JPH03133911A - Production of insecticide - Google Patents

Abstract

PURPOSE:To kill existing live endospores and live vegetative cells without causing denaturation of crystal toxins which are an insecticidal active ingredient by subjecting a culture solution of Bacillus.thuringiensis (bacterium BT) to sterilization treatment using isocyanuric chloride. CONSTITUTION:A bacterium BT is cultured in a natural culture medium rich in a nitrogen source, a carbon source, minerals and vitamins at 25-30 deg.C temperature under sufficient aerobic conditions for a period within 24hr from the time of releasing 90% enndospores to the outside of cells. Water-soluble intgredients in the resultant culture are then removal by centrifugation, filtration through a membrane, etc., and 0.05-0.20wt.% anionic or nonionic surfactant, as necessary, is then added or ultrasonic treatment is carried out to regulate dispersibility. Isocyanuric chloride is subsequently used to perform sterilization treatment at pH4-7, especially pH5-5.5. Not only insecticidal activity of crystal toxins can be prevented from deteriorating but also an insecticide of constant quality can stably be obtained. The insecticidal activity can be more enhanced than that of a conventional insecticide.

Description

Detailed Description of the Invention (a) Purpose of the Invention [Field of Industrial Application] The present invention
From a mixture of crystal-like insecticidal protein toxin (hereinafter referred to as crystal toxin) produced by culturing various strains of S. lus thuringiensis (hereinafter referred to as BT bacteria) and spores, etc., while retaining the insecticidal activity of the crystal toxin, spores are generated. Insecticides (hereinafter referred to as BT
It relates to a method for producing agricultural chemicals (referred to as agricultural chemicals) and is widely used in the agricultural chemicals industry and agricultural fields.

[Conventional technology]

Various strains of the BT bacterium produce toxins made of protein, such as summer-shaped, dice-shaped, and amorphous cubes, as they form spores, some of which are selective prephages for the larvae of lepidopteran insects. Others are known to have high insecticidal activity against the larvae of Diptera, such as Aedes and Culex, and those that are selectively toxic only to Coleopteran insects, such as the Colorado-bottomed beetle. It is being

As mentioned above, the crystalline toxin produced by the BT bacterium is characterized by its selectivity; it does not act on insects different from the target species, and of course is harmless to humans, livestock, fish, and birds. , its use as a selective insecticide has been pursued.

On the other hand, spores, which are living organisms for self-renewal produced through culture, are dormant cells.
It has a strong and durable structure, can withstand environmental conditions (including dry conditions) that are unfavorable to ordinary living things, and can survive for long periods of time. Therefore, when pesticides containing this compound are repeatedly sprayed outdoors, such as in fields, there are concerns that they may accumulate in the soil and disseminate through wind and rain. Strict precautions are being taken as this could lead to

Furthermore, this spore germinates into a vegetative cell, which secretes a water-soluble protein toxin during the process of logarithmic growth, which
It strongly immunochemically reacts with the cereus toxin that causes food poisoning.
563 (1988)), there is therefore concern that food poisoning may occur during the cooking and preservation process of food with spores attached. These concerns would disappear if a product containing no living spores could be obtained by separating live spores and crystalline toxins, but both spores and crystalline toxins are about a few micrometers in size and are microscopic. Moreover, the surface charge states are similar, and it is extremely difficult to separate the two physically and electrochemically, and it is not industrially practical.

On the other hand, the above concerns can also be eliminated by manufacturing insecticides that kill spores, but as mentioned earlier, spores have a durable structure and cannot be used for normal killing. It has the strongest resistance among living organisms to physical or chemical sterilization treatments such as heating, drying, and chemical treatments. Therefore, in order to completely kill the spores, severe sterilization conditions are required, such as heating at a high temperature exceeding 100°C.

However, when treated under such harsh conditions, the crystal toxin, which is the active insecticidal ingredient, may be denatured (heat denaturation of the protein), resulting in a loss of insecticidal efficacy, and there is a risk that an effective product may not be obtained. .

In order to solve this problem, we applied a slow chemical sterilization treatment to the bacterial cells and spores in the culture solution of BT bacteria containing the crystal toxin, which does not cause the crystal toxin to lose its insecticidal ability. A method for producing insecticides has been proposed that combines slow physical sterilization treatments and simultaneously performs them to kill bacterial cells and spores (Japanese Patent Publication No. 51-5
Publication No. 047).

