JPS63296696A - Production of natural type abscisic acid - Google Patents

Abstract

PURPOSE:To efficiently produce natural type abscisic acid useful as a plant hormone by a fermentation method, by inoculating excess specific spores of a specific bacterium belonging to the genus Botrytis into a nutrient culture medium and cultivating the bacterium in the culture medium. CONSTITUTION:Sterile water containing sporal parts of a microorganism, such as Botrytis.cinerea FERM P-615, belonging to the genus Botrytis and capable of producing natural type abscisic acid is dispersed in a stationery culture medium containing a carbon source, such as potato or bran, and further nitrogen source, inorganic salt, etc., added thereto so as to substantially provide >=1.0X10<3> number of spores per g culture medium present therein. Cultivation is carried out at 25-35 deg.C and pH 4-8 for 3-9 days to isolate the natural type abscisic acid from the obtained culture by operation, such as extraction.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for producing natural abscisic acid using the metabolic functions of microorganisms.

[Conventional technology]

Natural abscisic acid was first isolated in 1963 by Dr. Adikot et al. of the University of California as a substance that promotes fruit drop in young cotton fruit, and since then it has been discovered to have important physiological effects on plants, and today it is even considered a type of plant hormone. Confirmed.

The characteristic of natural abscisic acid's physiological effects on plants is that it antagonizes other plant hormones such as auxin, gibberellin, and cytokinin, canceling their effects, and suppressing plant growth. The most notable one is the closing effect on plant stomata. In the future, natural abscisic acid is expected to be applied to agricultural production as a substance that can respond to climate changes such as drought and cold damage by utilizing these physiological effects.

Furthermore, its use in brewing, such as improving quality and reducing costs in beer brewing (Japanese Patent Application Laid-open No. 101677/1983),
Progress is being made in its use as medicine and its intermediates.

Conventionally, organic synthesis methods have been considered as methods for producing abscisic acid, but it is extremely difficult to obtain optically active natural abscisic acid using this method, and the production cost is extremely high, making it difficult to produce the above-mentioned agricultural products. It was practically unusable for brewing.

In recent years, microbial methods using fermentation culture that utilize the metabolic functions of microorganisms have been considered as a promising alternative to organic synthesis methods, and production methods using Botrytis bacteria (Japanese Patent Application Laid-open No. 58-51895) and Cercospora・Cercospora rosi
kora) production method [εχp13rientia
33.1556 (1977)), JP-A-58-363
Publication No. 93, Japanese Unexamined Patent Application Publication No. 160996/1987, etc. are publicly known.

[Problem that the invention seeks to solve]

However, the biggest drawback of the above microbial method is that the amount of natural abscisic acid accumulated through fermentation production is extremely small, and the fermentation production rate is also extremely slow, making it extremely inefficient as a means of production on an industrial scale. Ta.

[Means and effects for solving the problems] The present inventors have conducted intensive studies on a method for efficiently producing natural abscisic acid, and as a result, they have developed a method for efficiently producing natural abscisic acid. When culturing a strain that belongs to the genus 1is) and has the ability to produce natural abscisic acid, by making the seed spores to be inoculated excessively present in the medium,
The inventors completed the present invention by discovering that the amount of natural abscisic acid accumulated in the medium increases significantly.

That is, the present invention relates to Botrytis
Bacteria belonging to the genus and having the ability to produce natural abscisic acid are inoculated and cultured in a medium to produce and accumulate natural abscisic acid, and when the natural abscisic acid obtained from the culture is isolated and collected, the above-mentioned medium This is a method for producing natural abscisic acid, which is characterized by inoculating seeds in excess with seed spores.

The present invention will be explained in detail below.

The bacteria used in the present invention are not particularly limited as long as they are natural abscisic acid-producing bacteria belonging to the genus Botrytis, and include normal mutant bacteria and bacteria produced by mutation treatment.

A specific example of a strain of naturally occurring abscisic acid-producing bacteria belonging to the Botrytis bacterium is Botrytis cinerea.
FERM P-6156. The mycological properties of Botrytis cinerea, which has the ability to produce natural abscisic acid, were already known in JP-A-58-5.
This method is discussed in Japanese Patent No. 1895.

Next, the culture medium used for producing natural abscisic acid in the present invention includes carbon sources such as bran, wheat, rice, canola, potato, glucose, maltose, malt extract, sucrose, dextrin, blackstrap molasses, starch, etc. A medium containing nitrogen sources such as soybean flour, soybean flour, gluten, yeast extract, peptone, meat extract, corn staple liquor, etc. may be used singly or in combination of two or more of them.

In addition, for example, inorganic substances such as magnesium salts, calcium salts, potassium salts, sodium salts, and phosphates, as well as vitamins, oils and fats, and the like can be added.

Further, the culture medium may be either a solid medium or a liquid medium, but it is advantageous to use a solid medium in terms of the yield of the target product.

The culture medium having the above composition is sterilized using an appropriate method to prepare a substantially sterile medium.

Sterilized water containing spores of the natural abscisic acid-producing strain belonging to the genus Botrytis is used to uniformly disperse the spores in the medium treated as described above. In this case, there is no problem even if mycelial parts other than spores are mixed in. In the conventionally known normal culture medium, the seed spores are 50% in 1 g of the medium.
Approximately 0 will be inoculated.

