JP2948936B2 - Control method of mold venom produced by mold belonging to genus Fusarium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[Industrial applications] Fusarium Graminearum (Fus
Some molds belonging to the genus Fusarium, such as arium graminearum, are known as "red mold" of agricultural products such as wheat and corn.
It is known as a pathogen. In other words, molds of the genus Fusarium produce trichothecene-based mold toxins such as bomitoxin, nivalenol, and 3-acetyldeoxynivalenol, and mold toxins such as zearalenone.

Zearalenone causes miscarriage and infertility of livestock such as pigs, and trichothecene fungus venom also causes a series of poisoning symptoms called "red mold poisoning". It has been reported.

[0003] Mold toxins have been found in agricultural products such as wheat and corn such as wheat and barley, and in processed foods using these agricultural products as raw materials. It is also conceivable that the area where such crops contaminated with mold venom are harvested is contaminated with mold of Fusarium genus which produces mold venom.

[0004] In fact, there have been reports of areas contaminated with fungus of the genus Fusarium which produces mold venom in the middle and high latitudes where the granary where these crops are harvested is distributed (P
roc. Jpn. Assoc. Mycotoxicol. No. 19, 8-9, 1984).

[0005] The present invention relates to a method for controlling mold venom of agricultural products contaminated with mold venom, and to a method for controlling mold of Fusarium spp. That produces the mold venom.

[0006]

2. Description of the Related Art An effective method for controlling a fungus of the genus Fusarium which produces mold venom has not been known. Furthermore, it is said that mold venoms such as bomitoxin and nivalenol are not easily decomposed and detoxified even in the processing steps of food production (Proc. Jpn. Assoc. Mycotoxico
l., No. 19, 13-19, 1984).

[0007] Accordingly, in the United States and Canada, the standard of administrative guidance and recommendation (Proc. Jp.
n. Assoc. Mycotoxocol., No19, 4-5, 1984).

An object of the present invention is to develop a method for controlling the contamination of agricultural products such as cereals and nuts with aflatoxin, a kind of mold venom, by using microorganisms having no hygiene problems in foods. Search for soil bacteria in various places, and from the strains belonging to Bacillus subtilis that have been used for food production and processing for a long time, the growth of bacteria that reduce aflatoxin in solution and produce aflatoxin and aflatoxin A strain that has the property of inhibiting the production of
Find {Bacillus subtilis NK-330 and Bacillus subtilis NK-C-3} and apply for a patent (Japanese Patent Application No. Sho 62-15)
No. 5998 and Japanese Patent Application No. 62-155999). They have also discovered that these strains produce iturin A, and disclosed a method for controlling aflatoxin contamination using iturin A or a strain producing iturin A (Japanese Patent Application No. 1-6041).
No. 4).

[0009] However, a strain belonging to Aitulin A or Bacillus subtilis that produces Aitulin A,
Inhibits the growth of Fusarium mold that produces mold venom,
It has not been reported that the production of the mold venom of the mold is reduced, and that it is effective in controlling the contamination of the mold venom produced by the Fusarium mold.

[0010]

An effective method for controlling the contamination of mold venom produced by Fusarium fungi has not been found so far. On the other hand, a method for predicting the occurrence of Fusarium mold in a field (Can. J. Plant.Pathol.,
Vol. 4, 20-26, 1982) and a method of separating fusarium-contaminated grains and non-contaminated grains by utilizing the difference in buoyancy between water and 30% sucrose solution (J. Food Protect, Vol. .48,41
6,1985) have been reported.

However, even if the occurrence of mold of Fusarium is predicted, there is no specific method for preventing the occurrence, and development of a method for controlling the occurrence is desired. In addition, even if the method of separating the grains contaminated by Fusarium is implemented, it does not reduce the total amount of the contaminated grains, and the complicated operation for removing the contaminated grains requires time and money, and The dried grains must be dried again in order to submerge them, and the quality is degraded. This is not practical and such a method has not been performed.

An object of the present invention is to prevent and eliminate the contamination of agricultural products with mold venom produced by Fusarium mold, and thereby to prevent the contamination of mold venom produced by Fusarium mold. The purpose is also to provide.

[0013]

DISCLOSURE OF THE INVENTION The present invention is to control mold venom, which is produced in agricultural products such as cereals by fungi belonging to the genus Fusarium, by treating it with a strain belonging to Bacillus subtilis that produces iturin A.

As used herein, the strain belonging to Bacillus subtilis refers to a strain that produces iturin A, and any strain that produces iturin A can be used. As such cells, for example, Bacillus subtilis ATCC10774 strain, Bacillus subtilis NK-330 strain (Deposit No. 1580 of Microtechnical Research Institute) or Bacillus subtilis NK deposited at the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology. -C
-3 strains (Microtechnical Lab. No. 1581).

