JPH05153996A - Method for producing anthocyanin using strawberry cultured cells - Google Patents

Abstract

(57) [Summary] [Structure] Cells obtained from strawberry leaves are cultured in a solid medium containing auxin or auxin and cytokinin to form callus, and then liquid medium containing auxin or auxin and cytokinin is added. Using callus, culturing callus cells under light conditions of illuminance 3000Lux or less, then culturing under light conditions of illuminance 3000Lux or more to produce anthocyanins in the cultured cells, and then extracting anthocyanins from the obtained cultured cells. Manufacturing method. [Effects] By using cells obtained from strawberry leaves, it is possible to produce a large amount of anthocyanins in a shorter period of time in culture for producing anthocyanins, as compared with the case of using cells from other parts of strawberry. it can.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a technique for mass-producing anthocyanins using cultured strawberry cells, and in particular, using cells obtained from strawberry leaves to improve productivity in the production of anthocyanins using the cultured cells. Technology for improving.

[0002]

2. Description of the Related Art Anthocyanins mean the color of flowers, 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., exhibit their color due to anthocyanins. Since this anthocyanin has a high practical value as a colorant or paint material, mass production is being considered. Recently, studies have been conducted on the blood pressure-lowering effect 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 for separating anthocyanins from this extract by ion exchange chromatography, or adding lead acetate to the extract to separate out lead chloride precipitates, 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 cultivation plant is used as a raw material, the raw material cost is high, and it is impossible to inexpensively produce the 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 production efficiency of anthocyanins 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,
A method for culturing a large amount of anthocyanin-producing cells derived from rose, apple, Jerusalem artichoke, etc. and extracting anthocyanin from the cultured cells has 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 and 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 anthocyanin producing ability.

[0008]

[Means for Solving the Problems] The present invention comprises culturing cells obtained from strawberry leaves in a solid medium containing auxin or auxin and cytokinin to form callus, and then auxin or auxin and cytokinin Using the added liquid medium, callus cells were cultivated under light conditions of illuminance of 3000 Lux or less, and then illuminance of 3000
It is a method for producing anthocyanins using strawberry cultured cells, which comprises culturing under an optical condition of Lux or higher to produce anthocyanins in the cultured cells and then extracting the anthocyanins from the obtained cultured cells.

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 amount of 2,4-dichlorophenoxyacetic acid added is preferably in the range of 0.1 to 5 mg / liter, and the amount of benzyladenine added is preferably 5 mg / liter or less.

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

Furthermore, the present inventors used the cells obtained from each part of strawberry to search for the optimal conditions for anthocyanin production, and compared the production amount when anthocyanins were produced from the cultured cells obtained from each site. I went. As a result, it was found that when cultured cells obtained by growing callus obtained from strawberry leaves in a liquid medium were to produce anthocyanins in the cultured cells under high illuminance, the amount of anthocyanins produced was highest. The present invention has been completed.

In the method for producing anthocyanins using cultured strawberry cells of the present invention, first, callus-forming cells are aseptically removed from strawberry leaves. The strawberry of the material is not limited to a particular variety and can be selected from various varieties and used, for example, the Dutch strawberry (Fragariachiloensis Duch var.
ananassa Bail) and four seasons strawberries (Fragaria x ananas)
sa) etc. can be used.

As a leaf cell suitable for callus formation, a fragment cut out from a young leaf or a young leaf of strawberry which is in the process of growing such as a leaf of a strawberry seedling is preferably used. Strawberry leaves are cut from plants and cut into a suitable size, sterilized by immersing in a bactericide normally used in tissue culture, transferred to a sterile environment, washed with sterile water, and then a thin blade cutter, etc. To make fragments of about 1 to several mm in size.

Next, this fragment is placed on a solid medium for callus formation. The solid medium used for this callus is MS (Murashige & Skoog) medium or LS (Lin & Stab
A) A solid medium obtained by adding auxin or auxin and cytokinin, sugar such as saccharose as a carbon source, and about 0.8 to 1% agar to a basic medium such as a medium, B5 medium and EM medium is preferably 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 low concentration promotes callus formation from leaf cells. .. On the other hand, if the amount of BA added is more than 5 mg / liter, callus formation is inhibited and the cells die.

The culture for callus formation of strawberry leaf fragments is preferably carried out under a light condition of a temperature of about 20 to 30 ° C. and an illuminance of 3000 Lux or less. Propagation of callus is good when the illuminance is about 3000 Lux, but when the illuminance is further increased, the growth ability of the callus is reduced, and anthocyanin is generated on the surface of the callus with the lapse of culture time, and thus anthocyanin is generated. The generated callus is unsuitable for forming a callus for large-scale culture, because the growth of cultured cells is poor even when liquid culture is subsequently performed.

