JPH10290699A - Production of oil and fat containing triglyceride containing gamma-linolenic acid at high concentration and/or triglyceride containing dihomo-gamma-linolenic acid at high concentration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for production by which a triglyceride containing γ-linolenic acid or dihomo-γ-linolenic acid excellent in preservation stability at a high concentration is continuously obtained for a long period. SOLUTION: A lipase acting only on ester bonds at the 1,3-positions of triglycerides is reacted with an oil and fat containing a γ-linolenic acid- containing triglyceride and/or a dihomo-γ-linolenic acid-containing triglyceride in the presence of a medium-chain fatty acid and 30-500 ppm water.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing highly γ-linolenic acid-rich triglycerides and / or highly di-homo γ-linolenic acid-rich triglycerides.

[0002]

2. Description of the Related Art In recent years, attention has been paid to the physiological activity of triglycerides containing highly unsaturated fatty acids. In particular, γ-linolenic acid-containing triglycerides and dihomo γ-linolenic acid-containing triglycerides are effective in improving and anticancer effects on adult diseases such as atopic dermatitis, rheumatoid arthritis, and high blood pressure.
It is known to have many physiologically active effects such as immunostimulatory effects. Various studies have been made on the use of γ-linolenic acid-containing triglycerides and dihomo γ-linolenic acid-containing triglycerides in pharmaceuticals and foods for specified health use.

[0003] Conventionally, there has been a demand for a method for producing triglycerides containing highly unsaturated fatty acids at a high concentration without reducing the loss of polyunsaturated fatty acids and by-producing diglycerides. For example, in JP-A-63-273485, a polyunsaturated fatty acid-containing oil and a saturated fatty acid and / or a saturated fatty acid alcohol ester are transesterified with a specific lipase to give a polyunsaturated fatty acid at the 2-position. A method for producing an oil / fat composition containing at least 40% by weight of a triglyceride having an acid and containing at least 50% by weight of a total saturated fatty acid having 16 or more carbon atoms is disclosed.
No. 7,594, using a transesterification reaction using a specific lipase, using fish oil and oleic acid as raw materials,
A method for producing a triglyceride containing oleic acid at the 1,3-position and docosahexaenoic acid at the 2-position is disclosed. Further, in Japanese Patent Application Laid-Open No. Hei 8-214891,
1,3 of triglyceride in the presence of fats and oils, medium chain fatty acids
There is disclosed a method for producing an oil or fat in which a lipase acting only on the -position ester bond acts.

[0004]

However, the method described in JP-A-63-273485 has a problem that a specific highly unsaturated fatty acid in triglyceride cannot be highly concentrated. 6-2875
No. 94, the oleic acid used for transesterification corresponds to the average molecular weight of the constituent fatty acids of fish oil, so that the content of γ-linolenic acid in triglycerides cannot be increased, and the content of triglycerides in the resulting fats and oils cannot be increased. The yield was not as high as about 90 mol% based on the raw material triglyceride. Further, in the method described in Japanese Patent Application Laid-Open No. 8-2144891, the amount of water (0 to 10
00%), the reaction system 20
As much as 2.5 g (12,300 ppm) of water is used per 2.5 g, and the present inventors have studied the amount of water. As a result, even if the amount of water is applied to γ-linolenic acid-containing triglyceride, It has been found that the concentration of polyunsaturated fatty acids in triglycerides is low and that no satisfactory ones can be obtained yet.

[0005]

Means for Solving the Problems Accordingly, the present inventors have:
In view of the above circumstances, as a result of intensive research, fats and oils containing γ-linolenic acid-containing triglycerides and / or dihomo γ-linolenic acid-containing triglycerides, medium-chain fatty acids, and 30 to 500 ppm of water, in the presence of triglycerides By reacting a lipase acting only on the 1,3-position ester bond, a medium-chain fatty acid having a smaller molecular weight than a highly unsaturated fatty acid such as γ-linolenic acid or dihomoγ-linolenic acid at the 1,3-position Is introduced, and the γ-linolenic acid or dihomoγ-linolenic acid present at the 2-position is retained as it is, resulting in an increased content of γ-linolenic acid and dihomoγ-linolenic acid. (Hereinafter referred to as highly γ-linolenic acid-rich triglyceride, dihomo γ-linolenic acid highly-containing triglyceride) with good yield and continuous Succeeded in producing a further found that good storage stability of the fat or oil containing the resulting triglyceride, thereby completing the present invention.

