JPH09140392A - Production of polysaccharides - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain a polysaccharide improved in productivity, useful as a thickening agent, a gelling agent, an emulsion stabilizer, etc., by culturing a plant cell such as a callus of a plant of the genus Polyanthus, adding a carbon source to a medium during the culture, capable of proliferating a large amount of the plant cell. SOLUTION: A flower bud of a plant belonging to the genus Polyanthus (e.g. tuberose) is cut out, sterilized, washed with sterilized water, cut into a proper size, laid as an explant on a callus inducing medium, cultured at 25 deg.C for 30 days and induced into a callus. Then the callus is inoculated into a liquid medium and subjected to shaking culture. In the middle of the culture on the 14th day of the culture, one or more of a monosaccharide (e.g. glucose) and a disaccharide (e.g. saccharose) are added as a carbon source to a medium and the callus is successively cultured until the 28th day to give a polysaccharide which is capable of proliferating a large amount of a plant cell and is useful as a thickening agent, a gelling agent, a foam stabilizer, a suspension/emulsion stabilizer, a film-forming agent, a tacky agent, a physiological activator, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a polysaccharide having improved productivity in the culture of plant cells.

[0002]

BACKGROUND OF THE INVENTION Plant-derived polysaccharides are widely used as thickeners, gelling agents, foam stabilizers, suspension / emulsion stabilizers, film-forming agents, adhesives, bioactive agents and the like. Such polysaccharides are conventionally generally seeds of natural or cultivated plants,
Manufactured by extraction or tapping from fruits, stems, trunks, leaves, roots, tubers, tubers and the like. However, the production from natural resources is easily affected by climatic conditions and the like, and there is a drawback that the production amount and the price are not constant. Therefore,
In recent years, many attempts have been made to produce naturally-derived substances under artificial control by culturing callus or organs using a plant tissue culture method, without being affected by climatic conditions.

In the production of plant-derived polysaccharides using these plant tissue culture methods, medium 1 is used for 30 days of culture.
It is disclosed that 3 to 4 g of polysaccharide is produced per liter (Japanese Patent Laid-Open No. 64-10997 and Japanese Patent Laid-Open No. 4-53).
495).

However, the plant cell culture period is 30
For a long period of about a day, specific nutrients in the culture medium are consumed for cell growth, production of polysaccharides, etc., resulting in a deficiency state during the culture period. Therefore, the cell growth ability and polysaccharide production ability originally possessed by plant cells have not been sufficiently exhibited by the conventional plant culture method.

[0005]

Accordingly, it is an object of the present invention to provide a method for improving callus growth and increasing polysaccharide productivity in culturing plant cells.

[0006]

Under such circumstances, the inventors of the present invention have conducted diligent studies, and as a result, when culturing plant cells, the carbon source in the medium became deficient with the progress of culturing, which resulted in cell growth ability. And the ability to produce polysaccharides are significantly affected, and even if the carbon source concentration in the medium at the start of culture is excessive, the growth ability and productivity do not improve, and additional addition of carbon source during culture is required. The present inventors have found that the proliferation ability and the production ability are improved for the first time, and have completed the present invention.

That is, the present invention provides a method for producing a polysaccharide characterized by adding a carbon source to the medium during the culture of plant cells.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the polysaccharide which is the object of the method of the present invention include various polysaccharides obtained by culturing plant cells. Specific examples thereof include acidic heteropolysaccharides obtained from callus cultures of plants belonging to the genus Polyantes (preferably tuberose).

The plant cells used in the method for producing a polysaccharide of the present invention are not particularly limited, but are preferably cells of a plant of the genus Polyanthes, particularly preferably callus derived from a tuberose plant of the genus Polyanthes. It is better to use. For such callus induction, it is preferable to use a part of an organ or tissue such as a tuberose flower, a flower bud, a stem, a leaf, a bulb, and a root, and it is particularly preferable to use a flower or a flower bud.

