JPS62195291A - Production of fats and oils containing gamma-linolenic ester by tissue culture of evening primrose - Google Patents

Abstract

PURPOSE:To obtain fats and oils rich in gamma-linolenic ester content, by subjecting a plant belonging to the genus Oenothera to give callus and subjecting it to tissue culture in an organic medium containing a plant hormone. CONSTITUTION:Murashige-Skoog medium is blended with 1ppm 2,4- dichlorophenoxybutyric acid (2,4-D) as auxin and 0.1ppm kinetin as cytokinin. Agar powder in an amount to give 0.9wt% solution is added to the medium to give an agar solution, which is sterilized at 120 deg.C for 15min. On the other hand sterily germinated evening primrose is collected three days after the germination, stem part having 1-5mm of evening primrose is cut into 5mm lengths and allowed to stand in solid medium. The cut stem part is kept in a dark place at 25 deg.C and cultivated for 2-3 weeks. When the steam is cultivated for three weeks, a white callus is developed. It is similarly transplanted to the medium and multiplied.

Description

[Detailed Description of the Invention] 3. Amount of (Industrial Application Field) The present invention provides a method for producing a γ-linolenic acid ester-containing fat or oil,
In particular, the present invention relates to a method for producing fats and oils with a high content of γ-linolenic acid ester by two-tissue culture.

(Prior art) γ-linolenic acid (cis, cis, cis-6/9
-12-octadecatrinenic acid)
is an 18-carbon cis-type fatty acid with unsaturated bonds at the 6°9 and 12th carbons from the carboxylic acid terminal, and is found in breast milk.
It exists as a glycerin ester in seeds such as evening primrose.

In the body, γ-lylinic acid is synthesized from linoleic acid, an essential fatty acid, by a desaturase (Δ-6-desaturase).
se). γ-Lilunic acid is bishomoγ-
It becomes linolenic acid, which is further converted in vivo to prostaglandins and leukotrienes that exhibit various physiological activities. The first rate-limiting factor in this metabolic pathway is the desaturase mentioned above. What is the function of this enzyme?

Diabetes, excess alcohol, aging, etc. can lead to a decline. As a result, the production of prostaglandins and the like is suppressed, which is known to cause health problems. γ-Linolenic acid and its salts and esters are effective as therapeutic nutrients for these health disorders. For example, premenstrual syndrome, heart disease.

It is known to be effective in lowering blood cholesterol, high blood pressure, eczema, allergies, and hangovers.

As mentioned above, γ-lylunic acid usually exists in the form of oil and fat, which is its glycerin ester, in the natural world, and γ-lylunic acid ester does not exist alone. Oils and fats containing are collected and used. Such gamma-lyrunic acid ester-containing oils and fats are usually obtained from plant seeds, especially evening primrose (evening primrose).
) is extracted from the seeds of plants of the evening primrose genus. but.

The amount of oil and fat contained in the seeds of plants of the genus Evening Primrose is small, and the content of γ-lylunic acid ester in the oil is also low. Normally, in such fats and oils, γ-linolenic acid, which constitutes γ-linolenic acid ester, accounts for 8 to 8% of the total fatty acid composition of fats and oils.
It is contained in a proportion of only 13% by weight. Therefore, γ
-To obtain linolenic acid ester-containing fats and oils, a large amount of raw materials (
seeds) and large-scale equipment. Furthermore, it takes one year to cultivate evening primrose, and the harvest period for the nine seeds is also limited, making stable production impossible. This is because there are variations in the oil and fat composition and content in the seeds.

There is also the drawback that γ-linolenic acid ester-containing fats and oils of constant quality cannot be obtained.

(Problem that the invention attempts to solve) The present invention solves the above-mentioned conventional drawbacks.

The purpose is to provide a method for effectively producing γ-linolenic acid ester-containing fats and oils with a high γ-linolenic acid ester content.

Another object of the present invention is to provide a method for inexpensively producing γ-linolenic acid ester-containing oil and fat of stable quality regardless of the harvest time or quality of evening primrose seeds.
Another object of the present invention is to provide a method for producing the above-mentioned excellent quality γ-linolenic acid ester-containing oil and fat by a tissue culture method.

(Means and effects for solving the problems) γ of the present invention
- The method for producing linolenic acid ester-containing fats and oils includes the steps of tissue culturing evening primrose (Oenothera) plants to obtain cell masses, culturing the cell masses in an organic medium containing plant hormones, and producing γ-linolenic acid ester-containing fats and oils. The above-mentioned objective is achieved by the process of producing.

