JPH0458894A - Production of anthocyanin - Google Patents

Abstract

PURPOSE:To produce a large amount of anthocyanin constantly throughout the year at a reduced production cost with easy and efficient cultivation method by separating a cell from strawberry, forming a callus and culturing the cell under a specific illumination condition. CONSTITUTION:Cells are separated from leaf, stalk, fruit, flower or root of strawberry. The cell is cultured on a solid medium added with (A) auxin or the component A and (B) cytokinin to form a callus. The callus cell is cultured in a liquid medium added with the component A or the component A and the component B under the illumination of <=3,000Lux and then cultured under the illumination of >=3,000Lux to form anthocyanin in the cell. The accumulated anthocyanin is extracted from the cell.

Description

DETAILED DESCRIPTION OF THE INVENTION "Industrial Application Field" The present invention relates to a method for producing anthocyanins using cultured strawberry cells, and particularly to a method for producing anthocyanins in large quantities using strawberry callus.

``Conventional technology'' Anthocyanin means the color of flowers, and most of the bright red, purple, and blue colors of flowers and fruits are due to this anthocyanin. Perilla, grapes, black beans, red turnips, roses, strawberries, etc. get their colors from anthocyanins.

Since this anthocyanin has high practical value as a material for colorants and paints, mass production is being considered. Recently, research has also been conducted on the antihypertensive effect of anthocyanins, and they have other interesting properties besides pigments.

Conventionally, the general method for producing anthocyanins is to extract the materials from various plants containing anthocyanins with 1-2% hydrochloric acid and acidic methanol, add lead acetate to strain out the lead chloride precipitate, and then Collect the resulting blue lead salt. 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 anthocyanins.

"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 anthocyanins at low cost.

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

Furthermore, the production of anthocyanin depends on the harvest time of cultivated plants, and there is also the problem that anthocyanin cannot be produced on average throughout the year.

Compared to conventional methods for producing anthocyanins from cultivated plants, methods that have higher anthocyanin production efficiency and can produce anthocyanins in large quantities and evenly throughout the year include carrots, grapes, roses, BACKGROUND ART Studies have been conducted on methods for mass culturing anthocyanin-producing cells derived from apples, Jerusalem artichokes, grapes, etc., and extracting anthocyanins from these cultured cells.

However, at this stage, it has not yet been possible to cultivate anthocyanin-producing cells in large quantities on an industrial scale. The main reason for this is that when removing anthocyanin-producing cells from the material and culturing them, it is difficult to scale up the cultured cells and grow them.

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

"Means for Solving the Problems" The method for producing anthocyanins of this invention includes strawberry leaves,
Cells are removed from any part of the stem, fruit, flower, or root, and the cells are cultured on a solid medium supplemented with auxin or auxin and cytokinin to form a callus, and then auxin or auxin and cytokinin are added. Using the added liquid medium, the illumination intensity was 3000.
The above problem was solved by culturing callus cells under light conditions of less than Lux, then culturing under light conditions of illuminance 3000 Lux or more to produce anthocyanins within the cells, and then extracting anthocyanins from these cells. .

The present invention will be explained in detail below.

The present inventors first used growing points obtained from strawberry runners and cultured the cells in a solid medium containing auxin and cytokinin to form a callus, and then in a liquid medium containing auxin and cytokinin. Using a medium, callus cells are cultured under light conditions with an illuminance of 3000 Lux or less, then cultured under light conditions with an illuminance of 3000 Lux or more to produce anthocyanin within the cells, and then anthocyanin is extracted from the cells. Invented a manufacturing method and filed patent application No. 1-22400.
A patent application was filed as No. 0. In this culture method, the cultured cells were used as the growth points of strawberry runners, but as a result of intensive research, the present inventors have carefully selected cells even at sites other than the growth points of the runners. found that anthocyanins can be produced by appropriately setting the hormonal composition (auxin, cytokinin concentration) of the culture medium in callus culture, and completed the present invention.

That is, in the present invention, cells extracted from any of the leaves, stems, fruit parts, flowers, and roots of strawberries are used for culture.

The strawberry material is not limited to a specific variety and can be selected from various varieties. For example, Dutch strawberries (Frag.
aria chiloensis Duch
Varanassa Ba1l) etc. can be used.

An example of a method for extracting cells suitable for culture from leaves, stems, fruit parts, flowers, and roots of strawberries is to sterilize the leaves, stems, or immature fruit parts of young seedlings using a method commonly used in tissue culture, etc. The materials are then sterilized using a disinfectant and then cut into small pieces using a thin-bladed knife or the like. Also, the roots are
The root tissue, including part of the stem, is cut out from the young seedling and used.

