JP2004135664A - Method for producing iturin a and its homologue - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing iturin A and its homologue, by culturing Bacillus genus bacteria producing the iturin A and its homologue and accumulating the iturin A and its homologue in a high concentration in a culture liquid. <P>SOLUTION: This method for producing the iturin A and its homologue comprises culturing the Bacillus genus bacteria having ability of producing the iturin A and its homologue in a liquid medium containing a soybean powder or an extract thereof in an amount of ≥ 2 wt% and accumulating the iturin A and its homologue in the culture liquid, wherein a concentration of the iturin A and its homologue reaches 1.5 g/L or more. A cultured material containing the iturin A and its homologue is given by accumulating the iturin A and its homologue by the method. A solid material containing the iturin A and its homologue is given by drying the cultured material. An agent for controlling plant disease contains the cultured material or the solid material. A method for controlling the plant disease comprises using the cultured material or the solid material. <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates to a method for producing iturin A and its homologues by culturing Bacillus microorganisms in a liquid medium containing soybean or an extract thereof as a nitrogen source and accumulating a high concentration of iturin A and its homologues in the culture solution. . The present invention also relates to the produced iturin A and its homolog-containing culture, dried iturin A and its homolog-containing solid, and methods of using them.

Bacillus (Bacillus) microorganism belonging to the genus, to be particularly Bacillus subtilis (Bacillus subtilis) is to produce iturin A and its homologues, have been conventionally known (for example, Besson (Besson) Other, "Journal of the anti-bio tick scan (Journal of Antibiotics) ", 1978, Vol. 31, p. 284-288 (Non-Patent Document 1).)

Iturin A and its homologues refer to a group of compounds having the structure represented by the following formula 1, and have attracted attention as an excellent plant disease controlling component because they have an antibacterial activity against fungi, especially plant pathogenic bacteria. Was.

（式中、Ｒは炭素数３ないし１０の直鎖型または分岐型アルキル基を示す。）
　アイチュリンＡおよびその同族体の製造方法としては、特開昭59-212416号公報（特許文献１）、特開平7-143897号公報（特許文献２）等がある。

(In the formula, R represents a linear or branched alkyl group having 3 to 10 carbon atoms.)
Methods for producing iturin A and its homologs include JP-A-59-212416 (Patent Document 1) and JP-A-7-143897 (Patent Document 2).

However, the productivity of iturin A and its homologues produced by microorganisms of the genus Bacillus is low, and sufficient productivity is obtained for industrial use of iturin A and its homologues or cultures containing them. As a result, many researchers have attempted to increase productivity of iturin A and its congeners.
Hatada et al., Bacillus subtilis no. It discloses a method for producing an iturin A-based substance by aerobically culturing an NA-apb-1 strain in a nutrient medium (see Japanese Patent Publication No. 63-20519 (Patent Document 3). Peptone, meat extract, yeast extract, casein hydrolysate, cornstarch, gluten meal, inorganic nitrogen source is taught, and culture obtained by culturing the same strain in a medium containing peptone, meat extract, yeast extract 30 liters of filtrate yield 270 mg of iturin A. Sandrin et al. Disclose that 480 mg / L of iturin A and its homologs can be obtained in a synthetic medium using proline as a nitrogen source ("Biotechnology and Applied"). -Biochemistry and Applied Biochemistry ", 1990, Vol. 12, p. 370-375 (Non-Patent Document 2).

Hbid et al. Disclose a method by which iturin A and its homologs yields 1388 mg / L in media containing peptone ("Applied Biochemistry and Biotechnology", 1996, No. 57). / 58, p.571-579 (Non-Patent Document 3)). Phae et al. Disclose how iturin A and its congeners yields of 620 mg / L are achieved in media containing peptone ("Journal of Fermentation and Bioengineering"). 1991, Vol. 71, pp. 118-121 (Non-Patent Document 4).)

On the other hand, as for a method for culturing Bacillus microorganisms in a liquid medium containing soybean flour or an extract thereof, the present inventors have disclosed a method for producing surfactin (Japanese Patent Application Laid-Open No. 2002-176993). (Patent Document 4)). However, this publication does not disclose anything about iturin A and its homologues. JP-A-59-212416 (Patent Document 1) discloses that culturing is carried out in a medium containing 1% soybean flour, and 300 mg of iturin A and a homolog thereof can be obtained from a 16-liter culture solution. However, the obtained amount of iturin A was almost equivalent to the amount obtained by a known culture method.