[Problem that the invention seeks to solve]

The above method is an excellent method for killing bacterial cells and spores and has been put into practical use; however, the crystal toxin obtained by this method is
Practical concentration (BT pesticide is -S to 1,0 against diamondback moth)
00 to 2.000 times (used as an aqueous suspension), the residual insecticidal activity tends to be reduced,
In order to completely kill bacterial cells and spores, this method has the problem that in some cases, it may be necessary to use it at a fairly high concentration, resulting in an insecticide that does not have sufficient insecticidal performance. There is also.

The present inventors have screened various fungicides that have excellent killing effects on spores and crystalline toxins, and that are particularly effective when used in the conventional method, and have evaluated the efficiency of killing bacterial cells and spores in the above method. We conducted extensive research to establish a manufacturing method that would improve the residual insecticidal activity and yield products of superior 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 used chlorinated isocyanuric acid or its salt as a chemical disinfectant. As a result, we discovered that not only can we stably obtain a crystalline toxin with insecticidal activity sufficient to produce a practical insecticide, but we can also obtain a crystalline toxin with dramatically improved insecticidal activity, and have developed the present invention. completed.

That is, the present invention relates to Bacillus thuringiensis (B.
The present invention relates to a method for producing an insecticide, characterized in that spores and biovegetative cells present in a culture solution of A. acillus thuringieusis are killed with chlorinated isocyanuric acid or a salt thereof.

Culture solution of oBT bacterium In the present invention, the culture solution containing crystal toxin, which is a raw material for the insecticide, is a culture solution containing crystal toxin, which is a raw material for the insecticide. It is obtained by culturing the BT strain of 100% using commonly known culture methods and conditions.

For example, by culturing BT bacteria using a culture solution consisting of meat extract, peptone, etc. in a conventional manner and under conditions, the culture solution containing spores and crystal toxins may be obtained, or the culture solution may be subjected to centrifugal sedimentation or membrane separation. An aqueous suspension containing crystalline toxin and spores obtained by purification or concentration through filtration, or a suspension obtained by diluting them with water is used.

More specifically, the culture is carried out in a natural medium rich in nitrogen sources, carbon sources, minerals and vitamins. The production of crystal toxins and bacterial cells is greatly influenced by aeration and agitation conditions, and the production amount of both increases when cultured under sufficient aerobic conditions. The culture temperature is preferably about 25-30°C. Examples of carbon sources used include sucrose, maltose, glucose, fructose, and molasses; examples of nitrogen sources include corn steep liquor, ammonium sulfate, ammonium chloride, cottonseed flour, yeast extract, soybean flour, and casein hydrolyzate. can be mentioned. Further, minerals and vitamins can be replaced with molasses and corn steep liquor yeast extract, and inorganic salts and vitamins may be further added as necessary. Especially when producing in large quantities,
Deep aeration agitation culture is preferable.

The culture period is preferably set within 24 hours after 90% of the spores are released outside the cells, and the release of spores is determined by microscopic examination of the culture fluid collected aseptically over time using a phase contrast optical microscope. This allows easy tracking. Spore release refers to the release of crystal toxins and spores formed within cells.
As a result of autolysis of the cell walls of BT bacteria as the culture progresses, they are released outside the cells and become suspended in the culture solution.

Spores are elliptical objects with a short axis of 1 to 3 μm x a long axis of 2 to 6 μm, and have light refractive properties, so within the field of view of a phase contrast optical microscope,
They can be distinguished from other granules because they emit a bluish-white light. Crystal toxins and spores can also be distinguished by known staining methods (Fadel A, 5harifet al., J.
Ind. Microbiol, 3. 227-229
(1988)).

Furthermore, the fact that 90% of the spores were released outside the cells means that the ratio of free spores to the total number of bacteria within the microscopic field (total number of cells containing crystal toxins/spores and free spores) is within the range. You can check it. Although there are some differences depending on the culture conditions, it takes 8 to 24 hours from when spores begin to be released outside the cells until the above state is reached.

If the sterilization treatment is performed outside the above range, the insecticidal activity of the crystal toxin will be impaired, making it difficult to regularly produce an effective insecticide at a practical concentration, and the residual insecticidal activity will not improve dramatically.

The culture medium cultured as described above contains components originating from the medium, metabolites excreted from the BT bacteria to the outside of the bacterial cells, or metabolites contained within the bacterial cells and released into the culture medium after autolysis. Water-soluble components exist, and 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 quite severe. It is preferable to remove water-soluble components before disinfection, as this would reduce the insecticidal activity of the crystal toxin, thereby facilitating the production of effective insecticides at practical concentrations. Furthermore, depending on the degree of removal of water-soluble components, it is also possible to produce insecticides with insecticidal activity of a level that could not be predicted up to now.

