JP4338080B2 - Method for producing iturin A and its homologs - Google Patents

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 high concentrations of iturin A and its homologues in the culture medium. . The present invention also relates to the produced iturin A and its homolog-containing culture, iturin A dried from it and its homolog-containing solid, and methods for 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 shown in the following formula 1, and have attracted attention as excellent plant disease control components because they have antibacterial activity against fungi, particularly plant pathogens. It 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.)
As methods for producing iturin A and its homologues, there are JP-A-59-212416 (Patent Document 1), JP-A-7-143897 (Patent Document 2) and the like.

However, the productivity of iturin A and its homologs produced by microorganisms belonging to the genus Bacillus is low, and sufficient productivity can be obtained for industrial use of iturin A and its homologs or cultures containing them. As a result, many researchers have made efforts to improve iturin A and its homologous productivity.
Hatada et al., Bacillus subtilis no. A method for producing an iturin A-based substance by aerobically cultivating NA-apb-1 strain in a nutrient medium (see Japanese Patent Publication No. 63-20519 (Patent Document 3)). Peptone, meat extract, yeast extract, casein hydrolyzate, cornstar chili car, gluten meal, inorganic nitrogen source is taught as a culture obtained by culturing the same strain in a medium containing peptone, meat extract, yeast extract 270 mg of iturin A is obtained from 30 L of filtrate, 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").・ Biotechnology and Applied Biochemistry ”, 1990, Vol. 12, p.370-375 (Non-Patent Document 2)).

  Hbid et al. Disclose a method in which iturin A and its homologue yield of 1388 mg / L is achieved in media containing peptone ("Applied Biochemistry and Biotechnology", 1996, 57th). / 58, p.571-579 (Non-Patent Document 3).) Phae et al. Disclose a method whereby iturin A and its homologue yield of 620 mg / L is achieved in media containing peptone ("Journal of Fermentation and Bioengineering"). ", 1991, Vol. 71, p. 118-121 (Non-Patent Document 4).).

On the other hand, regarding the 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 Laid-Open No. 2002-176993). (Patent Document 4)). However, this patent 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% soy flour, and 300 mg of iturin A and its homologue can be obtained from 16 L of the culture solution. However, the obtained amount of iturin A was almost the same as that obtained by a known culture method.

Iturin A at a concentration of 1.5 g / L or more in the culture solution by culturing iturin A and its homologue-producing microorganisms belonging to the genus Bacillus in a liquid medium containing 2% by mass or more of soybean flour or its extract And its congeners have not been known to accumulate.

Besson et al., “Journal of Antibiotics”, 1978, Vol. 31, p. 284-288 JP 59-212416 A Japanese Patent Laid-Open No. 7-143897 Japanese Patent Publication No. 63-20519 “Biotechnology and Applied Biochemistry”, 1990, Vol. 12, p. 370-375 “Applied Biochemistry and Biotechnology”, 1996, 57/58, p. 571-579 “Journal of Fermentation and Bioengineering”, 1991, Vol. 71, p. 118-121 JP 2002-176993 A

Conventional iturin A and its homologue productivity are not sufficient for industrial use, and a production method that further improves productivity has been desired.
Accordingly, an object of the present invention is to provide a method for producing iturin A and its homologs by culturing Bacillus microorganisms that produce iturin A and its homologs, and accumulating iturin A and its homologs in a high concentration in the culture medium. It is to provide.
Another object of the present invention is to provide iturin A and its homologs, containing cultures, solids thereof and methods for using them.

  As a result of intensive studies on various medium components to solve the above-mentioned problems, the inventors have found that iturin A and its homologues are contained in a medium containing 2% by mass or more of a soybean ground material or an extract thereof as a nitrogen source. By culturing the microorganisms of the genus Bacillus to be produced, it has been found that iturin A and its homologues are accumulated in a high concentration in the culture solution, and the present invention has been completed.

  That is, the present invention relates to the following method for producing iturin A and its homologs, iturin A and its homolog-containing cultures, iturin A and its homolog-containing solids, and the like.

