JP3191448B2 - Method for producing natural abscisic acid - 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 natural abscisic acid utilizing the metabolic function of microorganisms.

[0002]

2. Description of the Related Art Natural abscisic acid was first isolated by Adicott et al. As a substance promoting the fall of cotton seedlings in 1963, and is a kind of plant hormone whose important physiological effects on plants have been found one after another. .

[0003] The characteristics of natural physiological abscisic acid in plant physiology are auxin, gibberellin, and other plant hormones.
It is intended to antagonize cytokinins and the like to counteract their effects and to suppress plant growth, but there are also stomatal obstruction effects of plants. In the future, natural abscisic acid is expected to be applied to agricultural production as a substance that can respond to climate change such as drought and cold damage due to such physiological actions. In addition, it has recently been revealed that there are physiological functions such as promotion of fruit maturation 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)). Further, the use in the food field has been promoted, such as quality improvement and cost reduction in beer brewing (JP-A-58-101677).

Conventionally, an organic synthesis method has been mainly studied as a method for producing abscisic acid, but it is difficult to obtain optically active natural abscisic acid and the production cost is extremely high. Utilization in the agricultural and food sectors was virtually impossible.

[0005] In recent years, fermentation methods utilizing the metabolic function of microorganisms have been studied as a viable alternative to organic synthesis, and Cercospora rosicola (Cercospora.
rosicola) in a liquid culture method (JP-A-58-36393, JP-A-56-160996).
JP-A-61-1986) and a production method by a solid culture method using a microorganism belonging to the genus Botrytis.
No. 35838) is known. Thereafter, microorganisms belonging to the genus Botrytis can be produced by liquid culture (Japanese Patent Application Laid-Open No. 2-60590).

[0006]

However, when liquid culture of the above microorganisms is carried out, especially in the initial stage of the culture, bacteria are scattered from the culture solution by shaking or stirring, and the walls of the culture device (test tubes, flasks, jars, etc.) are disturbed. It is said that the bacteria do not grow stably in the culture solution, so that not only the growth is delayed but also the shape of the bacteria itself changes, and the fermentation production rate and accumulation amount of natural abscisic acid also decrease There was a problem.

[0007]

Means for Solving the Problems In view of the above, the present inventors carried out liquid shaking culture of a microorganism belonging to the genus Botrytis, a kind of imperfect bacterium, and having a natural abscisic acid-producing ability. As a result of intensive studies on a method for efficiently producing, when the viscosity of the medium increases due to the polysaccharide produced by the microorganism itself during culturing,
Since the scatter of bacteria is eliminated and the culture is stabilized, by adding a certain water-soluble polymer from the start of cultivation to give viscosity, it is possible to prevent the scatter of bacteria by shaking, and it is possible to grow well, resulting in It was found that natural abscisic acid accumulated in the medium significantly increased.

That is, the present invention relates to Botrytis (Botritis).
ytis) belonging to the genus and having a natural abscisic acid-producing ability in a culture solution of carboxymethylcellulose,
Polyethylene glycol and polyvinyl alcohol
A method for producing natural abscisic acid, comprising culturing in the presence of at least one selected from the group to produce and accumulate natural abscisic acid, and isolating and collecting the natural abscisic acid from the culture solution.

Hereinafter, the present invention will be described in detail.

The microorganism used in the present invention is not particularly limited as long as it is a microorganism belonging to the genus Botrytis and having a natural abscisic acid-producing ability (hereinafter, referred to as an ABA-producing bacterium).
It also includes ordinary mutant bacteria and bacteria generated by the mutation treatment.

A specific example of an ABA-producing strain belonging to the genus Botrytis is Botrytis cinerea.
s.cinerea) FERM P-6156. The mycological properties of Botrytis cinerea having the ability to produce natural abscisic acid have already been described in JP-B-61-35.
No. 8,38,838.

Next, in the present invention, a liquid medium is used as a medium used for producing natural abscisic acid.
In this liquid medium, bran, wheat, rice, sweet potato, potato, malt sugar, malt extract, sucrose, dextrin, waste sugar dense, carbon sources such as starch, defatted soy flour, soy flour, gluten, yeast extract, peptone, meat Nitrogen sources such as extract, corn steep liquor, ammonium sulfate, urea, and sodium nitrate are contained alone or in combination of two or more. In addition, for example, inorganic substances such as magnesium salts, potassium salts, sodium salts, and phosphates, as well as vitamins, fats, and the like can be added.

[0013] In the present invention, to stabilize the liquid culture of the ABA-producing bacteria, further Oh Luke Rubo <br/> carboxymethylcellulose effect of increasing the ABA productivity, polyethylene glycol, Oyo
And at least one selected from polyvinyl alcohol
Can be used . Among them, carboxymethyl cellulose (hereinafter abbreviated as CMC) is most preferable. The addition of CMC not only stabilizes the culture, but also has a growth promoting effect due to the CMC resolution and partial assimilation ability of the ABA-producing bacteria, and the viscosity increases due to the polysaccharide produced by the ABA-producing bacteria itself. By the time CMC
It has disappeared, and it is very convenient. Synthetic polymers such as polyethylene glycol and polyvinyl alcohol have the same effect, but since they are not metabolized by ABA-producing bacteria, CMC is more effective in producing natural abscisic acid. .

