JPH03183493A - Production of anthocyanin by cultured strawberry cell - Google Patents

Abstract

PURPOSE:To efficiently mass produce anthocyanin by culturing growth points taken from runners of a strawberry in a solid culture medium containing auxin and cytokinin and then culturing the resultant callous cells according to a specific method. CONSTITUTION:Growth points are initially taken from runners of a strawberry and then cultured in a solid culture medium containing auxin and cytokinin added thereto to form calli. The resultant callous cells are subsequently cultured in a liquid culture medium containing the auxin and cytokinin added thereto under condition of light at <=3000 lux illuminance and then cultured under condition of light at >=3000 lux illuminance to produce anthocyanin in the callous cells. The anthocyanin is finally extracted from the above-mentioned cells.

Description

Detailed Description of the Invention "Industrial Application Field" The present invention relates to a method for producing anthonanine using cultured strawberry cells, and particularly relates to a method for producing anthonanine in high yield using strawberry callus. .

``Conventional technology'' Antonanine means a flower pigment, and most of the bright red, purple, and blue colors of flowers and fruits are due to this Antonanine. Perilla, grapes, black beans, red turnips, roses, strawberries, etc. all get their colors from Anton Anine.

Since this antonine has high practical value as a material for colorants and paints, its production through mass cultivation is being considered both domestically and internationally. In addition, recent research has been conducted on the antihypertensive effect of antonine, and it has other interesting properties besides pigments.

Conventionally, the general method for producing Antonanine is to use various plants containing Antonanine as raw materials, extract the material with 1-2% hydrochloric acid and acidic methanol, add lead acetate, and strain out the lead chloride precipitate. Then collect the blue lead salt that forms. This is redissolved in 3% methanol-hydrochloric acid, precipitated by adding ether, and purified as a lead salt or precipitated as a picrate. Both are converted to chloride and crystallized to obtain andocyanin.

"Problems to be Solved by the Invention" However, in such conventional methods, since cultivated plants are used as raw materials, the cost of raw materials is high, and it has been impossible to produce anthonoamine at a low cost.

Furthermore, when cultivated plants are used as raw materials, the growth of the plants is slow, cultivation takes time and effort, and the production efficiency of antonanine is poor.

Furthermore, there is a problem in that the production of anthonanine depends on the harvest time of cultivated plants, and that only anthonanine can be produced on average throughout the year.

In addition, compared to conventional methods for producing anthonanine from plants, the production efficiency of anthonanine is higher;
As a method that can produce Antonanine in large quantities and on an average basis throughout the year, there is a method in which Antonanine-producing cells derived from carrots, grapes, roses, etc. are cultivated in large quantities, and Antonanine is extracted from these cultured cells. It is being considered.

However, at this stage, it has not yet been possible to culture antonanine-producing cells in large quantities on an industrial scale. The main reason for this is that it is difficult to scale up, culture, and proliferate the cultured cells when taking out the anthonanine-producing cells from the material and culturing them.

This invention was made in view of the above circumstances, and aims to provide a manufacturing method that can efficiently mass-produce anthonanine using strawberry cells that have a high ability to synthesize anthonanine.

"Means for Solving the Problems" The method for producing Antonanine using cultured strawberry cells of the present invention involves removing growing points from strawberry runners and culturing these growing points in a solid medium supplemented with auxin and cytokinin. After forming a callus, the callus cells are cultured using a liquid medium supplemented with auxin and cytokinin under light conditions of illumination of 3000 Lux or less, and then cultured under light conditions of illuminance of 3000 Lux or more to infiltrate Anton into the cells. The method for solving the above problem was to generate anin and then extract antonanine from the cells.

The present invention will be explained in detail below.

As the cultured strawberry cells used in this invention, growing points taken from strawberry runners are used. Growing point cells taken from strawberry runners grow well during callus culture, and callus with high antonanine synthesis ability can be easily obtained. On the other hand, cells taken from other parts such as strawberry leaves have poor callus formation and are unsuitable as materials.

Since a large number of strawberry runners occur after the fruit is harvested, it is desirable to easily obtain materials after the fruit is harvested, and to select and use those with good growth among the runners that have emerged in large numbers.

In addition, the strawberries used as ingredients are not limited to specific varieties;
It can be used by selecting from various varieties, for example, Dutch strawberry (Fr.
agaria chiloensis Duch
var, ananassa bail), etc. can be used as the material.

The tip of the strawberry runner is 30-50Il1m.
Collect and remove young leaves that can be seen with the naked eye.

To extract the growing points from the runner material collected in this way, first sterilize the surface of the runner material using a disinfectant commonly used in tissue culture, etc., and then dissect the growing point portion and extract the growing points. Extract. This dissection must be performed under sterile conditions.