These strains belonging to Bacillus subtilis are usually cultured and then used for treating agricultural products. An ordinary liquid medium can be used for the culture, and for example, a potato dextrose medium, a nutrient medium, and the like can be used. In addition, if necessary, inorganic salts such as dipotassium hydrogen phosphate and magnesium sulfate are added for culturing. Culture of these strains is performed at 20-35 ° C for 2-7
It is desirable to carry out in aerated aerobic conditions for days.

After the cultivation, the culture is separated from the cells by centrifugation or the like. The cells are dried by freeze-drying, spray-drying, or the like, and powdered if necessary. At this time, without separating the cells, the culture containing the cells may be concentrated or dried before use.

The target to be treated using the cells obtained in this manner is an agricultural product which is contaminated by the fungus belonging to Fusarium and is liable to be toxic to the mold, or an agricultural product which may be contaminated by the fungus. Yes, corn, wheat, barley,
Grains such as rice can be mentioned. In particular, corn and wheat are important in view of the current state of pollution.

In order to treat these agricultural products with a strain belonging to Bacillus subtilis that produces iturin A, not only a method of spraying and mixing cells with the agricultural products during storage,
For example, a method of sowing seeds after coating treatment with bacterial cells, a method of spraying or mixing the strain on a field where agricultural products are cultivated, a method of spraying the strain on a cultivated plant or a field, or a method after harvesting The method can be carried out by, for example, spraying and mixing the strain on agricultural products.

The amount of cells used at this time varies depending on the treatment method. That is, when seeding is performed after the seeds are covered with the cells, a solution containing the cells at 10 ⁶ to 10 ⁷ cells / ml is prepared, and the seeds are immersed in the solution so that the cells cover the surface of the seeds. It is desirable to sow after it. When the cells are directly sprayed and mixed on the field before sowing, it is preferable to use 10 ¹¹ to 10 ¹³ cells per hectare. Also, when the bacterial strain is sprayed on a cultivated plant or field, 10 ¹¹ to 10 ¹³ cells are used per hectare. Furthermore, when the strain is sprayed and mixed with agricultural products after harvest or during storage, 5 to 500,
000 cells, preferably 50 to 5,000 cells are used.

When seeds are seeded after being covered with cells, the cells are dried together with a diluent such as branched dextrin, and gums, methylcellulose, lignin sulfonate, dinaphthyl methanesulfonate are used. Sodium acid formalin condensate and other spreading agents are mixed, and when the agricultural products are processed, they are suspended in water so as to have a concentration of 10 ² to 10 ⁷ cells / ml. Good results are obtained.

[0022]

EXAMPLES Next, examples of cell culture and examples in which the effect on agricultural products is compared with comparative examples will be described.

[0023]

Example 1 200 g of potato was put in 1,000 g of distilled water, boiled in a boiling water bath for about 20 minutes, the potato was removed with a case, 20 g of glucose was added to the solution, and the pH was adjusted to 6.7 to 6.8. Was prepared. 50 ml of this medium was placed in a 100 ml flask and sterilized at 121 ° C. for 15 minutes. Next, one platinum loop of Bacillus subtilis NK-330 was inoculated into the sterilized medium and cultured at 30 ° C. overnight to obtain a seed.

Next, 7 l of the medium prepared in the same manner was placed in a 10 l jar fermenter, sterilized under the same conditions, and 50 ml of a seed mother was added thereto, followed by stirring (300 rpm) and aeration (1/2 v).
/ V · min) for 3 days at 30 ° C.
The cells were separated by centrifugation at 000 rpm for 10 minutes. The cells were suspended in an appropriate amount of water and freeze-dried overnight to obtain 9.5 g of dried cells of Bacillus subtilis NK-330.

In addition, dried cells of Bacillus subtilis NK-C-3 and Bacillus subtilis ATCC10774 were obtained in the same manner.

[0026]

Example 2 20 g of commercially available raw corn was placed in an Erlenmeyer flask, sterilized by heating in an autoclave at 121 ° C. for 15 minutes, and then a fungus venom-producing bacterium, Fusarium graminarum AT.
When the Bacillus subtilis NK-330 strain (10,000 spores / flask) was inoculated with the CC34909 strain (about 10,000 spores / flask) and cultured at 25 ° C., the amount of bomitoxin in the corn and the growth state of the Fusarium strain were determined over time. Table 1 shows the measurement results. Comparative Example 1 shows the results when Bacillus subtilis was not inoculated and only Fusarium graminearum was inoculated.

[0027]

The bomitoxin was extracted with 85% acetonitrile, passed through a column on which a cation exchange resin, activated carbon and alumina were layered, and the liquid obtained was evaporated to dryness. This was dissolved in methyl alcohol, and the activated carbon and zeolite were again layered. The solution was quantified by high performance liquid chromatography (JOAC, Vol. 70, No. 3, 47).
9 ■ 483, 1987).

