JPH067185A - Method for producing anthocyanin using strawberry cultured cells - Google Patents

Abstract

(57) [Summary] [Structure] A step of culturing cells obtained from strawberries in a solid medium to form callus, a step of liquid-culturing callus cells under a light condition of an illuminance of 3000 Lux or less, and a light of an illuminance of 3000 Lux or more. The nitrogen source concentration in the medium is in the range of 100 to 800 Nmg / liter in the anthocyanin production step in the production method including the step of producing anthocyanins in the cultured cells by culturing under the conditions and the step of extracting the anthocyanins from the cultured cells. Of producing the anthocyanin using the medium described above. [Effect] Compared to the case where a medium having a high nitrogen source concentration is used in the step of producing anthocyanin, the amount of anthocyanin produced per incubator can be increased, and the efficiency of anthocyanin extraction operation from cultured cells can be improved. It is possible to improve the production efficiency of anthocyanins.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a technique for mass-producing anthocyanins using strawberry-cultured cells, and particularly to controlling the amount of anthocyanins produced by adjusting the nitrogen source concentration in a medium for culturing strawberry-cultured cells. Regarding increasing technology.

[0002]

BACKGROUND OF THE INVENTION Anthocyanins have the meaning of flower color, and most of the bright red, purple and blue colors of flowers and fruits are derived from this anthocyanin. Perilla, grapes, black soybeans, red turnips, roses, strawberries, etc. are colored by anthocyanins. Since this anthocyanin has a high practical value as a colorant or paint material, mass production is being considered. In addition, recently, studies have been conducted on the blood pressure lowering action of anthocyanins, which have interesting properties other than pigments.

Conventionally, as a general method for producing anthocyanins, various plants containing anthocyanins are used as materials, and the anthocyanins are extracted from the materials with hydrochloric acid-methanol.
A method of separating anthocyanins from this extract by ion exchange chromatography, or adding lead acetate to the extract to separate out precipitation of lead chloride, and then collecting the resulting blue lead salt, which was added to hydrochloric acid-acidified methanol Melted again,
Ether is added to precipitate anthocyanin, which is then purified as a lead salt or precipitated as a picrate, and converted to chloride to obtain anthocyanin crystals.

[0004]

However, in the production of anthocyanins by the conventional method, since the anthocyanins are extracted from cultivated plants, the raw material cost is high, and it is impossible to produce the anthocyanins at low cost. 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.

As compared with conventional methods for producing anthocyanins from cultivated plants, carrots, grapes, and anthocyanins have high production efficiency and are capable of producing large amounts of anthocyanins on average throughout the year.
Methods for culturing a large amount of anthocyanin-producing cells derived from roses, apples, Jerusalem artichokes, etc. and extracting anthocyanins from the cultured cells have been studied.

However, at this stage, it has not been possible to cultivate a large amount of anthocyanin-producing cells on an industrial scale. The main cause of this is that when the anthocyanin-producing cells are taken out of the material and cultured, it is difficult to scale up the cultured cells to culture and proliferate them.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a production method capable of efficiently producing a large amount of anthocyanins by using strawberry cells having a high ability to produce anthocyanins.

[0008]

Means for Solving the Problem The present invention comprises a step of culturing cells obtained from strawberries in a solid medium containing auxin or auxin and cytokinin to form callus, and then, in which callus cells are auxin or auxin and cytokinin. Using a liquid medium containing and, the step of culturing under light conditions of illuminance 3000Lux or less, then the step of culturing the cultured cells under the light conditions of illuminance 3000Lux or more to generate anthocyanins in the cultured cells, then anthocyanin from the cultured cells A method for producing anthocyanin, comprising the step of extracting anthocyanin in the cultured cells, wherein the concentration of nitrogen source in the medium is 100 mg to 800 mg as nitrogen / liquid in the range of 1 liter of medium. A method for producing anthocyanin, which comprises using a medium.

As the auxin to be added to the solid medium and the liquid medium, 2,4-dichlorophenoxyacetic acid is preferable. Further, benzyladenine is preferable as the cytokinin. The addition amount of 2,4-dichlorophenoxyacetic acid is preferably in the range of 0.1 to 5 mg / liter, and the addition amount of benzyladenine is preferably 5 mg / liter or less.

