JPH03206891A - Collection of highly unsaturated fatty acid - Google Patents

Abstract

PURPOSE:To readily obtain a highly unsaturated fatty acid in high yield by selectively hydrolyzing phosphatidyl ethanolamine contained in phospholipid of egg yolk with phospholipase A2 derived from swine pancreas. CONSTITUTION:Phospholipid of egg yolk is fractionated to give phosphatidyl ethanolamin (A) of egg yolk comprising 16-18C fatty acid having 0-20 unsaturation degree. Then, 1 pt.wt. component A is dissolved in 10-500 pts.wt. organic solvent such as diethyl ether to give a solvent solution (B). Then phospholipase A2 (C) is added to the solution B in an amount to give 0.1-100mg phospholipase A2 based on 1g component A to give a reaction solution (D). Then the component D is reacted at 30-60 deg.C for about 3 hours with stirring at 60-150rpm while adjusting to a given pH with Ca salt, etc., to give a reaction product (E). Then the component H is washed with an ether, purified by reprecipitation with acetone to collect highly unsaturated fatty acids such as arachidonic acid and docosahexaenoic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for separating highly unsaturated fatty acids, particularly arachidonic acid and docosahexaenoic acid, which have attracted attention due to their physiological activity, from an egg yolk phospholipid raw material.

(Conventional technology) Highly unsaturated fatty acids exist in the phospholipid fraction in cell membranes,
In response to stimulation of the cell membrane, it is released from the membrane and converted into the desired physiologically active substance by various enzyme systems. Since this highly unsaturated fatty acid can easily impart physiological activity, its use in pharmaceuticals and pharmaceutical raw materials is being considered. However, currently, highly unsaturated fatty acids cannot be chemically synthesized for any purpose, so they are produced by concentrating natural raw materials. Moreover, the natural raw materials used include animal organs, bacterial cell lipids, fish oil, etc., which contain a large number of highly unsaturated fatty acids, and the content of each of these fatty acids is low.

Therefore, conventional enrichment of highly unsaturated fatty acids has been carried out in the form of triglycerides by crystal fractionation using acetone (Japanese Unexamined Patent Application Publication No. 59-1998).
No. 14792) or HPLC (Japanese Patent Application Laid-Open No. 192797/1983) with a reverse phase column, or fractional distillation in the form of free acid or ester, addition of urea, and H with a reverse phase column.
Method combining PLC, etc. (Japanese Patent Application Laid-Open No. 61-21004
No. 8, JP-A-61-192798, JP-A-1-180
No. 849) has been proposed. Furthermore, a method has been proposed in which fish oil triglycerides are decomposed with lipase to concentrate highly unsaturated fatty acids (Japanese Patent Application Laid-open No. 14793/1983), but
- The entire 3-series highly unsaturated fatty acids are concentrated, and it is not suitable for concentrating specific highly unsaturated fatty acids. A method of fractionating phosphatidylethanolamine from egg yolk phospholipids to separate highly unsaturated fatty acids (unexamined patent application) No. 120340/1982) had a low concentration ratio relative to the raw material fatty acid composition.

(Problems to be Solved by the Invention) Conventional raw materials used for fractionating highly unsaturated fatty acids are fatty acids recovered from fish oil, microbial lipids, and plant seed oils, but in many cases, a large number of highly unsaturated fatty acids are used. Saturated fatty acids coexist and their individual contents are also low. Therefore, specific highly unsaturated fatty acids cannot be isolated by a single operation. The urea addition fractionation method, low-temperature fractional crystallization method, fractional distillation method, etc. have been adopted as preparatory methods for highly unsaturated fatty acids, but their concentration rates are low. Furthermore, the specificity of the enzyme-based concentration method has not yet been applied in consideration of the fatty acid composition of the raw material. Currently, highly unsaturated fatty acids are fractionated using a combination of expensive analytical reverse-phase partition chromatography scaled up to an industrial scale and complicated pre-preparation methods. Therefore, the emergence of a method for concentrating highly unsaturated fatty acids by a simple single operation using inexpensive and easily available raw materials is awaited.

(Means for Solving the Problems) The present invention selectively hydrolyzes phosphatidylethanolamine contained in egg yolk phospholipids by utilizing the substrate specificity of phospholipase A2 derived from pig pancreas, and the resulting free It is characterized by separating arachidonic acid and highly unsaturated fatty acids from fatty acids.

The phosphatidylethanolamine used as a starting material in the present invention may be obtained from egg yolk as it is, or may be obtained by fractionation from egg yolk phospholipids. At this time, the higher the purity of phosphatidylethanolamine used, the higher the purity of the highly unsaturated fatty acid that can be obtained.