Iturin A at a concentration of at least 1.5 g / L in the culture medium by culturing the Bacillus (Bacillus) genus microorganisms having iturin A and its homologues ability to produce soybean flour or an extract thereof with a liquid medium containing more than 2 wt% And the accumulation of its congeners has not been previously known.

Besson et al., "Journal of Antibiotics", 1978, Vol. 31, p. 284-288 JP-A-59-212416 JP-A-7-143897 JP-B-63-20519 "Biotechnology and Applied Biochemistry", 1990, Vol. 12, p. 370-375 "Applied Biochemistry and Biotechnology", 1996, Vol. 57/58, p. 571-579 "Journal of Fermentation and Bioengineering", 1991, Vol. 71, p. 118-121 JP-A-2002-176993

Conventional productivity of iturin A and its homologues is not sufficient for industrial use, and a production method that further improves productivity has been desired.
Therefore, an object of the present invention is to provide a method for producing iturin A and its homologues, which comprises culturing a Bacillus genus producing iturin A and its homologues, and accumulating iturin A and its homologues in a culture solution at a high concentration. To provide.
Another object of the present invention is to provide iturin A and its homologues, containing cultures, its solids, and methods of using them.

The present inventors have conducted intensive studies on various medium components in order to solve the above-mentioned problems. As a result, it was found that iturin A and its homologues were obtained in a medium containing a ground material of soybean or an extract thereof as a nitrogen source in an amount of 2% by mass or more. By culturing the microorganism of the genus Bacillus to produce, it was found that iturin A and its homologs were accumulated at a high concentration in the culture solution, and the present invention was completed.

That is, the present invention relates to the following methods for producing iturin A and its homologues, iturin A and its homologue-containing culture, and iturin A and its homologue-containing solids.

[1] iturin A and B. (Bacillus) genus microorganisms having its homologues producing ability were cultured in soy flour or a liquid medium containing the extract 2 mass% or more, at a concentration of at least 1.5 g / L in the culture medium A method for producing iturin A and its homologues, which comprises accumulating iturin A and its homologues.
[2] iturin A and B. (Bacillus) genus microorganisms having its homologues producing ability, is viable Bacillus (Bacillus) microorganisms in a medium containing 1.5 g / L or more iturin A and its homologues 2. The method for producing iturin A and its homologues according to the above 1.
[3] iturin A and B. (Bacillus) genus microorganisms having its homologues producing ability, substantially free of surfactin producing ability have iturin A and its homologues producing ability in Bacillus (Bacillus) microorganisms of the genus 3. A method for producing iturin A and homologues thereof according to the above 1 or 2.
[4] The method for producing iturin A and the homologue thereof according to the above 1, wherein the liquid medium is a liquid medium in which the amount of a phosphate added is 0 to 3% by mass or less in terms of K ₂ HPO ₄ .
[5] The method for producing iturin A and homologs thereof according to any one of the above items 1 to 3, wherein the microorganism of the genus Bacillus is Bacillus subtilis .
[6] The method for producing iturin A and homologues thereof according to any one of the above items 1 to 3, wherein the microorganism of the genus Bacillus is Bacillus subtilis SD142 (FERM BP-8427).
[7] The method for producing iturin A and homologues thereof according to any one of the above items 1 to 3, wherein the microorganism of the genus Bacillus is a mutant strain of Bacillus subtilis SD142 (FERM BP-8427).
[8] The above-mentioned item 1, wherein the liquid medium containing 2% by mass or more of soybean powder or an extract thereof contains at least one selected from the group consisting of maltose, starch syrup, soluble starch, dextrin, glucose, sucrose, and fructose. For producing iturin A and homologues thereof.
[9] Iturin A and its homolog-containing culture, wherein iturin A and its homologs are accumulated in the culture solution according to any one of the above 1 to 8.
[10] Iturin A and its homolog-containing solid obtained by drying the culture containing iturin A and its homologs according to 9 above.
[11] A plant disease control agent comprising the culture or solid containing iturin A and its homologues described in 9 or 10 above.
[12] A method for controlling plant diseases, which comprises using the culture or solid containing iturin A and its homologues described in 9 or 10 above without purification.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in detail.

