JPH07313182A - Production of anthocyanin - Google Patents

Abstract

PURPOSE:To enable efficient mass production of anthocyanin which is useful as a coloring agent or a material for coatings by culturing the cells of a plant other than strawberry in a liquid medium containing an anthocyanin production- accelerating factor released from the strawberry culture cells. CONSTITUTION:In the extraction of anthocyanin from the cells which are subjected to cell culture and include anthocyanin, a filtrate from the strawberry cell culture liquid including the anthocyanin production-accelerating factor released from the strawberry cells, a dried product obtained by drying the culture residue after separation of the cells or an anthocyanin production- accelerating factor obtained by purification of this cell-free culture residue is added to a freshly prepared liquid culture medium. Then, the cells of a plant other than strawberry such as rose are cultured in this medium to produce a large amount of the objective anthocyanin efficiently in a large amount.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for culturing plant cells capable of producing anthocyanins to mass produce anthocyanins.

[0002]

2. Description of the Related Art Since anthocyanins have a high practical value as a material for coloring agents and paints, their mass production is being studied. In addition, recently, studies have been conducted for pharmacological use of anthocyanins such as antihypertensive action, and they have interesting properties for applications other than pigments. Conventionally, a general method for producing anthocyanins is to obtain anthocyanins by using various plants containing anthocyanins as materials and using an appropriate separation and purification method. However, in the method of extracting anthocyanins from cultivated plants in this way, the raw material cost is high, and it has been impossible to inexpensively produce anthocyanins.
Further, when a cultivated plant is used as a raw material, the growth of the plant is slow, cultivation takes time and labor, and there is a problem that the anthocyanin production efficiency is poor. Furthermore, the production of anthocyanins depends on the harvest time of cultivated plants, and there is a problem that anthocyanins cannot be produced on average throughout the year.

[0003] As compared with conventional methods for producing anthocyanins from cultivated plants, the production efficiency of anthocyanins is high, and as a method capable of producing a large amount of anthocyanins on average throughout the year, strawberry, carrot,
A method for culturing a large amount of anthocyanin-producing cells derived from grapes, roses, apples, Jerusalem artichokes, etc. and extracting anthocyanins from the cultured cells has been studied.

[0004]

However, at this stage, as described above, it has not been possible to cultivate a large amount of anthocyanin-producing cells on an industrial scale. The cause of this is that the anthocyanin-producing cells are removed from the material,
When culturing, it is difficult to scale up the cultivated cells to culture and proliferate them.

The present invention has been made in view of the above circumstances. Anthocyanins can be efficiently mass-produced using various plants such as roses, apples and other Rosaceae plants having the ability to produce anthocyanins, and other cells such as carrots and grapes. The purpose is to provide a manufacturing method that can be performed.

[0006]

The present invention provides a method for producing anthocyanins in which cultured cells are cultured, anthocyanins are produced in the cultured cells, and the anthocyanins are extracted from the anthocyanin-containing cells. This is to culture cultured cells of plants other than strawberries in a liquid medium containing an anthocyanin production promoting factor.

The above-mentioned cultured cells of plants other than strawberry include cultured cells of Rosaceae plants other than strawberry, for example, rose,
Apples and the like can be used, and carrots and grapes can be used in addition to the plants of the Rosaceae family.

The medium containing the anthocyanin production-promoting factor is a culture residual liquid obtained by separating cells from the culture liquid in which strawberry cultured cells are cultured, and a dried product obtained by drying the culture residual liquid into a newly prepared liquid medium. The liquid medium added, or the liquid medium added to the liquid medium newly prepared anthocyanin production-promoting factor obtained by purifying the culture residual liquid may be used, and further, as the dried product, the culture residual liquid is preferably vacuum. Lyophilized products are used. The medium containing the anthocyanin production promoting factor may be sterilized before use.

The present invention also includes a method for controlling the color tone of the obtained anthocyanin by changing the amount of the anthocyanin production promoting factor in the medium containing the anthocyanin production promoting factor.