Next, the extracted growing points are placed in a culture medium to form a callus from the growing point cells. The solid medium used for this culture is Murashige & Skoog (MS
) Medium or Lin&S Laba (LS)
The growth substances ouchinone and cytokinin are added to the medium,
As a carbon source, about 30gIQ of saccharose and 0.
A solid medium to which about 8 to 1% agar is added is preferably used.

As the auxin, 2,4-dichlorophenoquinoacetic acid (hereinafter referred to as 2.4-D) is particularly preferably used, and as the cytokinin, benzyladenine (hereinafter referred to as BA) and the like are preferably used.

The amounts of 2.4-D and BA added are approximately 0.5 to 2.0 pp+a for 2.4-D and 0.05 to 0.0 pp+a for BA.
It is desirable to set it within a range of about 2 ppm.

In addition, the culture for converting meristem cells into calluses requires 20 to 3
It is desirable that the temperature is about 0° C. and the illuminance is 3000 Lux or less. Callus proliferation pu degree 300
It is good when the illuminance is about 01, ux, but if the illuminance is higher than that, the proliferation ability of callus cells decreases, and as the culture time progresses, anthonanine is synthesized on the callus surface, and this anthonanine is synthesized. The callus cells are
Even if liquid culture is performed next, the proliferation ability of the cells decreases, making it unsuitable for forming callus for mass culture.

Then, the growing points are cultured under the above-mentioned culture conditions to form callus, and if necessary, the callus is subcultured.Furthermore, callus with high antonoanine synthesis ability is selected from the obtained calli, and the callus is selected as described above. Using a solid medium prepared at a temperature of 20~
Subculture is performed under conditions of 30° C. and illuminance of 3000 Lux or less to form callus for mass culture.

As a method for selecting calli with high andocyanin synthesis ability, cultured callus are placed on the above-mentioned solid medium, and the illuminance is 3000 Lux to 80001 Lux. UX level, illuminance 20
Cultivate at ~30°C to synthesize anthonanine on the surface of the callus, select calli with a strong degree of coloration and high ability to synthesize anthonanine, take out the uncolored part inside this callus and use it as cells for mass culture, or A method is used in which this uncolored part is taken out, placed on another fixed medium, and cultured to form a new callus.

Furthermore, calli with high anthonanine synthesis ability selected by this method have a white and soft appearance, whereas calli that are colored or hard during the callus formation stage cannot have sufficient anthonanine synthesis ability. Therefore, as a simple method for selecting calli with high antonoanine synthesis ability, it is possible to judge from the appearance of calli formed on the medium, select white and soft calli, and use them as calli for mass culture. be.

Next, the obtained callus for mass culture with high anthonoamine synthesis ability is placed in a liquid medium prepared by adding 2.4-D and BA to LS medium or MS medium and cultured with shaking. The amounts of 2.4-D and BA added in this liquid medium are:
Similar to the solid medium used in the previous callus formation, 2.4
-D 0.5-2, about 0 ppm, BA 0.05
It is desirable to set it to about 0.2 ppm. Further, the culture conditions are set to a temperature of 20 to 30°C and an illuminance of 3000 Lux or less. If the light conditions in culture medium 1 (early stage of culture) are set to an illuminance of 3000 Lux or more, anthonanine will be synthesized on the surface of the cultured cells as the culture time passes, and then the cells will turn brown and their proliferation ability will decrease. It is unsuitable for growing cultured cells.

By culturing under the above-mentioned conditions, callus cells proliferate in the liquid medium, and if necessary, the culture can be scaled up to obtain a desired amount of cultured cells.

Next, the cultured cells grown in the liquid medium were exposed to an illumination intensity of 3000.
Lux or more, preferably 3000-8000 Lux
The cultured cells are cultured under the following light conditions, and while the cultured cells are further proliferated, anthonanine is synthesized on the surface of the cultured cells (late stage of culture). The light conditions at this late stage of cultivation are illuminance 3000 Lux.
If it is below, the synthesis of Antonanine on the surface of the cultured cells will be poor, and if it is cultured under light conditions with an illuminance of 8000 Lux or more, the growth of the cultured cells will be poor and the ability of the cultured cells to synthesize Antonanine will be poor. In addition, even in this latter stage of culture, it is possible to scale up the culture as necessary.

Next, cells in which Antonanine synthesis has been completed are separated from the limb culture medium, Antonanine is extracted from the cells, and further purified to produce Antonanine.