ア イ The term iturin A and its homologues of the present invention mean derivatives represented by the following formula (1), and also include analogs thereof.

（式中、Ｒは炭素数３ないし１０の直鎖型または分岐型アルキル基を示す。）

(In the formula, R represents a linear or branched alkyl group having 3 to 10 carbon atoms.)

According to the present invention, pulverized soybean or an extract thereof is added as a nitrogen source in an amount of 2% by mass or more as a nitrogen source to a culture medium for culturing a microorganism that produces iturin A and its homologues. It can accumulate iturin A and its homologues at high concentrations. According to the inventors' knowledge, it has been known to culture Bacillus microorganisms in a medium containing a ground material of soybean or an extract thereof as a nitrogen source, but it produces iturin A and its homologues. The present inventors have newly found that microorganisms are cultured in a medium containing 2% by mass or more of a ground material of soybean or an extract thereof as a nitrogen source, and that iturin A and its homologs are accumulated at a high concentration in the culture solution. Things.

The microorganism of the genus Bacillus used in the present invention is not particularly limited as long as it produces iturin A and its homologs, but it accumulates iturin A and its homologs in a medium at a high concentration. Bacillus microorganisms capable of producing iturin A and its homologs and capable of growing in a medium containing 1.5 g / L of iturin A and its homologs Is preferred. Further, as another preferred example, a microorganism of the genus Bacillus, which has an ability to produce iturin A and its homologues and has substantially no surfactin-producing ability, is preferable. The phrase "substantially no surfactin-producing ability" means that the accumulated amount of surfactin is 50 ppm or less when cultured in a medium containing a ground material of soybean or an extract thereof as a nitrogen source. Iturin as A and viable Bacillus (Bacillus) microorganisms in a medium containing iturin A and its homologues having 1.5 g / L and its homologues producing ability, for example, Bacillus subtilis SD142 (FERM BP-8427) Is preferred.

Bacillus subtilis SD142 (FERM BP-8427) is isolated from compost and exhibits the following mycological properties.

Bacteriological properties (a) Morphology (1) Bacteria shape: rod-shaped (2) Bacteria size: 0.7-0.9 x 1.5-3.0 μm (3) Polymorphism: none (4) Motility: yes (5) Presence / absence of spores: Spore shape: Oval or cylindrical (6) Gram stain: Positive (7) Acid-fast: Negative (b) Growth status Gravy agar Plate culture: Colonies are circular and 1-2 mm in diameter , Peripheral shape is wavy, viscous and dull. (C) Physiological properties (1) Nitrate reduction: positive (2) VP test: positive (3) Indole formation: negative (4) Use of citric acid: positive (5) Use of succinic acid: negative (6) Use of propionic acid: negative (7) Use of tartaric acid: negative (8) urease: negative (9) oxidase: positive (10) catalase: positive (11) Growth range: pH 5-9 Temperature 20-50 ° C (12) 10% NaCl medium: Growth (13) Anaerobic culture: Negative (14) Egg yolk reaction: Negative (15) Hydrolysis of starch: Positive (16) Degradation of arginine: Positive (17) Degradation of tyrosine: Negative (18) Gelatin Liquefaction: Positive (19) Esculin degradation: Positive (20) OF test: Oxidative (21) Acid production from glucose: Negative

Bacillus subtilis SD142 was deposited on September 24, 2002 at 1-1, Higashi 1-1, Tsukuba, Ibaraki, Central No. 6 (Zip code 305-8566), by the National Institute of Advanced Industrial Science and Technology (accession number: FERM @ P- 19032), and transferred to the International Depositary on July 10, 2003 (International Accession No. FERM BP-8427).

に は Further, in the present invention, a mutant strain obtained by mutation from Bacillus subtilis SD142 can also be suitably used. Mutants can also be obtained by selecting, as spontaneous mutants, those in which the shape of colonies on the plate medium has changed, or by allowing a chemical or physical mutagen to act on Bacillus subtilis SD142, A and its homologues can be obtained by preparing a strain stock having altered yield and isolating a colony having an increased productivity from the stock.

As the chemical mutagen, for example, EMS (ethyl methanesulfonate), diethyl sulfate, NTG (N-methyl-N'-nitro-N-nitrosoguanidine) and the like can be used. As the physical mutagen, a mutagenic amount of ultraviolet ray, gamma ray, X-ray or the like can be used.

