JP2602743B2 - Process for producing eicosapentaenoic acid triglyceride - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a process for producing eicosapentaenoic acid triglyceride.

[0002]

2. Description of the Related Art Conventionally, ω-3 polyunsaturated fatty acids are often contained in marine foods such as fish oils such as mackerel and sardine, which are ingested by the human body and are useful for lowering cholesterol, antithrombosis and the like. Although it is used as a health food and is attracting attention for medical applications, it is easily oxidized, deteriorates, deteriorates, and is very expensive. In addition, although its ethyl ester is known, it is said that its bioactivity is lower than that of triglyceride. It has been reported that triglycerides are ideal for ingesting ω-3 polyunsaturated fatty acids.
(Carol M. Wojenski et al. B.
iochim. Biophys. Acta 1081
(1991) 33-38) On the other hand, ω-3 of a mixture of triglycerides such as eicosapentaenoic acid and linolenic acid.
Polyunsaturated fatty acid concentrates are also known, but contain ω-6 polyunsaturated fatty acid triglycerides such as oleic acid and linoleic acid, so that patients with adipose are treated with pure ω-3 highly unsaturated fatty acids. Unsaturated fatty acid triglycerides are required. In addition, glyceride synthesis of polyunsaturated fatty acids by lipase has been studied, and there is a research report. [Second
9th Oil Chemistry Symposium / Oil Chemistry Research Presentation Abstracts P19
2 (1990)]. According to this, the lipase TOYO,
Using two kinds of lipases of lipase OF in each oxygen solution, each highly unsaturated fatty acid such as α-lirenic acid, eicosapentaenoic acid, docosahexaenoic acid, etc. and glycerin are reacted with each enzyme to convert the respective glycerides. It is a composite.

[0003]

As described above, in the prior art, it was not possible to obtain a high concentration of triglyceride of ω-3 highly unsaturated fatty acid by the immobilized lipase. That is to say, even with the glyceride synthesis method reported above, even under optimal conditions, the immobilized lipase can produce one non-edible free fatty acid in the product.
It contains 0-20%. In addition, the lipase TOYO can achieve only a triglyceride synthesis rate of 26-38%, and the lipase OF can achieve only a triglyceride synthesis rate of 18-33%, which makes it impossible to use the triglyceride as a single product, and in functional foods and other fields. For example, it cannot be obtained as a product used as a food additive or the like for medical use. Especially,
It is desired that triglyceride of eicosapentaenoic acid, which is a typical ω-3 type highly unsaturated fatty acid, can be produced in high yield and can be isolated.

[0004]

The present invention SUMMARY OF THE INVENTION, the above conventional problems to solve, but which satisfies the above requirements, very effective eicosapentaenoic acid Toriguri <br/> glyceride conventional as such functional food significantly high yield obtained, follow compared to the synthetic methods
In addition, the present invention relates to a production method capable of easily separating and purifying the same , wherein eicosapentaenoic acid and glycerin are combined with eicosape.
A substrate is prepared by mixing glycerin at a ratio of 9 to 11% by weight with respect to pentaenoic acid, and the contact reaction between the substrate and immobilized lipase and the dehydration of the reaction solution are simultaneously performed. The dehydration treatment is performed by a vacuum dehydration method or a dry inert gas ventilation method, and maintains an ultrafine water system having a water concentration of 100 ppm or less in the reaction system.

[0005]

According to the preparation of the eicosapentaenoic acid triglycerides of the present invention, the eicosapentaenoic acid glyceryl
The substrate prepared by mixing phosphorus with the above specific ratio is reacted with the immobilized lipase, and at the same time, the reaction solution is dehydrated by a vacuum dehydration method or a dry inert gas ventilation method. Since the reaction is carried out while maintaining an ultra-micro water system with a water concentration of 70% or more, a reaction product containing 70% or more of eicosapentaenoic acid triglyceride, which can be easily separated and purified, can be obtained, and industrial production becomes possible. And 99%
Eicosapentaenoic acid triglyceride having a purity of

[0006] In the above production process, the initial moisture content of the immobilization lipase is from 1.3 to 8.3%, an initial water concentration of said substrate is about 50～8000Ppm, the reaction temperature is 40 ° C. As described above, the temperature is generally 30 to 60 ° C. in this case,
As the lipase of the immobilized lipase, a lipase of the genus Mucor or Candida is particularly suitable. Purification of eicosapentaenoic acid triglyceride from the above reaction product is performed by removing the contaminants from the reaction product using a basic alumina column or a liquid chromatograph made of silica gel, and evaporating the eluate. As a result, a purified product having a purity of 99% is obtained.