The present invention will be described in detail below. The inventors of the present invention used cells taken from each part of strawberry, and by appropriately setting the light conditions and the hormone composition (auxin, cytokinin concentration) in the medium, produced a large number of cultured strawberry cells and produced a large amount of anthocyanins. Invented the production method, Japanese Patent Application No. 1-224000, Japanese Patent Application No. 2-1651
Patent application was filed as No. 89 and Japanese Patent Application No. 2-165190.

Furthermore, the present inventors have investigated the effect of nitrogen source concentration in solid medium and liquid medium on anthocyanin production using cells obtained from strawberries, and as a result, in the process of producing anthocyanin in cultured cells, It was found that by using a liquid medium having a low nitrogen source concentration (concentration of nitrate nitrogen and ammonia nitrogen) in the liquid medium, the amount of anthocyanin produced per unit weight of cultured cells can be increased and the anthocyanin production efficiency can be improved. The present invention has been completed.

In the method for producing anthocyanins using cultured strawberry cells of the present invention, first, cells suitable for callus formation are aseptically taken out from strawberries. The strawberry of the material is not limited to a specific variety and can be selected from various varieties and used, for example, Dutch strawberry (Fragaria chiloensis Duchvar.
ananassa Bail) and four seasons strawberries (Fragaria x ananas)
sa) etc. can be used.

Suitable cells for callus can be obtained from various sites such as strawberry leaves, stems, roots and runners. These parts are cut to an appropriate size, immersed in a bactericide usually used in tissue culture to sterilize, transferred to a sterile environment, washed with sterilized water, and then using a thin blade cutter or the like. The growth point is extracted as a fragment with a size of 1 to several mm, or while observing with a stereomicroscope.

Next, the obtained cells are placed on a solid medium for callus formation. The solid medium used for this callus formation is MS (Murashige & Skoog) medium or LS (Lin &
Staba) medium, B5 medium, basic medium such as EM medium,
A solid medium containing auxin or auxin and cytokinin, sugar such as sucrose as a carbon source, and 0.8 to 1% agar is used.

2,4-dichlorophenoxyacetic acid (hereinafter referred to as 2,4-D) is particularly preferably used as the auxin, and benzyladenine (hereinafter referred to as BA) is preferably used as the cytokinin. . Two
The amount of 4-D added is preferably in the range of 0.1 to 5 mg / liter.
If the addition amount of 2,4-D is less than 0.1 mg / liter, callus formation hardly occurs, and if the addition amount exceeds 5 mg / liter, callus formation is inhibited and cells die. Also, BA
It is desirable to add 5 mg / liter or less. BA is not essential, and callus can be formed without addition of 2,4-D alone in the addition medium, but addition of a low concentration promotes callus formation. On the other hand, if the amount of BA added is more than 5 mg / liter, callus formation is inhibited and the cells die.

It is desirable that the culture for callus formation of cells taken out from strawberries is performed at a temperature of about 20 to 30 ° C. and a light condition of an illuminance of 3000 Lux or less. Callus multiplication is illuminance 30
It is good in the case of about 00 Lux, but when the illuminance is further increased, the growth ability of callus decreases, and anthocyanin is generated on the surface of the callus with the lapse of culture time,
The callus thus produced with anthocyanins is not suitable for forming a callus for large-scale culture because the growth of cultured cells is poor even when liquid culture is subsequently performed.

Callus is formed from strawberry cells under such conditions, and subculture is carried out if necessary to prepare callus for large-scale culture. Among the callus formed by this solid medium, white to cream-colored soft callus has a high proliferative ability at the time of culturing in a liquid medium next time, while on the other hand, anthocyanin or the like is generated on the surface of the callus to become colored or hard. Callus that has become less proliferative. Therefore, it is desirable to select and use white soft callus as the callus for mass culture.

Next, the obtained callus for large-scale culture is put into a liquid medium and shake-cultured at an illuminance of 3000 Lux or less. The liquid medium used here includes LS medium, MS medium, B
A liquid medium in which auxin or auxin and cytokinin and sugars such as saccharose as a carbon source are added to basal medium such as 5 medium, GA medium and EM medium is used.

The auxin and cytokinin added to the liquid medium are preferably 2,4-D and BA, and the addition amount thereof is 2,4-D, as in the solid medium used in the above-mentioned callus formation. It is preferable that D is in the range of 0.1 to 5 mg / liter and BA is in the range of 0 to 5 mg / liter. The amount of 2,4-D added is 0.
If the amount is less than 1 mg / liter, it is not preferable because the callus may cause tissue differentiation, or the cultured cells may become green and the growth rate may decrease. When the amount of 2,4-D added exceeds 5 mg / liter, the growth of cultured cells is inhibited. In addition, BA is not essential, and cultured cells can be grown using a medium containing 2,4-D alone without the addition of BA, but the growth of cultured cells is promoted by adding BA at a low concentration. be able to. On the other hand, the amount of BA added is
When it exceeds 5 mg / liter, the growth of cultured cells is inhibited.