As a method for preparing a mutant strain stock, for example, Bacillus subtilis grown in a nutrient medium such as NB (Nutrient Broth; manufactured by Difco Laboratories) until the logarithmic growth phase is collected, washed, suspended in physiological saline, A method for inducing mutation by adding a mutagenic amount of NTG, collecting cells again, washing to remove NTG, and culturing again in a nutrient medium such as NB (manufactured by Difco Laboratories) to prepare a mutant strain stock. And the like.

As a method for isolating a colony having an increased productivity, for example, a medium such as TBAB (Tryptose Blood Agar Base; manufactured by Difco Laboratories) supplemented with sheep blood is appropriately diluted on a plate medium prepared by adding agar. A mutant strain that can produce iturin A and its homologs at a high concentration by applying a mutant strain stock and selecting a colony having a larger clear zone around the colony of Bacillus subtilis that has grown and larger than the others. Can be sorted out.

Next, the productivity of iturin A and its homologues of the mutant strain of Bacillus subtilis thus isolated can be confirmed by test tube culture using Bacillus subtilis SD142 as a control.

Hereinafter, the method for producing iturin A and its homologues of the present invention will be described.
The method for producing iturin A and its homologues of the present invention is most conveniently carried out, for example, by adding Bacillus subtilis SD142 to a nutrient medium such as an L medium at a temperature of 25 to 42 ° C, preferably 28 to 38 ° C. After culturing for about 24 hours, the obtained culture solution is inoculated into a medium containing soybean powder or an extract thereof as a nitrogen source at 0.1 to 10% by mass, preferably 0.5 to 7% by mass, more preferably 1 to 5% by mass. . This can be achieved by culturing this at a temperature of 25 to 42 ° C, preferably 28 to 35 ° C for about 30 to 150 hours. If the temperature is out of the above range, production of iturin A and its homologues is remarkably reduced, which is not preferable.

In the present invention, soybean powder or an extract thereof is a coarse soybean powder obtained by crushing soybean or defatted soybean into granules, a crushed soybean powder obtained by crushing into powder, an extract thereof (for example, hot water extract), It refers to degradation products (for example, acid hydrolysis products, enzyme hydrolysis products) and the like. The concentration of soy flour or its extract is desirably 2% by mass or more. However, on the other hand, if the concentration of soybean flour or its extract becomes high, sterilization may be insufficient. Therefore, it is preferable that the concentration does not exceed 20% by mass. Therefore, the concentration of soybean flour or an extract thereof for obtaining a high production amount is 2 to 20% by mass, preferably 3 to 17% by mass, more preferably 4 to 14% by mass.

培 地 The medium used in the present invention may contain, in addition to soybean flour or its extract, commonly used dissimilar carbon sources, nitrogen sources, inorganic salts, and the like. If necessary, amino acids and / or vitamins can be added.

Catabolizable carbon sources include glucose, maltose, sucrose, fructose, soluble starch, starch syrup, dextrin, molasses, potato extract, malt, peat, vegetable oil, corn steep liquor, fructose, syrup, sugar, liquid Liquid sugars, invert sugars, alcohols, organic acids, organic acid salts, alkanes or other common carbon sources can be used and can be used alone or in combination. Of these, maltose, soluble starch, starch syrup, and dextrin are preferred. These can be used usually at a concentration of about 0.01 to 50% by mass, preferably about 1 to 40% by mass.

Examples of the catabolic nitrogen source include ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium carbonate and ammonium bicarbonate, ammonia, sodium nitrate, potassium nitrate, sodium glutamate, urea, peptone, meat extract, and corn. Those containing inorganic or organic nitrogen such as steep liquor, casein hydrolyzate, feather meal, yeast extract and the like can be used, and these can be used alone or in combination. These are usually used at a concentration of about 0.01 to 30% by mass, preferably about 0.1 to 10% by mass.

Further, as an inorganic component, potassium ion, sodium ion, magnesium ion, phosphate ion, iron ion, manganese ion, calcium ion, zinc ion, cobalt ion, nickel ion, copper ion, molybdenum ion, sulfate ion, chloride ion or Cations or anions such as nitrate ions can be added. The concentration of addition varies depending on the culture conditions, but is usually about 10 ppm to 2% by mass as a magnesium salt, and about 0.1 ppm to 1000 ppm for salts other than phosphate. Phosphate ions can be added as a phosphate, but if the addition concentration is higher than 3% by mass in terms of K ₂ HPO ₄ , the accumulated concentration of iturin A and its homologues decreases, so that it is 3% by mass or less. It is preferable that the medium be a medium to which phosphate ions are not added.