The egg yolk phospholipid used in the present invention accounts for 30% by weight of the lipid bone extracted from egg yolk, and the main component is about 70% by weight.
Phosphatidylcholine accounts for 15% to 20% of other
phosphatidylethanolamine, and contains very small amounts of plasmalogen, sphingomyelin, etc. The fatty acids constituting egg yolk phospholipids are mainly fatty acids with 16 to 18 carbon atoms and a degree of unsaturation of 0 to 2, but highly unsaturated fatty acids contain only arachidonic acid and docosahexaenoic acid at 4 to 6% by weight. Egg yolk phosphatidylethanolamine is contained in egg yolk phospholipids at a concentration of 15 to 20%.Egg yolk phospholipids are subjected to a preparative column packed with silica gel or a centrifugal liquid-liquid multi-stage fractionator, and easily reduced to 95% by alcohol-based solvent. A large amount of products with high purity of % by weight or more can be obtained. The constituent fatty acids of egg yolk phosphatidylethanolamine also have 1 carbon.
The main components are fatty acids with a degree of unsaturation of 6 to 18 and a degree of unsaturation of 0 to 2, but only the highly unsaturated fatty acids arachidonic acid and docosahexaenoic acid are contained at least twice as much.

The phospholipase A2 derived from pig pancreas used in the present invention includes:
For example, commercially available industrial phospholipase A2, Novo Industries A/S residase (phosphatide-2-azirubidolase, EC 3.1.1.4)
) etc. This enzyme is suitable because of its potency of phospholipase A2 activity, low price and easy availability, and ease of removal. This hydrolysis by enzyme f produces Sn
Arachidonic acid and docosahexaenoic acid can be selectively concentrated by utilizing the substrate specificity shown by the stereospecificity of the -2 position and the chain length specificity of high chain length highly unsaturated fatty acids.

The hydrolysis treatment in the present invention is carried out, for example, by dissolving 1 part by weight of egg yolk phospholipid or egg yolk phosphatidylethanolamine in 10 to 500 parts by weight of an organic solvent described below, and adding the above-mentioned enzyme to the solution. Enzyme amount is phospholipase A2
Although it varies depending on the activity titer and type of substrate, it is usually 0.1 to 100 mg per 1 g of substrate. Reaction temperature is 20-70
The temperature can be arbitrarily selected within the range of 0.degree. C., but 30 to 60.degree. C. is suitable from the viewpoint of reaction efficiency due to phospholipase A2 activity. The treatment time varies depending on the amount of enzyme and reaction temperature, but for example, 40
At °C, it takes about 3 hours, at which time the stirring speed is 60°C.
A low speed of ~150 rpm is sufficient. In the conventional water-based treatment, a calcium salt solution and additives for adjusting PO were required for the reaction to proceed, but in this solvent-based treatment, the minimum amount of water necessary for hydrolysis is added to the organic solvent at 0.00%. Add 01-1% by volume.

The organic solvent used in this treatment is selected from the group of ethers, hydrocarbons, and esters. Specific examples include diethyl ether, isopropyl ether, dioxane, tetrahydrofuran, etc. as ethers, n-hexane, n-hebutane, petroleum ether, cyclohexane, etc. as hydrocarbons,
Examples of esters include methyl acetate and ethyl acetate.

After completion of the treatment, the desired product can be easily isolated by known purification methods such as ether washing, reprecipitation with acetone, dry concentration, and deenzyme removal using a filter aid. Generally, the theoretical yield is 85
The theoretical yield is extremely good at 90%, especially by selecting the reaction solvent and enzyme amount.

The free fatty acids recovered by this treatment of egg yolk phospholipids contain 10% by weight each of arachidonic acid and docosahexaenoic acid.
On the other hand, free fatty acids from egg yolk phosphatidylethanolamine contain 15-20% by weight of each. In addition, the decomposition rate was 50 to 7 within 30 minutes to 1 hour after starting the treatment.
About 0% free fatty acids contain 20 to 25% of each.

(Effects of the Invention) According to the present invention, highly physiologically active polyunsaturated fatty acids can be concentrated by inexpensive enzyme treatment using easily available egg yolk phospholipids and egg yolk phosphatidylethanolamine. From these concentrates, arachidonic acid and docosahexaenoic acid can be separated easily and in high yield through a simple separation process, and can be effectively used as raw materials for pharmaceuticals.

(Examples) Hereinafter, the present invention will be specifically described based on Examples. In the examples, % indicates weight %.