According to the method of the present invention, first, September primrose, evening primrose,
Evening primrose (Qenothe)
2 using appropriate parts such as roots, stems, leaves, etc. of plants of the genus ra).
A cell mass (callus) is induced by a conventional method. The organic medium used does not need to be anything special; Murashige-Skoog, which is commonly used for plant tissue culture,
hige-5koog) medium, White
) culture medium, Linsmayer-Skoog (Linssai)
er-Skoog) medium, Ganth
eret's medium, Tulecke's medium, Motel's medium, etc. may be used. Among them, Murashige-Skoog's medium and White's medium are preferably used. Plant hormones (auxins, cytokinins, etc.) are added to this as necessary to further promote callus induction. Examples of auxins include 2,4-dichlorophenoxyacetic acid (2,4-D), indoleacetic acid (IAA),
There is naphthaleneacetic acid (N, AA).

Examples of cytokinins include kinetin and benzyladenine.

When the above-mentioned induction medium does not contain a plant hormone, the amount of γ-linolenic acid ester-containing oil and fat contained in the obtained callus is extremely small. Therefore, this callus has γ−
In order to produce fats and oils containing linolenic acid ester,
An organic medium containing plant hormones is used. As this medium, any of the above-mentioned media such as Murashige-Skoog's medium can be used.

The types of plant hormones added to the organic medium are not particularly limited, and one type may be added alone or two or more types may be added as a mixture.

Considering the production efficiency of γ-linolenic acid ester-containing fats and oils, it is preferable to add both auxins and cytokinins. In this case, it is particularly preferable to use naphthaleneacetic acid as the auxin and kinetin and/or benzyladenine as the cytokinin. The amounts of the above-mentioned plant hormones added may be small.9
Usually, the total amount is in the range of 0.01 to 1100 ppm, preferably 0.1 to 10 ppm. If the amount is too low, callus will proliferate, the production amount of γ-linolenic acid ester-containing oil and fat will be low, and the γ-linolenic acid ester content in the oil and fat will be low. If it is too much, the cost will only increase and the effect of producing γ-linolenic acid ester-containing fats and oils will be small.

The culture method may be a shaking culture method using a liquid medium or a static culture method using a solid medium. As a solid medium, for example, Mu containing about 0.9% agar
Rashige-Skoog's medium or the like is used.

The culture temperature is 15 to 36°C, preferably around 25°C. Callus cultured under such conditions produces a high proportion of γ-lylunic acid ester-containing fats and oils.

When the callus thus obtained is ground and extracted with an organic solvent, a γ-linolenic acid ester-containing fat (crude oil) is obtained. Dry the callus.

The water content thereof may be set to 10% by weight or less, preferably 5% by weight or less, and after being made into a powder, extraction with an organic solvent may be performed. As organic solvents, n-hexane, acetone,
Ethanol, chloroform, etc. are used. The obtained crude oil can be purified by conventional methods. The content of γ-lylunic acid ester in the oil thus obtained is usually 20 to 30% by weight of the fatty acid composition of the total fatty acid when converted to γ-lylphinic acid.

(Function) When callus derived from plants of the genus Evening Primrose by the method of the present invention is cultured in a medium containing plant hormones, the callus produces fats and oils with a high content of γ-linolenic acid ester. This callus proliferates rapidly, and its proliferation rate and fat content do not change even when it is subcultured over a long period of time. The composition of fats and oils also remains unchanged. Compared to the method of naturally cultivating evening primrose plants to obtain oils and fats, this method does not require a large amount of land for cultivation, is not affected by the seasons, and can produce a large amount of γ-linolene in a short period of time. It is possible to obtain acid ester-containing fats and oils.

γ-Linolenic acid ester-containing fats and oils extracted from callus are used as health foods, for example, to eliminate health problems caused by inactivation of desaturases. γ
- It is also useful as a raw material for medical products, such as by saponifying lylunic acid ester-containing fats and oils to produce γ-lylunic acid, and then converting it to methyl ester or ethyl ester, which is used as a raw material for producing vitamin F. In addition to extracting γ-lyrunic acid ester-containing fats and oils from callus, dried callus can also be used as it is or mixed with other foods after pulverizing if necessary.

(Example) The present invention will be described with reference to examples.

