl ester in the aqueous phase into the organic phase. Extracted. After the mixture was allowed to stand, the separated organic phase was collected and concentrated to obtain a fatty acid ethyl ester-containing composition.

(Comparative Example 1)
A fatty acid ethyl ester-containing composition was obtained in the same manner as in Example 1 except that the step (2) was not performed.

(Comparative Example 2)
A fatty acid ethyl ester-containing composition was obtained in the same manner as in Example 2 except that the step (2) was not performed.

  The composition ratio of each fatty acid with respect to the total fatty acids in the compositions of Examples 1-2 and Comparative Examples 1-2 was analyzed. Further, the impurity ratio, the recovery rate of EPA-E and DHA-E were determined, and the composition score, recovery rate score, and comprehensive evaluation of each composition were determined. The results are shown in Table 3.

  Compared to the composition of the comparative example obtained without performing the step (2), the composition of the example has a significantly reduced content of impurities AA and ETA, and the purity of the desired EPA and DHA is low. Improved. The composition of the comparative example had a high EPA and DHA recovery rate due to the small number of steps, but had a high content of AA and ETA and was inferior in purity of EPA and DHA.

(Production Examples 1-6)
A fatty acid ethyl ester-containing composition was obtained in the same manner as in Example 2 except that the temperature in the step (2) was as shown in Table 4.

Table 4 shows the composition ratio of each fatty acid to the total fatty acid, the impurity ratio, the recovery rate of EPA-E and DHA-E, and the evaluation results in the compositions of Production Examples 1 to 6. Table 4 shows the results of Example 2 again.
When the temperature of the step (2) was low, the content of EPA and DHA in the obtained composition was decreased and the content of impurities (AA and ETA) tended to increase. If the temperature of the step (2) was in the range of 5 ° C to 30 ° C, the impurity content was in an acceptable range. On the other hand, when the step (2) is performed at a high temperature, the EPA and DHA contents increase, but the recovery rate tends to decrease. In Production Examples 4 to 5 and Example 2 in which the temperature of the step (2) is in the range of + 5 ° C. to −10 ° C. with respect to the temperature of the step (1), the impurity ratio and the recovery rate of EPA-E and DHA-E The balance with was good.

(Production Examples 7 to 14)
The fatty acid ethyl ester containing composition was obtained like manufacture example 1-6 except having changed the temperature of process (1) and (2), respectively using the raw material of the reference example C.

Table 5 shows the composition ratio of each fatty acid to the total fatty acids, the impurity ratio, the recovery rate of EPA-E and DHA-E, and the evaluation results in the compositions of Production Examples 7 to 14.
In Production Example 7 where Step (1) and Step (2) were performed at 10 ° C., the content of impurities (AA and ETA) increased. On the other hand, in Production Example 8 in which the step (1) was performed at 10 ° C. and the step (2) was performed at 30 ° C., the recovery rate of EPA-E and DHA-E was decreased although the impurity content was decreased. Furthermore, in the manufacture example 14 which performed process (2) at 35 degreeC, the recovery rate fell significantly. If the temperature of the step (2) was in the range of 5 ° C to 30 ° C, the recovery rates of EPA-E and DHA-E were in an acceptable range. Furthermore, when the temperature of the step (2) was in the range of + 5 ° C. to −10 ° C. with respect to the temperature of the step (1), the recovery rate of EPA-E and DHA-E tended to be higher.

(Production Example 15)
Step (1): 15 mL of n-hexane was added to 30 g of the raw material B, and the mixture was thoroughly stirred and dissolved. 60 mL of an aqueous solution containing 50% by mass silver nitrate was added thereto, and the mixture was stirred at 20 ° C. for 1 hour. After allowing the solution to stand, the separated organic phase was removed, and the aqueous phase was recovered.
Step (2): While maintaining the aqueous phase obtained in Step (1) at 20 ° C., 50 mL of n-hexane was added and stirred for 30 minutes. After the mixture was allowed to stand, the separated organic phase was removed, and the aqueous phase was recovered. While maintaining the recovered aqueous phase at 20 ° C., 50 mL of n-hexane was added again, the mixture was stirred for 30 minutes, the organic phase separated after standing was removed, and the aqueous phase was recovered.
Step (3): The aqueous phase obtained in Step (2) is brought to 60 ° C., 150 mL of n-hexane is added, and the mixture is stirred for 30 minutes at 60 ° C. to convert the fatty acid ethyl ester in the aqueous phase into the organic phase. Extracted. After the mixture was allowed to stand, the separated organic phase was collected and concentrated to obtain a fatty acid ethyl ester-containing composition.

(Production Examples 16 to 17)
A fatty acid ethyl ester-containing composition was obtained in the same manner as in Production Example 15 except that the organic solvent used in Step (2) was changed to heptane (Production Example 16) or ethyl acetate (Production Example 17).

  Table 6 shows the composition ratio of each fatty acid to the total fatty acids, the impurity ratio, the recovery rate of EPA-E and DHA-E, and the evaluation results in the compositions of Production Examples 15 to 17. The purity of EPA and DHA in the compositions of Production Examples 15 to 17 were both high, but in Production Example 15 using hexane in step (2), EPA-E and DHA were compared to Production Examples 16 and 17. -E recovery rate was further improved.