Conventionally known methods can be used to extract and purify Antonanine from cultured cells. That is, cultured cells are separated from a liquid medium, preferably subjected to a drying process such as freeze-drying to obtain dry cells, and the dried cells are
The sample is immersed in hydrochloric acid and acidic methanol, and one or more extractions are performed to extract anthonanine. Lead acetate is added to this extract to strain out the lead chloride precipitate, and the resulting blue lead salt is collected. Next, it is dissolved again in methanol acidified with hydrochloric acid, and ether is added to precipitate it, and it is purified as a lead salt, or it is precipitated as a picrate salt, and either of these methods is converted into a chloride and crystallized.

In addition, the method for purifying andocyanin from the extract obtained by extracting Anton-anine from cultured cells is not limited to this, and for example, the above-mentioned extract is dried under reduced pressure, dissolved in water, and Anton-anine is added to an ion-exchange resin. It is possible to separate and purify it by adsorbing anine and eluting it with a hydrochloric acid acidic ethanol solution.

Through each of the above operations, Pel, which is a natural strawberry pigment,
Argonidin 3-glucoside is produced.

In this method, growing points taken from strawberry runners are cultured in a medium containing 2.4-D (auxin) and BA (cytokinin) to form callus.
．． Using a liquid medium containing 4-D and BA, and with an illuminance of 30
Mass culture of callus cells under light conditions of 00 Lux or less, then culturing under light conditions of illuminance of 3000 Lux or more to generate Antonanine within the cells, and then extract Antonanine from the cells to produce Antonanine. Therefore, when culturing calli with a high ability to synthesize Antonanine in a liquid medium, first set the illuminance to 30,001. By culturing the cultured cells under light conditions below ux and not allowing the cultured cells to synthesize Antonanine, the cells are allowed to proliferate without reducing the proliferation ability of the cultured cells, and then by changing the light conditions, Antonanine is added to the cultured cells. can be synthesized, and the scale-up of culture can be carried out extremely easily.

In other words, callus with high antonoanine synthesis ability is first grown in a small culture vessel such as a flask, and then the cultured cells are scaled up and cultured in a large tank culture vessel, and the cells are grown in large quantities in this tank. After setting the illuminance to 300
By culturing under conditions of 0 Lux or higher and allowing the cultured cells to synthesize anthonanine, a large amount of anthonanine can be easily obtained in a short period of time.

Furthermore, by subculturing the callus or cultured cells obtained by growing callus in a liquid medium under light conditions with an illumination intensity of 3000 Lux or less, mass culture of these cells can be started at any time. Antonanine can be produced on average throughout the year.

In addition, in culturing callus cells using a liquid medium, the production efficiency is much higher than in conventional cultivation of plants, and a large amount of anthonanine can be produced in a short period of time, reducing the production cost of anthonanine. It becomes possible to do so.

Furthermore, in callus culture using a solid medium and callus cell culture using a limb medium, the cell proliferation rate can be controlled by the light irradiation conditions. Therefore, by appropriately adjusting the moon irradiation conditions, it is possible to always culture at a constant growth rate, which facilitates culturing.
Efficiency can be improved.

Hereinafter, the effects of the present invention will be clarified by examples.

“Example” Strawberry (Shiki Soybean Strawberry)
Assa) runner was used as the material, 301 pieces of the tip of this runner were collected, the surface was sterilized, and the growing point was dissected under sterile conditions to extract the growing point.

Next, add 1p of 2.4-D to the extracted growing points in MS medium.
pm, BA 0, I pI) III, saccharose (as a carbon source) 30 g/Q, and 1% agar added to a solid medium, temperature 25°C, illuminance 800 Lu.
The cells were cultured under x conditions to form callus.

In addition, the leaves of sterile strawberry seedlings were implanted on a solid medium and cultured in the same way as the growing points from strawberry runners, but most of the plants either regenerated or turned brown and withered, leaving no callus. could not be formed.

On the other hand, the callus obtained from the growing points of the runners had good growth and was white and soft.

Since the growth of callus cells formed from the growing points of this strawberry runner is significantly affected by light, the relationship between callus growth and light conditions was investigated in the following experiment. One of the calluses formed on the solid medium was selected, it was incised and divided, these divided pieces were implanted on the solid medium, and the light conditions were changed to 800.3000 and 8000 LLIX. The amount of cell proliferation was measured. The results are shown in FIG.

As shown in Figure 1, the growth of strawberry callus cells depends on the light conditions! The best results were obtained when I was set to 6 degrees and 3000 Lu. Moreover, anthonanine was synthesized on the surface of the callus formed under this 3000 Lux light condition. In addition, the proliferative ability of callus cells in which anthonanine was synthesized subsequently decreased, and even when only these cells were transferred to a limb medium and cultured, almost no proliferation occurred. From this, it has been found that it is desirable for callus cells to be cultured under 3000 Lux or less of fish skin, where the synthesis of anthonanine does not occur.

