JPH067180A - Production of natural-type abscisic acid - Google Patents

Abstract

PURPOSE:To obtain the subject compound capable of suppressing the growth of plant and useful in agricultural field in high efficiency by using a bacterial strain belonging to the genus Botrytis and capable of producing natural-type abscisic acid, culturing the strain in a culture liquid containing a foamed carrier and separating the product from the culture liquid. CONSTITUTION:A bacterial strain belonging to the genus Botrytis and capable of producing natural-type abscisic acid [e.g. Botrytis cinerea (FERM P-6156)] is inoculated in a culture liquid containing a foamed carrier, left in a dark place at 25 deg.C for 4 days and subjected to standing culture for the following 4 days under irradiation with light having maximum intensity at 360nm wavelength to effect the production and accumulation of natural-type abscisic acid. The culture liquid is subjected to centrifugal separation to remove the bacterial cells and the product is extracted from the supernatant with an organic solvent such as ethyl acetate and chloroform. The extracted compound is adsorbed to active carbon, silica, synthetic adsorbent resin, etc., desorbed with a proper organic solvent and purified by concentration and crystallization to obtain the objective natural-type abscisic acid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing natural abscisic acid utilizing the metabolic function of microorganisms.

[0002]

2. Description of the Related Art Natural abscisic acid was first isolated as a fall-promoting substance for cotton young fruit by Adikot of the University of California in 1963. Since then, an important physiological action on plants has been found. It is also confirmed to be a kind of.

The characteristic of the physiological action of natural abscisic acid is that other plant hormones such as auxin, gibberellin,
By competing with cytokinins, etc. to cancel their effects,
Although it is intended to suppress the growth of plants, it also has a stomata obstruction effect on plants. Due to such physiological effects, natural abscisic acid is expected to be applied to agricultural production in the future as a substance that can cope with climate change such as drought and cold damage. In addition, it has been recently revealed that there are other physiological actions such as promotion of fruit maturity and control of flower bud formation, and it is expected to be used in the agricultural field (Bioindustry Vol. 9, No. 2, page 5 ( 1992)). Furthermore, utilization in the food field, such as utilization for improving quality and reducing costs in beer brewing (Japanese Patent Laid-Open No. 58-101677), is being promoted.

Conventionally, an organic synthesis method has been studied as a method for producing abscisic acid, but it is extremely difficult to obtain an optically active natural abscisic acid by this method.
In addition, the manufacturing cost is extremely high, and it is practically impossible to use it in the fields of agriculture and food.

[0005] In recent years, the microbial method by fermentation culture utilizing the metabolic function of microorganisms has been studied as a powerful alternative to organic synthesis, and Cercospora rosicola (Cerco
spora rosicola) (JP-A-58-363)
93, JP-A-56-160996) and a production method using Botrytis bacteria (Japanese Patent Publication No. 61-3).
No. 5838) and the like are known. The Botrytis bacterium has been conventionally studied by a solid culture method, but the study by the present inventors has made it possible to produce it by liquid culture (JP-A-2-60590).

[0006]

However, the greatest drawback of the above-mentioned microbial method is that the amount of natural abscisic acid accumulated by fermentation is very small and the fermentation production rate is also very slow. Was a very inefficient point.

[0007]

[Means for Solving the Problems] Therefore, as a result of intensive studies on the method for efficiently producing natural abscisic acid, the present inventors have found that Botrytis (B
It was found that the addition of a foaming carrier to the medium significantly increases the amount of natural abscisic acid accumulated in the medium during liquid shaking culture of a strain belonging to the genus otrytis) and having a natural abscisic acid-producing ability. It was In addition, the size of the foam carrier is in the range of 1 mm ³ to 150 mm ³ , the addition amount is in the range of 1.5 to 4 g per liter of the culture solution, or the foam carrier is mainly composed of cellulose. It was found that the production amount of natural abscisic acid in the medium can be significantly increased.

That is, the present invention relates to Botrytis.
s) a bacterium belonging to the genus and having a natural abscisic acid-producing ability,
A method for producing natural abscisic acid, which comprises culturing in a culture medium containing a foaming carrier to generate and accumulate natural abscisic acid, and isolating and collecting the natural abscisic acid from the culture medium. The foaming carrier is mainly composed of cellulose, and the size is in the range of 1 mm ³ to 150 mm ³ , and the addition amount is 1.5 to 4 g per liter of culture solution.
The method for producing natural abscisic acid is characterized in that

The present invention will be described in detail below.

The bacterium used in the present invention is not particularly limited as long as it is a natural abscisic acid-producing bacterium belonging to the genus Botrytis, and includes a normal mutated bacterium and a bacterium produced by a mutated treatment.

As a specific example of a strain of a natural abscisic acid-producing bacterium belonging to Botrytis, Botrytis cinerea (Bot
rytis • cinerea) FERM P-6156.

Next, a liquid medium is used as the medium used for the production of natural abscisic acid in the present invention.
This liquid medium includes bran, wheat, rice, sweet potato, potato, maltose, malt extract, sucrose, dextrin, waste sugar concentrate, carbon sources such as starch, defatted soybean powder, soybean powder, gluten, yeast extract, peptone, meat. Nitrogen sources such as extract, corn steep liquor, ammonium sulfate, urea and sodium nitrate are contained singly or as a mixture of two or more kinds. In addition to these, for example, inorganic substances such as magnesium salts, potassium salts, sodium salts, and phosphates, as well as vitamins, oils and fats, and the like can be added. Furthermore, adding a hot water extract of citrus to a culture medium, that is, a liquid medium is effective in increasing the production amount of natural abscisic acid.