Examples of methods for removing water-soluble components from the culture solution in which spores and crystal toxins have been formed include methods of removing the aqueous solution using conventional centrifugation, filtration, sedimentation, and the like. In particular, when processing a large amount, it is preferable to use a centrifugal separator or a membrane filtration material. In the former case, the amount of water-soluble components removed depends on operating conditions such as centrifugal force (gravitational acceleration), liquid flow interval, and washing interval.
In the latter case, although this will vary depending on the operating conditions such as membrane pore size, pressure, and liquid flow rate, it is preferable to avoid removing too much, which would impede the dispersibility of spores and crystal toxins.
If the dispersibility is inhibited, it is preferable to adjust the dispersibility by adding distilled water, especially a surfactant, or by applying ultrasonic treatment.

If materials with poor dispersibility are sterilized as is, the sterilization efficiency will be inconsistent, which may be due to contact with the sterilizer or poor heat transfer, and especially if complete sterilization is desired, sterilization conditions must be made quite severe. This may reduce the insecticidal activity of the crystal toxin.

The surfactant to be used is preferably an anionic or nonionic surfactant because the surfaces of particles to be dispersed, that is, vegetative cells, spores, and crystalline toxins, are negatively charged. , Sodium polyacrylate type, such as Aron A-20px (manufactured by Toagosei Kagaku Kogyo A), Sodium dodecylbenzenesulfonate type, such as Perex Nα6 (manufactured by Kao ■), Lebenol WZ (manufactured by Kao ■), Sodium lauryl sulfate type Examples of nonionic surfactants include Emar 2F (manufactured by Kao ■), dioctyl sulfosuccinate-based surfactants such as Perex OTP (manufactured by Kao II@), and nonionic surfactants such as polyoxyethylene ether-based surfactants such as Emulgen 910 (manufactured by Kao ■).
(manufactured by). The amount used is preferably in the range of 0.01 to 0.50% by weight, particularly preferably 0.05 to 0.20% by weight, based on the culture solution to be sterilized. It is preferable to set the setting according to the operating conditions, etc.

The culture solution of BT bacteria is sterilized preferably at pH 4 to 7, more preferably at pH 5 to 7, particularly preferably at pH 5 to 5.5.
Perform within the range of. Since the culture solution generally has a pH of about 8 to 9, it is desirable to adjust the pH to 4 to 7 with an acid such as sulfuric acid before sterilization. Also, during sterilization, if the pH changes due to the process and there is a risk of it deviating from the above range, it is desirable to add acid or alkali depending on the change to maintain the pH within the above range. .

If the sterilization treatment is performed outside the above range, the insecticidal activity of the crystal toxin will be impaired, making it difficult to regularly produce an effective insecticide at a practical concentration, and the residual insecticidal activity will not improve dramatically.

This effect is also shown when the culture supernatant is removed by centrifugation and resuspended in water.

0 Killing Killing in the present invention refers to chemical sterilization using a specific drug to kill vegetative cells and spores, and is described in the above-mentioned Japanese Patent Publication No. 51-5047. Preferably, it is carried out in conjunction with physical sterilization treatment methods, as disclosed. In other words, it is difficult to completely kill vegetative cells and spores while retaining the insecticidal ability of the crystal toxin with only one type of sterilization treatment, so the present invention also uses chemical sterilization treatment and physical sterilization treatment. It is preferable to perform these simultaneously in combination, and the drug that easily completes vegetative cells and spores by this method is chlorinated isocyanuric acid or its salt, and specific compounds include sodium dichloroisocyanurate, Examples include potassium dichloroisocyanurate and trichloroisocyanuric acid.

For comparison, sodium hypochlorite, sodium paratoluenesulfone chloramide, N-chlorosuccinimide, hydrogen peroxide, and formalin were used to demonstrate the effects of the present invention.

The physical sterilization method is a method of sterilizing the above-mentioned aqueous culture solution, etc. by heating, ultrasonic waves, radiation, or the like.

Industrially advantageous heating methods are the batch heating method, which heats while stirring in a reaction tank, or the continuous flow tube method, which flows into a long reaction tube from one end, heats it midway through, and discharges from the other end. The latter method is particularly useful in industrial implementation.