[1] A microorganism of the genus Bacillus having the ability to produce 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 the concentration in the culture solution is 1.5 g / L or more. A method for producing iturin A and its homologs, characterized by accumulating iturin A and its homologs.
[2] A microorganism belonging to the genus Bacillus having the ability to produce iturin A and its homologue is a microorganism belonging to the genus Bacillus that can grow in a medium containing 1.5 g / L or more of iturin A and its homologue. 2. A process for producing iturin A and its homologues according to 1 above.
[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 or a homologue thereof according to 1 or 2 above.
[4] The method for producing iturin A and the homologue thereof according to 1 above, wherein the liquid medium is a liquid medium in which the amount of phosphate added is 0 to 3% by mass in terms of K ₂ HPO ₄ .
[5] The method for producing iturin A and the homologue thereof according to any one of 1 to 3, wherein the microorganism belonging to the genus Bacillus is Bacillus subtilis .
[6] The method for producing iturin A and the homologue thereof according to any one of 1 to 3 above, wherein the Bacillus microorganism is Bacillus subtilis (FERM BP-8427).
[7] The method for producing iturin A and the homologue thereof according to any one of 1 to 3 above, wherein the Bacillus microorganism is a mutant of Bacillus subtilis (FERM BP-8427).
[8] The liquid medium containing 2% by mass or more of soybean 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. Of Iturin A and its homologues.
[9] Iturin A and its homolog-containing culture in which iturin A and its homolog are accumulated in the culture medium by the method according to any one of 1 to 8 above.
[10] Iturin A and its homolog-containing solid material obtained by drying the iturin A and its homolog-containing culture as described in 9 above.
[11] A plant disease control agent comprising the iturin A and its homolog-containing culture or solid as described in 9 or 10 above.
[12] A method for controlling plant diseases, comprising using the iturin A and its homolog-containing culture or solid product according to 9 or 10 without purification.

Detailed Description of the Invention

  Hereinafter, the present invention will be described in detail.

The iturin A and its homologues of the present invention mean a derivative represented by the following formula (1).

（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, by adding 2% by mass or more of soybean pulverized material or an extract thereof as a nitrogen source to a culture medium for cultivating microorganisms that produce iturin A and its homologues, the culture is performed. Iturin A and its homologues can be accumulated at high concentrations. According to the knowledge of the inventors, conventionally, it has been known to culture Bacillus microorganisms in a medium containing soybean ground or an extract thereof as a nitrogen source, but produces iturin A and its homologues. The inventor has newly found that microorganisms are cultured in a medium containing 2% by mass or more of soybean ground material or an extract thereof as a nitrogen source, and iturin A and its homologues are accumulated at a high concentration in the culture solution. Is.

The microorganism belonging to the genus Bacillus used in the present invention is not particularly limited as long as it produces iturin A and its homologues. However, since it accumulates iturin A and its homologues in a medium at a high concentration, Bacillus microorganisms that have the ability to produce iturin A and its homologues and can grow in a medium containing 1.5 g / L of iturin A and its homologues Is preferred. Further, as another preferred example, a microorganism belonging to the genus Bacillus which has the ability to produce iturin A and its homologues and which does not substantially have the ability to produce surfactin is preferable. The phrase “substantially having no surfactin-producing ability” as used herein means that the accumulated amount of surfactin is 50 ppm or less when cultured in a medium containing a pulverized soybean or extract thereof as a nitrogen source. Examples of microorganisms belonging to the genus Bacillus that have the ability to produce iturin A and its homologs and can grow in a medium containing 1.5 g / L of iturin A and its homologs include, for example, Bacillus subtilis (FERM BP-8427). Is preferred.

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

  Bacteriological properties (a) Morphology (1) Bacterial shape: Sponge (2) Bacterial size: 0.7-0.9 x 1.5-3.0 μm (3) Polymorphism: None (4) Motility: Yes (5) Presence or absence of spore: Shape of spore: Oval or cylindrical (6) Gram stainability: Positive (7) Antiacidity: Negative (b) Growth status Meat agar plate culture: Colony is circular and size is 1-2 mm in diameter The peripheral shape is wavy, viscous, and glossless. (C) Physiological properties (1) Nitrate reduction: Positive (2) VP test: Positive (3) Indole production: 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) Range of growth: pH 5-9 Temperature 20-50 ° C (12) 10% NaCl medium: Growth (13) Anaerobic culture: Negative (14) Egg yolk reaction: Negative (15) Starch hydrolysis: Positive (16) Arginine degradation: Positive (17) Tyrosine degradation: Negative (18) Gelatin Liquefaction: Positive (19) Degradation of esculin: Positive (20) OF test: Oxidative (21) Acid production from glucose: Negative

The Bacillus subtilis was deposited at the National Institute of Advanced Industrial Science and Technology (ATR), on September 24, 2002, 1st East, 1st Street, Tsukuba, Ibaraki, Japan. 19032), and transferred to an international deposit on July 10, 2003 (international deposit number FERM BP-8427).