The amino acids to be added include L-glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-serine, L-threonine, L-phenylalanine, L-tyrosine, L-cysteine, cystine, L-methionine, L-tryptophan, L-histidine, L-proline, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, L-arginine, L-lysine, D-valine, D-isoleucine and the like And one or more of these can be added. The addition concentration is about 0.001 to 5% by mass, preferably about 0.01 to 1% by mass.

As the vitamin, one or more kinds of biotin, thiamine, riboflavin, pyridoxine, nicotinic acid, nicotinamide, pantothenic acid, pyridoxal, pyridoxine, myo-inositol, choline, folic acid, cobalamin, cyanocobalamin and the like can be added. The concentration of the additive is 0.1 to 100 ppm, preferably 1 to 50 ppm.

培養 In the culturing in the present invention, the above-described medium is added to a container such as a test tube, a flask, or a fermenter, and the culturing is performed with strong aeration.

(4) In the case of culture using a test tube, flask, or other container, aeration is performed by shaking vigorously to adjust the initial pH of the medium to 6.5 to 8.0. When performing high-concentration production in a container such as a fermenter, cultivation is performed with aeration of sterile air and agitation. If culturing is difficult due to foaming, a commonly used general antifoaming agent is added. be able to.

The pH of the medium is maintained at 6.0 to 9.0, preferably 6.5 to 8.0, more preferably 6.8 to 7.3. The pH is adjusted by adding a basic aqueous solution such as an aqueous ammonia solution, an aqueous potassium hydroxide solution, an aqueous sodium hydroxide solution, an aqueous sodium carbonate solution, or an aqueous potassium carbonate solution. Among them, it is preferable to use an aqueous ammonia solution. The concentration of the aqueous ammonia solution is preferably about 8 to 25% by mass. By carrying out such culture under suitable conditions, iturin A and its homolog-containing culture containing iturin A and its homolog at a concentration of 1.5 g / L or more can be obtained in 30 to 150 hours.

The above culture can be dried by a known method such as freeze-drying or spray-drying to obtain iturin A and a solid containing a homologue thereof. The culture containing iturin A and its homologues, and the solid containing iturin A and its homologues can be used to control plant diseases by being applied to the soil of agricultural lands or sprayed on the leaves of agricultural crops. It is useful because it shows an effect.
These iturin A and its homolog-containing cultures and iturin A and its homolog-containing solids are also included in the present invention.
In addition, iturin A and its homologs can be recovered and purified from the culture containing iturin A and its homologs. Purification is performed by, for example, making the culture acidic by adding sulfuric acid, hydrochloric acid, nitric acid, or the like, and extracting the precipitated iturin A and its homologs with an organic solvent such as methanol, ethanol, chloroform, or the like, activated carbon treatment, crystallization, and other known methods. Can be done with

Iturin A and its homologues obtained according to the present invention can be used as, for example, detergents, emulsifiers, wetting agents, dispersants, solubilizers, antistatic agents, antifoggants, lubricants, in addition to plant disease control agents. It is useful as a compounding agent in the field of cosmetics, foods, pharmaceuticals, agricultural chemicals and the like.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 of obtaining a mutant strain of Bacillus subtilis SD142
Bacillus subtilis SD142 was inoculated into 5 mL of L medium (10 g of peptone, 5 g of yeast extract, 5 g of sodium chloride, and adjusted to 1 L with water), and cultured at 35 ° C. and 300 rpm for 16 hours. Subsequently, the obtained culture solution was inoculated into 5 mL of the same medium at 1 v / v%, and cultured at 35 ° C. and 300 rpm until the OD660 reached 0.2. Thereafter, the cells were collected by centrifugation, the supernatant was discarded, and PBS buffer (0.8 w / v% NaCl, 0.02 w / v% KCl, 0.144 w / v% Na ₂ HPO ₄ , 0.024 w / v% KH) was used. _The mixture was washed three times with 5 mL each of which was adjusted to pH 7.4 with ₂ PO ₄ and HCl, and resuspended in 0.5 mL of the same buffer.

