JP3126786B2 - Method for producing polysaccharide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a polysaccharide by culturing cells and secreting the polysaccharide into a liquid medium. More specifically, the present invention relates to a method for suppressing an increase in viscosity of a culture solution accompanying the production of a polysaccharide.
The present invention relates to a method for efficiently producing a polysaccharide by a simple operation by setting a low viscosity state.

[0002]

2. Description of the Related Art Polysaccharides are widely used as thickeners, gelling agents, foam stabilizers, suspension / emulsion stabilizers, film-forming agents, adhesives, bioactive agents and the like. Such polysaccharides have been produced by extraction or tapping from various sources (plants, animals, microorganisms, etc.), or separation and purification from various cell and / or tissue cultures.

[0003]

Among them, a method for producing a polysaccharide by secreting it into a medium by culturing,
Because polysaccharides and by-products secreted by the culture are macromolecules, the viscosity of the culture solution increases as the culture proceeds,
The following problems occurred. (1) At the time of culture, diffusion and mixing of the culture solution become insufficient due to an increase in viscosity, and as a result, the productivity of polysaccharide is not improved. (2) Since the viscosity of the culture solution is high, it is difficult to separate the cells and the like from the polysaccharide. As a result, the purity of the polysaccharide is not improved and the productivity is not improved.

[0004] As a method for solving these problems, in the case of producing hyaluronic acid by culturing microorganisms, addition of a culture medium (Japanese Patent Application Laid-Open No. 62-32893) and addition of sterilized water (Japanese Patent Application Laid-Open No. 63-94988). ) Is disclosed. However, in such a method, since the amount of the culture solution is remarkably increased, productivity per unit culture volume is reduced. Further, the method of performing the culture while removing the added medium or the like requires new equipment and is complicated in operation. There was a problem that. Accordingly, an object of the present invention is to provide a method for suppressing an increase in the viscosity of a culture solution during production of a polysaccharide by culturing by a simple operation and improving the productivity of the polysaccharide.

[0005]

Under such circumstances, the present inventors have conducted various studies on means for suppressing the increase in viscosity during culturing of plant cells, and as a result, it has been found that the concentration of the metal salt in the medium is higher than that in normal culturing. It has been found that the addition of glycerol to cultivation and the semi-continuous cultivation suppress the increase in viscosity during polysaccharide production and improve the productivity of the target polysaccharide, thereby completing the present invention.

That is, the present invention relates to a method for producing a polysaccharide by culturing plant cells and secreting the polysaccharide into a liquid medium, wherein semi-continuous cultivation is performed using a liquid medium containing 10 to 100 mM of a metal salt. And a method for producing a polysaccharide characterized by the following.

The type of the metal salt used in the present invention is not limited as long as the viscosity of the culture solution containing the desired polysaccharide is reduced and the cell salt does not adversely affect the polysaccharide-producing ability. Examples of such metal salts include chlorides, sulfates, carbonates, phosphates, and nitrates of monovalent metals such as sodium, potassium, calcium, magnesium, barium, iron, zinc, and aluminum. Specifically, sodium chloride, potassium chloride, calcium chloride, barium chloride, magnesium chloride, iron chloride, zinc chloride, aluminum chloride, sodium sulfate, potassium sulfate, calcium sulfate, magnesium sulfate, barium sulfate, zinc sulfate, sulfuric acid Iron, ferric sulfate, aluminum sulfate, sodium carbonate, potassium carbonate, calcium carbonate, barium carbonate, magnesium carbonate, sodium phosphate, potassium phosphate, calcium phosphate, magnesium phosphate, barium phosphate, sodium nitrate, potassium nitrate, calcium nitrate , Magnesium nitrate, barium nitrate and the like.

The concentration of the metal salt in the liquid medium is 10 to 1
It needs to be 00 mM. Usually, a liquid medium contains
In some cases, 1 to 3 mM of a metal salt is added, but at these concentrations, there is almost no effect of suppressing the increase in viscosity of the culture solution, and a sufficient effect of suppressing the viscosity can be seen only when the concentration is 10 mM or more. On the other hand, if it exceeds 100 mM, a decrease in polysaccharide productivity is observed, which is not preferable.

The other conditions in the method of the present invention differ depending on the cells used, and are not particularly limited as long as the cells differentiate and proliferate and produce polysaccharides. In the present invention, the plant cells include any of plant tissue fragments, calli, and plant cells themselves.

A particularly preferred example of such a plant cell is tuberose belonging to the genus Polyanthes L.
erosa L .; ) Callus (JP-A-64-10997). Examples of the target polysaccharide include an acidic heteropolysaccharide containing arabinose, mannose, galactose, xylose, and glucuronic acid, which are obtained by culturing the callus. Hereinafter, a case where this callus is cultured will be described as an example.