[0010]

[Function] In the study of producing anthocyanins using cultured strawberry cells by controlling the culture environmental conditions such as light, medium components, and pH of the medium, the inventors of the present invention established a cell inoculation amount even if optimal culture conditions were adjusted. Focusing on the phenomenon that anthocyanin production is not observed when the concentration is extremely low, it is presumed that an anthocyanin production promoting factor (conditioning factor), which has an action of increasing the anthocyanin production of cultured cells, is released in the strawberry culture solution. ,
We investigated the effect of strawberry cell culture on the culture filtrate after culturing and reported it earlier (Mie Sakurai, Junichi Shigeta, Moriji, The 26th Autumn Meeting of the Chemical Society of Japan.
Separate Volume p. 286 (1993)). That is, when the strawberry culture cells were added to the culture filtrate (conditioning medium) after culturing the strawberry culture cells, the anthocyanin accumulation amount and the total production amount could be increased.

The present invention was made based on the new finding that the anthocyanin production promoting factor contained in the culture filtrate is also effective for culturing plants other than strawberries. That is, the present invention is a liquid medium containing an anthocyanin production promoting factor contained in the residual liquid obtained by separating the cells from the culture liquid in which the strawberry cultured cells are cultured, by culturing the cultured cells of plants other than strawberry, It is possible to increase the amount of anthocyanins accumulated and the total amount of production in cultured cells of plants. In particular, when culturing using a plant other than strawberries, anthocyanins may not be produced in the cells even if the cells proliferate, but culturing using a liquid medium containing an anthocyanin production promoting factor. As a result, anthocyanins can be reliably produced in cultured cells, and anthocyanins can be produced even in plants where it was difficult to produce anthocyanins.

[0012]

The present invention will be described in detail below. The anthocyanin production-promoting factor used in the present invention is a substance released from strawberry-cultured cells when the strawberry-cultured cells are cultured in a liquid medium. It has not been completely identified. This anthocyanin production-promoting factor has heat resistance to such an extent that its anthocyanin production-promoting action is not impaired even when the culture filtrate containing it is heated at 121 ° C. for about 15 minutes, and is freeze-dried. It has freeze resistance to the extent that its anthocyanin production promoting action is not impaired. Then, the characteristic of the action of this anthocyanin production promoting factor is that when cultured cells of plants other than strawberries are cultured in a liquid medium containing this, the accumulated amount and total production amount of anthocyanins in the cultured cells can be significantly increased. There is a point that can be done.

The liquid medium containing the anthocyanin production promoting factor used in the present invention is a culture residual liquid obtained by separating cells from the culture liquid of strawberry cultured cells, or a liquid medium newly prepared with the culture residual liquid, and the culture residual liquid is dried. The obtained dried product, preferably a liquid medium obtained by adding a freeze-dried product to a freshly prepared liquid medium, or an anthocyanin production promoting factor fraction (low-molecular portion, high A liquid medium in which a portion of the molecule had no effect) was added to a freshly prepared liquid medium is used.

The culture residual liquid obtained by separating cells from the culture medium of strawberry cultured cells is callus derived from strawberry (cultured cells).
Is 2,4-dichlorophenoxyacetic acid (2,4-D),
Naphthalene acetic acid, indole acetic acid, indole butyric acid, auxin such as trichlorobenzoic acid, or auxin and benzyladenine (BA), cytokinin such as zeatin was added to a liquid medium and cultured for about 3 days to 2 weeks. The residual liquid obtained by separating the cultured cells by filtration with a mesh or by centrifugation is used. This culture residual liquid may be the residual liquid after repeating the culture twice or more. In addition, this culture residual liquid contains nutrients (carbon source, nitrogen source, inorganic salts, etc.), p
Since the amount of hormones such as H and auxin is changing,
When this culture residual liquid is used, or when the culture residual liquid and a newly prepared medium are used as a mixture, the nutrient components, pH and hormone amount are appropriately adjusted, and even if the insufficient components are added and used. good.