0.05 mL of a '2000 ppm N-methyl-N'-nitro-N-nitrosoguanidine aqueous solution was added to the suspension, and the mixture was allowed to stand at 30 ° C for 10 minutes. The suspension was centrifuged, the supernatant was discarded, and the cells were washed three times with 5 mL of the same buffer and resuspended in 1 mL of fresh L medium. The suspension was added to 4 mL of L medium and grown overnight at 35 ° C., and then 2.5 mL of a 50% by mass glycerin aqueous solution was added, dispensed little by little into a cryopreservation vial, and frozen at −135 ° C. The transformant stock was used.

Subsequently, an agar plate medium containing 5 w / v% sheep blood, 4 w / v% glucose, 0.1 w / v% NB (manufactured by Difco Laboratories), and 0.1 w / v% yeast extract (Applied Environmental Microbiology, 42: 408-412) (1981)) A 50,000 plate was coated with a stock of transformants stock diluted with sterile water to give approximately 200 colonies / plate. After incubation at 35 ° C. for 20 to 48 hours, the clear zone formed around the grown colony was observed, and 50,000 colonies forming a large clear zone were selected.

Bacillus subtilis SD142 and the resulting 50,000 colonies were streaked on an L plate medium and grown overnight at 35 ° C. 1 mL of the medium having the following composition A was dispensed into a test tube, one platinum loop was taken from the L plate medium, inoculated, and cultured at 35 ° C. for 72 hours.

[Composition A] (unit is% by mass)
Soy flour 8
K ₂ HPO ₄ 0.5
MgSO ₄ · 7H ₂ O 0.05
FeSO ₄ · 7H ₂ O 0.0025
MnSO ₄ · 5H ₂ O 0.0022
CaCl ₂ 0.0184
Maltose 6.7
Ion exchange water balance

The culture was centrifuged, and iturin A and its homologues contained in the supernatant were quantified by HPLC under the following conditions.
Sample volume: 10 μL,
Column: Shodex silica C18P4E, 4.6 mm x 250 mm (manufactured by Showa Denko KK),
Column temperature: 40 ° C,
Eluent: acetonitrile: 10 mM aqueous ammonium acetate solution = 35: 75 (v / v),
Flow rate: 1.5 mL / min,
Detector: UV detector,
Wavelength: 205 nm.
The quantification was measured by preparing a calibration curve using a standard sample of iturin A and its homologue (manufactured by Sigma-Aldrich).

(Iv) Iturin A and its homologue-producing mutants (mutant 1) in which the accumulated concentration of iturin A and its homologue were higher than that of Bacillus subtilis SD142 were obtained.

Example 2 of obtaining a mutant strain of Bacillus subtilis SD142:
The surfactin concentration in the culture supernatant of Acquisition Example 1 was quantified by the HPLC method under the following conditions.
Sample volume: 20 μL,
Column: Shodex
Silica C18P4E, 4.6 mm x 250 mm (manufactured by Showa Denko KK)
Column temperature: 40 ° C,
Eluent: acetonitrile: 19 mM trifluoroacetic acid aqueous solution = 80: 20 (v / v)
Flow rate: 1.0 mL / min,
Detector: UV detector,
Wavelength: 205 nm.
The quantification was performed by preparing a calibration curve using a standard sample of surfactin (manufactured by Sigma-Aldrich).

(4) A mutant (non-surfactin-producing mutant) which was not detected under the above conditions and had a surfactin accumulation concentration of 50 ppm or less was obtained.

Example 1 Effect of Nitrogen Source on Production of Iturin A and Its Homologs in Test Tube Culture Bacillus subtilis SD142 was streaked on an L plate medium and grown at 35 ° C. overnight. 1 mL of the medium having the following composition B was dispensed into a test tube, one platinum loop was taken from the L plate medium, inoculated, and cultured at 35 ° C. for 72 hours.

[Composition B] (unit is% by mass)
K ₂ HPO ₄ 0.5
MgSO ₄ · 7H ₂ O 0.05
FeSO ₄ · 7H ₂ O 0.0025
MnSO ₄ · 5H ₂ O 0.0022
CaCl ₂ 0.0184
Maltose 6.7
Nitrogen source * 2.0
Ion-exchanged water balance *: One selected from soy flour, potassium nitrate, ammonium nitrate, ammonium sulfate, sodium urea glutamate and peptone.

The culture was centrifuged, and iturin A and its homologues contained in the supernatant were quantified by HPLC.

