KR900001513B1 - Method for culture of bacillus thuringiensis - Google Patents

Abstract

The method for preparing a microbial insecticide by Bacillus thuringiensis isolated in soils is presented. Thus, B.thuringiensis is seed-cultured in TPB (Triptose Phosphate Broth) at 30oC for 12hrs. The main culture is carried out in the production medium containing 25g/l of glucose, 15g/l of corn starch, and trace elements consisted of 1g/l of KH2PO4, 1g/l of K2HPO4, 0.3g/l of MgSO4.7H2o, 1.0g/l of CaCO3 0.02g/l of CuSO4.5H2O, 0.04g/l of MnSO4, 0.01g/l of FeSO4.7H2O and 0.02g/l of ZnSO4.7H2O. The culture condition is as follows: Temp. is 28-32oC and initial Ph being 6.8 - 7.2. The initial aeration is 0.5 - 1.0v.v.m.

Description

Manufacturing method of microbial insecticide

The present invention relates to a method for preparing insecticides containing insecticidal substances capable of killing worms (mosquito larvae) and mosquito larvae by BACILLUS THURINGIENSIS obtained by separating soil samples.

In general, worms are also important in terms of national health because they are used as a carrier of many infectious diseases as larvae of mosquitoes. In addition, mosquito larvae are known as pests that are particularly problematic during the direct weaving of rice in the tideland.

On the other hand, the study of microbial insecticides has become more and more interested in serious problems such as increased pest resistance to insecticides due to the use of a wide range of chemical insecticides, the emergence of secondary pests, destruction of the ecosystem by the toxicity of the remaining pesticides. . Microorganisms mainly used as microbial insecticides include bacteria and viruses, and bacterial pesticides have been studied and put into practical use.

Bacillus thuringiensis is a Gram-positive bacterium that forms follicles and is known to produce various types of endotoxins and exotoxins along with follicle formation.It acts as a target pest, showing high lethal effect and being harmless to humans and animals and plants. Even known to be safe.

Until now, a number of methods for preparing pesticides using Bacillus thuringiensis are known. For example, US Pat. Nos. 3,073,749, 3,086,922, 3,087,865, 4,133,716, 4,206,281, 4,277,564, Japanese Patent Nos. 49-4364, and 52-17089.

The present inventors confirmed that the strain isolated from the soil is a very high strain, and in particular, the isolated strain in the soil has a strong insecticide against worms and mosquito larvae to develop a production medium and culture method thereof, insecticides The present invention has been completed, which is a method for producing a microbial insecticide containing.

The strain used in the present invention was isolated and named by the following method. (See US Patent No. 4,206,281.) That is, 1,684 isolates of soil isolates nationwide were separated by soil sample collection teams, and strains with high toxic effects on worms and mosquito larvae were isolated. The strain was selected and named Bacillus thuringiensis HB 101. This strain has been deposited with the Korean spawn association. (Deposit number 87.5.26, accession no. KFCC-10372)

The bacterium toxicity of Bacillus thuringiensis HB 101 of the present invention according to the Burgess manual is as follows.

1) Morphological Properties

(1) Form: Bacillus, (2) Gram Dyeing: Positive, (3) Mobility: Yes, (4) Endotoxin Production Capacity: Yes.

2) physiological properties

(1) Optimal growth temperature: 28-32 ℃

(2) Growth optimum pH: 6.8-7.2

(3) Oxygen Requirements: Aerobic

(4) Indole production capacity: negative

(5) Maryled: positive

(6) Acid production capacity from glucose: Yes

(7) Hydrolysis ability of casein, gelatin and starch: Yes

(8) urease: voice

(9) Catalase: Voice

3) Culture property

(2) Nutritional medium: Good growth

4) Biochemical Comparison of Known and Invented Bacillus Turingiensis Strains

TABLE 1

Comparison of culture and physiological characteristics

+: Positive,-: negative

On the other hand, the pesticides accumulated in the culture were assayed for titer by bioassay. The bioassay was performed with larvae and mosquito larvae, which are larvae of mosquitoes. It was.