Hereinafter, the present invention will be described in detail. In the present invention, fats and oils containing γ-linolenic acid-containing triglyceride and / or dihomo γ-linolenic acid-containing triglyceride (hereinafter abbreviated as raw material triglyceride) are used. It contains triglycerides containing γ-linolenic acid, and includes, for example, plants such as rapeseed oil, evening primrose oil, black currant oil, borage oil, chlorella, algae such as spirulina, oils and fats extracted from Mortierella spp. Can be mentioned.

[0007] The medium-chain fatty acid of the present invention has 6 carbon atoms.
These are selected from fatty acids having up to 12 fatty acids, such as caproic acid, caprylic acid, capric acid, and lauric acid. Of these, caprylic acid and capric acid are preferably used.

The lipase used in the present invention includes, for example, genus Rhizopus, genus Rhizomucor, and Aspergillus (Asp.)
porcine pancreatic lipase, and the like. As such a lipase, a commercially available lipase can be used. For example, Rhizopus delema
r) lipase (“Taripase”, manufactured by Tanabe Seiyaku Co., Ltd.), Rhizomuco
rmiei) (Novo Nortisk Co., Ltd., “Ribozyme IM”), Aspergillus
Aspergillus niger lipase (Amano Pharmaceutical Co., Ltd., “Lipase A”) and the like.

In the present invention, when an immobilized lipase is used as such a lipase, the water content can be reduced to zero, which is effective in adjusting the water content of the reaction system described later.

The carrier to be immobilized includes celite, ion exchange resin, ceramic and the like. Preferably, ceramic is used. As the type of ceramic, ceramic carrier SM-10 (manufactured by NGK Insulators, Ltd.) is used. Preferred but not limiting. When immobilized lipase is used, the amount of lipase is 100 per gram of the carrier.
~ 2,000,000 units, preferably 1,000
~ 300,000 units.

The method for immobilizing the lipase is not particularly limited. For example, 0.1 to 30% by weight, preferably 1.0 to 20% by weight, containing the above number of units of lipase.
1 to 100 ml, preferably 10 to 30 ml, of an aqueous solution of a protein (lipase) is suspended in 1 to 10 g of a ceramic carrier, and cooled to -20 to -80 ° C with gentle stirring, and 10 to 300 ml of acetone, ethanol or isopropanol is cooled. Is gradually added to adsorb the lipase to the immobilized carrier. The precipitated fraction is collected and dried sufficiently under reduced pressure.

Next, the immobilized lipase is activated. In the case of the batch method, the activation is 10 to 50 times the volume of the immobilized enzyme by the amount of oil / fat / medium chain fatty acid / water [30 to 40:60 to 70: 1 to 1
5 (weight ratio)] at 30 ° C. with shaking for 40 hours. In the case of the column method, the activation is carried out in an amount of 1 to 10 times the amount of the immobilized enzyme, such as fat / medium chain fatty acid / water [30 to
40:60 to 70: 5 to 10 (weight ratio)], and then pass the mixed solution through the column again. This operation is performed for 1 to 10 cycles in total. The flow rate at this time may be any value as long as there is no pressure loss.

In the production method of the present invention, the 1,3-position polyunsaturated fatty acid in the starting triglyceride is transesterified into a medium-chain fatty acid by lipase, and the amount of fats and oils containing the starting triglyceride in the reaction system is reduced. Is 10 to 50% by weight, preferably 15 to 50% by weight. The amount of the medium-chain fatty acid in the reaction system is 50 to 90% by weight, preferably 50 to 85% by weight, and the weight ratio of the raw material triglyceride / medium-chain fatty acid in the reaction system is preferably 1 to 10, and more preferably 1 to 10. ~ 5.