The accumulated concentrations of iturin A and its homologues for each nitrogen source were as follows:
Soy flour 1.5g / L
Potassium nitrate 0.03g / L
Ammonium nitrate 0.03g / L
Ammonium sulfate 0.03g / L
Urea 0.03g / L
Sodium glutamate 0.1g / L
Peptone 0.15g / L

Example 2: Effect of soy flour concentration on production of iturin A and its homologues in test tube culture Bacillus subtilis SD142 was streaked on L plate medium and grown overnight at 35 ° C. 1 mL of each of the media of the following compositions C, D, and E was dispensed into a test tube, one platinum loop was taken from the L plate medium, inoculated, and cultured at 35 ° C for 72 hours.

[Composition C] (unit is% by mass)
Soy flour 1
K ₂ HPO ₄ 0.5
MgSO ₄ · 7H ₂ O 0.05
FeSO ₄ · 7H ₂ O 0.0025
MnSO ₄ · 5H ₂ O 0.0022
CaCl ₂ 0.0184
Maltose 6.7
Ion exchange water balance

[Composition D] (unit is mass%)
Soy flour 2
K ₂ HPO ₄ 0.5
MgSO ₄ · 7H ₂ O 0.05
FeSO ₄ · 7H ₂ O 0.0025
MnSO ₄ · 5H ₂ O 0.0022
CaCl ₂ 0.0184
Maltose 6.7
Ion exchange water balance

[Composition E] (unit is mass%)
Soy flour 8
K ₂ HPO ₄ 0.5
MgSO ₄ · 7H ₂ O 0.05
FeSO ₄ · 7H ₂ O 0.0025
MnSO ₄ · 5H ₂ O 0.0022
CaCl ₂ 0.0184
Maltose 6.7
Ion exchange water balance

The culture was centrifuged, and iturin A and its homologues contained in the supernatant were quantified by HPLC. The amounts of iturin A and its homologues in each medium were as follows.
Composition C: 0.3 g / L
Composition D: 1.5 g / L
Composition E: 3.8 g / L

Example 3: Effect of carbon source on production of iturin A and its homologues in test tube culture Bacillus subtilis SD142 was streaked on L plate medium and grown at 35 ° C overnight. Each 1 mL of a medium obtained by adding the following carbon source to the following composition F was dispensed into a test tube, one platinum loop was taken from the L plate medium, inoculated, and cultured at 35 ° C. for 72 hours.

[Composition F] (unit is% by mass)
Soy flour 8
K ₂ HPO ₄ 0.5
MgSO ₄ · 7H ₂ O 0.05
FeSO ₄ · 7H ₂ O 0.0025
MnSO ₄ · 5H ₂ O 0.0022
CaCl ₂ 0.0184
Carbon source * 6.7
Deionized water Remainder *: One selected from maltose, soluble starch, starch syrup, dextrin, glucose, sucrose and fructose.

The culture was centrifuged, and iturin A and its homologues contained in the supernatant were quantified by HPLC. The accumulated concentrations of iturin A and its homologues for each carbon source were as follows.
Maltose 3.8g / L
Soluble starch 3.8g / L
3.8 g / L
Dextrin 3.8g / L
Glucose 2.8g / L
2.2 g / L sucrose
Fructose 2.3g / L

Example 4: Effect of phosphate on the production of iturin A and its homologues in test tube culture Bacillus subtilis SD142 was streaked on L-plate media and grown at 35 ° C overnight. 1 mL of each of the six types of media obtained by adding K ₂ HPO ₄ having the following concentrations (1) to (6) to the following composition G was dispensed into a test tube, and one platinum loop was taken from the L plate medium and inoculated. Cultivation was performed at 72 ° C for 72 hours.

