JPH10245585A - Method for concentrating n-6 series highly unsaturated fatty acids - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for obtaining glycerides containing n-6 series highly unsaturated fatty acids in high concentrations in good yields. SOLUTION: In fat and oil containing n-6 series highly unsaturated fatty acids, fatty acids other than the n-6 series highly unsaturated fatty acids are hydrolyzed in multiple stages by adding lipase derived from the genus Candida in divided portions to concentrate the n-6 series highly unsaturated fatty acids. It is desirable that the n-6 series highly unsaturated fatty acid in the first stage in which the degree of hydrolysis is 5-50% are γ-linolenic acid and/or dihomo-γ- linolenic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for obtaining a high-concentration n-6 series highly unsaturated fatty acid glyceride from fats and oils containing n-6 series unsaturated fatty acids, and furthermore, it has excellent storage stability. And a method for obtaining n-6 series polyunsaturated fatty acid glyceride.

[0002]

2. Description of the Related Art In recent years, attention has been paid to the physiological activity of highly unsaturated fatty acids. In particular, it has three unsaturated bonds,
Polyunsaturated fatty acids in which the first unsaturated bond is at position 6 (n-6 series) include γ-linolenic acid (cis, ci)
s, cis-6,9,12-octadecatrie
noic acid), dihomo γ-linolenic acid (ci
s, cis, cis-8, 11, 14-eicosat
For example, γ-linolenic acid has many physiological activities such as an ameliorating effect on adult diseases such as atopic dermatitis, rheumatoid arthritis, and hypertension, an anticancer effect, and an immunostimulating effect. It is known.

[0003] As a method for obtaining such n-6 series polyunsaturated fatty acids, fats and oils containing the raw material polyunsaturated fatty acids are converted into fatty acids or lower alcohol esters of fatty acids, and then (a) chromatographic separation. , (B)
A method by urea addition fractionation, (c) a method by fractional distillation,
(D) Concentration can be carried out by using a conventionally known technique such as a liquid separation method, followed by esterification with glycerin to obtain glyceride again. It is not practical because it produces fatty acid isomers.

As a solution to the above problem, for example, a method for obtaining γ-linolenic acid is disclosed in Japanese Patent Application Laid-Open No. 63-12289.
As described in the publication, a production method for partially hydrolyzing fats and oils containing γ-linolenic acid with a hydrolase and then removing free fatty acids has been proposed, and by using such a method, γ-linolenic acid glyceride is produced. It states that it can. On the other hand, Japanese Patent Application Laid-Open No. 58-165796 discloses a technique using fish oil, which is described in Japanese Patent Application Laid-Open No. 58-165796.
Hydrolysis by the lipase obtained from ea) is described. As a method for obtaining a highly unsaturated fatty acid glyceride, Japanese Unexamined Patent Publication (Kokai) No. 7-203979 discloses an oil or fat containing a polyunsaturated fatty acid as Geotricam (Geoticam).
(Richum) is hydrolyzed using a lipase obtained from the genus.

[0005]

However, in the technology disclosed in Japanese Patent Application Laid-Open No. 63-12289, the present inventors have conducted detailed studies and found that the concentration of γ-linolenic acid can be increased to only 47.4% at the maximum. However, the recovery rate of the glyceride fraction was 28.1%, which is still low as a recovery rate of γ-linolenic acid. Similarly, Japanese Patent Publication No. 165796 is not yet satisfactory with respect to the high concentration and recovery of docosahexaenoic acid and eicosapentaenoic acid. Further, in the technology disclosed in Japanese Patent Application Laid-Open No. 7-203979, geotricum (Ge) is used as a lipase for hydrolysis.
Although lipase obtained from the genus otrichum is used, the lipase is not a lipase for food supplementation, and oil produced using the lipase cannot be used as an edible oil.