[0007]

Next, embodiments of the present invention will be described in detail. As raw materials, eicosapentaenoic acid and glycerin isolated or commercially available by a desired method such as a conventional method are prepared. The mixing ratio of the two is usually at or near the stoichiometric amount. Usually, a substrate in which 9 to 11% by weight of glycerin is mixed with eicosapentaenoic acid is prepared.

The immobilized lipase to act on the substrate preferably has an initial water content of 1.3 to 8.3%, and the substrate preferably has an initial water concentration of about 50 to 8000 ppm. As the immobilized lipase, lipase SP382 (candi)
da lipase immobilized), lipase A (As
perrgillus sp. ), Lipase TOYO (Chromobacterium v)
isolum), a lipase of the genus Mucor, for example, lipozyme IM60 (Mucormie)
hei lipase immobilized) can be used. The lipozyme IM60 and lipase SP382 are manufactured by Novo, and can maintain their activity at a water content of 100 ppm or less. Depending on the type of lipase belonging to the genus Candida or Mucor, the activity can be maintained even in an ultra-micro water system by fixing the lipase in the presence of lecithin or polyol.

Thus, the substrate is allowed to act on the immobilized lipase. In this case, an appropriate device can be used. In addition, various devices used in the "method of refining high acid value oil" previously proposed by the inventor in Japanese Patent Application No. 2-307214 can be used. That is, the substrate is contact-reacted with the immobilized lipase using the desired device. In this case, it is important to simultaneously perform a dehydration treatment of the reaction solution, whereby about 70-85% Eicosapentaenoic acid triglyceride with a high content can be synthesized.

To ensure this synthesis, the rate of dehydration must be 100.1% of the rate of reduction of the eicosapentaenoic acid.
The reaction and the dehydration treatment are preferably performed continuously by repeatedly contacting the reaction solution with the immobilized lipase while dehydrating as described above. This dehydration treatment is performed by a vacuum dehydration method or by ventilation of a dry inert gas. Therefore, it is important to perform a dehydration treatment so as to maintain the inside of the reaction system at a water concentration of 100 ppm or less.

The reaction temperature in this case is generally 30 to
In the range of 60 ° C., the efficiency of the enzyme reaction is particularly good. The optimal temperature is between 40 and 60C. In such a temperature range, the transfer of the target product, the double bond of the eicosapentaenoic acid triglyceride, and the like can be prevented. The apparatus is preferably a closed-type continuous production apparatus, in which oxygen in the reactor is replaced with an inert gas such as nitrogen or argon, and the reaction is performed under an inert gas atmosphere so that oxygen in the atmosphere does not touch the reactants. Preferably, this prevents an increase in the peroxide value.

Next, a more detailed embodiment will be described. Example 1 0.3 g of eicosapentaenoic acid, 0.0305 g of glycerin, and 0.03 g of immobilized lipase SP382 were added to a reaction vessel in which the atmosphere in the vessel was replaced with nitrogen, and 10 mmH
g in a vacuum to about 100 g.
m and a shaking reaction at 60 ° C. Thereafter, the reaction was stopped with ether / ethanol, and the reaction product was measured by a high performance liquid chromatograph equipped with a GPC column. The reaction product after 46 hours of reaction contained 75.2
% Of eicosapentaenoic acid triglyceride was obtained.
The balance was 22.3% eicosapentaenoic acid diglyceride and 2.5% eicosapentaenoic acid monoglyceride.

Example 2 1 g of eicosapentaenoic acid, 0.1 g of glycerin and lipozyme IM were placed in a reaction vessel in which the atmosphere in the vessel was replaced with nitrogen.
Then, 0.05 g of 60 was added, degassed at about 750 mmHg, vacuum dried, and shaken at 60 ° C. while maintaining the water content in the reaction system at 100 ppm or less. Thereafter, 4 drops of the reaction product liquid was added to 5 ml of ether-ethanol (1: 1) to stop the reaction. The reaction product after the reaction was stopped was subjected to GP
The measurement was performed by a high performance liquid chromatograph equipped with a C column. In the reaction product having a reaction time of 48 hours, 81% of eicosapentaenoic acid triglyceride was detected, and the rest was eicosapentaenoic acid diglyceride. Reaction 72
In time, eicosapentaenoic acid triglyceride 83
% And eicosapentaenoic acid diglyceride 17%. [Example 3] In a reaction vessel in which the atmosphere in the vessel was replaced with nitrogen, 0.5 g of eicosapentaenoic acid, 0.05 g of glycerin, and 0.05 g of lipozyme IM60 were added, degassed by about 750 mmHg, and vacuum-dried. Was shaken at 40 ° C. while maintaining the water content at 100 ppm or less. In the sample with a reaction time of 94 hours, 76% of eicosapentaenoic acid triglyceride was detected, and the balance was 20% of eicosapentaenoic acid diglyceride and 4% of eicosapentaenoic acid.
In the reaction for 192 hours, eicosapentaenoic acid triglyceride 83%, eicosapentaenoic acid diglyceride 17%
Met.

