JPH0286790A - Production of tocopherol - Google Patents

Abstract

PURPOSE:To effectively obtain a large amount of natural tocopherol regardless of seasons without requirement of a vast field for cultivation by tissue culture of a Bryophyta plant and isolation of tocopherol from the cultured material. CONSTITUTION:A Bryophyta plant, preferably such as Marchantia polymorpha L. or Jungermannia amakawana Grolle is inoculated on a synthetic nutrient culture medium and subjected to tissue culture. The resultant cultured material is then subjected to extraction using a solvent such as n-hexane and purification by adsorption chromatography, etc., thus obtaining the objective tocopherol.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Industrial Application Field The present invention relates to a method for producing tocopherol by tissue culture of Bryophytina.

Tocopherol is naturally contained in plant oils and fats, and it is also possible to synthesize it chemically, and it is a compound with a romantic skeleton.
It consists of homologues such as α, β, γ, and δ, and its actions and activities differ depending on the type.

Generally, it has the physiological activity of vitamin E and also has an antioxidant effect, so it is used as an antioxidant in medicines and foods. In particular, in recent years, attention has been focused on the relationship between tocopherols and physiological functions such as anti-aging and prevention of adult diseases, and there has been a boom in health foods since then.Also, due to the trend towards natural products as food additives, tocopherols from natural products have become more popular. demand is rapidly increasing.

(2) Prior art As mentioned above, tocopherol is present in vegetable oils, such as soybean and wheat germ oil, cottonseed oil, palm oil, corn oil, etc., but since its content is generally quite low, it is usually found in natural oils. As a raw material for producing tocopherol, so-called deodorized scum of vegetable oil in which tocopherol is concentrated to a relatively high concentration is used.

However, the method of using deodorized vegetable oil scum is a natural product and is a by-product of specific products, so it is difficult to balance rapidly increasing demand, secure supply, and stabilize prices. is problematic.

As one way to solve these problems, for example,
0-149393.61-212294.62-589
No. 94 discloses a method of culturing tissues of safflower or their congeners and separating tocopherol from the culture. However, the content of tocopherol contained in the culture was not necessarily satisfactory when considering industrial production.

(3) Problems to be Solved by the Invention Therefore, when aiming at the industrial production of natural tocopherol by such tissue culture, it has been an important problem to further increase productivity.

(4) Means for Solving the Problems In view of the current situation, the present inventor investigated various plants and found that tocopherols, especially tocopherols, have been added to tissue cultures of moss plants, which have not been used as raw materials for producing tocopherols. It was discovered that α-tocopherol is contained in a high concentration, and the present invention was completed.

That is, the present invention provides a novel method for producing tocopherol, in which tocopherol is produced by tissue culturing a moss plant in a synthetic nutrient medium.

The method of the present invention will be explained in detail below.

Two pathways are considered for the biosynthesis of vitamin E: the tocotrienol pathway and the tocopherol pathway. In the former, homogentisic acid and geranylgeranyl birophosphate combine to form δ-tocotrienol, which undergoes methylation and reduction to become the respective γ, β, and α-tocopherols.

The latter is a pathway in which homogentisic acid and phytyl birophosphate combine to form δ-tocopherol, which is then methylated to form each tocopherol. Tocopherol is generally well known as α, β, γ, and δ, but the order of biological effects is α>β>γ>δ, and α-tocopherol is said to be vitamin E.

The normal intracellular biosynthesis site of α-tocopherol is said to be within chloroplasts. Therefore, when producing α-tocopherol by plant tissue culture, it is considered advantageous to use green callus having chloroplasts. However, mesophyll cells of higher plants usually lose chlorophyll when they become callus. On the other hand, the callus of moss plants has the characteristic of dividing and multiplying while maintaining its green color (Kanji Ono; Plant Biotechnology (Gendai Kagaku Supplementary Edition 5) 25 (1986)).

The present inventors focused on the characteristic that S'I liverwort plants can be cultured while retaining chloroplasts, an intracellular organelle unique to plants, and investigated the production of tocopherols through tissue culture of liverwort plants. We found that tocopherols can be efficiently produced by moss plants as shown below.

The moss plants subjected to tissue culture in the method of the present invention are a subphylum of green plants located between green algae and ferns. In the present invention, there is no particular restriction in principle on the tissue of the bryophyte plant to be cultured, and any cultured cells or cultured tissues induced by conventional methods may be used. However, from a practical standpoint, it is desirable to use green cultured cells that grow actively in the medium.

