KR101800330B1 - Culture method for improving spore formation of Beauveria bassiana - Google Patents

Abstract

The present invention relates to a liquid medium composition and a culture condition for improving spore formation of an insect pathogenic fungus ( Beauveria bassiana ). More specifically, the present invention relates to a liquid medium composition and a culture condition for culturing the fungus, wherein a conventional SDY (glucose, peptone, yeast extract) (NH ₄ ) ₂ SO ₄ ) extracted by sterilization of glucose, yeast extract, MgSO ₄ .7H ₂ O, K ₂ HPO ₄ and ERBM in water (1: 9) at 121 ° C for 15 minutes As a result of comparing the cultures, it was confirmed that the growth and spore formation of the germs were more than 10 times better than the SDY medium when cultured in the GYMK medium at pH 5.0 and 25 ° C. In order to improve the dispersibility in the culture, 2% glycerol ) Is stable to the growth of bacteria, has no effect on the color change of medium, and is effective for spore formation. Thus, it can be usefully used as the optimum liquid medium composition and culture condition of the fungus.

Description

TECHNICAL FIELD [0001] The present invention relates to a liquid medium composition for improving spore formation of an insect pathogenic fungus,

The present invention relates to a fungus culture medium composition and a culture method, and more particularly, to a fungus culture medium composition comprising a fungus belonging to the genus Beauveria bassiana ) and a culture method for improving spore formation using optimal culture conditions.

Beauveria bassiana (Beauveria bassiana) is a strain cultivated in the Department of Agricultural Microbiology, National Institute of Agricultural Science and Technology (RDA), and has been shown to have insecticidal effect against aphids.

In general, Beauveria bassiana is an incomplete generation of conidial fruiting body, which is attached to the surface of the outer wall of the insect and germinates after passing through the cuticle layer as an intruding tool, It passes through the cuticle layer and enters the inside of the insect. At this time, the action of lipase, protease, chitinase and other enzymes occurs simultaneously. Fungi that enter the inside of the insects may grow as mycelium or diapause of the mycelium from the extended germination tube to form spores and secrete toxic metabolites (cyclodepsipeptide, beauvericin) or deplete the nutrients of the bloodstream to kill the host. After the insect has died, insects such as mummies, which continue to grow and mycelium filled with insects, grow and store as mycelium in dry or cold environments. When the environment grows, they break through the cuticle of insects It grows and forms a spore layer on the carcass. The fungus that grows in this way infects other insects in the vicinity by water or wind. There is also a secondary insecticidal effect, in which the infected insects die but the fertility is reduced or the infected larvae are born (Korean Biotechnology Journal, 14 (3), 365 ~ 370, 1999; ~ 35, 1996; Korean Patent No. 10-2011-0094749).

Pesticides for control of pests of crops are known to cause side effects such as the development of resistance to pests and environmental pollution during prolonged or excessive use. In order to solve these problems, pest control using microorganisms has been suggested as an alternative. Fungicide insecticides registered for pest control worldwide have been used for controlling insects such as moths, beetles, aphids, and powdery moths. Three types of fungicide are registered in Korea. When insect pathogenic fungi are applied to crops for pest control, spray with high concentrations of spores. Therefore, mass production of insecticidal fungus spores is a key issue that should be solved first in developing mold insecticides. In general, three methods (solid culture, liquid culture, two-phase fermentation of liquid culture and solid culture) are used for mass culture of insect pathogenic fungus spores. Solid culture is the most commonly used method to produce spores of insect pathogenic fungi. Grains such as barley and rice are used as substrates. Liquid culture is a method mainly used for production of blastospore. It has been reported that addition of glucose, lecithin to the medium increases the production of spores of insect pathogenic fungi. In the case of Beauveria brongniartii , when the yeast extract and peptone were added to the liquid medium, spore production was increased, and in the medium supplemented with casamino acid, ( Metarhizium anisopliae ) not only increased spore production but also increased the virulence of spores. In addition, a representative strain of insecticidal microorganism, Beauveria bassiana ) are mainly used in laboratory scale cultures with PD (potato dextrose broth) medium. However, in a large-scale culture condition for commercialization, it is not economical to cultivate using a PD medium. Therefore, it is required to develop a medium that is excellent in economy and can be easily obtained.