0 Formulation method The aqueous culture solution that has undergone sporicidal treatment can be made into a suspension as it is, or after being concentrated and subjected to post-treatment in a separate facility, with or without the addition of appropriate auxiliaries. or further formulated into a wettable powder or granular product, preferably by spray drying or fluidized fluid drying.

The basic usage amount varies depending on the target insect, but it is usually used within a known range. Also, the main rule is that it can be used in combination with other insecticides.

0 Method of Measuring Insecticidal Activity A method for quantitatively understanding the insecticidal activity of the crystalline toxin produced by BT bacteria is to determine the half-lethal concentration through an insecticidal test using diamondback moths and Culex mosquitoes, and quantify the residual insecticidal activity after sterilization treatment. We adopted a method of measuring. That is, this method measures the mortality rate of test insects for each sample solution at an arbitrary dilution rate, determines 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.

[Action] By using chlorinated isocyanuric acid and its salts as a disinfectant, it is possible to make the disinfection process more gradual (for example, the heating temperature can be lowered), and as a result, the insecticidal effect of crystal toxins is achieved. The present invention has the effect of not only being able to prevent a decrease in activity, but also being able to stably provide an insecticide of constant quality and increasing insecticidal activity compared to conventional ones.

〔Example〕

Next, the present invention will be explained in more detail by giving examples. The methods for measuring the number of surviving cells/spores and residual insecticidal activity are as follows.

・ Take 1In1 sample solution of '1'''', dilute it appropriately with sterile water, and add Nut.
rient-Broth-agar medium (1% meat extract, 1% polypeptone, 0.5% sodium chloride, 1.5% agar)
%: pH 7.0), cultured at 30''c for 48 hours, count the number of colonies that develop, and calculate the number of surviving cells/spores (ke/d) in the sample.

Take 1 d of sample liquid and dilute it appropriately with sterile water to prepare 50 d each of test solutions with 5 to 7 concentrations.

Fresh cabbage leaves (200 cffl) are immersed in this test solution for 1 minute and then air-dried. This is spread on a large petri dish, 20 3rd instar diamondback moth larvae are released in each section (1 degree section), and after 72 hours, the number of dead and alive is counted to calculate the mortality rate (%). This result can be expressed using Finney's graphical method (Finney).
, D.J., (1974) Probit Analysis
sis+Cam-bridge Llniv, Pre
ss, Cambridge (London), pl)
-318) to determine the half-lethal concentration (concentration of the sample solution contained in the assay solution (expressed as ppml)).

[Culex Culex] The sample solution was appropriately diluted aseptically, and 2 samples of each of the test solutions with 5 to 7 concentrations were added to 30 3rd instar larvae of Culex mosquito.
It is added to the rearing solution of 00d, kept at 27°C, and after 48 hours, the number of dead and alive is counted to calculate the mortality rate (%). Analyze this result using Finney's graphical method described above to determine the half-lethal concentration.

Example 1 Bacillus thuringiensis varietus kurstakii HD-1 was grown in C medium (glucose 1
%, cone steeple force -1%, Mn 1 ppm:
pH 7,0) 50 rrdl was inoculated and cultured with shaking at 30"C for 10 hours. 120d of the same culture was used as a seed,
It was inoculated into a jar fermentor (total volume 10 f) containing 12 f of 2XC medium (twice the concentration of C medium) and incubated at 30°C for 4 hours while adjusting and maintaining the pH at 7.0.
8-hour aeration stirring culture (stirring speed 300 rpm, illumination!
0.5 vvm). Dispense 5 portions of the same cultured solution into test tubes and mix so that the concentration of sodium dichloroisocyanurate in the mixture is in the range of 0 to 0.25% in 0.05% increments. This is 20″C~70″
After heating for 10 minutes in 10°C increments, it is immediately cooled. This is a distal bone! (14,0
0 Orpm) at 4°C for 110 minutes), discard the supernatant, and add 5 ml of sterile water to the sediment to suspend. Repeat the procedure twice to remove sodium dichloroisocyanurate and extracellular soluble toxic substances. .

Table 1 shows the results of measuring the number of surviving cells and spores (k/-) of each sample solution thus obtained. These results show that when sodium dichloroisocyanurate is used, bacterial cells and spores can be completely killed under slow conditions of a drug concentration of 0.2% and a temperature of 40°C. When similar operations were performed using potassium dichloroisocyanurate and trichloroisocyanuric acid, the drug concentration was 0 for potassium dichloroisocyanurate.
．． Trichloroisocyanuric acid was able to kill bacterial cells and spores at a drug concentration of 0.15% and a temperature of 40'C, respectively.

