JPH02258706A - Preparation of insecticide - Google Patents

Abstract

PURPOSE:To sterilize bacterium cells or sprouts thereof for the preparation of a high quality insecticide without lowering the insecticidal ability of the crystalline toxin of Bacillus.thuringiensis by carrying out the sterilization of a culture solution containing the crystalline toxin produced by the Bacillus.thuringiensis after water-soluble components in the culture solution are removed. CONSTITUTION:Bacillus.thuringiensis is cultured at 25-30 deg.C for 2-4 days under a sufficiently aerobic condition and the culture solution containing sprouts and a crystalline toxin formed thus is subjected to a centrifugal treatment, filtration treatment, precipitation treatment, etc., to separate and remove water- soluble components (e.g. components caused by the medium, or metabolites discharged outside the cells) existing in the culture solution, followed by simultaneously performing a mild chemical sterilization treatment and a physical sterilization treatment to perfectly kill the bacterium cells and sprouts thereof, thereby providing an insecticide having an excellent insecticidal activity. The insecticide having such a remarkable insecticidal activity as having not been predicted can also be prepared.

Description

[Detailed description of the invention] (a) Object of the invention [Field of industrial application] The present invention is directed to the production of Bacillus thuringiensis
LIus thuringjensis: Hereinafter referred to as BT bacteria and 1. This article relates to a method for producing an insecticide (hereinafter referred to as BT pesticide) that contains crystalline toxin produced by 5) as an effective ingredient and is effective against Lepidoptera insects, etc. that do not contain BT sapling cells or spores, and is useful in the agrochemical industry and agriculture. It is widely used in the field of stuttering.

[Conventional technology]

The crystalline toxin produced by the BT bacterium exhibits a strong insecticidal effect against lepidopteran insects, and is harmless to humans, animals, fish, and shellfish.

It has been put into practical use as a biopesticide.

In general, in addition to crystalline toxins, BT pesticides contain spores (also called spores), which are living organisms for self-renewal.In the natural world, when sprayed as is, the spores germinate, and This is done in order to protect the domestic sericulture industry, as there is a risk that the BT bacteria will proliferate and cause chemical damage to the silkworms.

There is a need for a BT pesticide that does not cause secondary spore growth.

In order to solve this problem, we developed a method that can kill bacterial cells and spores in the culture solution of BT bacteria containing crystal toxin without losing the insecticidal ability of the crystal toxin. A method for producing insecticides has been proposed that combines slow chemical sterilization and slow physical sterilization and performs them simultaneously (Japanese Patent Publication No. 51-50).
Publication No. 47).

[Problem to be solved by the invention]

The above method is an excellent method for killing #1 bacterial cells and spores, and is a method that has been put into practical use.
The crystalline toxin obtained by this method remains at a practical concentration (BT concentrate is generally used as a suspension in water with a concentration of 1.000 to 2,000 times more effective against diamondback moths) through sterilization. The insecticidal activity tends to be reduced, and if you want to completely kill bacterial cells and spores, you may have to use it at a high concentration of Kanaυ to obtain an insecticide that does not show sufficient insecticidal performance. This method has some problems.

The present inventors have conducted intensive studies to improve the efficiency of killing bacterial cells and spores in the above method and to establish a manufacturing method that can yield products of excellent quality.

(B) Structure of the Invention [Means for Solving the Problems] In the process of studying to solve the above-mentioned problems, the present inventors discovered that the water-soluble components present in the culture solution kill bacterial cells and spores. It was discovered that the crystal toxin has sufficient insecticidal activity to produce a practical insecticide by removing the water-soluble components before sterilizing the culture solution by reducing the efficiency and ultimately the insecticidal activity of the crystal toxin. The present invention was completed based on the discovery that not only can a crystalline toxin be stably obtained, but also a crystalline toxin with dramatically improved insecticidal activity can be obtained.

That is, the present invention relates to a method for producing an insecticide, characterized in that sterilization of a culture solution containing crystalline toxin produced by Bacillus thuringiensis is carried out after a step of removing and separating water-soluble components present in the culture solution. It is.