Further, ND in the column of the amount of bomitoxin was not detected, and TR indicates detection of a trace amount (detection limit: 50 ppb).
In the column of Fusarium growth status,-indicates no growth, +-
Indicates growth at 25% or less of the surface of the substrate, + indicates growth at 50% or less, ++ indicates growth at 75% or less, and +++ indicates growth at all surfaces. (The growth status column in the table below is also displayed using the same criteria.)

[0030]

Example 3 In the same manner as in Example 2, 20 g of commercially available corn for raw food was placed in an Erlenmeyer flask, and autoclaved for 121 g.
After heat sterilization at 15 ° C for 15 minutes, Bacillus subtilis NK-C-3 strain (10,000 spores / flask) was inoculated together with the fungus venom-producing bacterium Fusarium graminearum ATCC34909 strain (about 10,000 spores / flask) and incubated at 25 ° C. Table 2 shows the results of measuring the amount of bomitoxin in the corn and the growth state of the Fusarium strain over time when cultured. Comparative Example 2 shows the results when only B. subtilis was not inoculated, but only Fusarium graminearum was inoculated.

[0031]

[0032]

Example 4 Similarly, 20 g of commercially available raw corn was placed in an Erlenmeyer flask and sterilized by heating at 121 ° C. for 15 minutes in an autoclave. The Bacillus subtilis NK-330 strain was then added to the fungus venom-producing Fusarium graminearum ATCC 34909 strain (approximately 10,000 spores / flask) in three different amounts of 10 spores / flask, 100 spores / flask and 1,000 spores / flask. And each flask was cultured at 25 ° C. for 14 days. Table 3 shows the results of measurement of the amount of bomitoxin in the corn and the growth state of the Fusarium strain at this time. Comparative Example 3 shows the results when only B. subtilis was not inoculated, but only Fusarium graminearum was inoculated.

[0033]

[0034]

Example 5 20 g of corn for raw consumption was placed in an Erlenmeyer flask, sterilized by heating at 121 ° C. for 15 minutes using an autoclave, and then a fungus venom-producing bacterium Fusarium graminerum ATCC34909 strain (about 10,000 spores / flask) was obtained. Together with Bacillus subtilis NK-C-3 strain 10 spores / flask,
100 spores / flask and 1,000 spores / flask were inoculated, and the results of measuring the amount of bomitoxin in corn and the state of growth of the Fusarium strain when each flask was cultured at 25 ° C. for 14 days are as shown in Table 4. . Comparative Example 4 shows the results when Bacillus subtilis was not inoculated and only Fusarium graminearum was inoculated.

[0035]

[0036]

Example 6 Commercially available raw corn (20 g) was placed in an Erlenmeyer flask, and sterilized by heating at 121 ° C. for 15 minutes in an autoclave, and then Fusarium graminearum ATCC34909 strain (about 10,000 spores / flask) producing mold venom was produced. Together with Bacillus subtilis NK-330 strain (10,000 spores / flask), and inoculate each flask at 18 ° C, 20 ° C, 25 ° C.
Table 5 shows the results of measuring the amount of bomitoxin in the corn and the growth state of the Fusarium strain when cultured at 14 ° C. and 30 ° C. for 14 days. Comparative Example 5 shows the results when only B. subtilis was not inoculated, but only Fusarium graminearum was inoculated.

[0037]

[0038]

Example 7 20 g of commercially available corn for raw food was placed in an Erlenmeyer flask, and sterilized by heating in an autoclave at 121 ° C. for 15 minutes, and then a fungus venom-producing bacterium, Fusarium graminearum.
Bacillus subtilis NK-C-3 strain (10,000 spores / flask) was inoculated with ATCC 34909 strain (about 10,000 spores / flask), and each flask was inoculated at 18 ° C, 20 ° C, 25 ° C, and 30 ° C.
Table 6 shows the results of measuring the amount of bomitoxin in corn and the growth state of the Fusarium strain after culturing for a day. Comparative Example 6 shows the results when Bacillus subtilis was not inoculated and only Fusarium graminearum was inoculated.

[0039]

[0040]

Example 8 After crushing wheat grains with a hammer, pieces of wheat having a size not passing through a 16-mesh sieve were collected and immersed in water overnight. Wheat immersed in water was placed in a 20 g Erlenmeyer flask and sterilized by heating to 121 ° C. for 15 minutes in an autoclave. This was inoculated with Bacillus subtilis NK-330 strain (10,000 spores / flask) together with the fungus venom-producing bacterium Fusarium graminearum ATCC34909 strain (about 10,000 spores / flask), and was cultured at 25 ° C for 14 days. Table 7 shows the results obtained by measuring the amount of bomitoxin and the growth state of the Fusarium strain. In addition, Comparative Example 7
The results are obtained when inoculating only Fusarium graminearum without inoculating subtilis.