The assay was performed by US Evotrap.

(Can. J. Microbiol. 28, 1089-1092 (1982)).

As described above, in order to powder the culture medium in which the insecticide is accumulated, a spray drying method or a freeze drying method may be used, but a spray drying method is industrially preferable.

In this method, a powder can be obtained by adding an electrodeposition agent, a stabilizer, a diluent, etc. at the time of drying.

In addition, it can be formulated as a powder, a liquid, or the like.

On the other hand, by synthesizing microbial insecticides with other pesticides, fungicides can be expected synergistic, it is possible to widen the pesticide range.

In addition, the pesticidal titers of Bacillus thuringiensis HD 567 (KFCC 11432), the standard strain, and Bacillus thuringiensis HB 101 (KFCC 10372), which are used in the present invention, are as follows.

Comparative Example 1

Species culture: Platinum is seeded in 500 ml Erlenmeyer flask using TPB, and cultured for 12 hours at 30 ℃ to make species culture.

Production medium composition and preparation method.

Medium soy flour: 5g / 1

Yeast Extract: 1g / 1

Magnesium Sulfate, Heptahydrate 0.3g / 1

Calcium Carbonate: 0.3g / 1

[Table 2]

Hereinafter, the gist of the present invention will be described in detail by way of examples.

Example 1

Use strain: Bacillus thuringiensis HB 101 of the present invention (KFCC 10372)

Species culture: TPB (Tryptose Phosphate Broth) is used as a medium. A 500 ml Erlenmeyer flask is used with 100 ml of spawn HB 101 and incubated at 30 ° C. for 12 hours, followed by 2% inoculation to make a secondary seed culture.

[Production medium composition and preparation method]

: 25 g of glucose

Corn starch powder 15g / 1

Soy flour 30g / 1

Trace ingredient

: Potassium monophosphate 1g / 1

Potassium Diphosphate 1g / 1

Magnesium Sulfate Heptahydrate 0.3g / 1

Potassium Carbonate 1.0g / 1

Copper sulfate pentahydrate 0.02 g / 1

Manganese Sulfate 0.04g / 1

Ferrous Sulfate Heptahydrate 0.01g / 1

Zinc Sulfate Heptahydrate 0.02g / 1

The heat stable medium component is autoclaved at 120 ° C. for 20 minutes, and the component which is unstable to heat or generates a complex compound is aseptically added to the fermenter after passing through a sterile filter paper having a pore size of 0.45 μm.

[Cultivation method]

: 10 liters of medium to be inserted into a 14 liter fermenter was inoculated with 200-300 ml of the seed culture solution at an initial pH of 6.8-7.2 at 29-32 ° C., aeration rate of 0.5-1.0vvm, and agitation at 500-700r.pm. Incubate with.

After 40 hours of inoculation, the culture is terminated.

The bioassay results over time are as follows.

Table 3

Bioassay results according to incubation time

Example 2

Same as Example 1, the production medium composition is as follows.

The maximum toxin activity values are as follows.

Caterpillar: 6.70

Mosquito larvae: 3.5

As described in Table 2, the insecticide of the present invention has excellent insecticidal effects on worms and mosquito larvae than known conventional strains, so that the titer is increased as an insecticide and can be widely used in related industries.

Claims (1)

Toxin accumulation medium using Bacillus thuringiensis HB 101 (KFCC 10372) is composed of glucose 25g / 1, corn starch powder 15g / 1, mostly powder 1g / 1 Potassium diphosphate 1g / 1, magnesium sulfate heptahydrate 0.3g / 1, calcium carbonate 1.0g / 1, copper sulfate pentahydrate 0.02g / 1, manganese sulfate 0.04g / 1, iron sulfate heptahydrate 0.01g / 1, zinc sulfate 7 Hydrate 0.02g / l and incubation conditions at 28-32 ℃ initial pH is 6.8-7.2 aeration rate 0.5-1.0vvm rotational agitation is incubated at 500-700r.pm, so that the worms and mosquito larvae than known fungi Method for preparing microbial insecticides with improved insecticide titers.