Reference example Purified egg yolk lecithin PLi00 (manufactured by Kewpie Corporation,
Dissolve 100g of the product (product name) in 500ml of benzene and perform fully automatic preparative high performance liquid chromatography HL C8.
37 (manufactured by Tosoh Corporation) with a particle size of 10! Preparative column packed with Jm spherical silica (column length x column diameter: 60
cm
The raw material solution was automatically injected. The eluent had a flow rate of 4 (1 mf/
The column was kept at 40°C in a constant temperature bath, and a refractive index detector was used for peak detection. 32 immediately after benzene elution
The phosphatidylethanolamine fraction eluted after ~35 minutes was collected using a fraction collector. The purity of the fractionated phosphatidylethanolamine fraction is 95% or more as determined by the Iatroscan method.

The fatty acid composition of the egg yolk phospholipid in this experiment and the phosphatidylethanolamine fractionated from this egg yolk phospholipid was determined using Carbowax 20M Scotto (
The contents of arachidonic acid and docosahexaenoic acid were determined using a gas chromatograph equipped with a SCOT column (X25m). The contents were as follows.

Egg yolk phospholipids: arachidonic acid 5.6%, docosahexaenoic acid 6.2% Fractionated egg yolk phosphatidylethanolamine: arachidonic acid 9.7%, docosahexaenoic acid 10.9% The cycle time for one hatch is 67.5 minutes, and the raw material 7 of the solution
Automatic filling was carried out every 0 minutes, and 13 g of fractionated phosphatidylethanolamine was fractionated from 100 g of egg yolk phospholipid in 25 cycles over about 29 hours.

Example 1 Purified egg yolk lecithin PL-100 (manufactured by Kewpie Corporation) was subjected to cold acetone treatment three times to remove fatty acids. Defatted egg yolk phospholipids (in an eggplant-shaped flask)
Phosphatidylcholine 0%, phosphatidylethanolamine: 15%, Iatroscan method) 10g, ethyl ether 100M, and porcine pancreatic origin phospholipase A2 (manufactured by Novo Industries A/S) powder 50
0 mg was added, 1 yd of distilled water was added while stirring at 12 Orpm with a stirrer, and the mixture was treated at 40°C for 3 hours. After a period of time, ethyl ether was recovered by decantation, 300 ml of fresh ethyl ether was added and stirred, and ethyl ether was recovered by decantation again. Plus 300m7 of cold acetone! The stirring extraction and decantation were repeated twice, and the recovered solvent was collected and concentrated to 300 ml.

3 g of a filter aid (manufactured by Dicalide Orient Co., Ltd., trade name: Dicalide Perlite) was added to this concentrated solution, stirred, filtered, and the solvent was distilled off from the mother liquor.
After drying, 3.1 g (88% yield) of pale yellow oil was obtained.

The analytical values of the oily substance were as follows.

■TLC (Thin Layer Chromatography) Merck TLC (Plates 5ilica)
Gel 60), 20X20cm, thickness 0.25mm Developing solution: Chloroform/methanol/water 65/25/4
(C/C/v'1 Coloring agent: Ditmar Lester reagent Rf value: Weak blue color at 0.10, strong black color at 0.95 ■TL, C-FID (Iatoscan method) Developer: T
Thin layer bar similar to LC: chromarosodosn Lysophospholipid group 4% Fatty acid 96% ■GC'' (capillary column method) Column: Carbowax 2OM liquid phase, 5CO
T type, x 25m temperature 2 columns (180~210°C1
1°C/m1n) Arachidonic acid 10.8% Docosahexaenoic acid 11.9% Example 2 10 g of egg yolk phosphatidylethanolamine (phosphatidylethanolamine content 98%, Iatoscan method) fractionated from the egg yolk phospholipid of the reference example, Dissolved in 1,000 mj of ethyl acetate, then added 250 mg of phospholipase AZ (manufactured by Novo Industries A/S) powder derived from porcine pancreas, and added 5 ml of distilled water Q while stirring at 12 orpm with a stir bar. It was treated at ℃ for 3 hours. After time, ethyl acetate was collected by decantation, 300ml of ethyl ether was added and stirred, and then
Collected by decantation.

Furthermore, repeat stirring extraction and decantation twice with 300 mf of cold acetone, collect the recovered solvent, and add 300 mff1
Concentrated into A filter aid (manufactured by Dicalide Orient Co., Ltd., trade name Dicalide Perlite) is added to this concentrated solution.
After stirring, the mother liquor was filtered and the solvent was distilled off, followed by drying to obtain 3.5 g (yield: 90%) of a pale yellow oil.