The amount of lipase added to the reaction system is 1 g of the reaction solution.
Is preferably 4 to 80,000 units, and more preferably 40 to 8,000 units. Here, one unit refers to the amount of lipase required to produce 1 μmol of fatty acid per minute using olive oil as a substrate.

In the present invention, 30 to 5
The most characteristic feature is that the reaction is carried out in the presence of 00 ppm of water, preferably 50 to 150 ppm.
If the amount of water is less than 30 ppm, transesterification hardly proceeds, while if it exceeds 500 ppm, the stability of the enzyme is deteriorated and the hydrolysis of triglyceride occurs, which is not preferable. Water may be contained in fats and oils containing lipase, medium-chain fatty acid, and raw material triglyceride, but it is necessary to control the total amount of water to be 30 to 500 ppm. ,
In advance, the water content of each component is measured by the Karl Fischer method, a method of controlling the total water content, a method of completely dehydrating the reaction components and adding a predetermined amount of water later, and the like. The method is preferred because the handling of powdery lipases and other hygroscopic materials is simple. The amount of water held by the immobilized lipase is not included in the amount of water of the present invention.

As the reaction method, both the batch method and the column method can be applied, but the column method is preferred because the reaction can be continuously performed in large amounts and the solid-liquid separation is easy.

Hereinafter, a column method using an immobilized lipase will be described, but the present invention is not limited thereto. First, the column is filled with the immobilized enzyme,
0 volumes of fats / oils / medium chain fatty acids / water [30-40: 60-
70: 5 to 10 (weight ratio)] As described in the section of the activation of the immobilized lipase, the mixture is passed through for a total of 4 cycles to activate. Then, each water content of fats and oils and medium-chain fatty acids containing raw material triglyceride dehydrated by vacuum distillation, dehydration treatment, etc. is measured, and if necessary, a predetermined amount of water is added so that the water content becomes 30 to 500 ppm, A homogeneous solution is prepared by stirring uniformly. This reaction stock solution was added at a linear velocity of 0.5
１０００1000 ml / hr, preferably 1-10 ml / h
r, space velocity 0.01 to 10 / hr, preferably 0.1
Pass through the column at １ ／ 1 / hr. Reaction temperature 10-60
° C, preferably 15 to 45 ° C.

[0018] An alkali is added to the obtained passing solution to neutralize an excess medium-chain fatty acid which has not been exchanged with free fatty acids produced by transesterification to form a fatty acid salt, and then water is added. The fatty acid salt is extracted into the aqueous layer, and an organic solvent is added to recover the triglyceride (oil layer). The aqueous layer can be recycled for use in the reaction system.

By the above ester synthesis reaction, the triglyceride was 93 to 97 mol% based on the starting triglyceride.
It can be recovered, and γ-linolenic acid in triglyceride is 28 to 35% by weight and dihomo γ-linolenic acid is 2%.
It can be 5 to 36% by weight.

In the present invention, it is also preferable to coexist with vitamin E in the above-mentioned lipase reaction, which contributes to the improvement of the storage stability, handleability and the like of the produced fat and oil containing triglyceride. As the vitamin E, α-
Any or a mixture of tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol and the like are used, and preferably, wheat germ oil and the like are used.

The triglyceride produced according to the present invention is 2-triglyceride.
Γ-linolenic acid or dihomoγ-linolenic acid, which contains a large amount of γ-linolenic acid or dihomoγ-linolenic acid, contains a high amount of γ-linolenic acid or dihomoγ-linolenic acid, and the production method of the present invention uses the above-described column method. When the transesterification reaction is continuously performed, 95% of the starting triglyceride is used.
The reaction to obtain triglyceride with a recovery of at least
It can be operated continuously for about 0 to 200 days. Furthermore, even if the obtained fat or oil containing triglyceride is left at room temperature for a long period of time, the increase in the acid value is small and the storage stability is good.