0 Culture solution containing crystal toxin In the present invention, the culture solution containing crystal toxin, which is a raw material for the insecticide, is a culture solution containing BT bacteria that produces crystal toxin, such as Bacillus thuringiensis varietus kurstaki HD-
1 (Bac i I l usthuringiens
is var kurstaki HD-1), etc., by culturing them using known culture methods and conditions.

The BT bacterium used in the present invention may be any known BT bacterium, and the culturing method and conditions are specifically described below.

BT can be cultured using a natural medium rich in nitrogen sources, carbon sources, minerals and vitamins; however, the production of crystalline toxins and bacterial cells is highly dependent on the aeration and agitation conditions; It is preferable to culture under atmospheric conditions, thereby increasing the production amount of both. The culture temperature may be about 25 to 30°C, and the number of culture days may be 2 to 4 days. As the carbon source, for example, sucrose, maltose, glucose, 7-lactose, and tangmi are used, and as the nitrogen source, for example, corn steep liquor, ammonium sulfate, ammonium chloride, cottonseed flour, yeast extract, soybean flour, and casein ice melt are used. Examples include things. In addition, minerals and vitamins can be substituted with minerals, corn steep liquor, and yeast extract, and if necessary, inorganic salts and vitamins may be further added. In particular, when performing mass production, K
There are water-soluble components such as Ifi, components originating from the culture medium, metabolites excreted outside the bacterial cells by BTIii, or metabolites contained within the bacterial cells and released into the culture solution after autolysis, and the present inventors Which of these components is K?
Although it is not clear why, the presence of these water-soluble components reduces the sterilization efficiency, and in order to completely sterilize as it is, that is, in the presence of water-soluble components, the sterilization conditions must be made stricter. vinegar.

Furthermore, the present invention was completed by discovering that the insecticidal activity of crystalline toxins can be reduced.

Removal of water-soluble components before disinfection not only makes it possible to produce effective insecticides at practical concentrations, but also makes it possible to predict This makes it possible to produce insecticides with insecticidal activity to a degree that was previously impossible.

Methods for removing water-soluble components in the cultured solution in which spores and crystalline toxins have been formed (the progress of the culture can be easily tracked using a phase contrast microscope are 1) ordinary centrifugation method, r-proper method,
An example is a method of removing an aqueous solution using a sedimentation method or the like. In particular, when processing a large amount, it is preferable to use a centrifugal separator or a membrane filtration material. In addition, the amount of water-soluble components removed depends on operating conditions such as centrifugal force (gravitational acceleration), liquid passing interval, and washing interval in the former case, and in the latter case, operating conditions such as membrane pore diameter, pressure per liquid passing rate, etc. Although it differs depending on the situation, it is preferable to avoid removing too much amount so that the dispersibility of spores and crystal toxins is inhibited, if this is not possible.

O Sterilization The sterilization in the present invention is carried out to kill bacterial cells and spores, and the following chemical sterilization treatment and physical sterilization treatment methods are available.
As proposed in Publication No. 047, simply one type of sterilization treatment does not reduce the insecticidal activity of the crystal toxin.
#1 Since it is difficult to completely kill bacterial cells and spores, it is preferable in the present invention to combine slow chemical sterilization treatment and physical sterilization treatment and perform them simultaneously. It is possible to completely kill bacterial cells and spores, and it is possible to obtain an industrially extremely useful insecticide with excellent insecticidal ability.

Q Chemical sterilization treatment Chemical sterilization treatment methods include formalin, para-toluenesulfone chloramide sodium, para-toluenesulfonic acid dichloramide, azobischloroformamidine, acrizivin, methylene blue, benzalkonium chloride,
This method involves adding an appropriate amount of a drug such as cetylpyridinium chloride to the above-mentioned culture solution for sterilization.

O Physical sterilization treatment The physical sterilization treatment method is a method of sterilizing the above-mentioned culture solution by heating, ultrasonic waves, radiation, etc.