These fragments and tissues are sterilized using a disinfectant commonly used in tissue culture and the like.

Next, the extracted fragments and tissues are placed in a culture medium to form a callus. The solid medium used for this culture is M u
Rashige & Skoog (M S ) medium or Lin & Staba (L S ) medium contains auxin or auxin and cytokinin, about 30 g IQ of saccharose as a carbon source, and 0.8 to 1
A solid medium to which about 10% agar is added is preferably used.

As the auxin, 2,4-dichlorophenoxyacetic 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 5.0 ppm for 2.4-D and 0.05 to 2 ppm for BA.
It is set within a range of about m.

In addition, the culture for turning fragments and tissues into calluses takes 20 to 30 minutes.
It is desirable that the temperature is about 0° C. and the illuminance is 3000 Lux or less. Callus growth requires illuminance of 3000
It is good when the illuminance is about Lux, but if the illuminance is higher than that, the proliferation ability of callus cells decreases, and anthocyanin is produced on the callus surface as the culture time passes, and the callus cells that have produced this anthocyanin are Even if liquid culture is subsequently performed, the cell proliferation ability is reduced, making it unsuitable for forming callus for mass culture.

Then, the cells are cultured under the above-mentioned culture conditions to form a callus, and if necessary, the callus is subcultured, and further,
Calli with high anthocyanin-producing ability were selected from the obtained calli and incubated at a temperature of 20 to 3
Subculturing is performed under conditions of 0° C., illuminance of 3000 LLIX or less to form callus for mass culture.

As a method for selecting callus with high anthocyanin-producing ability, cultured callus is placed on the solid medium described above, and the illuminance is about 3,000 Lux to 8,000 Lux, and the temperature is about 20 to 3
Cultivate at 0°C to produce anthocyanin on the surface of the callus, select highly colored calli with high anthocyanin production ability, take out the uncolored part inside this callus and use it as cells for mass culture, or use this uncolored callus as cells for mass culture. A method is used in which a portion is removed, placed on a separate fixed medium, and cultured to form a new callus.

Furthermore, calli with high anthocyanin-producing ability selected by this method have a white and soft appearance, and callus that is colored or hard during the callus formation stage cannot have sufficient anthocyanin-producing ability. As a simple method for selecting calli with high anthocyanin-producing ability, it is also 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.

Next, the obtained callus for mass culture with high anthocyanin-producing ability is placed in a liquid medium prepared by adding auxin or auxin and cytokinin to LS medium or MS medium and cultured with shaking.

The amounts of 2.4-D and BA added in this liquid medium were the same as in the solid medium used in the previous callus formation.
D (auxin) 0.5-5, Oppm5B
A (cytokinin) is set to about 0.05 to 2 pptr. The culture conditions were a temperature of 20-30°C and an illuminance of 3.
000 Lux or less. If the light conditions for this culture (first stage of culture) are illuminance of 3000 Lux or more, anthocyanins will be produced 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 cells.

By culturing under the above-mentioned conditions, callus cells proliferate in the liquid medium, and if necessary, the culture is scaled up to produce 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 the cultured cells are allowed to produce anthocyanin while further proliferating (late stage of culture).
. If the light conditions in the latter stage of the culture are illuminance of 3000 Lux or less, the anthocyanin production of the cultured cells will be poor,
Furthermore, when cultured under light conditions with an illuminance of 8000 Lux or more, the proliferation of the cultured cells becomes poor and the anthocyanin production ability of the cultured cells also becomes poor. In addition, even in this latter stage of culture, it is possible to scale up the culture as necessary.

Next, cells that have finished producing anthocyanins are separated from the liquid medium, and anthocyanins are extracted and purified from these cells to produce anthocyanins.

Conventionally known methods can be used to extract and purify anthocyanins 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 immersed in hydrochloric acid acidic methanol and extracted one to multiple times to extract anthocyanins. , Add lead acetate to this extract, strain out the lead chloride precipitate, and then collect the blue lead salt that forms. 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 in either case, it is converted into a chloride and crystallized.

In addition, the method for extracting and purifying anthocyanins from cultured cells is not limited to this, but for example, the above extract is dried under reduced pressure, dissolved in water, and then the anthocyanins are adsorbed on an ion exchange resin, followed by hydrochloric acid acidic ethanol. It is also possible to separate and purify using a method of elution with a liquid.

Anthocyanin is produced by each of the above operations.