[0041]

[0042]

Example 9 A crushed product obtained by crushing wheat grains with a hammer was used for 16
The pieces of wheat were sieved through a mesh sieve and sized so as not to pass through the eyes of the sieve. After immersing it in water overnight,
g was placed in an Erlenmeyer flask and sterilized by heating in an autoclave at 121 ° C. for 15 minutes. In addition to this, Fusarium
Graminearum ATCC34909 strain (about 10,000 spores / flask)
Together with Bacillus subtilis NK-C-3 strain (10,000 spores /
Table 8 shows the results of measuring the amount of bomitoxin in the wheat chips and the growth state of the Fusarium strain when inoculated and cultured at 25 ° C for 14 days. Comparative Example 8
Is the result when only B. subtilis was not inoculated but only Fusarium graminearum was inoculated.

[0043]

[0044]

Example 10 20 g of commercially available corn for raw food was placed in an Erlenmeyer flask, and sterilized by heating in an autoclave at 121 ° C for 15 minutes. In addition to this, along with the fungus venom-producing bacterium Fusarium graminearum ATCC34909 strain (about 10,000 spores / flask),
Bacillus subtilis NK-330 strain (10,000 spores / flask)
Table 9 shows the results of measuring the amounts of bomitoxin, 3-acetyldeoxynivalenol and zearalenone in the corn and the growth state of the Fusarium strain when inoculated and cultured at 25 ° C. for 14 days. Comparative Example 9 shows the results when Bacillus subtilis was not inoculated and only Fusarium graminearum was inoculated.

[0045]

After extracting 3-acetyldeoxinvalenol and zearalenone with 85% acetonitrile, impurities in the extract were removed with hexane and dried. The dried product is applied to a Florisil column, the column is washed with hexane, then the mold venom is eluted with a mixed solution of chloroform and methyl alcohol (9: 1), and gas chromatography (electron capture detector) and liquid chromatography are performed. (Pro
c. Jpn. Assoc. Mycotoxicol No. 26, 17-18, 1987).

[0047]

Example 11 20 g of commercially available raw corn was placed in an Erlenmeyer flask, and sterilized by heating to 121 ° C. for 15 minutes using an autoclave. The fungus venom-producing bacterium Fusarium graminearum ATCC34909 strain (about 10,000 spores / flask) was inoculated with Bacillus subtilis NK-C-3 strain (10,000 spores / flask), and the corn was cultured at 25 ° C for 14 days. Table 10 shows the results of measurement of the amounts of bomitoxin, 3-acetyldeoxynivalenol and zearalenone in the medium and the growth state of the Fusarium strain. Comparative Example 10 is the result when only B. subtilis was not inoculated, but only Fusarium graminearum was inoculated.

[0048]

[0049]

Example 12 20 g of commercially available corn for raw food was placed in an Erlenmeyer flask, and sterilized by heating at 121 ° C. for 15 minutes in an autoclave. Along with the fungus venom-producing bacterium Fusarium graminearum ATCC 34909 strain (about 10,000 spores / flask), Bacillus subtilis ATCC 10774 strain (10,000
Table 11 shows the results of measuring the amounts of bomitoxin, 3-acetyldeoxynivalenol and zearalenone in corn and the growth state of the Fusarium strain when inoculated and cultured at 25 ° C. for 14 days. Comparative Example 12 shows the results when Bacillus subtilis was not inoculated and only Fusarium graminearum was inoculated.

[0050]

[0021]

Industrial Applicability According to the present invention, it is possible to inhibit the growth of fungus venom-producing Fusarium spp. And the production of mold venom by treating agricultural products with a strain belonging to Bacillus subtilis that produces iturin A. Was known. Therefore, by treating an agricultural product or a field producing it with cells of a strain belonging to Bacillus subtilis, it has become possible to prevent the agricultural product from being contaminated with mold belonging to the genus Fusarium. That is, the cells of the strain belonging to Bacillus subtilis are sprayed on the field where the agricultural product is planted, the seed to be sown, the plant growing in the field, or the field where the plant is growing, or the harvested agricultural product. Alternatively, it has become possible to control the fungus toxin produced by Fusarium spp. By spraying and mixing the cells with the stored agricultural products. The same effect can be obtained by using a culture solution from which the cells have been separated or purified Aitulina A.

Claims (2)

(57) [Claims] 1. A fungus belonging to the genus Fusarium, wherein the fungus toxin produced in agricultural products such as cereals by the fungus belonging to the genus Fusarium is controlled by treating it with a strain belonging to Bacillus subtilis that produces iturin A. How to control mold venom. 2. The method for controlling mold venom produced by mold belonging to the genus Fusarium according to claim 1, wherein the agricultural product such as cereals is selected from corn, wheat and barley.