In the present invention, the liquid medium used for culturing the callus is not particularly limited as long as it is a medium usually used for culturing plant cells.
urashige-Skoog's medium, Linsmaie
r-Skoog's medium, Gamborg's medium, Whi
te medium, Tulecke medium, Nitach & N
Itach's medium and the like can be used.
medium of size-Skoog, Linsmeier-S
Koog's medium is preferred.

[0012] These liquid media may further contain a plant hormone, a carbon source and the like. The type and concentration of plant hormones are related to polysaccharide productivity, such as 2,4.
Auxins such as dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA); kinetin;
Benzyladenine (BA), dimethylaminopurine (2
cytokinins such as ip); gibberellin A ₃ (GA ₃ )
Gibberellins and the like are used. Among them, 2,4-
It is preferable to use D and NAA alone or in combination with NAA and BA or kinetin. Its concentration is
5 × 10 ⁻⁴ when 2,4-D or NAA is used alone
M to 1 × 10 ^-7 M, especially 5 × 10 ^-5 M to 5 × 10 ^-6
When M is used in combination with NAA and BA or NAA and kinetin, the concentration of NAA is 1 × 10 ⁻⁴ M to 1
× 10 ^-7 M, especially 1 × 10 ^-4 M to 5 × 10 ^-6 M, BA
Alternatively, the concentration of kinetin is 5 × 10 ⁻⁵ M to 1 × 10 ^5
-9 M, especially 1 × 10 ⁻⁵ M to 1 × 10 ⁻⁷ M is preferred.

As a carbon source, glucose, fructose, mannose, xylose, saccharose, rhamnose, fucose, starch and the like are used. Polysaccharide production is not so strongly affected by the type of carbon source added, and saccharose is usually used. Although there is not a deep relationship between the concentration of the carbon source and the production amount of the polysaccharide, generally, 1 to 6% is preferable.

[0014] The culture method is not particularly limited.
It is preferably performed at a temperature of ３０30 ° C. for 15 to 30 days, and shaking culture is preferred.

[0015]

In the present invention, as a result of suppressing the increase in the viscosity of the culture solution, it is extremely easy to separate the polysaccharide from the callus (cells) after completion of the culture. That is, after completion of the culture, the callus sediments quickly only by leaving the culture solution alone, so that the target polysaccharide can be easily separated by extracting the supernatant containing the polysaccharide. In addition, centrifugation can also be used to make such separation faster. in this way,
Because calli (cells) and polysaccharides can be easily separated,
The semi-continuous cultivation is industrially advantageous as shown by.

[0017]

According to the present invention, it has been possible to solve various problems which occur conventionally because the viscosity of a culture solution increases with the production of polysaccharides. That is, as a result of suppressing the increase in the viscosity of the culture solution, (1) the diffusion and mixing of the culture solution are improved, and the productivity of polysaccharide is improved. (2) The supernatant containing the callus and cells and the target substance is used. Separation becomes easier and productivity improves.
(3) There is no lag time after the second medium change after adding the medium, and semi-continuous culture can be performed efficiently.

[0018]

The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples. Reference example (a) Induction of callus: Tuberose buds 2 to 7 days before flowering were cut off, sterilized with a 70% ethanol solution for 1 minute, further sterilized with a 1% sodium hypochlorite solution for 10 minutes, and then sterilized with water. Washed. The sterilized explants were cut to an appropriate size and inoculated into a callus-inducing culture. As the callus induction medium, a Linsmeier-Skoog medium (LS medium) containing 0.8% agar was used as a base medium. As plant hormones, 10 ^-5 M NAA as auxin and 10 ^-6 MB as cytokinin
A was added. 3% saccharose was added as a carbon source. The medium was adjusted to pH 5.7 with 0.1 NKOH and then autoclaved at 120 ° C. and 1.2 atm.
Sterilized for 5 minutes. The culture was performed at 25 ± 1 ° C. under light.
After 30 to 60 days of culture, calli were induced from each explant. (B) Passage of callus: Each callus induced in (a) was cultured under the same conditions using the same medium as the induction medium, and transplanted to a new medium every 30 days.

Example 1 Using the callus subcultured for 10 or more passages in Reference Example (b); culturing was carried out under the following conditions, and the concentration of the produced polysaccharide and the viscosity of the culture solution after culturing for 30 days were measured. That is, 500
In a conical flask with a volume of 20 ml, the callus inoculum was set to 20 w / v%,
The mixture was charged with 00 ml and cultured with shaking at 26 ° C. and 90 rpm. As a medium, 5% w / v sucrose, 2,4-
It was used as the addition of D 10 ^-5 M and 10~50mM metal salts. The normal LS medium contains 3 mM of calcium chloride and 1.5 mM of magnesium sulfate. The results are shown in FIG. From FIG. 2, calcium salt or magnesium salt (added as CaCl ₂ .2H ₂ O or MgSO ₄ .7H ₂ O)
It can be seen that the addition of 0 mM or more greatly reduces the viscosity of the culture solution and increases the polysaccharide production.