The conditions for large-scale culture (first stage of culture) using this liquid medium are a temperature of 20 to 30 ° C., and a light condition of illuminance of 3000.
Lux or less. If the illuminance is higher than 3000 Lux in this culture, the cultured cells may become green and the growth may deteriorate, which is not preferable. By culturing under these conditions, callus cells grow in the liquid medium, and the cultured cells are scaled up and cultured as necessary to produce a desired amount of cultured cells.

The type of the liquid medium used in the first half of the culture and the concentration of nitrogen source are not limited, and both the medium having the same composition as the liquid medium used in the next step of producing anthocyanin and the liquid medium having a different composition are used. Although it can be used, a medium having a high growth rate of cultured cells is usually used.
For example, in the early stage of culture, LS medium or MS medium in which the growth of cultured cells is good or B5 medium with an increased nitrogen source is used, and the nitrogen source concentration is increased in the next anthocyanin production step.
Culture can be performed by transferring to a liquid medium in the range of 100 mg to 800 mg Nmg / liter.

Next, the cultured cells grown in the liquid medium are
The nitrogen source concentration in 1 liter of culture medium is nitrogen (N) (hereinafter referred to as Nm
Expressed as g / liter. ) Using a liquid medium in the range of 100 mg to 800 mg Nmg / liter, an illuminance of 3000 Lux or more, preferably an illuminance of 3
The cells are cultured under a light condition of 000 to 8000 Lux, and the cultured cells are further grown while anthocyanins are produced in the cultured cells (the latter stage of culture).

If the nitrogen source concentration of the liquid medium used in the latter stage of the culture is less than 100 Nmg / liter, the growth of the cultured cells deteriorates in the latter stage of the culture, which is not preferable. The nitrogen source concentration is 80
When a liquid medium having a concentration higher than 0 Nmg / liter is used, the method conventionally performed by the present inventors (Japanese Patent Application No. 1-22).
No. 4000, Japanese Patent Application No. 2-165189, Japanese Patent Application No. 2-1
No. 65190) gives the same culture result. That is, although the growth rate of cultured cells is high, the amount of anthocyanin produced per unit weight of cultured cells is lower than that in the case of using a liquid medium having a relatively low concentration in the latter stage of culture as in the present invention.

In addition, the light condition in the latter half of the culture is illuminance 3000 Lu.
When it is lower than x, anthocyanins are not sufficiently produced in the cultured cells, and when the illuminance is higher than 8000 Lux, the growth of the cultured cells is deteriorated and the anthocyanin producing ability of the cultured cells is deteriorated.

Tables 1 and 2 show the compositions of LS medium, MS medium, B5 medium, GA medium and EM medium (the amount of each component in 1 liter of medium) as the medium generally used in tissue culture.

[0026]

[Table 1]

[Table 2]

Calculating the nitrogen source concentration of these media, that is, the total amount (N mg / liter) of nitrate nitrogen (NO ₃ -N) and ammonia nitrogen (NH ₄ -N), the LS medium contained 1174 Nmg / liter.
Liter, B5 medium 375 Nmg / liter, MS medium 1169 Nmg
/ Liter, GA medium 444Nmg / liter, EM medium 444Nmg
/ Liter. Therefore, B5 medium, GA medium and EM medium are used as the liquid medium in the latter stage of the culture, and the nitrogen source concentration in the range of 100 mg to 800 mg Nmg / liter is B5 medium, GA medium and EM medium.
Note that these media are merely examples, and liquid media other than those having a nitrogen source concentration in the range of 100 mg to 800 mg Nmg / liter can be used. Further, the nitrogen source concentration in the LS medium and the MS medium can be adjusted so as to be in the range of 100 mg to 800 mg Nmg / liter and used.

The nitrogen source concentration of the B5 medium or the like is 100 mg to 800
Illuminance 3000Lu using liquid medium in the range of mgNmg / liter
By culturing with shaking at 20 to 30 ° C. for x or more, anthocyanins are produced in the cultured cells. It should be noted that the culture can be scaled up as needed even in the latter stage of the culture.