[0006]

Means for Solving the Problems In order to solve such problems, the present inventors have intensively studied on increasing the concentration of n-6 series highly unsaturated fatty acids such as γ-linolenic acid and dihomo γ-linolenic acid. As a result, surprisingly, in fats and oils containing n-6 series polyunsaturated fatty acids, fatty acids other than n-6 series polyunsaturated fatty acids were replaced by Candida (Can.
lipase derived from the genus dida) is dividedly charged and hydrolyzed in multiple stages to increase the concentration of the n-6 series polyunsaturated fatty acids by adjusting the hydrolysis in the first stage to 5 to 50%. The present inventors have found that a glyceride containing a high concentration of n-6 series polyunsaturated fatty acids having excellent storage stability can be obtained in a high yield. still,
As a lipase for hydrolysis, Candida (Candi)
da) In place of lipase derived from the genus
No. 979, Geotrich (Geotrich).
When a lipase obtained from the um) genus is used, the stability of the highly unsaturated fatty acid glyceride upon standing is not preferable, and turbidity is likely to occur.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present invention, the n-6 series polyunsaturated fatty acids are not particularly limited, and include γ-linolenic acid and dihomo γ-linolenic acid, and examples of fats and oils containing the polyunsaturated fatty acids include evening primrose oil, Borage herb oil, black currant, seed oil such as saxifrage seed oil, oils and fats obtained from algae such as Spirulina, Mortierella spp., Cunninghamera spp.,
Fats and oils obtained from microorganisms of the genus Fycomyces, Dilperella, and Tamnidium are exemplified. Preferably, vegetable oils such as evening primrose oil, borage oil, black currant oil, and oils and fats obtained from microorganisms such as Mortierella are used.

[0008] In the present invention, in the fats and oils containing the above-mentioned polyunsaturated fatty acid, the first step is a hydrolysis rate of 5 to 5
Hydrolysis is performed to 0%, preferably 10 to 40%. However, in the present invention, if the hydrolysis rate is less than 5%, the concentration cannot be sufficiently increased, and if it exceeds 50%, the recovery rate decreases. But not preferred.

As such a lipase for hydrolysis, those produced by microorganisms of the genus Candida, those derived from other animals, and those derived from plants are used. As such a lipase, a commercially available lipase can be used. For example, candy dasylindrache (Ca
lipase (manufactured by Meito Sangyo Co., Ltd., "Lipase OF"), lipase of Rhizopus delemar (manufactured by Tanabe Seiyaku Co., Ltd.), Pseudomonas aeruginosa
osa) lipase (manufactured by Toyobo Co., Ltd.), Candida antractia
Lipase (Novozyme manufactured by Novo Nordicx)
435 "), and pancreatic lipase of pigs, cows, etc., and particularly, candy dacillin drache (Cand).
Lipase (ida Cylindracea) (“Lipase OF”, manufactured by Meito Sangyo Co., Ltd.) is preferred.

For the hydrolysis, n-6 in the reaction system is used.
The amount of the series of highly unsaturated fatty acid-containing fats and oils is 1 to 90% by weight, preferably 10 to 80% by weight, and the amount of water is 1 to 90% by weight.
It is 0 to 99% by weight, preferably 20 to 90% by weight.
The fat / oil containing highly unsaturated fatty acid / water (weight ratio) is 0.01 to 9.0, preferably 0.11 to 4.0.

The use form of the lipase for hydrolysis is not particularly limited, and lipase immobilized on celite, ion exchange resin, ceramic carrier or the like may be used, but it is preferable to use it as it is. The addition amount of lipase is preferably 2 to 5000 units per 1 g of the reaction solution, and more preferably 5 to 10 units.
00 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, it is necessary to control the hydrolysis rate in the first stage of hydrolysis in the range of 5 to 50%, preferably 10 to 40%, and the amount of lipase added, reaction temperature, reaction time, The hydrolysis rate can be controlled within the above range by adjusting the reaction conditions such as the stirring speed and the weight ratio of fats and oils containing highly unsaturated fatty acids and water in the reaction system. Specifically, in the presence of fats and oils containing polyunsaturated fatty acids, water, and lipase for hydrolysis (the order of addition is not particularly limited), the reaction temperature is 10 to 55 ° C, preferably 20 to 40 ° C. ° C, the reaction time is 0.5 to 72 hours,
Preferably, the stirring speed is 0 to 800 rpm for 1 to 48 hours.
It is good to adjust. The hydrolysis rate in the present invention is a ratio (%) when the fatty acid in the fat or oil is liberated as a free fatty acid by hydrolysis. Hydrolysis rate = (acid value of oil layer after reaction / oil and fat before reaction) Saponification value) × 100.

