JP2524964B2 - Method for producing antibacterial substance 2,4-diacetylphloroglucinol - 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 an antibacterial substance, 2,4-diacetylfluoroglurucinol, and more specifically, a shoot Monas fluorescens (Pseudomona) having an ability to produce 2,4-diacetylfluoroglurucinol.
s fluorescens) is co-cultivated with plant cells to obtain a high-yield yield of antibacterial substance, 2,4-diacetylfluoroglycurinol.

[0002]

2. Description of the Related Art In recent years, a natural-ecosystem-utilizing agricultural method has been proposed in order to protect the environment and utilize the ecosystem, and there is interest in bacteria that inhabit the rhizosphere of agricultural crops. That is, among such rhizosphere bacteria, there are bacteria that are considered to be useful for plant disease suppression or plant growth, and this is an attempt to utilize such bacteria. However, when specific microorganisms are introduced into the soil, these introduced microorganisms are inferior to the indigenous bacteria already inhabiting the soil. Therefore, such a method has not yet been put to practical use because it becomes difficult to settle in the soil before the purpose is achieved.

Under these circumstances, the present inventors have focused on the relationship between microorganisms that live in soil or in plant roots and antagonists produced by useful bacteria in such microorganisms. Among them, the attention was paid to 2,4-diacetylfluorologlucinol, which is an antibacterial substance produced by Chlamydomonas fluorescens, which has been conventionally known. That is, this antibacterial substance was isolated in 1953 as an antibacterial substance produced by Chlamydomonas fluorescens. Since then, starting with the evaluation of antibacterial activity against Gram-positive bacteria, actinomycetes, and filamentous fungi, to date the evaluation of in vitro antibacterial activity for the purpose of disease control of crops, etc.
It is carried out using barley wilt fungus, corn seedling wilt fungus, wheat leaf wilt fungus and the like.

[0004] As a method for obtaining this 2,4-diacetylfluoroglucurinol, a production method by culturing Chlamydomonas fluorescens has been conventionally known. For example, as a method, examination of production conditions using sugar substrates (P. Shanahan et al., Appl. Environ.
Microbiol., 58 (1), 353 (1992)), or examination of production conditions in King B medium (E. Levy et al., Plant Pathol., 4
1 (3), 335 (1992)) have been conducted.

However, according to such a usual culture method, the maximum production amount of this antibacterial substance is 200 mg / L.
Since the production amount is low, the evaluation of the efficacy of the antibacterial substance is limited to the in vitro evaluation as described above, and the application of this substance to the actual field has not yet been put to practical use. Is the current situation. Therefore, this antibacterial substance 2,
Today's challenge is how to efficiently obtain large amounts of 4-diacetylfluoroglycinol.

[0006]

Under the above circumstances, the present inventors have repeatedly studied a method for efficiently obtaining a large amount of 2,4-diacetylfluoroglurucinol, and as a result, it is an endophytic microorganism. Attention was paid to the relationship between Chlamydomonas fluorescens and this substance. Then, it was found that a large amount of the antibacterial substance 2,4-diacetylfluoroglurucinol can be obtained by separating this bacterium from the roots of plant bodies and co-culturing it with plant cells, and based on such findings The present invention has been completed.

[0007]

[Means for Solving the Problems] That is, the present invention is a method of co-cultivating chlamydomonas fluorescens, which has the ability to produce 2,4-diacetylfluoroglurucinol, with plant cells, and from this culture solution, 2,4-diacetylfluoroglurucinol The present invention relates to a method for producing an antibacterial substance 2,4-diacetylfluoroglurcinol, which is characterized in that Further, the present invention is characterized in that after producing the co-cultivated producing bacteria and plant cells as immobilized cells, 2,4-diacetylfluorogrucinol is fractionated from the culture solution cultured using the immobilized cells. The present invention relates to a method for producing the antibacterial substance 2,4-diacetylfluoroglycinol, which provides a method capable of obtaining a larger amount of this substance.