Insect pathogenic fungus spores are generally cultured in solid or liquid culture to form spores while growing hyphae on the surface. The spore concentration of the insect pathogenic fungi varies depending on the species of each species and the composition of the medium. Conventionally, a lot of studies have been conducted on the solid culture by the method of spore production of fungi (J. Life Science, 23 (8) Korea Institute of Bioscience and Biotechnology, Korea Institute of Bioscience and Biotechnology, 14 (3), 365 ~ 370, 1999, Kangwon National University, Doctoral thesis, Shin Sang Chul, 2001, Korean J. Microbiol. Biotechnol. 41 (4), 398-406, 2013), there is little known about the prior art for the conditions under which spore formation is good in liquid culture.

Generally, it is easy to obtain spores by cultivating solid from cereals such as rice bran, which is a byproduct of agricultural use. However, in order to develop as an agricultural pest control agent, problems such as clogging of the spray nozzle occur, so development of powder wettable powder have.

In addition, in the conventional liquid medium, it is generally aimed at the growth of mycelium rather than the formation of spores, so it is common to cultivate the spore by mixing the liquid medium with the mycelium in a solid medium such as rice bran. However, in order to formulate into a wettable powder, it is impossible to cultivate in the above manner, so that a high concentration of spores is produced in a liquid medium, and cultivation of a single mycelium is carried out.

In order to commercialize a large number of insect pathogenic fungus strains, the conventional culture medium will have an economic burden on raw material costs, which will hinder industrialization. It is difficult to use the existing medium such as PDB or SDB because the existing medium has a longer incubation period as compared with the medium developed in the present invention. In this case, if the culture days of the culture medium depend on the difference of the culture medium, the additional cost is not negligible. Accordingly, the present invention provides a spore culture suitable for culturing an insecticidal strain in a liquid medium in which spore formation is optimized by using high-efficiency components capable of inducing a low spore concentration without using an existing medium (preparation medium) The aim of this study was to select the optimal culture medium for growth.

In addition, spore formation is not easy because the existing medium has a problem that the concentration of spores unsuitable for mass culture and single hyphae are not easy to cultivate.

Thus, the present inventors have baekganggyun (Beauveria In order to establish a culture medium for mold spores during liquid cultivation of shaking culture method, conventional SDY (glucose, peptone, yeast extract) medium and GYMK glucose, yeast extract, MgSO ₄ .7H ₂ O, K ₂ HPO ₄ ) and ERBM (1: 9) at 121 ° C for 15 minutes, (NH ₄ ) ₂ SO ₄ ) As a result, it was confirmed that the growth of bacteria and spore formation were excellent when cultured at pH 5.0 and 25 ° C in GYMK medium at 130 rpm for 5 days, and the present inventors completed the present invention by establishing the optimal culture conditions and culture conditions for the fungus bacterium .

It is an object of the present invention to provide an optimal liquid medium composition and culture conditions for improving spore formation of Beauveria bassiana in order to overcome ineffective and uneconomical problems in the mass production of insect pathogenic fungus spores under existing culture medium and culture conditions , And a method for mass production of spore bacterium using the same.

In order to achieve the above object, the present invention provides a liquid medium composition for improving spore formation.

Yet another object of the present invention is to provide a method for producing a yeast extract which comprises culturing yeast extract in a liquid culture medium (GYMK (glucose, yeast extract, MgSO ₄ .7H ₂ O, K ₂ HPO ₄ ) medium according to the present invention at pH 5.0 and 25 ° C A culture method for improving spore formation is provided.

The invention baekganggyun (Beauveria bassiana ) were cultured at pH 5.0 and 25 ℃ using GYMK medium (glucose, yeast extract, MgSO ₄ .7H ₂ O, K ₂ HPO ₄ ), and compared to conventional SDY medium (glucose, peptone, yeast extract) (NH ₄ ) ₂ SO ₄ ) than in the ERBM medium (water (1: 9) for 15 min at 121 ° C for 15 min.). And 2% glycerol was added to improve the dispersibility during culture, and it was stable to the growth of the microorganism, had no effect on the color change of the medium, and was also effective for spore formation. Thus, the present invention can be used to obtain a large amount of fungus spores by establishing the optimum liquid medium composition and culture conditions of the fungus.

Brief Description of the Drawings Fig. 1 is a graph showing the change of medium color on three days and four days after incubation of three kinds of GYMK medium on Beauveria bassiana .

Hereinafter, the present invention will be described in detail.

The present invention relates to a pharmaceutical composition comprising 1 to 3% (w / v) glucose, 0.1 to 0.3% (w / v) yeast extract, 0.004 to 0.006% (w / v) MgSO ₄ .7H ₂ O, K ₂ HPO _{4 in an amount of} 0.04 to 0.06% (w / v), in order to improve the spore formation of Beauveria bassiana .