[0022]

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples. Note that “%” indicates the peak area% obtained by analyzing the fatty acid composition by gas chromatography. Example 1 A ceramic carrier SM-10 (manufactured by NGK Insulators, Ltd.) was used on Rhizopus delmera.
r) Lipase (Talipase 5,0 manufactured by Tanabe Seiyaku Co., Ltd.)
After immobilizing 8 g of (00 units / g of carrier), a cylindrical column (1.5 cm in diameter, 6.2 cm in length, capacity of 10.
After filling to 95 cm ³ ), borage oil [γ-linolenic acid in triglyceride: 22.2%, water content: 200 ppm, wheat germ oil as vitamin E (Eisai, “Emix”) 0 .2% by weight]
And caprylic acid, which is a medium-chain fatty acid having 8 carbon atoms (water content 2
Transesterification at 30 ° C. while charging a 1: 2 (weight ratio) mixture [raw material triglyceride: caprylic acid = 1: 2 (weight ratio)] at a linear velocity of 4 ml / hr and a space velocity of 0.589 / hr. A continuous reaction was performed. 1 day and 90 days after the start of the reaction, 3 g of the passing solution was collected, neutralized by adding a 1N aqueous solution of sodium hydroxide, allowed to stand, and the aqueous layer (lower layer) was removed to remove the triglyceride layer ( The upper layer was extracted with hexane, and the hexane was removed to obtain a glyceride fraction. The glyceride fraction was applied to an ODS column (A
M120 S-50, manufactured by YMC), and the triglyceride fraction was calculated to be 0.79 g. The obtained triglyceride fraction was subjected to methyl esterification by a conventional method, and the fatty acid composition in the triglyceride was analyzed by capillary chromatography. As a result, γ-linolenic acid was 29.4% [raw material triglyceride (γ-linolenic acid 22). .2%)
1 g to 0.79 g of triglyceride (γ-linolenic acid 2
9.4%), so the recovery of triglycerides was 96 mol%.] Also, 1 g of the fat or oil is placed in a sealed test tube, and a storage test is performed at room temperature for one month. Before and after storage, the acid value is measured by a standard fat and oil analysis test method, and the increase in acid value is measured as follows. evaluated. ◎ ・ ・ ・ 1mgKOH / less than oil g ○ ・ ・ ・ 1-5mgKOH / less than oil g △ ・ ・ ・ 5-10mg KOH / less than oil g × ・ ・ ・ 10mgKOH / g or more oil Table 2 shows the results after 90 days.
Shown in

Example 2 The same reaction as in Example 1 was carried out except that borage oil containing no wheat germ oil (“Emix” manufactured by Eisai Co., Ltd.) was used. 0.0%
The contained triglyceride was obtained at a recovery of 94 mol% based on the raw material borage oil. A storage test was performed in the same manner. The results are shown in Tables 1 and 2.

Example 3 The same procedure as in Example 1 was repeated except that caprylic acid (water content: 200 ppm) was used in place of caprylic acid, and the same operation was performed, and the same evaluation was performed. The results are shown in Tables 1 and 2.

Example 4 The procedure of Example 1 was repeated, except that instead of borage oil, Mortierella extracted oil (containing 16% of dihomo γ-linolenic acid) was used and reacted in the same manner as in Example 1. The results were evaluated and the results are shown in Tables 1 and 2.

Example 5 In Example 2, a reaction was carried out in the same manner as in Example 1 except that an extract oil of the genus Mortierella (containing 16% of dihomo γ-linolenic acid) was used instead of borage oil, and the reaction was carried out in the same manner as in Example 1. The results were evaluated and the results are shown in Tables 1 and 2.

Example 6 In Example 3, a reaction was carried out in the same manner as in Example 1 except that an extract oil of the genus Mortierella (containing 16% of dihomo γ-linolenic acid) was used instead of borage oil, and the reaction was carried out in the same manner as in Example 1. The results were evaluated and the results are shown in Tables 1 and 2.