[0008]

The method for producing the antibacterial substance 2,4-diacetylfluoroglurcinol of the present invention will be described in more detail below. Used in the present invention 2,
Chlamydomonas fluorescens, which has the ability to produce 4-diacetylfluoroglucurinol, is isolated from the inside of the plant, especially within the root of the plant. The means for separating such chutemonas fluorescens is
The surface of the plant root may be sterilized and then separated and cultured. For example, a method may be used in which the surface of the plant root is washed and surface-sterilized with ethanol or the like, and then the plant root is homogenized and then directly pour-cultured with bare agar. Separation culture is performed by such a method, and then, colonies that emit fluorescence are selected from the generated colonies. Most of the isolated colonies composed of fluorescent bacteria are chlamydomonas fluorescens having the ability to produce 2,4-diacetylfluoroglucurinol.

The present invention has the greatest feature in using such a Chlamydomonas fluorescens and performing co-cultivation with plant cells. According to the usual culturing means as means other than co-cultivation, The purpose cannot be achieved at all.
As plant cells, aseptic cultured roots, hairy roots, callus, and other cultured tissues can be used. Furthermore, although it is possible to use the plant itself such as a sterile seedling,
In this case, the yield of 2,4-diacetylfluoroglycinol is slightly reduced.

The method for obtaining such plant cells is as follows. For example, a cultured root can be obtained by culturing a sterile root tip. Further, hairy roots can be obtained by a direct inoculation method, a leaf disc method, a co-culture method, etc., but the Agrobacterium tergenes used for this must be sterilized in advance, After sterilization, it is desirable to culture in a hormone-free medium. It is desirable to use dicotyledonous plants for the preparation of these hairy roots, and it is difficult to prepare them when monocotyledonous plants are used. Furthermore, for callus, the stem of the plant,
Culture may be performed using either root or leaf tissue, and the type of medium is not particularly limited. For example, MS medium (Murashige-Skoog medium), white medium and the like are desirable as the type of this medium. In addition, for the preparation of this callus, the cell mass generated during the preparation of hairy roots described above can also be used.

The co-cultivation of plant cells with Chlamydomonas fluorescens by using such plant cells is carried out using a basic medium for plant tissue culture. For example, using white medium or the like, plant cells are transferred to it, and further, a colony composed of the above-mentioned isolated Chlamydomonas fluorescens is inoculated to carry out co-cultivation. In this case, Chlamydomonas fluorescens grows in the plant cells in the culture medium or on the cell surface while being influenced by the plant cultured cells. The culture is usually carried out at a temperature of 23 to 30 ° C. for about 2 weeks, although it depends on the mycological characteristics of the Chlamydomonas fluorescens used.

After the completion of the culture, the culture solution is lyophilized, and the dried product is extracted and separated with a mixed solvent of chloroform and methanol (chloroform: methanol = 5: 1).
A crude separation of diacetylfluoroglycinol is performed. Next, 2,4-diacetylfluoroglurucinol is separated from this crude product by silica gel column chromatography. According to the method of the present invention, it is possible to obtain the amount of 2,4-diacetylfluoroglurucinol obtained in the culture solution at about 1000 mg / L, and the yield of 2,4-diacetyl fluoroacetyl is about 50%. Fluoroglucinol can be produced.

Next, the more preferred method for producing 2,4-diacetylfluoroglurcinol of the present invention will be described in detail. INDUSTRIAL APPLICABILITY As described above, the present invention co-cultivates chlamydomonas fluorescens having the ability to produce 2,4-diacetylfluoroglucurinol with plant cells, and from this culture solution
The antibacterial substance 2,4-diacetylfluoroglurucinol is produced by a method of fractionating 2,4-diacetylfluoroglurucinol, and after the co-cultivation producing bacteria and plant cells are fixed cells, this immobilization is performed. 2,4-from the culture medium in which activated cells were cultured in a microorganism culture medium
By fractionating diacetylfluoroglucurinol, it becomes possible to obtain this substance in a higher yield. That is, according to the above-mentioned method, 2,4-diacetylfluoroglucurinol is approximately 1000 mg in the culture medium.
According to the culture method using this fixed cell, in the example using the 5-fold diluted potato-dextrose medium, the concentration can be obtained at a high yield of about 3000 mg / L, while the concentration of The yield reaches up to about 75%.