In the present invention, the glyceride fraction is further recovered from the reaction solution obtained by the hydrolysis by a method such as solvent extraction, distillation, ion exchange resin, membrane, chromatography and the like, and based on the glyceride fraction. It is necessary to carry out the second-stage hydrolysis using the above-mentioned lipase for hydrolysis.
However, the method for removing fatty acids is not limited to these methods. In the present invention, not only a one-stage hydrolysis reaction but also
It is characterized by performing a multi-stage hydrolysis reaction of more than one stage.
Examples of the solvent used for the solvent extraction include acetone, petroleum ether, hexane and the like, and hexane is preferably employed.

In the second and subsequent stages of hydrolysis,
The same hydrolysis can be carried out using the same lipase as in the first stage of the hydrolysis, but the reaction conditions are preferably 2 to 5000 units, more preferably 5 to 1000 units of lipase to 1 g of the reaction solution. Reaction temperature is 1
0 to 55 ° C, preferably 20 to 40 ° C, and the reaction time is 0.
The stirring speed is 0 to 5 to 72 hours, preferably 1 to 48 hours.
Preferably, it is ~ 800 rpm. The hydrolysis rate in the second and subsequent hydrolysis reactions is appropriately selected,
The high concentration that is the object of the present invention is performed.

Thus, according to the present invention, a fat containing an n-6 series polyunsaturated fatty acid such as γ-linolenic acid or dihomo γ-linolenic acid is subjected to a specific multi-step hydrolysis by a specific lipase as described above. As a result, n-6
Glycerides containing a series of highly unsaturated fatty acids can be obtained.

[0016]

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples. Example 1 (1) First-stage hydrolysis 100 g of borage vegetable oil (containing 22.2% of γ-linolenic acid) was placed in a 500 ml stirred tank equipped with a magnetic stirrer.
, Water 100g, candy dasylindrache (C
Lipase (manufactured by Meito Sangyo Co., Ltd., “Lipase OF”) 40 of andida cylindraces)
00 units were added and stirred at 500 rpm for 3 hours.
The reaction was performed at 5 ° C. for 16 hours. Then, at 100 ° C, 1
Heat treatment was performed for 0 minutes to inactivate the enzyme, thereby terminating the hydrolysis reaction (hydrolysis rate: 41%). The reaction mixture was alkalified by adding 0.5N potassium hydroxide and extracted with hexane, and the glyceride fraction was recovered, to obtain a glyceride fraction of 59 g.The concentration of γ-linolenic acid in the glyceride was as follows: 35% (93% recovery of γ-linolenic acid from fatty acid composition).

(2) Second stage hydrolysis To 59 g of the glyceride fraction, 59 g of water was added to make the water concentration 50%, 2360 units of the same lipase were added, and the mixture was stirred at 500 rpm at 35 ° C. The hydrolysis reaction was performed for 8 hours. The hydrolysis rate at this time is 35
%Met. Thereafter, when the glyceride fraction was recovered, 38.4 g of a glyceride fraction was obtained. The recovery of the glyceride fraction from the two reactions was 38.4%, the concentration of γ-linolenic acid was 48.0%, and the recovery of γ-linolenic acid was 82.9%. Further, the storage stability of the obtained γ-linolenic acid-containing glyceride was evaluated as described below. As a result, no turbidity was generated even when left at 0 ° C. for 1 hour.