0Measurement method of insecticidal activityAs a method for quantitatively understanding the insecticidal activity of crystal toxins,
A method was adopted in which the half-lethal concentration was determined through an insecticidal test using diamondback moths, and the residual insecticidal activity was quantitatively measured.

Namely.

This method measures the mortality rate of test insects for each appropriately diluted sample solution, calculates the half-lethal concentration from the relationship between the concentration of the sample solution and the mortality rate, and compares the level of insecticidal activity.

After removing the water-soluble components from the culture solution of the 0-preparation BT bacteria, the above-mentioned sterilization treatment is performed, if necessary.

Through solid-liquid separation and purification, an insecticide consisting of dead spores containing crystalline toxins can be obtained.

Such insecticides are usually made into powders by known methods. Alternatively, it can be prepared by suspending it in a fi bath agent.

The amount of the active ingredient varies depending on the target insect trap, but it is usually used within a known range, and it can also be used in combination with other insecticides.

[Effect]

It is unknown which water-soluble components reduce the sterilization efficiency, but by removing water-soluble components from the culture solution,
It is possible to sterilize the culture solution more slowly (for example, the amount of chemical disinfectant used is reduced), which not only prevents a decrease in the insecticidal activity of the crystal toxin, but also increases the insecticidal effect to a degree that cannot be predicted by conventional methods. The present invention has the effect of dramatically increasing activity.

〔Example〕

The present invention will be explained in more detail below using Examples.

Example 1. (When part of the culture supernatant is replaced with water: use formalin) Bacillus thuringiensis varietus kurstaki HD-1
Nsisvar kurstaki HD-1) is cultured with shaking in a Sakaro flask at C medium C for 3 days. After 6 days of culture, the culture solution was transferred to a tabletop centrifuge (14,000 rpm, 4
℃.

10 minutes), and the supernatant (aqueous solution) portion is collected by decantation. At that time, measure the volume of the supernatant obtained. Next, a part of this culture solution supernatant was replaced with water (100%, 90%, 80%, 60%).
%, 40%.

20% and 0%) are prepared respectively, and an amount equal to the soil volume obtained after centrifugation is returned to the residue in the centrifuge tube and redispersed. At that time, following dispersion using a stirrer, ultrasonic treatment (output 40W).

B) (Using the ANSON 5ONIFIER450 ultrasonic generator: While cooling the sample in ice water, irradiate the sample for 60 minutes.
The treatment was carried out for a total of 5 minutes with intervals of 30 seconds and 30 seconds of rest.

In this way, some or all of the culture supernatant is replaced with water, and various preparations (bacterial cells, etc.) are redispersed.
The amounts of spores and crystal toxins are the same, but the amounts of water-soluble components are different) are dispensed into test tubes, and each concentration of formalin (5-) is added and mixed to reduce the formalin concentration in the mixture to 0.
The mixture was heated to 70°C for 10 minutes, cooled to room temperature, centrifuged at 14,000 rpm for 10 minutes, and the supernatant liquid was drained. Add 10 g of sterile water to the sediment.

Resuspend and repeat the centrifugation and subsequent operations.

Next, the number of surviving cells/spores and residual insecticidal activity are measured for the final solution suspended in sterile water (the measurement method is as follows).

Take the sample solution 11111 for measuring the number of surviving cells and spores, dilute it appropriately with sterile water, and add Nu.
trient-Broth-agar medium (meat extract 1%
.

Polypeptone 1%, sodium chloride 0.5%, agar 1.5%: pH 7.0) was poured onto the sample, cultured at 30°C for 48 hours, the number of colonies generated was counted, and the number of surviving spores in the sample was counted. The minimum concentration of formalin in the mixed solution for which the number (ke/punishment) is 0ke/1 (corresponds to the critical concentration that completely kills bacterial cells and spores) and the residual insecticidal activity of the same treatment solution are expressed. Shown in 1.