In general, a reaction product containing about 70 to 85% of eicosapentaenoic acid triglyceride is obtained by the treatment of the present method, and as a contaminant, most of eicosapentaenoic acid diglyceride, eicosapentaenoic acid monoglyceride, It is a trace amount of peroxide. The purified product of eicosapentaenoic acid triglyceride having a high purity of 99% is obtained from the reaction product as described below.

The reaction product obtained by stopping the above reaction is dissolved in ether, and the impurities are adsorbed and removed by liquid chromatography comprising a basic alumina column, while eicosapentaenoic acid triglyceride is eluted and the eluate is evaporated. Thus, a highly purified 99% purified product was obtained. Alternatively, the reaction product is dissolved in hexane, and the contaminants are adsorbed by liquid chromatography comprising a silica gel column, and then eluted with a mixed solution of hexane and ether (95: 5).
The eluate was evaporated to give a highly purified product of 99% purity.

Next, examples of the purification method will be described in detail. The reaction product obtained in Example 2 was dissolved in diethyl ether, the immobilized enzyme was separated by filtration, and then applied to a basic alumina column. Three basic alumina columns were prepared by packing 4 g of aluminum oxide 90 (Merck product number 1076) with adjusted activity, and chromatographed three times. The eluate was blown with nitrogen to remove ether, and stored at -60 ° C in argon gas. The obtained eicosapentaenoic acid triglyceride was analyzed by capillary gas chromatography (WCOT CP-Si188) and HPLC.
Column (Shodex GPC KF-802 300
x3), it was confirmed that the purity was 99% or more.

The isolated product was analyzed by ¹ HNMR (JEOL)
JNM-GSX500) and IR analysis (JEOL)
(JIR-100). As a result, the ¹ H NMR spectrum shown in FIGS. 1 and 2 and the FT-IR shown in FIG.
It was found to have a spectrum. That is, it can be seen that the assignment relationship between the main peak ah of the NMR spectrum shown in FIG. 1 and the chemical bond shown in FIG. 1 completely matches that of eicosapentaenoic acid triglyceride. When the number of hydrogen atoms determined from the peak size was calculated with the size of f and g set to 4,000, this also almost coincided with the theoretical value. More specifically, CD of eicosapentaenoic acid triglyceride
In ¹ HNMR using CL ₃ as a solvent, the main peak is shown in FIG.
As shown. That is, the chemical shift δ 5.37, 4.2
9, 4.15, 2.84, 2.80, 2.33, 2.0
There are peaks at 8, 1.69 and 0.97 ppm, and their assignments are as shown in FIG. On the other hand, in the FT-IR spectrum applied to the NaCl plate, as shown in FIG.
⁻¹ at CH absorption, 1745 cm ⁻¹ at ester C ＝ O
Absorption, C = C absorption at 1653 cm ⁻¹ , 1144c
m- ¹ had absorption of CO, confirming their presence. The proton peak area intensity was as shown in Table 1 below.

[0018]

[Table 1]

As is apparent from Table 1, the other peaks are 1-3% weaker than the theoretical values on the basis of f + g. Since the theoretical error of the integrated intensity is usually considered to be several to 5% or less,
It can be seen that this range is within the range of the error. From the above measurement results, it was confirmed that the purified product was eicosapentaenoic acid triglyceride having the structural formula shown in the following Chemical Formula 1 and FIG. 1 and specified in FIG.