(Stability during Storage) When the obtained glyceride containing γ-linolenic acid was allowed to stand at 0 ° C., the time until turbidity appeared was determined by the standard method for analysis of fats and oils 2.2.7-1996.
Evaluation was performed according to (Measurement of cloud point).

Example 2 In Example 1, the second stage of hydrolysis was carried out by adding 531 g of water to 59 g of the glyceride fraction to give a water concentration of 90 g.
%, The same lipase as above was added, 11800 units were added, and the same procedure was performed except that the temperature was changed to 35 ° C. for 26 hours while stirring at 500 rpm. The hydrolysis rate at this time is 63%
Met. Then, when the glyceride fraction was collected,
21.8 g of a glyceride fraction were obtained. The recovery of the glyceride fraction from the two reactions was 21.8%, the concentration of γ-linolenic acid was 61.0%, and the recovery of γ-linolenic acid was 60.0%. Furthermore, as a result of similarly evaluating the storage stability of the obtained γ-linolenic acid-containing glyceride,
Even when left at 0 ° C. for 1 hour, no turbidity occurred.

Example 3 (1) First Stage Hydrolysis The same procedure was followed as in Example 1 except that borage oil was replaced by 100 g of black currant oil (containing 15.8% of γ-linolenic acid). A first-stage hydrolysis reaction was performed.
Then, the enzyme was inactivated to terminate the hydrolysis reaction (hydrolysis rate: 47%). The reaction mixture was alkalified by adding 0.5N potassium hydroxide and extracted with hexane to collect a glyceride fraction.
And the concentration of γ-linolenic acid in the glyceride is 27.
4%. (Recovery rate of γ-linolenic acid 92% from fatty acid composition)

(2) Second Stage Hydrolysis To 53 g of the glyceride fraction, 477 g of water was added to adjust the water concentration to 90%, 10600 units of the same lipase were added, and the mixture was stirred at 500 rpm at 35 ° C. for 1 hour.
The hydrolysis reaction was performed for 6 hours. The hydrolysis rate at this time was 58.6%. Then, when the glyceride fraction was recovered, 22.0 g of the glyceride fraction was obtained. The recovery of the glyceride fraction from the two reactions was 22.0%,
The concentration of γ-linolenic acid was 50.3%, and the recovery of γ-linolenic acid was 71.8%. Further, the obtained γ
-The storage stability of the linolenic acid-containing glyceride was evaluated in the same manner.

Example 4 (1) First Stage Hydrolysis The procedure of Example 1 was repeated, except that 100 g of evening primrose oil (containing 8% of γ-linolenic acid) was used in place of borage oil. An eye hydrolysis reaction was performed. Then, the enzyme was inactivated to terminate the hydrolysis reaction (hydrolysis rate 50
%). The reaction mixture was alkalified by adding 0.5N potassium hydroxide and extracted with hexane, and the glyceride fraction was collected.Accordingly, 50 g of a glyceride fraction was obtained, and the concentration of γ-linolenic acid in the glyceride was as follows: 15.5% (the recovery rate of γ-linolenic acid from the fatty acid composition was 97%).
%).

(2) Second stage hydrolysis To 50 g of the glyceride fraction, 450 g of water was added to make the water concentration 90%, and the same lipase as above was used for 40,000.
A unit was added, and a hydrolysis reaction was performed at 35 ° C. for 30 hours while stirring at 500 rpm. At this time, the hydrolysis rate was 76%. Thereafter, when the glyceride fraction was recovered, 12 g of the glyceride fraction was obtained. The recovery of the glyceride fraction from the two reactions was 12%, the concentration of γ-linolenic acid was 46.0%, and the recovery of γ-linolenic acid was 69%. Further, as a result of similarly evaluating the storage stability of the obtained γ-linolenic acid-containing glyceride, 0 ° C.
Turbidity did not occur even after leaving for 1 hour.