! Soil application (A) Callus induction and culture: Murashige
-2,4-dichlorophenoxyacetic acid (2,4-D) as auxin was added to Skoog's medium at 1 ppm + kinetin as cytokinin at o, tppm. Add agar powder to 0.9wt% to make an agar solution, and autoclave at 125℃ for 1 hour.
Sterilized for 5 minutes. A solid medium was prepared with this agar solution.

Separately, aseptically germinated evening primrose (Oenoth)
era biennis) were collected on the third day after germination.

Measure the part of the evening primrose stem with a diameter of 1 to 51 mm to a length of approximately 51 mm.
It was cut into pieces and placed on the solid medium described above. When this was cultured in the dark at 25° C. for 2 to 3 weeks, white callus was generated from the cut surface of the stem. The evening primrose stems in which callus had developed were transplanted to a new medium similar to the above, and culture was continued under the same conditions to further propagate callus. Next, only the proliferated calli were transplanted to a new medium and subcultured until generation 12.

(B) Culture of callus containing γ-linolenic acid ester-containing oil and fat extraction: Murashige-
2 ppm NAA as auxin in Skoog's medium
+ Kinetin as cytokinin 0. lppm+
It was added at a ratio of This was sterilized in the same manner as in section (A).

Pour 300++1 of this medium into 11 Erlenmeyer flasks.

The 12th generation callus subcultured in section (A) was then transplanted into this medium, and cultured with shaking in the dark at 30°C using a reciprocating shaker (90 strokes/sin) for 4 weeks.

The culture solution was filtered to obtain callus, which was dried at 60° C. for 24 hours. 0.6 g of dry callus was obtained. When this was crushed in a mortar and extracted by adding n-hexane, 0.02 g of oil and fat was obtained.

This was methyl esterified using a conventional method, and the fatty acids constituting the oil and fat were analyzed. The results are shown in Table 1. In Table 1, the number of carbon atoms is 18. A fatty acid with three double bonds indicates γ-linolenic acid.

Table 1 (^) Callus induction and culture II: Example 1 Same as section (^).

(B) Cultivation of callus containing γ-linolenic acid ester-containing oil and fat extraction A one-hite liquid medium containing IAA as nioxin at ippm and benzyladenine as cytokinin at tpp was prepared and carried out. Example 1 Sterilization was performed in the same manner as in section (1). 2501 Ill of this medium was placed in a 500 ml Erlenmeyer flask, the callus obtained in Example (A) was added thereto, and the mixture was heated in the dark at 25°C using a reciprocating shaker (90 strokes/si).
Shaking culture was performed using the following method.

After continuing the culture for 4 weeks, the callus was filtered and freeze-dried to obtain 0.9 g of dry callus. A chloroform-methanol mixture (2:1 (v/v)) was added to the dried callus and extracted using a homogenizer. The amount of oil and fat obtained was 0.04 g. This fat and oil was methyl esterified in the same manner as in Example 1.

Fatty acid analysis was performed. The results are shown in Table 2.

Table 2 (Effects of the Invention) According to the method of the present invention, a γ-linolenic acid ester-containing fat or oil containing a high proportion of γ-linolenic acid ester can be effectively obtained. Since plant tissue culture is used, it is possible to obtain a large amount of γ-lyrunic acid ester-containing oil and fat in a short period of time without being affected by the season and without requiring a large area of land for cultivation. Such γ-lylunic acid ester-containing fats and oils contain, for example, desaturase (Δ-6-d
It is used in health foods for the purpose of resolving various health disorders caused by a decrease in the activity of esaturase.

Furthermore, it can be used in a wide range of fields as an industrial raw material for pharmaceuticals and other products.

that's all

Claims (1)

[Scope of Claims] 1. A step of tissue culturing a plant of the genus Oenothera to obtain a cell mass, and culturing the cell mass in an organic medium containing a plant hormone,
A process of producing γ-linolenic acid ester-containing oil and fat. A method for producing a γ-linolenic acid ester-containing oil and fat by tissue culture of evening primrose. 2. The production method according to claim 1, wherein the plant hormone contains auxins and/or cytokinins. 3. The production method according to claim 1, wherein the auxin is at least one of naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, and indoleacetic acid, and the cytokinin is kinetin and/or benzyladenine. . 4. The manufacturing method according to claim 1, wherein the γ-linolenic acid ester-containing fat or oil is extracted from the cultured cell mass. 5. The manufacturing method according to claim 1, wherein the γ-linolenic acid constituting the γ-linolenic acid ester is contained in a proportion of 20% by weight or more of the fatty acid composition of the total oil and fat.