Dilute the sample thickness appropriately with sterile water, and prepare 50+ ul of assay solution at 5 to 7 concentration levels. Fresh cabbage leaves (200 cd) are immersed in this test solution for 1 minute and then air-dried. This was spread in a large petri dish, 20 3rd instar diamondback moth larvae were released in each area (# area), and the number of dead and alive was counted after 72 hours.
Calculate the mortality rate (%). This result is Finny()'1
Pinney, D. J. (19
47) probit Al'1alysis.

Cambriage Univ, Press, Cam
ridge (Lordan).

318 pp) to determine the half-lethal n degree (expressed as the concentration (ppm) of the sample solution contained in the test solution).

This treatment sterilized a prepared solution in which some or all of the culture supernatant was replaced with water, and it was shown that the number of surviving spores and residual insecticidal activity increased.

Further, the substitution ratio was 80% or more, giving almost constant results.

Example 2 (Part of the supernatant part of the culture solution is replaced with water.) Suspension solution of paratoluenesulfone chloride spores/crystalline toxin (part of the supernatant part of the culture solution is replaced with water) Dispense 51 portions of the redispersed material into test tubes, add and mix 5 portions of each degree of aqueous solution of sodium paratoluenesulfone chloramide (paratoluenesulfonechloramide) in the mixed solution. -)-)jlmu[tfO70
Suspended at a level of o, o1s in the range of 3% to 0.1 inch,
The number of surviving cells/spores and residual insecticidal activity were measured. The results are shown in Table 2.

＼ Collect the precipitate from which paratoluene 7-sulfone chloramide sodium has been removed, and add it to sterile water. As shown in Table 2 above, by replacing the upper tube of the culture solution with water, para-toluene sulfone chloramide in the mixed solution can be removed. It was observed that the sodium concentration could be reduced and the residual insecticidal activity increased.

Furthermore, the substitution ratio was approximately constant at 90 or more.

As is clear from Example 1.2, by removing water-soluble components during culture, the concentration of disinfecting chemicals can be reduced, and as a result, the residual insecticidal activity is increased (Table 1.2
However, assuming actual industrial production, first, the centrifuged precipitate (consisting of bacterial cells, spores, and crystalline toxins) should be soaked in water or electrolyte. It is not necessarily a good idea to return the culture solution to its original level and sterilize it using clear water.

Therefore, in order to reduce the burden of separating and concentrating the liquid after sterilization, we attempted to sterilize the viscous liquid concentrate from which the water-soluble components in the culture liquid had been removed.

Example 6 0.8 t of the culture solution prepared in the same manner as in Example 1 was placed in a Hitachi continuous centrifuge rotor (capacity 211), and the rotation speed was varied (gravitational acceleration 1.000x, p-7, QOOXg).
Perform batch centrifugation (4°C, 10 minutes). After centrifugation,
Carefully remove the supernatant portion so as not to include the precipitate, and perform ultrasonication at the removal rate of water-soluble components (concentration ratio: Xl, X2°).

Dispense 5d of this treatment solution into test tubes, add and mix 51R1 of each formalin concentration solution, set the formalin concentration in the mixed solution to 5 as appropriate, and heat it at 70°C for 10 minutes. The precipitate from which formalin has been removed in the same manner is collected, suspended in sterile water, and then mixed with surviving cells and
The number of spores and residual insecticidal activity were measured. Table 3 shows the minimum formalin concentration in the mixture at which the number of viable spores is 0/nil and the residual insecticidal activity of Kaisei.