Example 5 (1) First Stage Hydrolysis In Example 1, microbial oil (Mortierella oil (dihomo γ-linolenic acid 1) was used instead of borage oil.
5.8%), except that 100 g was used.
A stage hydrolysis reaction was performed. Then, the enzyme was inactivated to terminate the hydrolysis reaction (hydrolysis rate: 46%). The resulting reaction solution was alkalified by adding 0.5N potassium hydroxide and extracted with hexane, and the glyceride fraction was collected. As a result, 54 g of a glyceride fraction was obtained, and the concentration of dihomo γ-linolenic acid in the glyceride was obtained. Was 27.8% (the recovery of dihomo γ-linolenic acid from the fatty acid composition was 95%).
%).

(2) Second stage hydrolysis To 54 g of the glyceride fraction, 54 g of water was added to adjust the water concentration to 50%, 2160 units of the same lipase were added, and the mixture was stirred at 500 rpm at 35 ° C. 30
The hydrolysis reaction was performed for a time. The hydrolysis rate at this time is 5
It was 5.6%. Thereafter, when the glyceride fraction was recovered, 24 g of the glyceride fraction was obtained. The recovery rate of the glyceride fraction from the two reactions was 24.0%, the concentration of dihomoγ-linolenic acid was 48.1%, and the dihomoγ-linolenic acid concentration was 48.1%.
The recovery of linolenic acid was 73.0%. Further, the storage stability of the obtained dihomo γ-linolenic acid-containing glyceride was evaluated in the same manner. As a result, no turbidity was generated even when left at 0 ° C. for 1 hour.

Comparative Example 1 A hydrolysis reaction was carried out in the same manner as in Example 1, except that the amount of lipase added was changed to 100,000 units and the water concentration was changed to 90% in the first stage of hydrolysis. Then, the enzyme was inactivated to terminate the hydrolysis reaction (hydrolysis rate 65
%). The reaction mixture was alkalified by adding 0.5N potassium hydroxide and extracted with hexane, and the glyceride fraction was recovered, to obtain 35 g of a glyceride fraction.The concentration of γ-linolenic acid in the glyceride was as follows: 46.2% (the recovery rate of γ-linolenic acid from the fatty acid composition was 73%).
%). When the storage stability of the obtained γ-linolenic acid-containing glyceride was expressed in the same manner as described above, turbidity was generated even when left at room temperature.

(2) Second stage hydrolysis To 35 g of the glyceride fraction, 315 g of water was added to adjust the water concentration to 90%, and the same lipase was used as described above for 35,000.
A unit was added, and a hydrolysis reaction was performed at 35 ° C. for 16 hours while stirring at 500 rpm. At this time, the hydrolysis rate was 54%. Thereafter, when the glyceride fraction was recovered, 16.1 g of a glyceride fraction was obtained. The recovery of the glyceride fraction from the two reactions was 16.1%, and γ
-The concentration of linolenic acid was 60.2%, and the recovery of γ-linolenic acid was 43.7%. In the above comparative example, γ
-The storage stability of linolenic acid-containing glycerides is poor, and γ-
The recovery of linolenic acid was also low.

[0028]

According to the present invention, a high-concentration of n-6 series polyunsaturated fatty acid is obtained by subjecting fats and oils containing n-6 series polyunsaturated fatty acid to multi-stage specific hydrolysis as described above. The polyunsaturated fatty acid thus obtained has an excellent effect on 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 (2)

[Claims] 1. A fat and / or oil containing an n-6 series polyunsaturated fatty acid, in which a fatty acid other than the n-6 series polyunsaturated fatty acid is divided and charged with a lipase derived from the genus Candida in multiple stages. Hydrolyzing to increase the concentration of the polyunsaturated fatty acids of the n-6 series, wherein the hydrolysis in the first stage is 5 to 50%.
Six series of methods for increasing the concentration of highly unsaturated fatty acids. 2. The method for increasing the concentration of highly unsaturated fatty acids according to claim 1, wherein the n-6 series highly unsaturated fatty acids are γ-linolenic acid and / or dihomo γ-linolenic acid.