(Production Examples 18-22)
Step (1): 5 mL of cyclohexane was added to 30 g of the raw material A, and the mixture was thoroughly stirred and dissolved. 80 mL of an aqueous solution containing 50% by mass of silver nitrate was added thereto, and the mixture was stirred at 20 ° C. for 1 hour. After allowing the solution to stand, the separated organic phase was removed, and the aqueous phase was recovered.
Step (2): While maintaining the aqueous phase obtained in Step (1) at 20 ° C., cyclohexane was added in the amount shown in Table 7 and stirred for 30 minutes. After the mixture was allowed to stand, the separated organic phase was removed, and the aqueous phase was recovered.
Step (3): The aqueous phase obtained in Step (2) is brought to 60 ° C., 150 mL of fresh cyclohexane is added, and the mixture is stirred for 30 minutes at 60 ° C. to extract fatty acid ethyl ester in the aqueous phase into the organic phase. . After the mixture was allowed to stand, the separated organic phase was collected and concentrated to obtain a fatty acid ethyl ester-containing composition.

(Production Examples 23 to 27)
Step (1): 15 mL of cyclohexane was added to 30 g of the raw material B, and the mixture was thoroughly stirred and dissolved. 50 mL of an aqueous solution containing 50% by mass of silver nitrate was added thereto, and the mixture was stirred at 15 ° C. for 1 hour. After allowing the solution to stand, the separated organic phase was removed, and the aqueous phase was recovered.
Step (2): While maintaining the aqueous phase obtained in Step (1) at 15 ° C., cyclohexane was added in the amount shown in Table 7 and stirred for 30 minutes, and the organic phase separated after standing was removed, The aqueous phase was collected.
Step (3): The aqueous phase obtained in Step (2) is brought to 60 ° C., 150 mL of fresh cyclohexane is added, and the mixture is stirred for 30 minutes at 60 ° C. to extract fatty acid ethyl ester in the aqueous phase into the organic phase. . After the mixture was allowed to stand, the separated organic phase was collected and concentrated to obtain a fatty acid ethyl ester-containing composition.

  Table 7 shows the composition ratio of each fatty acid to the total fatty acids, the impurity ratio, the recovery rate of EPA-E and DHA-E, and the evaluation results in the compositions of Production Examples 18 to 27. In Production Example 18 in which the amount of the organic solvent added to the aqueous phase was small in Step (2), the impurity content increased.

(Production Examples 28-30)
In the step (3), the remaining aqueous phase after collecting the organic phase, that is, an aqueous silver nitrate solution was repeatedly used. Steps (1) to (3) were performed in the same manner as in Example 1, and the aqueous silver nitrate solution separated from the organic phase was recovered. The recovered solution was added to the raw material newly prepared as the silver nitrate aqueous solution in step (1), and steps (1) to (3) were similarly performed to recover the silver nitrate aqueous solution again. This was repeated the number of times shown in Table 8 to obtain a fatty acid ethyl ester-containing composition.
Table 8 shows the composition ratio of each fatty acid to the total fatty acid, the impurity ratio, the recovery rate of EPA-E and DHA-E, and the evaluation results in the obtained composition. Table 8 shows the results of Example 1 again. Repeated use of silver nitrate did not change the purity of EPA and DHA and the recovery of EPA-E and DHA-E in the resulting composition. According to the method of the present invention, high-purity EPA-E and DHA-E can be efficiently produced even when a silver nitrate aqueous solution is repeatedly used.

Claims (8)

A process for producing an eicosapentaenoic acid and / or docosahexaenoic acid-containing composition comprising:
(1) contacting a raw material containing an alkyl ester of a highly unsaturated fatty acid containing eicosapentaenoic acid and / or docosahexaenoic acid with an aqueous solution containing a silver salt;
(2) contacting the organic solvent while maintaining the aqueous solution at 5-30 ° C., and then recovering the aqueous phase; and (3) recovering the organic solvent phase after adding the organic solvent to the aqueous phase. To do,
Only including,
The method wherein, in the step (2), the amount of the organic solvent is 3 to 300 parts by volume with respect to 100 parts by volume of the aqueous solution .   The method according to claim 1, wherein the raw material contains eicosapentaenoic acid and / or docosahexaenoic acid in an amount of 15% by mass or more based on the total amount of fatty acids.   The method according to claim 1 or 2, wherein the organic solvent in the step (2) is at least one selected from hexane and cyclohexane. Wherein in the step (2), the temperature of the aqueous solution is maintained at 10 to 25 ° C., any one method according to claim 1-3. In the step (2), the temperature of the aqueous solution is maintained in the range of +. 5 to-10 ° C. relative to the temperature of step (1), any one method according to claim 1-4. The method of any one of Claims 1-5 whose reaction temperature of the said process (1) is 5 to 80 degreeC.   The method includes repeating steps (1) to (3) using an aqueous silver salt solution containing a silver salt contained in an aqueous phase remaining after recovering the organic solvent phase in the step (3). The method of any one of -6. A method for separating a complex of eicosapentaenoic acid and / or alkyl ester of docosahexaenoic acid and silver from a complex of silver and an alkyl ester of highly unsaturated fatty acid containing eicosapentaenoic acid and / or docosahexaenoic acid, Contacting an aqueous solution containing a complex of an alkyl ester of a highly unsaturated fatty acid containing eicosapentaenoic acid and / or docosahexaenoic acid with silver with an organic solvent while maintaining the temperature at 5-30 ° C., and then recovering the aqueous phase only it contains the amount of the organic solvent is 3 to 300 parts by volume aqueous solution 100 parts by volume, method.