Example 2 A culture solution obtained by culturing under the same conditions as in Example 1 while changing the calcium salt concentration was allowed to stand at 26 ° C., and the callus sedimentation rate was measured. As a result, as shown in FIG.
The culture solution obtained by adding 10 mM or more of calcium salt (added as CaCl ₂ · 2H ₂ O) has a high callus sedimentation rate, and the supernatant can be easily and quickly separated. Understand.

Example 3 Sucrose 3 w / v%, NA 10 ^-5 M in normal LS medium
And BA10 ^-6 M added medium (calcium chloride 3 mM)
And 1.5 mM of magnesium sulfate), and cultured for 4 weeks under the following culture conditions using an LS medium as a basal medium, and the polysaccharides were separated again under the same conditions. Cultured. FIG. 4 shows the results of measuring the polysaccharide concentration in the culture solution over time during this culture. Culture conditions Callus inoculation amount: 20 w / v% Medium preparation amount: 500 ml / 2 l Sucrose concentration: 5 w / v% Plant hormone: 2,4-D 10 ^-5 M Calcium chloride added: 33 mM Revolution: 80 rpm As a result, the growth medium In the first 3-4 days after conversion from (without addition of calcium salt) to the production medium, polysaccharides are not produced and there is a lag time, whereas after the second medium conversion there is no lag time and half It can be seen that continuous culture can be performed efficiently.

Example 4 A culture solution obtained in the same manner as in Example 1 was used at 3,500 ×
g, the centrifugation was performed under the conditions of 2 minutes, and the transmittance of the centrifuged supernatant was measured. The higher the permeability, the clearer the supernatant and the lower the content of callus in the supernatant. As a result, as shown in FIG. 4, it can be seen that the greater the amount of calcium salt added, the higher the permeability and the better the callus separation efficiency.

Example 5 A centrifugal supernatant obtained in Example 4 containing 1.6 g / l of a polysaccharide was applied to a cartridge filter (TCP
Callus fragments were removed by filtration using D-03A). FIG. 5 shows the permeation flux in that case. As a result, it was found that the filtration of the culture supernatant to which 33 mM of the calcium salt was added was completed in a shorter time.

[Brief description of the drawings]

FIG. 1 is a diagram showing a schematic explanatory diagram of semi-continuous culture.

FIG. 2 is a drawing showing the relationship between the concentration of a metal salt added to an LS medium, the concentration of a produced polysaccharide, and the viscosity of a culture solution.

FIG. 3 is a graph showing the relationship between the calcium salt concentration added to the LS medium, the callus sedimentation rate, and the relative polysaccharide production (relative value assuming that the case where no calcium salt is added is 1).

FIG. 4 is a drawing showing the relationship between the culturing time and the produced polysaccharide concentration in Example 3. The bar graph shows the amount of polysaccharide production per week.

FIG. 5 is a graph showing the effect of adding a calcium salt on the permeability of a centrifuged supernatant.

FIG. 6 is a graph showing the effect of the addition of a calcium salt on the permeation flux in the filtration of the centrifuged supernatant.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-49-75792 (JP, A) JP-A-63-94988 (JP, A) JP-A-1-10997 (JP, A) Journal of Textile Studies 7 (1976), 341-351 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 19/00-19/64

Claims (5)

(57) [Claims] 1. A method for producing a polysaccharide by culturing a plant cell and secreting the polysaccharide into a liquid medium, wherein the metal salt comprises
A method for producing a polysaccharide, comprising semi-continuous culturing using a liquid medium containing 0 to 100 mM. 2. The method for producing a polysaccharide according to claim 1, wherein the metal salt is a salt of a monovalent to trivalent metal. 3. The method for producing a polysaccharide according to claim 1, wherein the metal salt is a salt of one or more metals selected from sodium, potassium, calcium, magnesium, barium, iron and aluminum. 4. The method according to claim 1, wherein the cell is a Polyantes family.
hes L. Tuberose belonging to (Polian)
the tuberosa L. 2. The method for producing a polysaccharide according to claim 1, wherein the callus is derived from the plant of (1). 5. The method according to claim 5, wherein the polysaccharide is tuberose (Polia).
nthes tuberosa L. Arabinose, obtained by culturing callus derived from
The method for producing a polysaccharide according to claim 1, which is an acidic heteropolysaccharide containing mannose, galactose, xylose and glucuronic acid as constituents.