In the present invention, the method of culturing plant cells is not particularly limited, but a step (1) of culturing a part of plant cells or tissue to induce callus, subculturing the induced callus A method including a step (2) of growing and a step (3) of producing a polysaccharide in which the callus obtained by the subculture is shake-cultured in a liquid medium is preferable. In the present invention, the step (2) is a plant cell growth culture period, and the step (3) and the subsequent steps are a polysaccharide production culture period. Examples of the basal medium for inducing callus include Murashige-Skoog's medium, Rinsmeier-Skoog's medium, Gamborg's medium, White's medium and the like, which are usually used for plant tissue culture. This basal medium preferably further contains a plant hormone and a saccharide as a carbon source. Examples of such a plant hormone include 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (NAA), Auxins such as indole acetic acid (IAA) and indole butyric acid (IBA); cytokinins such as furfurylaminopurine (kinetin), benzyladenine (BA) and dimethylaminopurine (2iP). Among them, 2,4-D alone or NAA
The combination of BA with BA or the combination of NAA with kinetin gives good results. The plant hormone concentration necessary for callus induction is 5 × 10 ⁻⁴ M to 1 × when 2,4-D is used alone.
In the case of a combination of 10 ⁻⁷ M, NAA and BA or NAA and kinetin, the concentration of NAA is 5 × 10 ⁻⁴ M to 1 × 10 5.
^-7 M, BA or kinetin concentration is 1 × 10 ^-4 M to 1 ×
10 ⁻⁷ M is preferred. Examples of the saccharide as a carbon source include monosaccharides or disaccharides such as glucose, fructose, mannose, arabinose, xylose, saccharose and rhamnose, and saccharose is particularly preferable. Callus induction can be performed in a solid medium or a liquid medium, but a solid medium is usually used. In addition, the culture is 20
Callus is induced if it is performed under bright or dark conditions of -30 ° C.

The induced callus can be subcultured for 10 or more generations while maintaining the same morphology in the above callus induction medium. As a medium for subculture, it is preferable to use Rinsmeier-Skoog's medium as a basic medium and Murashige-Skoog's medium containing a plant hormone and sugar as a carbon source.
10 ⁻⁴ M to 1 × 10 ⁻⁷ M 2,4-D or 1 × 10 ⁻⁴ M
~ 1 x 10 ^-7 M NAA and 1 x 10 ^-4 M to 1 x 10 ^-7 M
Examples of BA and carbon source include the same as those in the above-mentioned callus induction medium. The subculture is preferably carried out at 20 to 30 ° C for 15 days to 1 month.

In the production of polysaccharides from callus obtained by subculture, the same basic medium as the callus induction medium can be used, and plant mediums such as Murashige-Skoog's medium or Rinsmeier-Skoog's medium Those containing a sugar as a carbon source are preferred. Such a medium is
Liquid media are preferred.

The type and concentration of the plant hormones used in the polysaccharide production medium are related to the large-scale growth of callus and the production of polysaccharides, such as 2,4-D, NAA, IAA and I.
Auxins such as BA, kinetin, BA and cytokinins such as 2iP are used. Among them, 2,4-D,
NAA is preferably used alone or in combination with kinetin and BA. Its concentration is 2,4-D, and when NAA is used alone, it is preferably 5 × 10 ⁻⁴ M to 1 × 10 ⁻⁷ M, and particularly preferably 5 × 10 ⁻⁵ M to 1 × 10 ⁻⁶ M. Also 2,4
When -D and NAA are used in combination with kinetin and BA, the concentration of 2,4-D and NAA is from 1 x 10 ^-4 M to
1 × 10 ⁻⁷ M, particularly 5 × 10 ⁻⁵ M to 1 × 10 ⁻⁶ M is preferable, and the concentration of kinetin and BA is 5 × 10 ⁻⁵ M to ⁵ × 1.
0 ^-7 M, especially 1 x 10 ^-5 M to 1 x 10 ^-7 M are preferred.

The type of sugar as a carbon source at the start of the culture does not affect strongly the mass growth of callus and the production of polysaccharides, and examples thereof include those similar to the above-mentioned callus induction medium. Among them, glucose and fructose are included. , Mannose and sucrose are preferred. Although the concentration of sugar as a carbon source at the start of culture varies depending on the type of sugar,
In the medium, 0.5 to 7% by weight, preferably 1 to 6% by weight, and particularly 1.5 to 5.5% by weight is preferable from the viewpoint of mass growth of callus and improvement of polysaccharide productivity. Further, the culture is preferably carried out at a temperature of 20 to 30 ° C. for 15 to 30 days, and further, it is preferably carried out 2 to 10 times by replacing with a new medium under the same conditions.

In the present invention, when culturing plant cells,
It is necessary to add a carbon source to the medium during the culture.
The timing of addition of the carbon source is not particularly limited as long as it is during the culture, and it is preferable to add once or twice or more during the growth culture period of callus or the production culture period of polysaccharide. Of these, it is particularly preferable to set it to 5 to 25 days from the start of callus subculture or 5 to 25 days from the start of the polysaccharide production process. For example, if the polysaccharide production culture period is 15 days, start the culture. In the case of 30 days after the 7th day, it is more preferable that the time is 10 days and 20 days after the start of the culture. The carbon source may be added once or twice or more in each of the callus growth phase and the polysaccharide production phase.