In the present invention, a mutant obtained by mutation from Bacillus subtilis (FERM BP-8427) can also be preferably used. Mutants can be obtained as natural mutants by selecting those in which the shape of the colonies on the plate medium has changed, and chemical or physical mutagens in Bacillus subtilis (FERM BP-8427). To obtain a strain stock having altered yields of iturin A and its homologues, from which a colony having an increased production ability is isolated.

  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, mutagenic amounts of ultraviolet rays, gamma rays, X-rays and the like can be used.

  As a method for producing a mutant strain stock, for example, Bacillus subtilis grown to a logarithmic growth phase in a nutrient medium such as NB (Nutrient Broth; manufactured by Difco Laboratories) is collected, washed and suspended in physiological saline, A method of creating a mutant strain stock by adding a mutagenic amount of NTG to induce mutation, collecting and washing again, removing NTG, and culturing again in a nutrient medium such as NB (Difco Laboratories) Etc.

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

Subsequently, iturin A and its homologous productivity of the mutant strain of Bacillus subtilis isolated in this manner can be confirmed by test tube culture using Bacillus subtilis (FERM BP-8427) as a control. it can.

Hereafter, the manufacturing method of the iturin A of this invention and its homologue is demonstrated.
The most convenient method for producing iturin A and its homologues of the present invention is, for example, Bacillus subtilis (FERM BP-8427) in a nutrient medium such as L medium at 25 to 42 ° C, preferably 28 to 38 ° C. For 5 to 24 hours at a temperature of 0.1 to 10% by mass, preferably 0.5 to 7% by mass, more preferably 1 to 5% in a medium containing soybean powder or an extract thereof as a nitrogen source. Inoculate mass%. This can be achieved by culturing at a temperature of 25 to 42 ° C., preferably 28 to 35 ° C. for about 30 to 150 hours. When the temperature is out of the above temperature range, iturin A and its homologues are remarkably reduced, which is not preferable.

  In the present invention, soybean powder or an extract thereof is a coarse soybean powder obtained by pulverizing soybean or defatted soybean, a pulverized soybean powder pulverized into a powder, an extract thereof (for example, hot water extract), water It refers to a decomposed product (for example, an acid hydrolyzate or an enzyme hydrolyzate). The concentration of soybean powder or an extract thereof is preferably 2% by mass or more. However, on the other hand, since the sterilization may be insufficient when the soybean powder or the extract thereof has a high concentration, it is desirable that the concentration does not exceed 20% by mass. Therefore, the concentration of soybean flour or its extract 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.

  The medium used in the present invention may contain, in addition to soy flour or an extract thereof, commonly used catabolizable carbon sources, nitrogen sources, inorganic salts, and the like. If necessary, amino acids and / or vitamins can be added.

  Catabolic 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 are available 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 nitrogen sources that can be catalyzed include ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium carbonate or ammonium bicarbonate, ammonia, sodium nitrate, potassium nitrate, sodium glutamate, urea, peptone, meat extract, corn Those containing inorganic or organic nitrogen such as steep liquor, casein hydrolyzate, feather meal and yeast extract can be used, and these can be used alone or in combination. These are usually used in 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. Although the addition concentration varies depending on the culture conditions, it is usually about 10 ppm to 2% by mass as a magnesium salt, and about 0.1 ppm to 1000 ppm for other salts other than phosphate. Phosphate ions can be added as phosphates, 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 is reduced, so that it is 3% by mass or less. It is preferable that the culture medium does not contain phosphate ions.

  Examples of 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, etc. 1 or 2 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 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 addition concentration is 0.1 to 100 ppm, preferably 1 to 50 ppm.

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

  In the case of culturing using containers such as test tubes and flasks, aeration is performed by shaking vigorously, and the initial pH of the medium is adjusted to 6.5 to 8.0. When producing a high concentration in a vessel such as a fermenter, sterilize air and incubate with stirring. If foaming is difficult and culture is difficult, add a commonly used antifoaming agent. 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, an aqueous ammonia solution is preferably used. The concentration of the aqueous ammonia solution is preferably about 8 to 25% by mass. By performing 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.