The method of culturing with fixed cells will be described in detail. First, the plant cells in the culture obtained by the above-mentioned co-cultivation are separated by means such as filtration. Next, this plant cell is used as an immobilized cell, and this method is performed by a commonly used plant cell immobilization technique. For example,
First, the plant cells are homogenized, then a sodium alginate solution is added thereto, and the mixture is stirred. This plant cell dispersion liquid is added dropwise to a solution of calcium chloride to obtain plant cell immobilized cells containing Chlamydomonas fluorescens having a diameter of about 3 to 6 mm.
Immobilized cells are inoculated into microbial culture medium at a temperature of 23-30 ° C.
Incubate for about 2 weeks. After co-cultivation, the above-mentioned method of collecting and fractionating 2,4-diacetylfluoroglucinol may be followed. By this method, it is possible to obtain 2,4-diacetylfluoroglurucinol in a higher yield.

[0015]

EXAMPLES The examples of the present invention will be further described below. In this example,% means% by weight unless otherwise specified.

(Example 1) 1. Separation of chlamydomonas fluorescens , culture Tomato strains and bent grass at the late stage of harvest were used as sources for separation of chlamydomonas fluorescens. Chlamydomonas fluorescens having the ability to produce lucinol was separated. Isolate the collected plant roots with sterile water and 0.005
% Aerosol OT aqueous solution, and the root surface was sterilized by immersing this in a 75% ethanol aqueous solution. Next, after thoroughly washing this root with sterilized water to remove ethanol, 1 g of the root is added to 100 ml of sterilized water, and 8,000 to 10,000
The roots were homogenized at rpm for 10 minutes. For homogenized roots, PDA medium (Potato Dextrose agar medium) and P-1 medium (Chutemonas selective medium, Aono and Katoh, 1979) were used.
Each was pour-cultured at 25 ° C. for 4 weeks. Then, colonies that emit fluorescence were selected from the colonies formed in the culture. The selected colonies were subcultured on a PDA slant medium to obtain a strain producing a granule producing bluish white in the medium under irradiation with UV 365 nm. Table 1 shows the mycological characteristics of the strains isolated from the roots of tomato and turf. Based on the morphology and physiological properties of these strains, they were all identified as Pseudomonas fluorescens.

[0017]

[Table 1] Note) Notation; +: Positive,-: Negative, O: Oxidative acid formation

2. Preparation of plant cells a. Preparation of culture roots Tomato seeds (variety: House Momotaro) were surface sterilized with a 1% sodium hypochlorite aqueous solution and a 75% ethanol aqueous solution, and then 0.8% Seed on agar and in dark aerobic condition at 25 ° C for 5
The seeds were germinated by holding for a day. Approximately 1 cm is cut off from the root end of the germinated seed root, and 5 to 6 pieces of this are cut into 300 ml of white liquid medium.
The root tip culture was carried out under dark aerobic conditions at 25 ° C for 10 days.

B. Preparation of Callus After surface sterilization of tomato seeds (variety: Mizuken) with a 1% sodium hypochlorite aqueous solution and a 75% ethanol aqueous solution,
This was sown on 0.8% bare agar and kept at 25 ° C. in the dark and aerobic for 5 days to germinate the seeds. After germination, the seedlings were continuously grown under aerobic conditions for about 1 week. The root end of this germinated seed, the stem,
Each cotyledon is cut to about 5 mm, and these are MS medium (containing hormone agent, naphthalene acetic acid 0.2 mg / l, benzyl adenine 0.2 mg / l) 300
Each was placed in a 1 L Erlenmeyer flask containing ml, and cultivated for 4 weeks at 25 ° C. under dark aerobic conditions. One-fifth of the culture broth after culturing is sampled, inoculated into MS medium, and stored under dark aerobic condition for
Culture was performed at 5 ° C for 4 weeks. This operation was repeated twice more to obtain a cultured callus.