Next, cells for which anthocyanin production has been completed are separated from the liquid medium, and anthocyanins are extracted and purified from the cells to produce anthocyanins. A conventionally known method can be used as a method for extracting and purifying anthocyanins from cultured cells. For example, the cultured cells are separated from the liquid medium, preferably subjected to a drying treatment such as lyophilization, crushed if necessary, and this is immersed in an extraction solvent such as hydrochloric acid acidic methanol and the extraction is performed 1 to several times. Anthocyanin is extracted with water, the extract is dried under reduced pressure, dissolved in water, and then poured into a column filled with an ion exchange resin to adsorb anthocyanin on the resin, and an effluent such as hydrochloric acid ethanol solution is passed. A method of separating and purifying anthocyanins is preferably used. Anthocyanins are produced by the above operations.

In the method for producing anthocyanin according to the present invention, cells obtained from strawberries are cultured in a solid medium containing auxin or auxin and cytokinin to form callus, and then callus having a high anthocyanin-producing ability is added to auxin or Using a liquid medium added with auxin and cytokinin, cultured under light conditions of illuminance 3000Lux or less, then cultured under light conditions of illuminance 3000Lux or more, to generate anthocyanins in the cultured cells,
Next, since anthocyanins are produced by extracting anthocyanins from the obtained cultured cells, it is possible to extremely easily scale up the culture.

That is, callus derived from strawberry cells is first grown in a small incubator such as a flask and then
The grown cultured cells are scaled up in a large tank incubator and cultured.
A large amount of anthocyanins can be easily obtained in a short period of time by growing the cells under a light condition of 00 Lux or less and then switching to a condition of illuminance of 3000 Lux or more and culturing to cause the cultured cells to produce anthocyanins.

Furthermore, by subculturing the above-mentioned callus or cultured cells obtained by growing the callus in a liquid medium under a light condition of illuminance of 3000 Lux or less, large-scale culture of these cells can be started at an arbitrary time. Therefore, unlike the production of anthocyanins using cultivated plants, it is possible to produce anthocyanins evenly throughout the year.

Further, in callus cell culture using a liquid medium, the production efficiency is remarkably higher than that in normal cultivation of plants, and a large amount of anthocyanins can be produced in a short period of time, thus reducing the production cost of anthocyanins. Can be converted.

Further, in the method for producing anthocyanin according to the present invention, the nitrogen source concentration is in the range of 100 to 800 Nmg / liter in the culture for producing anthocyanin in the cultured cells (late stage of culture). Concentration is 80
Compared with the case of using a liquid medium higher than 0 Nmg / liter,
The amount of anthocyanins produced per unit weight of cultured cells is increased, and the production efficiency of anthocyanins can be improved.

[0035]

[Examples] Examples of the method for producing anthocyanins using strawberry cultured cells according to the present invention will be described below.

(Preparation of Sample) Strawberry (Four Seasons Strawberry:
Fragaria x ananassa) seedlings 10% with 5% neutral detergent solution
After stirring and washing for a minute, the mixture was placed in 70% ethanol and ultrasonically washed for 30 seconds. Then, it was immersed in a 5% NaClO aqueous solution for 8 minutes for sterilization, and each sample was placed in a clean bench and washed three times with sterile water. From this seedling, leaves were cut out and cut into pieces of 1 mm to several mm.

(Formation of Callus) LS having the composition shown in Table 1
2,4-D was added to the medium at 1 mg / liter, BA was added at 0.5 mg / liter, and a solid medium containing 1% agar was used. A leaf fragment sample was placed on this solid medium at a temperature of 25 ° C and an illuminance of 25 ° C. 800 Lux 16
After culturing for 2 weeks under the condition of time-day length, the callus formation state was examined. For comparison, callus formation was performed by placing stem and stalk fragment samples of strawberry seedlings and growing point cells extracted from runners on a fixed medium in the same manner as the leaf fragment samples. As a result, callus was formed in each sample.

A leaf fragment sample was used in LS medium for 2,4-
Using various solid media in which D was added in the range of 0 to 7 mg / liter and BA in the range of 0 to 5 mg / liter, leaf fragment samples were placed on these media to examine the optimal medium hormone composition for callus formation. The results are shown in Table 3.

[0039]

[Table 3]

As is clear from Table 3, the concentration of 2,4-D in the callus-forming medium is 0.1-5 mg / liter, preferably
The range is 1 to 2 mg / liter, and the BA is preferably 0 to 5 mg / liter, preferably 2 mg / liter or less.