In the present invention, the carbon source added during the culturing is not particularly limited, but is preferably one or more selected from monosaccharides and disaccharides, glucose,
Examples thereof include fructose, mannose, arabinose, xylose, saccharose and rhamnose, and among them, glucose, fructose, mannose and sucrose are particularly preferable.

The amount of carbon source added during the culture is
There is no particular limitation, and it is preferable that the amount consumed in the culturing process is 0.01 to 5% by weight based on the whole culture.
Preferably 0.05 to 4% by weight, particularly preferably 0.1
~ 3% by weight.

To collect the polysaccharide from the culture thus obtained, for example, cells are removed from the culture by centrifugation or filtration, and then the culture solution is concentrated using a rotary evaporator or the like and concentrated. It is carried out by adding ethanol to the liquid to cause precipitation, and freeze-drying the precipitate. The polysaccharide can be purified according to a general polysaccharide purification method. For example, the crude polysaccharide can be dissolved in water, centrifuged to completely remove insolubles, and a highly purified product can be obtained by dialysis or a method using an ion exchange resin.

[0019]

According to the present invention, a large amount of plant cells can be grown and the productivity of polysaccharides is improved. Therefore, it does not require large farms and can overcome price fluctuations due to the effects of climate, which is the weakest point of natural resources.

[0020]

EXAMPLES The present invention will now be described in more detail by way of examples, which are merely examples and do not limit the present invention.

Example 1 (a) Induction of callus Tubulose buds were cut out 2 to 7 days before flowering, and 70
% Ethanol solution and 1% sodium hypochlorite solution were used for sterilization and then washed with sterile water. The sterilized bud was cut into an appropriate size and placed as an explant in a callus induction medium. The callus induction medium contained 0.
Using Rinsmeier-Skoog medium containing 8% agar, 1 × 10 ^-5 M NAA and 1 × 1 as plant hormones were used.
The 0 ^-6 MBA was added, was added 3% saccharose as a carbon source. After culturing at 25 ± 2 ° C. for 30 days, callus was induced from each explant. (B) Callus culture Each callus induced in (a) was separated from the explants and cultured at 25 ± 2 ° C for 30 days using a medium having the same composition as the callus induction medium, and every 30 days. It was transferred to a new medium. The transplantation was repeated 10 times to grow the callus. (C) Shaking culture The callus grown in (b) was inoculated into the above callus induction medium in a liquid medium containing no agar, and shaking culture was performed. For culture, put 80 ml of medium in a 200 ml Erlenmeyer flask and inoculate 2 g of callus with a fresh weight of 25 ± 2.
It was carried out at 120 rpm at 28 ° C for 28 days, and transplanted to a new medium every 28 days. This transplant was repeated 5 times.

Next, using the callus obtained by the shaking culture of (c) above, the culture was carried out under the same conditions as the shaking culture of (c) above. On the 14th day of culture, 5 ml of a 25% sucrose solution was added as sugar, and the culture was continued until the 28th day. At the end of the 14th and 28th day from the start of the culture, the culture solution and cells were separated by centrifugation, and the cells were freeze-dried.
Cell weight was measured. As a result, the cell dry weight on day 14 of culture was 0.38 ± 0.06 g, and on day 28 it was 1.
It was 45 ± 0.09 g.

Comparative Example 1 Using the callus obtained by the shaking culture of Example 1 (c),
Culture was performed under the same conditions as the shaking culture of (c), and was continued until the 28th day. At the end of culture on the 14th and 28th days from the start of the culture, the culture solution was separated into cells by centrifugation, the cells were freeze-dried, and the cell weight was measured. as a result,
The cell dry weight on day 14 of culture was 0.40 ± 0.05 g,
On day 28, it was 0.92 ± 0.08 g.