Under these conditions, callus is formed from strawberry leaf fragments and, if necessary, subcultured to prepare a callus for mass culture. Among the callus formed by this solid medium, white to cream-colored soft callus has a high proliferative ability when it is subsequently cultured in a liquid medium, 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 was treated with M
The S medium, LS medium, B5 medium, EM medium and the like are used as the basic medium, and the medium is added to auxin or auxin and cytokinin and a liquid medium containing sugar such as saccharose as a carbon source and shake-cultured.

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 be differentiated from the tissue or the callus may be green and the growth rate may be decreased. Also, the amount of 2,4-D added
Above 5 mg / l, callus growth is inhibited. Also, BA is not essential, and even if BA is not added,
Callus can be grown using a medium containing 4-D alone, but the growth of callus can be promoted by adding a low concentration of BA. On the other hand, if the amount of BA added exceeds 5 mg / liter, callus growth is inhibited.

The conditions for large-scale culture (first period 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 callus 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 culture is scaled up as necessary to produce a desired amount of cultured cells.

Next, the cultured cells grown in the liquid medium are
The cells are cultured under light conditions with an illuminance of 3000 Lux or more, preferably 3000 to 8000 Lux, and anthocyanins are produced in the cultured cells while further proliferating the cultured cells (late stage of culture). If the light condition in the latter half of the culture is lower than illuminance of 3000 Lux, anthocyanin is not sufficiently produced in the cultured cells, and the illuminance is 8000.
If it is higher than Lux, the growth of the cultured cells deteriorates and the anthocyanin production ability of the cultured cells also deteriorates. Even in the latter stage of the culture, the culture can be scaled up if necessary.

Next, cells in 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 freeze-drying, crushed if necessary, and this is immersed in an extraction solvent such as acidic methanol hydrochloric acid, and the extraction is performed 1 to several times. Anthocyanin is extracted with, 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 ethanol solution of hydrochloric acid is passed to flow the anthocyanin. A method of separating and purifying the is preferably used.

Anthocyanins are produced by the above operations. In the method for producing anthocyanins according to the present invention, cells obtained from strawberry leaves are cultured in a solid medium containing auxin or auxin and cytokinin to form callus, and then callus having high anthocyanin-producing ability is auxin or auxin. Using a liquid medium added with 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, then the obtained cultured cells Since anthocyanins are produced by extracting anthocyanins from, it is possible to extremely easily scale up the culture.

That is, callus having a high anthocyanin-producing ability derived from strawberry leaf cells was first grown in a small incubator such as a flask, and then the expanded cultured cells were scaled up to a large tank incubator. After culturing by culturing, the cultured cells are grown in this tank incubator under the light condition of illuminance of 3000 Lux or less, and then the culture is switched to the condition of illuminance of 3000 Lux or more and cultured, and a large amount of anthocyanin is produced in the cultured cells. Can be obtained quickly and easily.

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

In addition, in the culture of callus cells 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, so the production cost of anthocyanins is reduced. Can be converted.

Furthermore, in the callus culture in the solid medium and the callus cells in the liquid medium, the growth rate of the cells can be controlled by the light irradiation conditions. Therefore, by appropriately adjusting the light irradiation conditions, it is possible to always culture at a constant growth rate, and it is possible to facilitate and increase the efficiency of the culture.

[0028]

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

(Preparation of Sample) Strawberry (Four Seasons Strawberry:
Fragaria x ananassa) seedlings with 10% 5% neutral detergent solution
After stirring and washing for a minute, the mixture was placed in 70% ethanol and ultrasonically washed for 30 seconds. Thereafter, the sample 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.

(Callus formation) Saccharose (as a carbon source) was added to LS (Lin & Staba) medium at 30 g / liter and 1%.
The solid medium containing the agar of was used as the basic medium.
Using a medium supplemented with 4-D at 1 mg / liter and BA at 0.5 mg / liter, a leaf fragment sample was placed on this solid medium at a temperature of 25
Culturing was carried out for 2 weeks under the conditions of 16 hours photoperiod at 800 ° C and illuminance of 800 Lux, and the state of callus formation was examined. For the purpose of comparison, callus formation was carried out 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.

Using a leaf fragment sample, a basal medium was prepared with 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 1.

[0032]

[Table 1]

As is clear from Table 1, 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 indolebutyric acid (IBA), naphthaleneacetic acid (NAA), and indoleacetic 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 2.

[0035]

[Table 2]

As shown in Table 2, callus formation is possible 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.
Place in a 0 ml flask and add 2,4-D to this at 1 mg / liter,
BA 0.1 mg / liter, saccharose as carbon source 30 g /
Add 100 ml of LS medium (liquid medium) with liter added, and set the illuminance to 800Lux, 3000Lux, 8000Lux, culture temperature 25 ℃, 80r.
The culture was performed with shaking at pm, and the growth of callus under each light condition was examined.