In the present invention, it is important to inoculate an excessive amount of seed spores into the medium. Preferably, the spores are present in the medium so that the number of spores is substantially 1.0×10 3 or more per 1 g of the medium. The inoculation operation is thus completed.

By inoculating an excessive amount of seed spores into the medium, culture can be performed in the presence of an excessive amount of spores. Under such conditions, natural abscisic acid can accumulate at high concentrations and at high rates.

The culture conditions are as follows: culture temperature is usually 10-40°C, preferably 25-35°C, medium pH is usually 3-12, preferably 4-8, and culture period is usually 1-15 days, preferably 3-35°C.
The culture is carried out for about 9 days using a culture system that excludes the contamination of contaminants.

Next, after completion of the culture, a conventional method can be used to isolate natural abscisic acid from the culture, for example, the method described below is used.

First, the culture is extracted using an extraction solvent.

In this case, the extraction solvent used is, for example, acetone, ethyl acetate, benzene, chloroform, diethyl ether, methanol, or ethanol. The natural abscisic acid transferred into the extract solution can be extracted using conventional fractional extraction,
Isolation and purification are possible by applying general purification methods such as adsorption, thin layer chromatography, column chromatography, and distillation, and ordinary purification methods for organic compounds.

〔Example〕

The present invention will be specifically illustrated by examples below.

Example 1 Boil 11 distilled water and 200 g of potatoes for 1 hour,
The liquid was passed through gauze. Water was added to the filtered liquid to make 11, and 20g of glucose and 2Or of agar were added to it at 100°C.
Heat treatment was performed for 20 minutes to obtain a potato agar medium solution.

(Method for preparing Botrytis cinerea seed spores) A seed strain for preservation was inoculated into a veterinary dish containing 20 ml of the potato agar medium, and cultured stationary at 28°C. In this case, the cells were kept in the dark for 4 days after inoculation, cultured for the next 2 days while irradiated with light having a maximum wavelength of 360r+n+, and cultured still in the dark again for 1 day. The cultured cells were suspended in sterilized water and filtered to obtain an aqueous solution containing substantially only Botrytis spores.

Example 1 (Production of natural abscisic acid) The above-mentioned sterilized potato agar medium was placed in a vetri dish at 20:1
A solid plate medium was prepared by dispensing the mixture in batches.

Next, an aqueous spore solution of Botrytis cinerea (FERMP-6156) prepared by the above-mentioned sterile procedure was sprayed onto the plate medium in the amounts shown in Table 1 to complete the inoculation.

This Petri dish was statically cultured at 28°C for 7 days.

After the completion of the culture, each culture was disrupted and immersed in acetone to extract the product. After repeating the acetone oil field twice, the extract was concentrated and subjected to silica gel thin layer chromatography as a fixed amount (1 [111>). Both preparations were dispensed onto the top and developed with a solvent of 1 part ethyl acetate and 1 part n-hexane, and the accumulated amount of natural abscisic acid was quantified.

The results are shown in Table-1.

Table 1 *) The number of inoculated spores is the value obtained by counting the number of spores present in a certain amount of solution using an optical microscope and converting it.

Example 2 The potato agar medium shown in Example 1 was transformed into a wheat bran medium (
A similar experiment was conducted by changing the ratio to 1 part bran and 1 part water.

The results are shown in Table-2.

Table 2 Example 3 After culturing a spore aqueous solution (1.5 x 10' spores/g medium) on the same potato agar medium 21 as in Example 1, the culture was extracted with acetone.

This acetone solution was concentrated under reduced pressure, and the water-soluble residue was adjusted to pH 9.0.
The mixture was extracted and washed with ethyl acetate.

After adjusting the remaining aqueous solution to pH 3.0, it was extracted with ethyl acetate, and the obtained ethyl acid solution was dried with anhydrous sodium sulfate.
Concentrate to obtain an ethyl acetate-soluble acidic fraction (natural abscisic acid target purified product). This product is subjected to silica gel chromatography, and n-hexane/ethyl acetate (n-hexane: ethyl acetate is 5:1 first) (The ratio was changed sequentially so that the final ratio was 1:1). The purified natural abscisic acid fraction was separated and concentrated to obtain pure natural abscisic acid (>0.3 g of crystals).

The optical rotation of the crystal of natural abscisic acid obtained is [α)2
5=+432±2' (C=0.23, ethanol)
Met.

〔Effect of the invention〕

According to the present invention, natural abscisic acid can be obtained in a high accumulation amount and at a far fermentation production rate by fermentation culture using microorganisms, and production on an industrial scale can be made possible.

Patent applicant Higashi Shikikai Co., Ltd. Shin Nippon Chemical Industry Co., Ltd.

Claims (1)

[Claims] Bacteria belonging to the genus Botrytis and capable of producing natural abscisic acid are inoculated into a medium and cultured to produce and accumulate natural abscisic acid, and the natural abscisic acid obtained from the culture is isolated and collected. A method for producing natural abscisic acid, which comprises inoculating seed spores into the medium in excess.