[0014] Carboxymethylcellulose and polyethyleneglycol used as factors that determine the viscosity of the culture solution
The molecular weight of the alcohol or polyvinyl alcohol is not particularly limited. The optimum concentration differs depending on the type of the water-soluble polymer and the viscosity of the same water-soluble polymer due to the difference in the molecular weight and the like.
The range is 0 wt%. In general, the higher the added amount and the higher the viscosity, the higher the effect.
Examples include polyethylene glycol and polyvinyl alcohol. The conditions of the water-soluble polymer to be added include not only increasing the viscosity of the culture medium but also increasing the affinity between the bacteria and water, and it is important that the ABA-producing bacteria do not inhibit the ABA productivity. It is.

The same effect can be obtained in any case from the small scale of test tubes and flasks to the large scale of jar fermenters and tanks.

The medium subjected to the sterilization treatment may be inoculated with either a hypha (including a precultured bacterial solution) or a seed spore of an ABA-producing strain belonging to the genus Botrytis, but depending on the water-soluble polymer to be added, Each may have different effects. For example, alginic acid is more effective when seed spores are used as an inoculation source than when precultured cells are used as an inoculation source. Also, the amount of inoculation may greatly affect the stability of the culture. At an inoculum amount above a certain level, there is a tendency to stabilize slightly even without adding a water-soluble polymer compound. Although it has a remarkable effect on productivity, the effect of stabilizing the culture is small when the amount of inoculation is low. For example, in the case of CMC, there is always an effect regardless of the amount of inoculation.

The culturing conditions are usually such that the culturing temperature is 10 to 32.
° C, preferably 25 to 30 ° C, the pH of the medium is usually 3 to 12, preferably 3 to 8, and the culture period is usually 1 to 3
The culture is performed for 0 day, preferably about 5 to 20 days, using a culture system in which contamination by various bacteria is excluded.

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

First, cells are removed from the culture solution by centrifugation, and natural abscisic acid is 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. Can be isolated and purified by a method such as desorption with a suitable organic solvent and concentration and crystallization.
Isolation and purification can be performed by applying a general organic compound purification method.

[0020]

The present invention will be described in more detail with reference to the following examples.

Example 1 Each of various water-soluble polymers shown in Table 1 was added in an amount of 1%.
Add 10 ml of potato dextrose medium (Difco) with a diameter of 25
The mixture was placed in a test tube having a diameter of 120 mm, and sterilized by autoclaving at 120 ° C. for 15 minutes. Botrytis cinerea (FERM P
-6156), and inoculated with a seed spore or a precultured bacterial solution.
Reciprocal shaking culture was performed at 5 ° C., 2 cm amplitude and 250 spm for 7 days.

After completion of the culture, the cells are removed by centrifugation, and the supernatant is subjected to high performance liquid chromatography (column: Shiseido CA
PCELL PAK C18 4.6 × 250mm, developing solvent: 0.1M
Analysis was performed with phosphoric acid / methanol = 1/1) to determine the amount of natural abscisic acid. The residual glucose amount is glucose CII-
It was quantified by Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). Table 1 shows the results.

[0023]

[Table 1]

Example 2 As a water-soluble polymer to be added, three kinds of CMs having different viscosities were used.
The same procedure as in Example 1 was carried out except that C was used and the addition concentration was changed as shown in Table 2. Table 2 shows the results.

[0025]

[Table 2]

Example 3 As a water-soluble polymer to be added, 500 ml of the medium of Example 1 containing 0.6% of CMC was evenly dispensed into 10 500 ml baffled flasks, and pressurized at 120 ° C. for 15 minutes. Steam sterilized. The precultured bacterial solution was inoculated at 2% each and cultured at 25 ° C. for 7 days at a rotation speed of 180 rpm and an amplitude of 7 cm. After completion of the culture, the supernatant liquid from which the cells were removed by centrifugation was adsorbed to a column filled with Amberlite XAD7 resin, washed with water and eluted with 50% methanol. Concentrate the eluate to pH
Adjusted to 3 and extracted with ethyl acetate. The extract was dehydrated with anhydrous sodium sulfate and concentrated, followed by silica gel column chromatography, and n-hexane / ethyl acetate = 3/1.
The fraction distilled under the above conditions was concentrated to obtain 0.14 g of pure crystals of natural abscisic acid.

[0027]

According to the present invention, in the fermentation culture production of natural abscisic acid using a microorganism, the production rate and the amount of accumulation can be significantly increased.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12P 7/42 CA (STN) REGISTRY (STN) JICST file (JOIS)

Claims (2)

(57) [Claims] A microorganism belonging to the genus Botrytis and having a natural abscisic acid-producing ability is prepared by culturing a carboxymethylcellulose or polyethylene glycol in a culture solution.
Less selected from coal and polyvinyl alcohol
A method for producing natural abscisic acid, comprising culturing in the presence of one of them, producing and accumulating natural abscisic acid, and isolating and collecting the natural abscisic acid from the culture solution. Wherein the presence of carboxymethyl cellulose culture
The method for producing a natural abscisic acid according to claim 1, which is carried out below .