Using leaf fragment samples and stem fragment samples, 0.1-5 mg / liter of indole butyric acid (IBA), naphthalene acetic acid (NAA), and indole acetic acid (IAA) were added as auxins other than 2,4-D. Using the medium added in the above range, the above leaf and stem fragment samples were placed and examined for callus formation. The results are shown in Table 4.

[0042]

[Table 4]

As shown in Table 4, calluses can be formed by using auxins (IBA, NAA, IAA) other than 2,4-D. The formation was poor.

(Primary callus cell culture) According to the above test, white soft callus obtained from a leaf fragment sample was used in a solid medium containing 2,4-D at 1 mg / liter and BA at 0.1 mg / liter. This callus is incised and divided into 50 pieces.
Put it in a 0 ml flask, and in it, 2,4-D 1 mg / liter,
100 ml of LS medium (liquid medium) containing 0.1 mg / liter of BA
Was added, and the illuminance was set to 800 Lux, 3000 Lux, and 8000 Lux, and shake culture was performed at a culture temperature of 25 ° C. and 80 rpm, and the growth of callus under each light condition was examined.

As a result, the growth of callus was good when the illuminance was 800 Lux and 3000 Lux. But 3000Lux
After 10 to 14 days, the cultured cells of the callus were discolored and their subsequent growth was reduced. On the other hand, callus cultured at an illuminance of 800 Lux showed a high proliferation rate without discoloration. This 80
Callus cultured under 0 Lux light condition could be subcultured in a culture cycle of 1 to 2 weeks, and it was possible to mass-produce cultured cells by scale-up culture under the same condition. on the other hand,
Callus cultivated at 8000 Lux showed poor growth from the beginning of culture, discolored within a few days after the start of culture, and almost no callus growth was observed.

From these results, callus derived from strawberry cells can be grown by liquid culture under the temperature condition of illuminance of 3000 Lux or less, and the callus can be grown by scale-up culture under this condition. It turns out that mass production is possible. The type of auxin or auxin and cytokinin to be added to the liquid medium and their addition amount are 2,4-D as auxin, as in the case of callus formation on a solid medium.
Is preferable, and the addition amount is 0.1 to 5 mg / liter, preferably
A range of 1-2 mg / liter is desirable. BA can be used as the cytokinin, and the amount of BA added is 0 to 5 mg /
Conditions of 1 liter, preferably 2 mg / liter or less were suitable.

(Callus Secondary Culture) Callus obtained from a leaf fragment sample was subjected to primary culture for 2 weeks under a light condition of illuminance of 800 Lux, and then 2 g (wet weight) of the cultured cells were put into a 500 ml flask, and Table 1 was prepared. B5 medium having the composition shown in
375 Nmg / liter), 2,4-D at 1 mg / liter and BA at 0.
Add 100 ml of liquid medium containing 1 mg / liter and adjust the light condition to 80.
It was irradiated with 00Lux continuous light and shake-cultured at a culture temperature of 25 ° C. and 80 rpm. For comparison, LS medium (nitrogen source concentration
1175 Nmg / liter) and MS medium (nitrogen source concentration 1169 Nmg /
2 g of the cultured cells was added to 100 ml of a liquid medium containing 1 mg / liter of 2,4-D and 0.1 mg / liter of BA in (1 liter), and the cells were similarly shake-cultured.

After culturing these for 2 weeks, the cultured cells in each incubator were taken out and weighed. The weight of cultured cells per bottle for each liquid medium is shown in FIG.
Next, 0.1 g of cultured cells cultured in each liquid medium was collected, anthocyanins were extracted with hydrochloric acid acidic methanol while crushing the cells, and the extract obtained by centrifugation was diluted to a specified amount, and at 520 nm. Absorbance was measured by performing colorimetric determination. The result is shown in FIG. The amount of anthocyanin per 0.1 g of each cultured cell was determined from a calibration curve prepared using an anthocyanin standard product, and multiplied by the weight of cultured cells per bottle in FIG. 1 to calculate the amount of anthocyanin produced per bottle. The result is shown in FIG.

As is clear from FIG. 1, the culture cell weight per bottle shows that the LS medium and the MS with a high nitrogen source concentration were used.
The culture medium has a higher increase in cultured cells than the B5 medium having a low nitrogen original concentration. However, as is clear from FIG.
The amount of anthocyanin per unit weight of cultured cells was significantly higher when 5 medium was used, and the amount of anthocyanin produced per bottle was twice that of LS medium or MS medium. It is the above production amount.