[Composition G] (unit is mass%)
Soy flour 8
MgSO ₄ · 7H ₂ O 0.05
FeSO ₄ · 7H ₂ O 0.0025
MnSO ₄ · 5H ₂ O 0.0022
CaCl ₂ 0.0184
Maltose 6.7
K ₂ HPO ₄
(1) 0% by mass (not added)
(2) 0.1 mass%
(3) 0.5% by mass
(4) 1.5% by mass
(5) 3.0% by mass
(6) 4.5% by mass
Ion exchange water balance

After adjusting the pH to 7 with sodium carbonate, the culture was centrifuged, and iturin A and its homologues contained in the supernatant were quantified by HPLC. The cumulative concentrations of iturin A and its homologues for each K ₂ HPO ₄ concentration were as follows:

(1) 0 mass% 3.8g / L
(2) 0.1 mass% 3.6g / L
(3) 0.5 mass% 3.5g / L
(4) 1.5 mass% 3.0g / L
(5) 3.0% by mass 2.2g / L
(6) 4.5 mass% 1.5g / L

Example 5: Production of iturin A and its homologues in fermenters Bacillus subtilis SD142, mutant 1 and mutant 2 were streaked on L plate medium and grown overnight at 35 ° C. From there, one platinum loop was inoculated into a baffled flask containing 50 mL of L medium, and cultured at 35 ° C. and 150 rpm for 8 hours. A medium having the following composition H was prepared in a 5 L fermenter, a culture solution of the L medium was added, and culture was performed at 35 ° C. for 150 hours while adjusting the pH to 6.5 to 7.5 with 20% aqueous ammonia.

[Composition H]
160g soy flour
MgSO ₄ · 7H ₂ O 5g
FeSO ₄ · 7H ₂ O 0.25g
MnSO ₄ · 5H ₂ O 0.22g
1.84 g of CaCl ₂
450g syrup
Ion exchange water 1383g

Iturin A and its homologues contained in the supernatant obtained by centrifuging the culture solution were quantified by HPLC. Aitulin A and its homologue amounts were as follows.
Bacillus subtilis SD142 3.8g / L
Mutant 1 6.7 g / L
Mutant 2 6.7 g / L
From these results, it is clear that all of these strains can grow in the presence of 1.5 g / L or more of iturin.

According to the present invention, iturin A and its homologues useful in a wide variety of industrial fields such as pharmaceuticals, agrochemicals, foods, cosmetics, and chemicals are dramatically compared with conventional methods using inexpensive medium materials. Can be produced in high concentrations.

In the field of pesticides and plant disease control, iturin A and its homologues can be produced in high concentrations in the culture solution. It is also possible to do.

Claims (12)

A Bacillus genus microorganism capable of producing iturin A and its homologues is cultured in a liquid medium containing 2% by mass or more of soybean flour or an extract thereof, and iturin A and 1.5 g / L or more are contained in the culture solution. Iturin A and a method for producing the homologue, wherein the homologue is accumulated. Iturin A and B. (Bacillus) genus microorganisms having its homologues producing ability, claim 1 is viable Bacillus (Bacillus) microorganisms in a medium containing 1.5 g / L or more iturin A and its homologues The method for producing iturin A and its homologues according to the above. Iturin A and B. (Bacillus) genus microorganisms having its homologues producing ability, iturin A and claims a Bacillus (Bacillus) microorganism having substantially no surfactin producing ability has its homologues producing ability 3. The method for producing iturin A and its homologues according to 1 or 2. Liquid medium, iturin A and preparation of its homologues as claimed in claim 1, which is a liquid medium that the amount of phosphate was 0-3 wt% or less K ₂ HPO ₄ terms. 4. The method for producing iturin A and its homologues according to claim 1, wherein the microorganism belonging to the genus Bacillus is Bacillus subtilis . The method for producing iturin A and homologues thereof according to any one of claims 1 to 3, wherein the microorganism belonging to the genus Bacillus is Bacillus subtilis SD142 (FERM BP-8427). The method for producing iturin A and homologues thereof according to any one of claims 1 to 3, wherein the microorganism belonging to the genus Bacillus is a mutant strain of Bacillus subtilis SD142 (FERM BP-8427). The iturin according to claim 1, wherein the liquid medium containing 2% by mass or more of soy flour or an extract thereof contains at least one selected from the group consisting of maltose, starch syrup, soluble starch, dextrin, glucose, sucrose, and fructose. Method for producing A and its homologues. {Circle around (5)} Iturin A and its homolog-containing culture, wherein iturin A and its homologs are accumulated in the culture solution by the method according to any one of claims 1 to 8. [11] An iturin A and its homolog-containing solid obtained by drying the culture containing iturin A and its homolog according to claim 9. 植物 A plant disease control agent comprising the culture or solid containing iturin A and its homologues according to claim 9 or 10. A method for controlling plant diseases, which comprises using the culture or solid containing iturin A and its homologues according to claim 9 or 10 without purification.