In the present invention, it is important to add the foaming carrier to the liquid medium and culture it.

Here, the foam carrier is not particularly limited, and any other material such as urethane foam can be used.
In particular, those mainly composed of cellulose are preferable. The size of the foam carrier is preferably a granular material in the range of 1 mm ³ to 150 mm ³ , and more preferably 1 to 5
A cubic shape with a size of about mm square is used, but other shapes such as spherical shape may be used. The amount of the foaming carrier added is 1
It is preferably in the range of 1.5 to 4 g per liter,
More preferably, about 2.5 to 3.5 g is added.

A foaming carrier is added to the liquid medium and sterilized by a conventional method to make a substantially sterile medium, and then inoculated with bacteria.

The sterilized medium may be inoculated with either the hyphae or spores of a natural abscisic acid-producing strain belonging to the genus Botrytis or both.

The culturing condition is that the culturing temperature is usually 10 to 35.
℃, preferably 20 to 30 ℃, pH of the medium is 3 to 1
2, preferably 4 to 8, culture period is usually 1 to 30
The culture is carried out for a day, preferably about 5 to 15 days, using a culture system free from contamination by various bacteria. Further, in the present invention, it is preferable to perform aeration stirring culture. Natural type abscisic acid is produced even in static culture, but its production is remarkably promoted by aeration and agitation culture.

After completion of the culture, a conventional method can be employed for isolating and collecting natural abscisic acid from the culture medium, and for example, the method described below is used.

First, bacterial cells were removed from the culture solution by centrifugation, and natural abscisic acid was extracted from the supernatant with an organic solvent such as ethyl acetate or chloroform and adsorbed on activated carbon, silica gel, synthetic adsorption resin or the like. After that, it can be isolated and purified by a method such as desorption with an appropriate organic solvent and concentrated crystallization, and it can be isolated and purified by applying a general method for purifying an organic compound.

[0020]

EXAMPLES The present invention will be specifically described below with reference to examples.

Example 1 (Preparation of medium) 100 ml of the liquid medium shown in Table 1 was added to 500
The mixture was placed in a ml-volume Erlenmeyer flask, 3.5 g of each of the cellulose foams (manufactured by Toray Fine Chemical Co., Ltd.) shown in Table 2 was added as a foaming carrier, and steam sterilization was performed at 120 ° C. for 15 minutes.

[0022]

[Table 1]

(Preparation of Botrytis cinerea spores) Botrytis cinerea (FERM P) was added to the medium shown in Table 2.
-6156) was inoculated with the inoculum for storage, and statically cultured at 25 ° C. In this case, the cells were left in the dark for 4 days after the inoculation, cultivated while irradiating with the wavelength light having the maximum value at 360 nm for the following 4 days, and further cultivated in the dark for the following 1 day. The cultured cells were sterilized with 0.1% “Twe
It was suspended in an en "80 solution and filtered to obtain an aqueous solution containing substantially only Botrytis spores.

[0024]

[Table 2]

(Production of Natural Type Abscisic Acid) Botrytis cinerea (FERM) obtained above in the above liquid medium
P-6156) inoculated with spore solution at 25 ° C, amplitude 3c
The cells were cultured with aeration and stirring at m, 172 rpm for 10 days.

After the completion of the culture, the cells are removed by centrifugation,
The supernatant was subjected to high performance liquid chromatography (column: Shiseido CAPCELL PAK C18 4.6 × 250 mm,
The developing solvent: 0.1 M phosphoric acid / methanol = 1/1) was used for analysis to quantify natural abscisic acid.

The results are shown in Table 3.

[0028]

[Table 3]

Example 2 Preparation of medium, culture method, and preparation of seed spores were all performed in Example 1.
I went the same way. Cellulose foam having a cubic shape of 3 mm square was added in the amount shown in Table 4.

The results are shown in Table 4.

[0031]

[Table 4]

Example 3 To the medium shown in Example 1, 1 g / l of 3 mm square cubic cellulose foam was added, cultivated by the method shown in Example 1, and the obtained culture broth was centrifuged. The body was removed, and the supernatant was adsorbed on a column filled with Amberlite XAD7 resin, washed with water, and then eluted with 50% methanol.
The eluate was concentrated and the pH was adjusted to 3, then extracted with ethyl acetate, dehydrated with anhydrous sodium sulfate, and the concentrate was subjected to silica gel chromatography to distill off with n-hexane / ethyl acetate = 3/1. The purified natural abscisic acid fraction was separated and concentrated to obtain 80 mg of pure natural abscisic acid crystals.

The optical rotation of the obtained crystals of natural abscisic acid is

[Equation 1] Met.

[0034]

EFFECTS OF THE INVENTION According to the present invention, natural abscisic acid can be obtained in a high accumulation amount by fermentation culture using microorganisms, and production on an industrial scale can be realized.

Claims (4)

[Claims] 1. A bacterium belonging to the genus Botrytis and having a natural abscisic acid-producing ability is cultivated in a culture medium containing a foaming carrier to produce and accumulate natural abscisic acid. A method for producing natural abscisic acid, which comprises isolating and collecting abscisic acid. 2. The size of the foam carrier is from 1 mm ³ to 150 mm
^The method for producing natural abscisic acid according to claim 1, which is in the range of ³ . 3. The method for producing natural abscisic acid according to claim 1, wherein the amount of the foamed carrier added is in the range of 1.5 to 4 g per liter of the culture solution. 4. The method for producing natural abscisic acid according to claim 1, wherein the foam carrier is mainly composed of cellulose.