Iturin A and its homolog-containing solids can be obtained by drying the culture by known methods such as freeze drying and spray drying. These iturin A and its homolog-containing cultures, and iturin A and its homolog-containing solids are applied to the soil of farmland or sprayed on the leaves of crops to control plant diseases. This 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.
Iturin A and its homologues can be recovered and purified from iturin A and its homolog-containing cultures. For purification, for example, the culture is acidified by adding sulfuric acid, hydrochloric acid, nitric acid, etc., and the precipitated iturin A and its homologues are extracted with an organic solvent such as methanol, ethanol, chloroform, etc., activated carbon treatment, crystallization, and the like. Can be done.

  In addition to plant disease control agents, iturin A and its homologs obtained by the present invention can be used as, for example, cleaning agents, emulsifiers, wetting agents, dispersing agents, solubilizers, antistatic agents, antifogging agents, and lubricants. It is useful as a compounding agent in the field of cosmetics, foods, pharmaceuticals, agricultural chemicals and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited at all by these Examples.

Example of obtaining mutant strain of Bacillus subtilis (FERM BP-8427) 1:
Bacillus subtilis (FERM BP-8427) was inoculated into 5 mL of L medium (10 g of peptone, 5 g of yeast extract, 5 g of sodium chloride, and 1 L with water), and cultured at 35 ° C. and 300 rpm for 16 hours. Next, 1 v / v% of the obtained culture solution was inoculated into 5 mL of the same medium, and cultured at 35 ° C. and 300 rpm until OD660 reached 0.2. Thereafter, the cells are collected by centrifugation, the supernatant is 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) is collected. ₂ Adjusted to pH 7.4 with PO ₄ , HCl) Washed 3 times with 5 mL and resuspended in 0.5 mL of the same buffer.

  0.05 mL of an aqueous 2000 ppm N-methyl-N′-nitro-N-nitrosoguanidine solution was added to the suspension and allowed to stand at 30 ° C. for 10 minutes. The suspension was centrifuged, the supernatant was discarded, the cells were washed 3 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 allowed to grow overnight at 35 ° C., then 2.5 mL of 50% by mass glycerin aqueous solution was added, dispensed in small portions into cryopreservation vials, frozen at −135 ° C., A transformant stock was used.

  Then, agar plate medium (Applied Environmental Microbiology, 42: 408-412) containing 5 w / v% sheep blood, 4 w / v% glucose, 0.1 w / v% NB (Difco Laboratories), 0.1 w / v% yeast extract. (1981)) A stock of transformant stock diluted with sterile water to about 200 colonies / plate was applied to 50000 plates. After incubating at 35 ° C. for 20 to 48 hours, the clear zone formed around the grown colonies was observed, and 50000 colonies forming a large clear zone were selected.

Bacillus subtilis (FERM BP-8427) and the resulting 50000 colonies were streaked on L plate medium and grown overnight at 35 ° C. 1 mL of a medium having the following composition A was dispensed into a test tube, 1 platinum loop was inoculated from the L plate medium, and cultured at 35 ° C. for 72 hours.

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

Iturin A and its homologue high production mutant strain (mutant strain 1) were obtained in which the accumulated concentration of iturin A and its homologue was improved from Bacillus subtilis (FERM BP-8427) .

Example 2 of obtaining mutant strain of Bacillus subtilis (FERM BP-8427) :
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, 4.6mm x 250mm (made by Showa Denko KK)
Column temperature: 40 ° C
Eluent: Acetonitrile: 19 mM aqueous trifluoroacetic acid = 80: 20 (v / v)
Flow rate: 1.0 mL / min,
Detector: UV detector,
Wavelength: 205 nm.
Quantification was performed by preparing a calibration curve using a standard sample of surfactin (manufactured by Sigma-Aldrich).

  A substantially non-surfactin-producing mutant strain (mutant strain 2) having a surfactin accumulation concentration of 50 ppm or less that was not detected under the above conditions was obtained.

Example 1 Effect of Nitrogen Source on Production of Iturin A and its Congeners in Tube Culture Bacillus subtilis (FERM BP-8427) was streaked on L plate medium and grown overnight at 35 ° C. 1 mL of a medium having the following composition B was dispensed into a test tube, 1 platinum loop was inoculated from the L plate medium, and cultured at 35 ° C. for 72 hours.

[Composition B] (unit: 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 remainder *: Select one from soy flour, potassium nitrate, ammonium nitrate, ammonium sulfate, urea sodium glutamate and peptone.