3. Co-cultivation and 2,4-diacetyl fluoro from culture
Table 1 strain No.T-32 separated cultured by preparative above 1. grayed lucinol and (PDA slant passage culture) using cultured roots prepared above 2.a. were co-cultivation. In the co-cultivation method, the above culture root was first added to a 10-fold diluted white liquid medium. Next, 1 platinum loop of the above-mentioned isolated culture was inoculated into this, and co-cultivation was carried out at 25 ° C. for 2 weeks.

After completion of the culture, the culture solution was freeze-dried, and the dried product obtained by drying was dispersed in 50 ml of a chloroform / methanol mixed solution (chloroform: methanol = 5: 1). After the dispersion, the solid content was removed by filtration. The mixed solution after the solid content was removed was concentrated using a rotary evaporator, and then this solution was put into a column packed with Wakogel C-100 (manufactured by Wako Pure Chemical Industries) for fractionation. In this case, the developing solvent used was a mixed solvent of benzene / chloroform / methanol (benzene: chloroform: methanol = 4: 2: 1). A second fraction containing a blue-white fluorescent component was collected and dried to remove the solvent, thereby obtaining 2,4-diacetylfluoroglycurinol. In order to identify the component of this compound, it was subjected to precision instrumental analysis and measured. The results are shown in Table 2. Further, from the results, this product could be identified as 2,4-diacetylfluoroglurucinol, and the yield was 1097 mg / L when converted to the concentration in the above-mentioned coculture medium.

[0022]

[Table 2]

Comparative Example 1 300 ml of a 10-fold diluted white liquid medium was placed in a 1 L Erlenmeyer flask, and the strain No. T-
One platinum loop of 32 (PDA slope subcultured bacteria) was inoculated, and shake culture was performed at 25 ° C for 2 weeks. After the culturing, the 2,4-diacetylfluoroglurucinol was collected from the culture solution in the same manner as in Example 1, and the result was 2,4-
The yield of diacetylfluoroglycinol was 30 mg / L when converted into the concentration in the above culture solution.

(Example 2) The callus obtained in the preparation of the plant cells of Example 1 was used, and this was added to 5% of MS medium (without hormone).
The mixture was placed in a 1 L flask containing 300 ml of double-diluted medium. Then, the strain No. T-33 of Table 1 which was separately cultivated in Example 1 was added thereto.
One platinum loop of (PDA slope subcultured bacterium) was put in and statically cultured at 30 ° C. for 2 weeks. After the culturing, 2,4-
As a result of fractionation of diacetylfluoroglucurinol, the yield of 2,4-diacetylfluoroglurucinol was 828 mg / converted to the concentration in the above culture solution.
It was L.

(Example 3) From the culture solution in which the co-cultivation was carried out in Example 1, the culture root was filtered off. After washing the surface of this culture root with sterilized water, add it to 100 ml of a 2% sodium alginate aqueous solution, and use a homogenizer at 10000 rpm for 1
It was homogenized for 0 minutes to give a gel solution. The gel-like solution was added dropwise to a 1% calcium chloride aqueous solution to obtain immobilized cells having an outer diameter of about 5 mm. Then, add 1 volume of 300-fold of 5-fold diluted potato dextrose liquid medium.
ml was added, and the above fixed cells were added to this so as to have a volume of 1%. This medium was placed at 30 ° C. for 2 weeks under dark aerobic conditions and statically cultured. After the culturing, the 2,4-diacetylfluoroglurucinol was fractionated from the culture broth in the same manner as in Example 1, and as a result, the yield of 2,4-diacetylfluoroglurucinol was 2700 mg / in terms of the concentration in the above-mentioned culture broth. It was L.