(Margins below) Example 2 Dispense 5 d of the same cultured solution as in Example 1 into test tubes, add sodium dichloroisocyanurate (so that the concentration at the time of mixing is 0.2%), and dichloromethane. Potassium isocyanurate (0.2%) and trichloroisocyanuric acid (0.15%) were mixed together for 50 minutes.
After heating for 10 minutes in the range of 10'C to 80°C in increments of 10'C, the mixture is immediately cooled. This solution is centrifuged in the same manner as in Example 1.

The numbers of surviving cells and spores and residual insecticidal activity of each sample solution obtained in this way are shown in Table 2.The results show that for these three types of drugs, the number of surviving cells and spores is θ
It can be seen that under the conditions of a1, the lower the heat treatment is performed, the higher the residual insecticidal activity can be obtained.

- (Margins below) - Table 2 Values in the table...Residual insecticidal activity (half lethal concentration for diamondback moth (ρpm)) 1 Example 3 Bacillus thuringiensis varietus kurstaki HD-1 was added to the meat extract polypeptone in a Sakaro flask. Inoculate 50 d of culture medium (1% meat extract, 1% polypeptone, 0.5% sodium chloride: pH 7.0) and incubate at 30°C.
Culture with shaking for 3 days.

Collect the culture solution (about pH 8.2) and measure its solid content (culture solution at 14,000 rpm, 4°C, 11
Centrifuge for 0 minutes, dry the residual scent at 100°C until it reaches a constant weight, and calculate its weight percentage) and add H, S at an appropriate concentration.
o, using a solution and sterile water containing the same amount of cultured solid content per unit weight and with a pH in the range of 4.0 to 8.0.
．． Adjust the level in increments of 5. This solution 5Id
Dispense each into test tubes, and add sodium dichloroisocyanurate or potassium dichloroisocyanurate to them so that the concentration in the mixture is 0.2%, respectively, at 40°C.
After heating for 10 minutes at C, cool immediately. This solution is subjected to the same centrifugation operation as in Example 1.

The number of surviving cells/spores and residual insecticidal activity of the sample solution thus obtained are shown in Table 3. Based on this result, when the above two agents are used as bactericidal agents, the pH after culturing is 5.
．． It can be seen that the highest residual insecticidal activity is obtained when adjusted to 5. Similar results were also obtained when trichloroisocyanuric acid was used.

(Left below) PDCI Potassium dichloroin cyanurate Example 4 The same cultured solution as in Example 3 was heated to an appropriate concentration of Hz S0.
After adjusting the pH to 5.5 using 4 solution and sterile water, it was placed in a Hitachi centrifugal rotor (capacity If), and the rotation speed was varied (power acceleration: 1.000Xg to 7.000X).
g) Perform batch centrifugation (4°C for 110 minutes). After centrifugation, remove the supernatant and calculate the removal rate of water-soluble components (concentration ratio = ×11 ×2, ×5, ×10, ×15, ×20)
After obtaining different concentrated solutions, ultrasonication (output 40 WSBRANSON
Using a SONIFIER 450 ultrasonic generator: While cooling the sample in ice water, the test was performed for a total of 5 minutes with an interval of 30 seconds of irradiation and 30 seconds of rest). Mix them with 5d of trichloroisocyanuric acid solution, set the concentration of trichloroisocyanuric acid in the mixture appropriately, and heat at 40°C.
After heating for 10 minutes, the same centrifugation operation as in Example 1 is performed.

The number of surviving cells/spores and residual insecticidal activity of the sample solution obtained in this way are measured, and the minimum value of the concentration of trichloroisocyanuric acid in the mixed solution at which the number of surviving cells/spores is Oke/d is determined. The residual insecticidal activity of the same method is shown in Table 4. From this result, compared to when the culture solution is sterilized as it is, the amount of drug used in terms of culture solid content is 1/9.4 when concentrated 20 times.
Since this can be significantly reduced, the residual insecticidal activity per culture solid content can be dramatically increased.

(Left below) Example 5 Bacillus thuringiensis varietus kurstaki HD-1 was placed in a medium in a Sakaro flask (meat extract 0.5
%, polypeptone 0.5%, corn steeple force -1%
, glucose 1%: pH 7,0) inoculated into 50Id and 30
Incubate for 54 hours at °C. The same culture solution was adjusted to pH 5.5 in the same manner as in Example 3, and 5d was added with a 0.4% solution of sodium dichloroisocyanurate, and for comparison, 0.2% paratoluenesulfone chloramide. Melting W1. , N-chlorosuccinimide 0.4% solution, sodium hypochlorite 0.08% solution (effective chlorine concentration conversion)
, a 0.5% hydrogen peroxide solution, and a 2% formalin solution were added at sId each and heated (see Table 5 for treatment conditions), followed by centrifugation in the same manner as in Example 1.