In this method, cells taken from strawberry leaves, stems, fruits, flowers, and roots are cultured in a medium containing auxin or auxin and cytokinin to form a callus, and then treated with auxin or auxin and cytokinin. Mass culture of callus cells is performed using a liquid medium containing cytokinin under a light condition of illuminance of 3000 Lux or less, and then cultured under a light condition of illuminance of 3000 Lux or more to produce anthocyanin within the cells. Since anthocyanins are produced by extracting anthocyanins from the following, the scale-up of culture can be carried out extremely easily. In other words, callus with high anthocyanin 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 then grown in large quantities in this tank. After that, a large amount of anthocyanin can be easily obtained in a short period of time by culturing the cells under illuminance conditions of 3000 Lux or more and causing the cultured cells to produce anthocyanin.

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. Anthocyanins 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 anthocyanin can be produced in a short period of time, reducing the production cost of anthocyanin. becomes possible.

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 adjusting the light irradiation conditions appropriately, 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" (Preparation of sample) Strawberry (Shikisou strawberry: FragariaX an
Anassa) seedlings, immature fruit parts, and pre-flowering hole parts were washed with stirring in a 5% neutral detergent solution for 10 minutes, and then placed in 70% ethanol and ultrasonically cleaned for 30 seconds. Thereafter, the sample was sterilized by immersion in a 5% NaCl0 aqueous solution for about 8 minutes, and each sample was placed in a clean bench and washed three times with sterile water.

The seedlings were cut into leaves, a root portion including the lower part of the stem, and the remaining stem portion. Each of the leaf, stem, fruit, and pore samples was cut into pieces of 1II to several square centimeters. The roots were used as samples.

(Formation of callus) Add 30gIQ of sucrose (as a carbon source) to MS medium.
, a solid medium supplemented with 1% agar was used as the basic medium,
To this, 0.1 to 5.0 ppm of 2.4-D and 0.0 ppm of BA were added.
Using media with different hormone concentrations ranging from 0.05 to 0.05 ppm, fragments and tissues of each part were placed on each solid medium and incubated for 2 weeks at a temperature of 25°C, illuminance of 800 Lux, and a 16-hour photoperiod. The cells were cultured and the state of callus formation was examined.

The results are shown in Table 1.

Margin below: Table 1
Callus formation on the stem, root, and leaves △3... Callus formation on the stem and leaves △4... Callus formation on the stem △5... Callus formation on the leaves It is clear from Table 1 By appropriately setting the concentration of hormones (2,4-D and BA) in the callus-forming medium, callus can be formed using cells from various parts of strawberries, such as leaves, stems, and roots. was possible. Furthermore, the optimal concentration range of 2.4-D and BA in the callus-forming medium is 2.4-D and BA.
D is 0, 5 ppm to 2 ppm SBA is 0, 1 p
pm to 2 ppm.

Using leaf and stem fragments of the above samples, the presence or absence of callus formation was examined in a medium containing only 2°4-D and a medium containing only BA. The results are shown in Tables 2 and 3.

Table 2 Table 3 Table 3 As shown in Tables 2 and 3, 2 as a hormone.
Even in the medium containing 4-D alone, callus formation was possible from leaf and stem fragment samples. On the other hand, callus formation was not possible in a medium containing BA alone.

Moreover, the optimal concentration range of 2.4-D in a medium containing 2,4-D alone was 1 to 2 ppm. This 2゜4-D
A comparison of callus-forming culture in a medium containing only 2,4-D and callus-forming culture in a medium containing both 2,4-D and BA shown in Table 1 shows that both 2.4-D and BA Callus formation was better in the medium containing .

In addition, as auxins other than 2,4-D, naphthalene acetic acid (NAA), indole acetic acid (IAA), and indole butyric acid (IBA) were added at a concentration of 0.1 to 5 ppm. The presence or absence of callus formation was examined by placing fragment samples on the bed. The results are shown in Table 4.

Table 4: X: No callus formation △: Poor callus formation
Callus formation was also possible using NAAS I AAS I BA), but callus formation was poorer with these auxins than with 2.4-D.

(Primary culture of callus cells) According to the above test, 2.4-D 2ppm, BA
Using a solid medium containing 0 spp-, white and soft calli obtained from leaf and stem fragment samples were incised and divided, and the divided pieces were placed in a 500-liter flask.2.4- D ippm, BA Ol p
Add LS medium (liquid medium) to (1+1, and set the illuminance to 800 Lux, 3000 Lux, 8
000 Lux, a culture temperature of 25° C., and shaking culture at 80 rpm, and the growth of callus under each condition was examined.