[0020]

Embedded image

Further, the purified product has the following structure and physicochemical properties. That is, its elemental composition is carbon, oxygen, and hydrogen. From the elution pattern of the capillary column of gas chromatography, it was confirmed that the constituent fatty acid was only eicosapentaenoic acid. In addition, the following was confirmed. That is, GPC of high performance liquid chromatography
Elution position by column (Kelpertion column) and N
From the structural formula estimated from the proton intensity and assignment of MR,
The molecular weight is 945.4. A colorless and transparent substance soluble in hexane, ether and acetone even at −40 ° C. and a neutral fat because it passes through a basic alumina column. The silica gel thin layer chromatography spot develops a brown color under iodine vapor. In addition, eicosapentaenoic acid triglyceride isolated by the above production method of the present invention has better absorbability than eicosapentaenoic acid ethyl ester, and studies the physiological functions of eicosapentaenoic acid, lowers cholesterol, antithrombosis, etc. Is effective for prevention of circulatory diseases, medical experiments, etc., and furthermore, its physiological activity is elucidated, so that it can be used as a pharmaceutical. It can be added as a functional food accurately, and if necessary, is useful for balancing with ω-6 highly unsaturated fatty acid triglyceride.

[0022]

As described above, according to the production method of the present invention,
The mixing ratio of eicosapentaenoic acid and glycerin, stingray
9-11% by weight of glycerin with respect to osapentaenoic acid
The substrate thus formed is brought into contact with the immobilized lipase and, at the same time, the reaction solution is dehydrated by a vacuum dehydration method or a dry inert gas ventilation method.
since as will maintain the ultrafine water of less water concentration m, it is easily considerably and separated and purified in high yield compared with the conventional 7
It has the effect that 0% or more of eicosapentaenoic acid triglyceride can be produced. Thus, a reaction product containing such a high concentration of eicosapentaenoic acid triglyceride can be obtained, and it is easy to separate and purify impurities from the reaction product. It has the effect of obtaining industrial production. Further, in the above production method, the initial moisture concentration of the substrate to about 50 to 8000
When the lipase of the genus Mucor or Candida is used as the lipase of the immobilized lipase, the above-mentioned high-yield production can be accurately obtained. In addition, as a purification method, when the adsorption of contaminants by a basic alumina column or a liquid chromatograph comprising silica gel, the elution of the eicosapentaenoic acid triglyceride by a solvent, and the evaporation of the eluate, a highly efficient 99% purified product is used. Has the effect of being obtained.

[Brief description of the drawings]

FIG. 1 and

FIG. 2 is a diagram showing the ¹ HNMR spectrum of eicosapentaenoic acid triglyceride obtained by the production method of the present invention and the assignment of its main peak.

FIG. 3. F of the eicosapentaenoic acid triglyceride
It is a figure of a T-IR spectrum.

 ────────────────────────────────────────────────── ─── Continuing from the front page Judge Masahide Sakai Judge Toshiko Otaka Judge Junko Uchida Judge (56) Reference JP-A-61-43143 (JP, A) JP-A-1-257485 (JP, A) Kaihei 1-104184 (JP, A)

Claims (2)

(57) [Claims] 1. An eicosapentaenoic acid and glycerin
9-11% by weight of glycerol based on eicosapentaenoic acid
The substrate was prepared by mixing in a ratio of emission performs a catalytic reaction with the immobilized lipase and said substrate and the dehydration of the reaction mixture at the same time, dehydration processing Tsu reaction solution, vacuum dehydration method or drying The reaction is carried out by an inert gas ventilation system, and the inside of the reaction system is 10
A process for producing eicosapentaenoic acid triglyceride, characterized by maintaining an ultrafine water system having a water concentration of 0 ppm or less. 2. A process for removing eicosapentaenoic acid triglyceride comprising subjecting a reaction product containing 70% or more of eicosapentaenoic acid triglyceride obtained by the production method according to claim 1 to removal and purification of contaminants. Manufacturing method. 3. The initial water content of the immobilized lipase is as follows:
3-8.3%, and the initial water concentration of the substrate is about 50
The method for producing eicosapentaenoic acid triglyceride according to claim 1, wherein the reaction temperature is 30 to 60 ° C. Wherein the immobilized lipase, Mucor or preparation of eicosapentaenoic acid triglyceride according to claim 1 or 3, wherein at the genus Candida lipase obtained by immobilizing. 5. The process of removing and purifying impurities of the reaction product comprises adsorbing the impurities by a basic alumina column or a liquid chromatograph comprising silica gel, eluting the eicosapentaenoic acid triglyceride by a solvent, and evaporating the eluate. The method for producing eicosapentaenoic acid triglyceride according to claim 2, wherein