Table 5 shows the number of surviving cells/spores and residual insecticidal activity of each sample solution thus obtained. These results show that among these six types of chemicals, the use of sodium dichloroisocyanurate has the highest residual insecticidal activity.

Example 6 Bacillus thuringiensis varietus israelensis strain HD-522 was inoculated into heat-sterilized culture solution 1042 containing 1% meat extract, 1% polypeptone, 0.5% yeast extract and pH 7.0, and the culture temperature 30°C1 ventilation rate 0
．． 5 vvm under the conditions of stirring speed 350 rpa+
Cultivate for 4 hours. Dispense 5 d of this cultured solution (about pH 8.4) into test tubes, and the concentrations of sodium dichloroisocyanurate, potassium dichloroisocyanurate, and trichloroisocyanuric acid in the mixture are 0 to 0.
．． The mixture is mixed in a range of 3% in 0.05% increments, heated for 10 minutes in a range of 20 to 70°C in 10°C increments, and then cooled immediately.

The same centrifugation operation as in Example 1 is performed on these treated solutions.

The number of surviving cells and spores in each sterilized solution thus obtained is measured. As a result, the conditions for the number of surviving cells/spores to be 0 cells/spores were as follows: sodium dichloroisocyanurate concentration in the mixed solution was 0.15%, heating temperature was 40°C, heating time was 10 minutes, and sodium dichloroisocyanurate was Potassium was 0.15% at 40°C for 110 minutes, and trichloroisocyanuric acid was 0.1% at 40°C for 110 minutes.

Example 7 The same procedure as in Example 3 was carried out using the same culture-finished solution as in Example 6 to adjust the pH to a level in the range of 4.0 to 8.0 in 0.5 increments. Dispense 5 ml of the above culture solution into test tubes, add sodium dichloroisocyanurate to the mixture to give a concentration of 0.15%, and incubate at 40°C for 10 ml.
Heat for a minute. Similarly, for comparison, N-chlorosuccinimide was added to the mixture so that the concentration was 0.2%.
Heat at 0°C for 10 minutes.

Each of the sterilized liquids thus obtained was diluted with sterile water to prepare various graded diluted liquids, and the residual insecticidal activity was measured using third instar larvae of Culex mosquito. Table 6 shows the results.
Shown below. Based on this result, the pH of the culture solution on both sides was adjusted to 5.
．． It can be seen that the highest residual insecticidal activity is obtained when adjusted to 5.

It can also be seen that the residual insecticidal activity is considerably higher when sodium dichloroisocyanurate is used than when N-chlorosuccinimide is used.

(Left below) Table 6 SI N-Chlorsuccinimide Example 8 The same culture solution as in Example 6 was adjusted to pH in the same manner as in Example 4.
5,5, the removal rate of water-soluble components (411 ratio:
×1, ×5, ×20) different concentrated culture solutions for 5 d
Dispense each into test tubes. To this, trichloroisocyanuric acid and sodium paratoluenesulfone chloramide were added for comparison, and sterilization was performed at the levels shown in Table 7.

Table 7 shows the measurement results of the residual insecticidal activity of the same treatment solution (using Culex pipiens). From this result, compared to when the culture solution was sterilized as it was, the amount of drug used in terms of culture solid content was 1/8.3 of trichloroisocyanuric acid and 1/7 of sodium paratoluenesulfone chloramide when concentrated 20 times. .1, both of which can be significantly reduced, making it possible to dramatically increase the residual insecticidal activity per culture solid content. Note that higher residual insecticidal activity was obtained when trichloroisocyanuric acid was used than when sodium paratoluenesulfone chloramide was used.

(C) Effects of the Invention The method of the present invention can completely kill bacterial cells and spores in a culture solution containing crystal toxin of BT bacteria while maintaining high insecticidal activity, and also prevents secondary damage caused by living spores. This has the excellent effect of making it possible to industrially and stably produce a safe and highly effective BT pesticide.

Claims (1)

[Claims] 1. Bacillus thuringiensis
1. A method for producing an insecticide, which comprises killing viable spores and biovegetative cells present in a culture solution of P. thuringiensis using chlorinated isocyanuric acid or a salt thereof.