Further, a nitrogen source (NH ₄ NO ₃ ) was added to the B5 medium to give a nitrogen source concentration of 600 Nmg / liter, 800 Nmg / liter,
Liquid medium (2, 1000 Nmg / l, 1500 Nmg / l)
4-D 1 mg / liter, BA 0.1 mg / liter was added), and the same operation as in the secondary culture was performed. As a result, the medium having a nitrogen source concentration of 600 Nmg / liter produced the same amount of anthocyanin as the B5 medium. In the medium having a nitrogen original concentration of 800 Nmg / liter, the anthocyanin production amount obtained was several% higher than that in the LS medium. Original nitrogen concentration 1000 Nmg / liter and 1500 Nmg /
The liter medium produced less anthocyanin than the LS medium.

[0051]

As described above, in the present invention, cells obtained from strawberries are cultured in a solid medium containing auxin or auxin and cytokinin to form callus, and then auxin or auxin and cytokinin are added. Illuminance 3000Lux using the added liquid medium
Callus cells were cultured under the following light conditions and then
Anthocyanins are produced by culturing under an optical condition of 3000 Lux or more to generate anthocyanins in the cultured cells, and then anthocyanins are extracted from the obtained cultured cells to produce anthocyanins. When liquid-culturing callus formed from strawberry cells, it is possible to extremely easily scale up the culture, and by performing the scale-up of culture, it is possible to obtain a large amount of cultured cells and to mass-produce anthocyanins. You can

Further, by subculturing the above-mentioned callus or cultured cells obtained by growing callus in a liquid medium under light conditions of an illuminance of 3000 Lux or less, large-scale culture of callus cells can be started at any time. Therefore, it is possible to plan the manufacturing time and the manufacturing amount arbitrarily, and it is possible to surely carry out the manufacturing according to the plan.

Further, the culture of callus cells using a liquid medium has a markedly higher production efficiency than ordinary culture of raw material plants such as strawberries, and a large amount of cultured cells can be produced in a short period of time using equipment such as a tank incubator. Since it can be produced and anthocyanin can be produced, the production cost of anthocyanin can be reduced.

Furthermore, in the present invention, in the step of producing anthocyanins in the cultured cells, the concentration of nitrogen source in the medium is
By using a liquid medium in the range of 100 to 800 Nmg / liter, it is possible to increase the amount of anthocyanin produced per unit weight of cultured cells as compared with the case of using a liquid medium having a nitrogen source concentration higher than 800 Nmg / liter. it can. Therefore, compared with the case of using a medium with a high nitrogen source concentration in the step of producing anthocyanin, it is possible to increase the amount of anthocyanin produced per incubator and improve the efficiency of anthocyanin extraction operation from cultured cells. The production efficiency of anthocyanins can be improved.

[Brief description of drawings]

FIG. 1 is a graph showing the results of Examples of the present invention and showing the callus weight (cultured cell weight) for each medium.

FIG. 2 is a graph showing the amount of anthocyanins per unit weight of each medium.

FIG. 3 is a graph showing the amount of anthocyanins produced in each incubator.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:91)

Claims (3)

[Claims] 1. A step of culturing cells obtained from a strawberry in a solid medium containing auxin or auxin and cytokinin to form callus, and then forming a callus in a liquid medium containing auxin or auxin and cytokinin. Use, illuminance 3000Lu
A step of culturing under the light conditions of x or less, then a step of culturing the cultured cells under the light conditions of illuminance of 3000 Lux or more to generate anthocyanins in the cultured cells, and then a step of extracting anthocyanins from the obtained cultured cells. A method for producing anthocyanin, comprising the step of producing anthocyanin in the cultured cells,
The nitrogen source concentration in the medium is 100 mg to 800 mg as nitrogen / medium
A method for producing anthocyanins using strawberry cultured cells, which comprises using a liquid medium in the range of 1 liter. 2. The strawberry cultured cells according to claim 1, wherein the auxin added to the solid medium and the liquid medium is 2,4-dichlorophenoxyacetic acid, and the cytokinin added is benzyladenine. Method for producing anthocyanin. 3. Addition to solid medium and liquid medium 2,4
The method for producing anthocyanins using strawberry cultured cells according to claim 2, wherein the amount of dichlorophenoxyacetic acid added is 0.1 to 5 mg / liter and the amount of benzyladenine added is 5 mg / liter or less.