There is no need for the medium to be anything special; synthetic nutrient media that are generally used as tissue culture media for moss plants,
That is, those containing inorganic salts, organic components, carbon sources, and vitamins, amino acids, growth-promoting substances, etc. as necessary are used. For example, Knop's medium + Kunds
On's medium 1 Murashjge &Skoog's medium, White's medium + Linsmaior &
Skoog's medium, N1jsch's medium, GaIIIb
Org culture medium can be used. Furthermore, based on these media, plant hormones (auxins, cytokinins, etc.) are added as necessary, but plant hormones are not necessarily essential. Examples of auxins include 2,4-dichlorophenoxyacetic acid, indoleacetic acid, indolebutyric acid, and naphthaleneacetic acid, and examples of cytokinins include kinetin, benzyladenine, and zeatin. Furthermore, coconut milk, yeast extract, natural extracts such as casamino acids, etc. may be added as appropriate.

The culture conditions of the present invention are basically the same as those for normal plant tissue culture, and are carried out as appropriate according to conventional methods. For example, the temperature is 15-35℃, preferably 2
Appropriate ranges are 0 to 30°C and Pl+ of 4 to 8, preferably 5 to 6. The culture may be static culture using a solid medium or shaking culture using a liquid medium, and if necessary, aerated culture may be performed with stirring.

There are no particular restrictions on light irradiation, and cultivation is carried out under light irradiation with an appropriate wavelength and intensity or in a dark place.
Preferably, 100,001 ux of white light is irradiated during the culture.

There are no particular restrictions on the separation and purification method for tocopherol accumulated in the culture (including the medium), and it can be extracted from the culture using a suitable solvent such as n-hexane, chloroform/methanol, etc. according to a conventional method. Then, it is further purified by methods such as adsorption chromatography and molecular distillation.

(5) Examples Below, typical examples of the method of the present invention will be shown and explained in more detail. However, these are merely examples for explanation, and it goes without saying that the present invention is not limited to these examples and can be implemented in various ways, and is not limited in any way by these examples.

Example 1 Murasige carbon source modified to 2% glucose
Pour 100 ml of &Skoog's medium into a 300 ml flask and add Aurasige &Skoog's medium containing 0.9% agar.
Cultured liverwort A18 cells (Plant Sci, Lett.
, 14.225 (1979)) was inoculated into the above-mentioned modified Murasige &Skoog's medium, and irradiated with 3000 lux white light at 10 min.
After 3 weeks of shaking culture at 25° C. in an orbital shaker at 0 rotations, the culture was plated. After lyophilization of the resulting culture.

The mixture was heated and moistened with 50 ml of n-hexane under a N2 stream for about 8 hours. After passing, n-hexane was distilled off under reduced pressure to obtain a brown oily substance. A portion of this oily substance was taken, dissolved in n-hexane, and subjected to high performance liquid chromatography (HPL).
In C), the tocopherol content was analyzed under the following conditions. In addition, for comparison, the tocopherol content in safflower tissue culture used in the conventional method was analyzed.

(Analysis conditions) Column: 5hodex 5ilicapak E-1
14 Temperature: Room temperature (approx. 25°C) Eluent 2 〇-hexane/dioxane/ethanol=
97.6: 2.0: 0.4 Flow rate: 1.
Oml/min Detector: Fluorescence; Ex 289 nm, Em 3
25 nm results are shown in Table 1.

Table 1 By the same procedure as in Example 1 except that
T.

Lab., 56.201 (1984)),
Its tocopherol content was analyzed.

The results are shown in Table 2.

Table 2 Example 2 Using this medium of Murasige & 5koo with the carbon source modified to 4% glucose and 0.1% calcium carbonate added (6) Effects If the 'IBM method of tocopherol according to the present invention is adopted, Compared to conventional methods, natural tocopherols are more efficiently produced.
It can be built. By using plant tissue culture, it is possible to obtain large amounts of tocopherols in a short period of time without being affected by the seasons and without requiring vast amounts of land for cultivation.

Claims (1)

[Scope of Claims] 1. A method for producing tocopherol, which comprises culturing tissue of a moss plant and separating tocopherol from the culture. 2. The method for producing tocopherols according to claim 1, wherein the moss plant is liverwort or lichen.