When a liquid medium in which the dried product of the culture residual liquid is added to the newly prepared medium is used, the dried product obtained by freeze-drying the culture residual liquid is added to 100 ml of the newly prepared medium.
The amount may be 0.01 g to 5.0 g, preferably 0.1 to 0.5 g. The dried product of the culture residual liquid is not limited to the freeze-dried product, and may be dried using another drying method such as spray drying or hot air drying. Further, as the newly prepared medium, a liquid medium generally used in the field of plant tissue culture such as LS medium is basically used, and hormones such as auxin and carbon sources such as sucrose and nitrogen such as potassium nitrate and ammonium nitrate are used as necessary. source,
A liquid medium supplemented with vitamins may be used. In the case of 2,4-D, the amount of auxin in this liquid medium is 0.1 to 5.0 ppm, preferably about 0.5 to 2.0 ppm, and cytokinins such as BA are 0 to 3.0 pp.
m, preferably about 0 to 0.2 ppm.

In order to obtain the anthocyanin production promoting factor fraction by purifying the culture residual liquid, a purification means such as gel filtration or ion exchange chromatography is used. The anthocyanin production promoting factor purified by these means is added to a newly prepared liquid medium. The purified anthocyanin production promoting factor may be dried as a dried product by freeze-drying or the like.

In the present invention, a liquid medium containing any of the above-mentioned anthocyanin production promoting factors is subjected to steam pressure sterilization treatment at 115 ° C. or higher, and more preferably 121 ° C. used for sterilization treatment of general liquid medium. , A liquid medium sterilized by steam pressure sterilization for 10 minutes or more is used, and cultured cells (callus) of plants other than strawberry are added to the sterilized liquid medium and cultured.

In the present invention, the cultured cells of plants other than strawberries used for the production of anthocyanins may be cultured cells (callus) derived from plants having anthocyanin-producing ability, and plants other than Rosaceae Preferred as are roses, apples, and the like, and plants other than Rosaceae plants are carrots, grapes, and the like.

As the plant cultured cells other than strawberry, callus derived from cells isolated from the plant other than strawberry may be used, and the callus may be used in a liquid medium for 3000 Lu.
Cultured cells cultured under light conditions of x or less, preferably 1000 Lux or less, or subcultured many times are used.

When strawberry cultured cells are cultured in a sterilized liquid medium containing an anthocyanin production promoting factor, 3
Cultivation is carried out for 1 day or more, preferably 3 to 10 days under light conditions of 000 Lux or more, preferably about 8000 Lux. By this culture, anthocyanins are accumulated in the cultured cells. After completion of the culturing, the cultured cells are separated from the culture broth by filtration or centrifugation, taken out, dried, and then extracted with a suitable extraction solvent such as acidic methanol such as hydrochloric acid to extract and purify the anthocyanins. The culture residual liquid after separating the cultured cells can be used again as a source of the liquid medium containing the anthocyanin production promoting factor.

(Experimental Example) Rose-cultured cells were cultured with a culture filtrate of strawberry-cultured cells, and the anthocyanin production promoting effect of rose cells was confirmed. As the strawberry culture cells, callus derived from leaves of a cultured seedling of strawberry (four seasons) was used. About 2g
Callus of 2,4-D 1ppm, BA 0.1pp
m, culturing was carried out in an LS liquid medium containing 3% of sucrose at 800 Lux, a temperature of 25 ° C., and a rotational stirring condition of 80 rpm.
The culture broth precultured 3 times or more at intervals of about 1 week was filtered with a nylon mesh (30 μm) to obtain a culture filtrate (culture residual liquid). Callus derived from roses was cultured using this culture filtrate, and its anthocyanin-producing ability was examined.