The analytical values of the oily substance were as follows.

■TLc (Thin Layer Chromatography) Merck's TLC (Plates 5ilica)
Gel 60), 20X20cm, thickness 0.25mm Developing solution: Chloroform/methanol/water 65/25/4
(v/v/ν) Coloring agent: Ditmarrester reagent Rf value: weakly blue at 0.08, strongly black at 0.96 ■TLC-FID (Iatroscan method) developing solution:
Thin layer bar similar to TLC: chromarosodosen lysophosphatidylethanolamine 3% fatty acid
97%■GC (capillary column method) Column: Carbowax 2OM liquid phase, 5CO
T-type, x 25m temperature 2 columns (180-210℃, 1
°C/m1n) Narachidonic acid 19.3% Docosahexaenoic acid 20.6% Example 3 Egg yolk phosphatidylethanolamine fractionated from egg yolk phospholipid of Reference Example (phosphatidylethanolamine content 98%, Iatroscan method) 10 g, n - Dissolved in 500 ml of hexane, then added 200 mg of phospholipase Am derived from porcine pancreas (manufactured by Novo Industries A/S) powder, added 1.0 ml of distilled water while stirring at 200 rpm with a stirrer, and heated at 30°C. Processed for 30 minutes. After time, n-hexane was collected by decantation, 300rnl of ethyl ether was added and stirred,
It was collected again by decantation. Further, stirring extraction and decantation were repeated twice using 300 jaws of cold acetone, and the recovered solvent was collected and concentrated to 300 ml. To this concentrated solution, 3 g of a filter aid (manufactured by Dicalide Orient Co., Ltd., trade name: Dicalide Perlite) was added, stirred, filtered, the solvent was distilled off from the mother liquor, and 2.5 g ( A pale yellow oil was obtained (yield: 63%).

The analytical values of the oily substance were as follows.

■TLC (Thin Layer Chromatography) Merck TLC (Plates 5ilica)
Gel 60), 20x20cm, thickness 0.25 Seal Developing solution: Chloroform/methanol/water 65/25/4
(C/C/ν) Color forming agent: Ditmarrester reagent Very weak blue color at Rf value of 70.12, strong black color at 0.96 ■TLC-FID (Iatroscan method) developing solution:
Thin layer bar similar to TLC: chromarosodo-sn lysophosphatidylethanolamine 2% fatty acid
98% GC (capillary column method) Column: Carbowax 20M night phase, 5C
OT type, x 25m Temperature: Column (180-210℃,
1°C7mln) Arachidonic acid 23.2% Docosahexaenoic acid 24.8% Example 4 3g of fatty acid obtained in Example 2 (Arachidonic acid 19.3%)
%, docosahexaenoic acid 20.6%, capillary column method) to obtain 2.7 g of fatty acid methyl by the 0.1% sulfuric acid methanol method. This fatty acid methyl was dissolved in methanol, a methanol solution saturated with 2.1 g of urea was added, and after stirring for 1 hour, the precipitated crystals were separated by filtration. 1.4 g of urea was added to the filtrate and dissolved by heating to 45°C. Next, the mixture was gradually cooled to precipitate crystals, and the crystals were separated by filtration to obtain a filtrate. The filtrate was concentrated to obtain 0.6 g of fatty acid methyl. This fatty acid methyl contained 38.6% arachidonic acid and 34.2% docosahexaenoic acid (the analysis conditions are
(same as C).

0.6 g of urea-added fatty acid methyl was dissolved in 12-methanol and processed using the above-mentioned fully automatic preparative high performance liquid chromatography. The preparative column used had the column length×column diameter, cross-sectional area, and column volume as described above, but the packing material was a high-retention ODS chemically bonded type with a particle size of 10-. Sample solution 6- was injected twice, 5% aqueous methanol was used as the eluent at a flow rate of 20 mf/min, the column temperature was kept at 40°C, and peak detection was performed using an ultraviolet absorption spectrum detector (detection wavelength 225 nm). I monitored it. The first peak and second peak after solvent elution were collected using a fraction collector. After removing the solvent from both fractions, the weight and purity were measured. 310 mg of docosahexaenoic acid (GC purity 97%) was obtained from the first fraction, and 197 mg of arachidonic acid (GC purity 95%) was obtained from the second fraction.

Claims (1)

[Claims] (1) Selectively hydrolyze phosphatidylethanolamine contained in egg yolk phospholipids using the substrate specificity of phospholipase A_2 derived from pig pancreas, and separate arachidonic acid and docosahexaenoic acid from the resulting free fatty acids. A method for preparative separation of highly unsaturated fatty acids.