As a result, callus from both leaves and stems proliferated best when the light condition was set to an illuminance of 3000 Lux. However, callus cultured under these conditions
After 0 to 14 days, anthocyanin was produced, the cultured cells turned brown, and the subsequent proliferation ability decreased.

On the other hand, each callus of the leaves and stems cultured at an illuminance of 800 Lux showed a high proliferation rate without producing anthocyanin in the cultured cells. Callus cultured under these light conditions could be subcultured in a culture cycle of 1 to 2 weeks, and it was possible to perform scale-up culture under the same conditions to mass-produce cultured cells.

In addition, calli cultured under a light condition of 8,000 Lux showed poor growth in both leaf and stem callus from the beginning of culture, and turned brown within a few days.

From these results, callus derived from cells such as strawberry leaves and stems can be grown in liquid culture under illuminance conditions of 3000 LLIX or less, and the cultured cells can be grown without producing anthocyanin. It was found that mass production of cultured cells is possible by scale-up culture under the following conditions.

(Secondary culture of callus) After culturing the callus obtained from the cells of the leaf and stem parts for two weeks under a light condition of 800 Lux, 2 g (wet weight) of the cultured cells was placed in a 500 ml flask, and 2.4-Dlppm
, BAO, ippm or S medium (liquid medium)
Pour 100ml and set the light condition to 3000 Lux.

Continuous light irradiation of 8000 Lux, culture temperature 25°C,
Culture was performed with shaking at 80 rpm.

When the illuminance was set to 3000 Lux, cell proliferation was observed in both leaf and stem samples, and anthocyanin was produced within the cultured cells in 10 to 14 days.

When the illuminance was set to 8000 Lux, cell proliferation was slight in both the leaf and stem samples, and anthocyanins were produced in the cultured cells within one to several days.

As a result, anthocyanins can be produced in the cultured cells by primary culturing at an illuminance of 3000 Lux or less and then secondary culturing at an illuminance of 3000 Lux or more, especially at an illuminance of 3000 Lux to 8000 LLI.
By culturing with K, it was possible to produce anthocyanins while growing the cultured cells.

(Separation of anthocyanin) In the above secondary culture, the illumination intensity was 3000 Lux and 2
Using a culture sample that had been cultured for a week, the cultured cells were strained from the culture solution and qualitative analysis of anthocyanin pigments was performed. After boiling the cultured cells separated from the culture solution for 5 minutes, the cultured cells were suction-filtered, the filtrate was adsorbed on PVP (polyvinylpyrrolidone), washed with methanol, and then soaked in 1% H
It was eluted with Cl-methanol, and the absorbance was measured and analyzed by TLC. As a result of these analyses, the anthocyanin obtained from cultured cells was found to be pelargonidin-3-glucosid, a natural strawberry pigment.
It turned out to be E and cyanidin3-glucoside.

From the above culture results, it is clear that large-scale culture of callus, which is the preliminary stage for producing anthocyanins, can be carried out in the dark or at 800 L.
carried out under the light conditions of LIK, -3 after proliferation of skin cells.
By culturing at an illuminance of 000 Lux or higher,
It has been found that it is possible to mass produce anthocyanins using cultured strawberry cells.

"Effects of the Invention" As explained above, the present invention extracts cells from any of the leaves, stems, fruits, flowers, and roots of strawberries and culture them in a solid medium supplemented with auxin or auxin and cytokinin. to form a callus, and culture the obtained callus in liquid at an illuminance of 3000 Lux or less to perform mass culture of callus cells, and then reduce the illuminance to 3000 Lux.
By producing anthocyanin in cells as described above and then extracting anthocyanin from the cells to produce anthocyanin, it is possible to scale up the culture extremely easily during culture before anthocyanin is synthesized. By scaling up, a large amount of cultured cells can be easily obtained and anthocyanins can be produced in large quantities.

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

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 anthocyanin can be produced in a short period of time, reducing the production cost of anthocyanin. I can do it.

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 is possible to always perform the culture at a constant culture rate, making the culture easier and more efficient.

Claims (1)

[Claims] Cells are removed from any part of the leaves, stems, fruits, flowers, and roots of strawberries, and the cells are cultured in a solid medium supplemented with auxin or auxin and cytokinin to form callus. Then, a liquid medium containing auxin or auxin and cytokinin was used, and the illumination intensity was 3000 Lux.
A method for producing anthocyanin, which comprises culturing callus cells under the following light conditions, then culturing under light conditions with an illuminance of 3000 Lux or more to produce anthocyanins within the cells, and then extracting anthocyanins from the cells. .