Comparative Example 1 In Example 1, the water content of borage oil was changed to 10 ppm.
In addition, using a water content of caprylic acid of 10 ppm,
The reaction was performed in the same manner as in Example 1, and the evaluation was performed in the same manner as in Example 1.
The results are shown in Tables 1 and 2.

Comparative Example 2 In Example 1, the water content of the borage oil was changed to 1000 pp.
The reaction was carried out in the same manner as in Example 1 by using caprylic acid having a water content of 1000 ppm for m, and evaluated in the same manner as in Example 1. The results are shown in Tables 1 and 2.

[0030]

[Table 1] Triglyceride preservation test GLA (%) DGLA (%) triglyceride raw material After reaction Raw material After reaction Recovery (mol%) Example 1 22.2 29.4----96 ◎ Example 2 22.2 29.0----- 94 ○ Example 3 22.2 29.0-----96 ◎ Example 4----16.0 25.3 95 ◎ Example 5----16.0 25.0 95 ◎ Example 6-----16.0 25.0 95 ◎ Comparative example 1 22.0 28.5 −− −−− 98 ○ Comparative Example 2 22.0 28.0 −− −−− 90 ○

[0031]

[Table 2] Triglyceride storage test GLA (%) DGLA (%) triglyceride raw material After reaction Raw material After reaction Recovery (mol%) Example 1 22.2 29.2----95 ◎ Example 2 22.2 29.0----- 94 ○ Example 3 22.2 29.0-----96 ◎ Example 4----16.0 25.3 95 ◎ Example 5----16.0 25.0 95 ◎ Example 6-----16.0 25.0 95 ◎ Comparative example 1 22.0 22.2 −− −−− 100 ◎ Comparative Example 2 22.0 23.0 −− −−− 75 ○

[0032]

According to the present invention, medium-chain fatty acids and 30 to 500 g of fats and oils containing γ-linolenic acid-containing triglyceride and / or dihomo γ-linolenic acid-containing triglyceride are added.
In the presence of water of ppm, the lipase acting only on the ester bond at the 1,3-position of triglyceride is reacted, so that the triglyceride can be obtained in good yield, and the column can be continuously used for a long time using immobilized lipase. And the resulting oils and fats containing triglycerides have good storage stability.

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Hiroyuki Fujita, Inventor 2- 13-1, Muroyama, Ibaraki-shi, Osaka, Japan Inside the Central Research Laboratory of Nippon Gosei Chemical Industry Co., Ltd. (72) Inventor Nobuhiro Fukushima 2--13, Muroyama, Ibaraki-shi, Osaka No. 1 Nippon Kasei Chemical Co., Ltd. Central Research Laboratory (72) Inventor Tomohide Yamagami 9-6 Nozaki-cho, Kita-ku, Osaka

Claims (4)

[Claims] (1) a γ-linolenic acid-containing triglyceride and / or
Or fats and oils containing dihomo γ-linolenic acid-containing triglyceride, in the presence of medium-chain fatty acid and 30 to 500 ppm of water, reacting a lipase acting only on the 1,3-position ester bond of triglyceride, Γ
-A method for producing fats and oils containing highly linolenic acid-rich triglycerides and / or highly di-homo-γ-linolenic acid-rich triglycerides. 2. The method according to claim 1, wherein the reaction is carried out in the presence of vitamin E. 3. The method for producing fats and oils containing highly γ-linolenic acid-rich triglycerides and / or highly di-homo γ-linolenic acid-rich triglycerides. 3. The method according to claim 1, wherein immobilized lipase is used as the lipase. 3. The method according to claim 1, wherein the lipase comprises a highly γ-linolenic acid-rich triglyceride and / or a highly di-homo γ-linolenic acid-rich triglyceride. 4. A γ-linolenic acid-containing triglyceride and / or
Or the fat or oil containing dihomo-γ-linolenic acid-containing triglyceride is extracted from plants, algae or fungi, and the gamma-linolenic acid-rich triglyceride and / or dihomoγ according to any one of claims 1 to 3 -A method for producing an oil or fat containing a highly linolenic acid-containing triglyceride.