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

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 (FERM BP-8427) was streaked on L plate medium and grown overnight at 35 ° C. 1 mL of each medium of the following composition C, D and E was dispensed into a test tube, 1 platinum loop was inoculated from the L plate medium, and cultured at 35 ° C. for 72 hours.

[Composition C] (unit: 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: 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: 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 solution was centrifuged, and iturin A and its homologue contained in the supernatant were quantified by HPLC method. The amounts of iturin A and its homologue for 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 (FERM BP-8427) was streaked on L plate medium and grown overnight at 35 ° C. 1 mL of each medium in which the following carbon source was added to the following composition F was dispensed to a test tube, 1 platinum loop was inoculated from the L plate medium, and cultured at 35 ° C. for 72 hours.

[Composition F] (unit: 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
Ion-exchanged water balance *: Select one from maltose, soluble starch, starch syrup, dextrin, glucose, sucrose and fructose.

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

Example 4: Effect of phosphate on production of iturin A and its homologues in test tube culture Bacillus subtilis (FERM BP-8427) was streaked on L plate medium and grown overnight at 35 ° C. Dispense 1 mL of each of the six types of medium in which K ₂ HPO ₄ having the following concentrations (1) to (6) is added to the following composition G into test tubes, inoculate one platinum loop from the L plate medium, inoculate 35 Culturing was performed at 72 ° C. for 72 hours.

[Composition G] (unit: 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 (no addition)
(2) 0.1% by 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 to pH 7 with sodium carbonate, the culture solution was centrifuged, and iturin A and its homologue contained in the supernatant were quantified by HPLC. The accumulated concentrations of iturin A and its homologues for each K ₂ HPO ₄ concentration were as follows:

(1) 0% by mass 3.8g / L
(2) 0.1% by mass 3.6 g / L
(3) 0.5% by mass 3.5 g / L
(4) 1.5% by mass 3.0 g / 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 a fermentor Bacillus subtilis (FERM BP-8427) , Mutant 1 and Mutant 2 were streaked on an L plate medium and grown overnight at 35 ° C. From there, 1 platinum ear was inoculated into a baffled flask to which 50 mL of L medium was added, and cultured at 35 ° C. and 150 rpm for 8 hours. A medium having the following composition H was prepared in a 5 L fermentor, a culture medium of L medium was added, and the mixture was cultured 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
CaCl ₂ 1.84g
Minamata 450g
Ion exchange water 1383g

Iturin A and its homologue contained in the supernatant obtained by centrifuging the culture solution were quantified by HPLC. The amounts of iturin A and its homologues were as follows.
Bacillus subtilis (FERM BP-8427) 3.8g / L
Mutant 1 6.7g / L
Mutant 2 6.7g / L
From these results, it is clear that any 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, agricultural chemicals, foods, cosmetics, and chemicals are drastically compared with conventional methods using inexpensive medium raw materials. Can be produced at high concentrations.

In the field of pesticide and plant disease control, iturin A and its homologues can be produced at high concentrations in the culture solution, so the culture solution can be used as it is for applications where the concentration of the culture solution itself has been insufficient. It is also possible to do.

Claims (7)

Bacillus microorganisms that have the ability to produce iturin A and its homologues and have a surfactin accumulation of 50 ppm or less when cultured in a medium containing soybean ground or an extract thereof as a nitrogen source. Or a method for producing iturin A and its homologues, comprising culturing in a liquid medium containing 2% by mass or more of the extract and accumulating iturin A and its homologues in a culture solution at a concentration of 1.5 g / L or more , The production method, wherein the microorganism is Bacillus subtilis (FERM BP-8427) or a mutant thereof . Liquid medium, iturin A and preparation of its homologues as claimed in claim 1, which is a liquid medium in which the amount of phosphate in 0-3% by weight K ₂ HPO ₄ terms.   The iturin according to claim 1, wherein the liquid medium containing 2% by mass or more of soybean 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. A method for producing A and its homologues. Iturin A and its homolog-containing culture in which iturin A and its homolog are accumulated in the culture solution by the method according to any one of claims 1 to 3 . The iturin A and its homolog containing solid substance which dried the iturin A and its homolog containing culture of Claim 4 . A plant disease control agent comprising the iturin A according to claim 4 or 5 and its homolog-containing culture or solid matter. A method for controlling plant diseases, comprising using the iturin A and its homolog-containing culture or solid matter according to claim 4 or 5 without purification.