Cells aseptically removed from rose leaves are
It was placed in an MS solid medium containing 5 ppm of NAA, 0.5 ppm of BA and 3% of sucrose, and cultured at 25 ° C. in the dark for about 1 to 2 months to form callus. The obtained callus was pre-total-cultured in a liquid medium having the same composition at 8000 Lux, 25 ° C. and 80 rpm. The cultured cells, which have been cultivated 3 times or more at intervals of about 2 weeks, were added to the culture filtrate obtained by the above strawberry culture, and 8000 Lu
The culture was carried out under the conditions of rotary stirring at x, temperature 25 ° C. and 80 rpm. Also, for comparison, 2 g of the above callus was added to 2,4-
The cells were cultured under the same culture conditions in a newly prepared LS liquid medium (newly prepared medium) containing 1 ppm of D, 0.1 ppm of BA and 3% of sucrose.

After culturing for one week, the cultured cells of the culture filtrate and the newly prepared medium were compared with each other. As a result, the cells cultured in the newly prepared medium were white without showing red due to anthocyanins. On the other hand, the cultured cells using the culture filtrate had a bright red color due to anthocyanins. A comparison of the weight of cells cultured in each medium is shown in FIG.
Further, 0.1 g of each cultured cell was taken, anthocyanin was extracted with methanol of hydrochloric acid, and the results of comparing the absorbances of the solutions at 528 nm are shown in FIG. From these results, by culturing rose culture cells using a culture filtrate containing an anthocyanin production-promoting factor, for cultured cells in which anthocyanins are hardly produced when cultured in a new conditioned medium, large-scale production of anthocyanins It was confirmed that

[0024]

Industrial Applicability As described above, in the present invention, a liquid medium containing an anthocyanin production promoting factor contained in the residual liquid obtained by separating cells from the culture medium in which strawberry cultured cells are cultivated, By culturing the cultured cells, the amount of anthocyanins accumulated and the total amount of production of the cultured cells of the plant can be increased. In particular, when culturing using a plant other than strawberries, anthocyanins may not be produced in the cells even if the cells proliferate, but culturing using a liquid medium containing an anthocyanin production promoting factor. As a result, anthocyanins can be reliably produced in cultured cells, and anthocyanins can be produced even in plants where it was difficult to produce anthocyanins. Therefore, according to the present invention, it is possible to mass-produce anthocyanins from cultured cells of various anthocyanin-producing plants, and it is possible to produce various anthocyanins having different colors and components.

[Brief description of drawings]

FIG. 1 is a graph comparing cell weights of rose cultured cells cultured with a culture filtrate and a newly prepared medium as a result of an experimental example according to the present invention.

FIG. 2 is a graph which similarly compares the amount of pigment produced in rose cultured cells.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:91) (C12N 5/04 C12R 1:91) C12R 1:91)

Claims (4)

[Claims] 1. A method for producing anthocyanins, which comprises culturing cultured cells, producing anthocyanins in the cultured cells, and extracting anthocyanins from the anthocyanin-containing cells, wherein a liquid medium containing an anthocyanin production promoting factor released from strawberry cultured cells. A method for producing anthocyanins, which comprises culturing cultured cells of a plant other than strawberry in the above. 2. A liquid medium containing an anthocyanin production promoting factor, wherein a dried product obtained by drying a culture residual liquid obtained by separating cells from a culture liquid in which strawberry cultured cells are cultured is added to a newly prepared liquid medium. The method for producing anthocyanin according to claim 1, wherein 3. A liquid medium containing an anthocyanin production promoting factor, which is a liquid medium newly prepared with an anthocyanin production promoting factor obtained by purifying a culture residual liquid obtained by separating cells from a culture medium in which strawberry cultured cells are cultured. The method for producing anthocyanins according to claim 1, wherein the anthocyanin is added to. 4. The method for producing anthocyanin according to claim 1, wherein the color tone of the obtained anthocyanin is controlled by changing the content of the anthocyanin production promoting factor in the liquid medium containing the anthocyanin production promoting factor.