Example 2 Using the callus obtained by the shaking culture of Example 1 (c),
Cultivation was performed under the same conditions as the shaking culture of (c) except that a liquid medium containing 2,4-D 1 × 10 ⁻⁵ M as a plant hormone was used. On the 14th day of culture, 5 ml of a 25% sucrose solution was added as sugar, and the culture was continued until the 28th day. At the end of the 14th and 28th days from the start of the culture, the culture solution was separated into cells by centrifugation, and the culture solution was concentrated using a rotary evaporator. To this concentrated culture solution, 3 volumes of ethanol was added, and the mixture was allowed to stand at 5 ° C. for 24 hours to obtain a precipitate. The precipitate was recovered by centrifugation and 70%
After washing with ethanol and freeze-drying, the amount of polysaccharide produced was measured. As a result, the polysaccharide production amount on the 14th day of culture was 1.25 ± 0.05 g / l / 30 days, and on the 28th day was 3.
It was 00 ± 0.11 g / l / 30 days.

Comparative Example 2 Using the callus obtained by the shaking culture of Example 1 (c),
Culture was performed under the same conditions as the shaking culture of (c) except that a liquid medium containing 2,4-D 1 × 10 ⁻⁵ M as a plant hormone was used, and the culture was continued until day 28. At the end of the 14th and 28th days from the start of the culture, the culture solution was separated into cells by centrifugation, and the culture solution was concentrated using a rotary evaporator. To this concentrated culture solution, 3 volumes of ethanol was added, and the mixture was allowed to stand at 5 ° C. for 24 hours to obtain a precipitate. The precipitate was collected by centrifugation, washed with 70% ethanol and then freeze-dried to measure the amount of polysaccharide produced. as a result,
Polysaccharide production on day 14 of culture 1.24 ± 0.08 g / l
/ 30th day, 28th day: l. 70 ± 0.13 g / l / 3
Day 0.

Example 3 Using the callus obtained by the shaking culture of Example 1 (c),
Culturing was carried out under the same conditions as the shaking culture of (c) above, except that a liquid medium containing 2,4-D 1 × 10 ⁻⁵ M as a plant hormone was used and the carbon source was glucose 3%. Culture 1
On day 0 and day 20, 2 ml of a 25% glucose solution was added as sugar and the cells were cultured until day 30. On the 10th and 20th days after the start of the culture and at the end of the culture, the culture solution was separated into cells by centrifugation, and the culture solution was concentrated using a rotary evaporator. To this concentrated culture solution, 3 volumes of ethanol was added, and the mixture was allowed to stand at 5 ° C. for 24 hours to obtain a precipitate.
The precipitate was collected by centrifugation, washed with 70% ethanol and then freeze-dried to measure the amount of polysaccharide produced.
As a result, the polysaccharide production amount on the 10th day of culture was 0.85 ± 0.
12g / l / 30 days, 1.73 ± 0.09 on day 20
g / l / 30 days, 282 ± 0.15 g / on day 30
It was 1/30 days.

Comparative Example 3 Using the callus obtained by the shaking culture of Example 1 (c),
A liquid medium containing 2,4-D 1 × 10 ⁻⁵ M as a plant hormone was used, and the culture was performed under the same conditions as the shaking culture of (c) except that the carbon source was glucose 3%, and the culture was performed for 30 days. I went to my eyes. On the 10th and 20th days after the start of the culture and at the end of the culture, the culture solution was separated into cells by centrifugation, and the culture solution was concentrated using a rotary evaporator. To this concentrated culture solution, 3 volumes of ethanol was added, and the mixture was allowed to stand at 5 ° C. for 24 hours to obtain a precipitate. The precipitate was collected by centrifugation, washed with 70% ethanol and then freeze-dried to measure the amount of polysaccharide produced. As a result, the polysaccharide production amount on the 10th day of culture was 0.80 ± 0.20 g / l / 30 days, the 20th day was 1.32 ± 0.15 g / l / 30 days, and the 30th day was 1.72 ±. It was 0.11 g / l / 30 days.

From the examples and comparative examples, by adding a carbon source to the culture medium during the culture, it becomes possible to mass-produce cells and improve the polysaccharide productivity.

Claims (5)

[Claims] 1. A method for producing a polysaccharide, which comprises adding a carbon source to a medium during the culture of plant cells. 2. The method for producing a polysaccharide according to claim 1, wherein the carbon source is added during the growth culture period of the plant cell and / or the production culture period of the polysaccharide. 3. The method for producing a polysaccharide according to claim 1, wherein the plant cell is a callus derived from a plant belonging to the genus Polyanthes. 4. The method for producing a polysaccharide according to claim 3, wherein the plant belonging to the genus Polyanthes is tuberose. 5. The method for producing a polysaccharide according to claim 1, wherein the carbon source is one kind or two or more kinds selected from monosaccharides and disaccharides.