Next, among the calli formed on the solid medium, calli with high antonanine synthesis ability were selected, and 2.4-DI ppm
, BA O, I Subculture was performed in LS medium (liquid medium) supplemented with Plum.

Next, in order to obtain a large amount of strawberry cells with high synthetic ability, we attempted to culture a large amount of cells in a liquid medium with the same 1:Lumon condition by changing light conditions.

Antonanine synthesis was carried out by placing 0.5 g of liquid cultured cells (Nishigesato) in a 500 m Erlenmeyer flask and changing the light conditions.
The cells were cultured with shaking at 25° C. and 80 rpm, and the synthesis of antonanine was examined. The results are shown in FIG.

As shown in Figure 2, even in liquid culture, the light conditions are set to an illuminance of 300.
Proliferation was best when the setting was 0 Lux. However, in cells cultured under these conditions, anthonanine was synthesized after about 14 days, after which the cells turned brown and their proliferation ability decreased.

On the other hand, cells cultured in the dark (in OLu) or under light conditions of 800 Lux exhibited stable growth, although no synthesis of anthonanine was observed, and subculture was possible.

Furthermore, cells cultured under light conditions of 8000 Lux had poor growth from the beginning of culture.

Next, the sample cultured under the light condition of 800 Lux used in the previous experiment was cultured under the light condition of 3000 Lux.

When anthonanine was synthesized in the cultured cells, the cultured cells were strained from the medium and qualitative analysis of the anthonanine pigment was performed. This qualitative analysis was performed by boiling the removed cultured cells for 5 minutes, filtering the cells with suction, and using PVP to collect the filtrate.
(polyvinylpyrrolidone), washed with methanol, then eluted with 1% I (CI-methanol), measured the absorbance, and then analyzed with TI and C. As a result of these analyses, the The anthonanine produced is Pelargonid, a natural strawberry pigment.
It was found that in 3g1ucoside.

From the above culture results, it is clear that the mass culture of cells, which is the preliminary step for synthesizing anthonanine, should be carried out in the dark or at 800 Lu.
carried out under light conditions of -300 x after growing the skin cells.
It has been found that it is possible to mass-produce antonanine from cultured strawberry cells by culturing under light conditions of 0 Lux.

"Effects of the invention" This invention extracts growing points from strawberry runners,
2. Form a callus by culturing in a solid medium supplemented with auxin such as 4-D and cytokinin such as BA, and then cultivate the callus using a limb medium supplemented with auxin and cytokinin under light conditions of illuminance of 3000 Lux or less. By culturing cells in large quantities, then culturing them under light conditions with an illuminance of 3000 Lux or more to produce anthonanine within the cells, and then extracting anthonanine from these cells to produce anthonanine, it is possible to produce anthonanine during limb culture. , J.
The light conditions are set to 3000 Lux or less, and the cultured cells are allowed to proliferate without synthesizing anthonanine, and then the light conditions are changed to allow the synthesis of anthonanine. Scaling up can be performed extremely easily, and a large amount of cultured cells can be easily obtained by scaling up the culture, so a large amount of anthonanine can be easily obtained.

Furthermore, by subculturing the above-mentioned callus or cultured cells grown from callus in a liquid medium under light conditions with an illuminance of 3000 Lux or less, it is possible to start mass culture of callus cells at any time. , the production of antonanine can be carried out on average throughout the year.

In addition, in culturing callus cells using a liquid medium, the production efficiency is much higher than in conventional cultivation of plants, and a large amount of anthonanine can be produced in a short period of time, reducing the production cost of anthonanine. can do.

Furthermore, in callus culture using a solid medium and callus cell culture using a liquid medium, the growth rate of cells can be controlled by the light irradiation conditions. Therefore, by appropriately adjusting the light irradiation conditions, it becomes possible to always perform the culture at a constant culture rate, thereby making the culture easier and more efficient.

[Brief explanation of drawings]

Figures 1 and 2 are graphs showing the culture results in Examples of the present invention, where Figure 1 is a graph showing the growth amount of strawberry callus on a solid medium, and Figure 2 is a graph showing the amount of strawberry callus grown in a liquid culture medium depending on light conditions. It is a graph showing the growth amount of.

Claims (1)

[Claims] A growing point is extracted from a strawberry runner, and the growing point is cultured in a solid medium containing auxin and cytokinin to form a callus, and then a liquid medium containing auxin and cytokinin is used to form a callus. , and culturing the callus cells under a light condition with an illuminance of 3000 Lux or less, then culturing under a light condition with an illuminance of 3000 Lux or more to produce anthonanine within the cells, and then extracting anthonanine from the cells. A method for producing antonanine using cultured strawberry cells.