As is clear from Table 6, the rate of increase in the amount of formalin (critical concentration) required to completely kill bacterial cells and spores decreases as the rate of removal of water-soluble components from the culture solution increases. Therefore, it is possible to reduce the amount of formalin used in terms of culture solid content (formalin critical concentration in the mixed solution/solid content concentration in the mixed solution), and as a result, the culture solid content (
IThK makes a leap forward in the residual insecticidal activity of atashi (the main pesticide)
It was possible to increase it. In addition, it is desirable to set the removal rate of water-soluble components, that is, the concentration ratio of the culture solution, at least 10 times so that the residual insecticidal activity in terms of culture solid content remains unchanged. If the concentration exceeds 15%, the viscosity of the liquid will increase significantly and there is a risk of losing fluidity, so it is particularly desirable to adjust the concentration of cultured solids in the concentrated solution to a range of 4 to 15%. . Table 3
Although it is not stated in , if the concentration of culture solids in the concentrate is 1
When 5% of the sample was subjected to sterilization treatment, the residual insecticidal activity in terms of culture solid content was 10 times the vertical ratio (open solid content concentration: 4
It was almost the same as that of C).

Therefore, depending on the performance of the centrifugal separator used, water-soluble components may be removed and concentrated so that the solid content concentration falls within the above range. Furthermore, the results in Table 6 show that by 10-fold concentration, the amount of chemical (formalin in this case) used in terms of culture solid content is significantly reduced (approximately 3A, x
*) This means that it shows what is possible and contributes to reducing manufacturing costs.

Example 4 Five portions of a concentrated solution prepared in the same manner as in Example 6 (including ultrasonication) were dispensed into test tubes, and 5N of each concentration solution of sodium paratoluenesulfone chloramide was added and mixed.
After heating the mixed solution with an appropriately set concentration of sodium paratoluenesulfone chloramide at 60°C for 10 minutes, the number of surviving spores and the concentrated solution from which sodium paratoluenesulfonechloramide was removed in the same manner as in Example 3 were determined. Residual insecticidal activity was measured. The results are described in Section 2.4.

In Table 4 above, as in Table 6, by removing water-soluble components and sterilizing the concentrated culture solution, the use of chemicals (in this case, sodium paratoluenesulfone chloramide) in terms of culture solid content is achieved. The amount can be significantly reduced (approximately 1./6.7 with 10 times concentration).

It was confirmed that the residual insecticidal activity in terms of culture solid content (which becomes the pesticide itself) was also dramatically increased (approximately 6.3 times by 10 times 7J reduction).

Example 5 Bacillus thuringiensis varietus cristaki H,D-1 was grown in a meat extract/peptone medium in a Sakaro flask (meat extract 1%, polypeptone 1%, sodium chloride 0825%: pH, -, j O) Inoculate at 50M and culture with shaking at 30°C for 6 days. .

The same culture solution was concentrated by removing water-soluble components in the same manner as in Example 3.
Ultrasonic treatment was performed. A sterilization treatment was performed using Kaisei in the same manner as in Examples 6 and 4, and the number of surviving spores and residual insecticidal activity after the 8 bacteria treatment were determined.

As a result, the concentration of cultured solids in the concentrate was 10 times
6%), significantly reducing the use of chemicals in terms of culture solid content compared to no treatment (#, #man!7: approx.
+15.5. Halato A en sulfone chloramide sodium: approx. 1/'5).

Residual insecticidal activity also increased (Hollow FW: approximately 1.5 times).

Balatruuni/Sulfone Chloramide Sodium: Approximately 1
, 8 times) L 7'': The results of the 6-piece knot were in good agreement with the results in Tables 3 and 4.

e→ Effects of the Invention The present invention relates to a method for producing an insecticide that is effective against the larvae of lepidopteran insects such as the flying moth, the armyworm moth, the armyworm moth, and the armyworm, and which does not cause secondary growth due to viable bacterial bodies and spores. (7. After removing water-soluble components from the culture solution of Bacillus thuringiensis containing crystal toxin,
Sterilized! This has the excellent effect of greatly reducing the amount of chemicals used, enabling slower sterilization, and dramatically increasing the residual insecticidal activity after sterilization. It can contribute widely to the agrochemical industry and agricultural fields because it greatly contributes to improving productivity and quality.

Claims (1)

[Claims] 1. Bacillus thuringiensis
A method for producing an insecticide, which comprises sterilizing a culture solution containing a crystalline toxin produced by P. thuringiensis after a step of removing and separating water-soluble components present in the culture solution.