As a result, when the illuminance was 800 Lux and 3000 Lux, the growth of callus was good. 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 cultivated 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 conditions. 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 culturing, and almost no callus growth was observed.

From these results, it is possible to grow callus by liquid-culturing callus derived from strawberry leaf cells under the temperature condition of illuminance of 3000 Lux or less,
It was found that large-scale production of cultured cells is possible by performing scale-up culture under these conditions. Also, the type of auxin or auxin and cytokinin added to the liquid medium and the amount of those added are 2,4 as auxin, as in the case of callus formation on a solid medium.
-D is preferred, and the addition amount is 0.1 to 5 mg / liter, preferably 1 to 2 mg / liter. In addition, BA or the like can be used as cytokinin, and the amount of BA added is 0 to 5
Conditions of mg / liter, preferably 2 mg / liter or less were suitable.

(Secondary Callus Culture) After the callus obtained from leaves, stalks, and runner growth points was subjected to primary culture for 2 weeks under a light condition of illuminance of 800 Lux, 2 g (wet weight) of these cultured cells was placed in a 500 ml flask. Put, 100ml of liquid medium of the same composition as the primary culture into it, the light condition was 8000Lux continuous light irradiation, shaking culture at a culture temperature of 25 ° C, 80r.pm, and the amount of anthocyanins in the cultured cells was measured every few days. .. For the amount of anthocyanin, remove the cultured cells from the medium and dry under reduced pressure.
Anthocyanin was extracted while adding methanol with hydrochloric acid and crushing, and this was centrifuged to take out an extract, and methanol with hydrochloric acid was added to the residue to perform extraction three times.
Collect the extract after centrifugation, dilute to a specified amount, measure the absorbance at 528 nm using a spectrophotometer, determine the anthocyanin content from the anthocyanin calibration curve prepared in advance, and calculate the anthocyanin content in one culture flask. Calculated as amount. FIG. 1 shows the relationship between the number of days of culturing and the amount of anthocyanin produced when the cultured cells derived from the cells of leaves, stalks, and growth points were secondarily cultivated at an illuminance of 8000 Lux.

As is clear from FIG. 1, cultured cells derived from strawberry leaf cells have a higher anthocyanin production amount than cultured cells derived from runner growth points or stems. Moreover, it was found that the amount of anthocyanin produced reached a peak in about 20 days from the start of the secondary culture in this cultured cell, and the secondary culture period was shorter than that of the cultured cell derived from the growing point.

From the above culture results, cells taken out from strawberry leaves were callus in a solid medium and the illuminance was 30 in a liquid medium.
00Lux Primary culture under the conditions below, scale-up culture if necessary, and after culturing cells, illuminance 3000Lux
As described above, it was found that the secondary culture can produce a large amount of anthocyanins in a short period of time as compared with the case of cells in other strawberry parts.

[0043]

INDUSTRIAL APPLICABILITY As described above, in the present invention, cells obtained from strawberry leaves are cultured in a solid medium containing auxin or auxin and cytokinin to form callus, and then auxin or auxin and cytokinin. Using liquid medium with and added, illuminance 3000
Callus cells are cultivated under a light condition of Lux or less, then cultivated under a light condition of illuminance 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 leaves, 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 mass-produce anthocyanins. You can

Further, by subculturing the above-mentioned callus or cultured cells obtained by growing the callus in a liquid medium under a light condition 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 the normal culture of raw material plants such as strawberries, and a large amount of cultured cells can be produced in a short period of time by 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, by using cells obtained from strawberry leaves, culture for anthocyanin production after callus growth culture in a liquid medium at an illuminance of 3000 Lux or less (illuminance of 3000 Lux or more) In, in comparison with the case where cells from other parts of strawberry (runner growth point, stem, stalk, etc.) are used, a large amount of anthocyanins can be produced in a short period of time.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the number of days of culturing and the amount of anthocyanin produced when the cultured cells obtained from each part of strawberries are subcultured.

Claims (3)

[Claims] 1. Cells obtained from strawberry leaves are cultured in a solid medium containing auxin or auxin and cytokinin to form callus, and then auxin or a liquid medium containing auxin and cytokinin is used to obtain illuminance. It is characterized in that callus cells are cultured under light conditions of 3000 Lux or less, then anthocyanin is produced in the culture cells by culturing under light conditions of illuminance of 3000 Lux or more, and then anthocyanins are extracted from the obtained culture cells. A method for producing anthocyanin using strawberry cultured cells. 2. The strawberry cultured cell 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.