(Example 4) Callus was separated from the culture solution in which co-cultivation was carried out in Example 2. After washing the surface of this callus with sterilized water, add it to 100 ml of a 2% sodium alginate aqueous solution, and use a homogenizer at 10000 rpm for 1
It was homogenized for 0 minutes to give a gel solution. The gel-like solution was added dropwise to a 1% calcium chloride aqueous solution to obtain immobilized cells having an outer diameter of about 5 mm. Then, add 1 volume of 300-fold of 5-fold diluted potato dextrose liquid medium.
ml was added, and the fixed cells were added to this so that the volume became 0.5%. This medium was placed under dark aerobic conditions at 25 ° C. for 2 weeks, and static culture was performed. After the culturing, the 2,4-diacetylfluoroglurucinol was collected from the culture solution in the same manner as in Example 1, and the result was 2,4-
The yield of diacetylfluoroglucurinol was 2320 mg / L in terms of the concentration in the culture medium.

<Evaluation of antibacterial property> 2,4-Diacetylfluoroglurcinol obtained by the above culture was dissolved in acetone to prepare 1, 1
Solutions with concentrations of 0, 100 and 1000 μg / ml were prepared. These solutions were impregnated into antibacterial property test paper discs (outer diameter 8 mm, thick, manufactured by Toyo Roshi Paper Co., Ltd.), dried to remove acetone, and used as antibacterial substance test discs. Further, tomato wilt disease fungus (MAFF-03-01485) was subcultured to a PDA slant medium and used as a test strain. 50 ml of sterile water with 1 platinum loop of test bacteria
1 ml was mixed with potato-dextrose agar medium, and this was used as a plate culture medium. Place the antibacterial substance-containing disc on this agar plate and place it at 30 ° C.
Culture was performed for 96 hours. The minimum growth inhibitory concentration (MIC) against the bacterial wilt of tomato was assayed by the presence or absence of the growth inhibitory circle of the bacterial wilt of tomato bacterial around the disc after the culture. As a result, the MIC of this substance was 1 μg / ml.

[0028]

EFFECTS OF THE INVENTION The method for producing the antibacterial substance 2,4-diacetylfluoroglurucinol of the present invention provides a method capable of obtaining a large amount of 2,4-diacetylfluoroglorucinol useful as an antibacterial drug. To do. According to a conventionally known culture method, the obtained concentration was limited to 200 mg / L, but in the method of the present invention, about 1000 mg / L was obtained, and according to the immobilized cell method of the present invention, about It is possible to obtain 2,4-diacetylfluoroglurucinol at a concentration as high as 3000 mg / L. Therefore, 2,4-diacetylfluoroglurcinol which can be obtained in high yield by such a method is
It becomes possible to use it for multiple purposes as an antibacterial substance,
For example, it can be used not only as an antibacterial material for the purpose of controlling diseases of plants such as agricultural crops, but also as a fungicide and fungicide for various industrial or building materials.

Front page continued (72) Inventor Yoshio Maekawa 3-491 Higashi Jiyugaoka, Shizen-cho, Miki-shi, Hyogo Prefecture (72) Inventor Taizo Akiyama 331-9 Kamisume, Yoneda-cho, Takasago-shi, Hyogo Prefecture (72) Inventor Yoshinori Hayashi, Kako, Hyogo Prefecture 2689-1 Oka, Inami Town, Gunma Examiner Reiko Imamura

Claims (2)

(57) [Claims] 1. A method of co-cultivating shoot Monas fluorescens, which has the ability to produce 2,4-diacetylfluoroglucinol, with plant cells, and collecting 2,4-diacetylfluoroglucinol from this culture solution. A method for producing the antibacterial substance 2,4-diacetylfluoroglucurinol. 2. A chlamyza monas fluorescens capable of producing 2,4-diacetylfluoroglucurinol is co-cultured with a plant cell, and after the culturing, the producing bacterium and the plant cell are fixed cells, and the fixed cells are then fixed. A method for producing the antibacterial substance 2,4-diacetylfluoroglucinol, which comprises collecting 2,4-diacetylfluoroglucinol from a culture broth cultured using