The liquid medium in the composition glucose 2% (w / v), 0.2% yeast extract _{(w / v), MgSO 4} .7H 2 O 0.005% (w / v), K 2 HPO 4 0.05% (w / v) More preferably, it is configured.

Further, it is preferable to further include 1.5 to 2.5% (v / v) of glycerol, more preferably 2%.

In addition,

1) the glucose 1 to 3% (w / v baekganggyun), yeast extract from 0.1 to 0.3% (w / v), MgSO 4 .7H 2 O 0.004 to 0.006% (w / v), K 2 HPO 4 0.04 to 0.06 % (w / v); And

2) culturing the liquid medium of step 1) at pH 4.7 to 5.2 and 22 to 28 ° C at 100 to 150 rpm for 3 to 7 days; The present invention provides a culture method for enhancing spore formation of a white germ strains.

Liquid medium of step 1) is glucose 2% (w / v), 0.2% yeast extract _{(w / v), MgSO 4} .7H 2 O 0.005% (w / v), K 2 HPO 4 0.05% (w / v).

In a particular embodiment of the invention, baekganggyun (Beauveria Mass production selected for the medium and culture conditions, the results for the bassiana), GYMK medium (glucose 2% than the conventional SDY medium (glucose, peptone, yeast extract) , yeast extract 0.2%, MgSO 4 · 7H 2 O 0.005%, K 2 HPO ₄ 0.05%) and ERBM medium (the extract, (NH ₄ ) ₂ SO ₄ , which was extracted with sterilized water (1: 9) for 15 minutes at 121 ° C) (See Table 2). ≪ tb >< TABLE >

Also, when comparing GYMK medium and ERBM medium, it was confirmed that GYMK medium was suitable as a culture medium for massive culture of Bacillus thuringiensis as a result of culturing using GYMK medium.

In addition, 2% treatment of glycerol to improve the dispersibility of fungal spores was found to be effective for spore formation without affecting the color change of the medium (see Table 4).

Thus, the optimum liquid medium composition and culturing method of the present invention was pH 5.0, 25 (g / 100 ml) with a composition of GYMK medium (glucose 2%, yeast extract 0.2%, MgSO ₄ .7H ₂ O 0.005%, K ₂ HPO ₄ 0.05% Lt; 0 > C and 130 rpm for 5 days, this can be usefully used to obtain large quantities of insecticidal fungus spores.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example  1> Bulk Culture medium  And establishment of culture conditions

Beauveria bassiana ) for the mass production and culture conditions. The Bacillus thuringiensis was distributed from the Department of Agricultural Microbiology, National Institute of Agricultural Science, RDA.

Specifically, the spore forming ability of GYMK medium and ERBM medium was examined by cultivating an insect pathogenic fungus, Bacillus subtilis, using SDY medium as a control group. Each medium was placed in a 500 ml Erlenmeyer flask in the composition shown in Table 1 below, and the medium was sterilized at 121 ° C for 15 minutes. Spore suspension was prepared by scraping the spores with sterilized distilled water in advance in a medium in which platelets were cultured on a plate, and 80 쨉 l of each spore suspension was inoculated to each sterilized liquid medium. The suspension was cultured with shaking at 25 캜 and 130 rpm for 8 days. The pH of the culture medium was adjusted by adjusting the pH to 5 and the pH of the pure medium.

In addition, the final culture medium was selected by measuring the number of viable cells (cfu / ml) and measuring the amount of cells (g) by the method of measuring the number of spores using a hemocyte counter, the step dilution plate method.

The hemocyte counting method was performed by observing the number of spores present in the compartment displayed in the hemocyte counter by a microscope and measuring the number of spores by diluting the culture with 10 times the amount of the culture solution and then culturing the appropriate dilution concentration on the PDA medium Colonies were measured. For the amount of cells, the cultured medium was filtered using a filter paper, and the cells were completely dried and weighed.

badge Furtherance Content (g or%) Whole unit SDY Glucose
Peptone
Yeast extract 40
10
10 L GYMK Glucose
Yeast extract
MgSO ₄ .7H ₂ O
K ₂ HPO ₄ 20
2
0.05
0.5 L ERBM Extract of rice bran by sterilization with water (1: 9) at 121 ℃ for 15 minutes
(NH _{4) 2} SO ₄ 95.5%

0.5% %

As a result, as shown in Table 2 below, it was found that the growth of the bacteria was good in the GYMK medium and the ERBM medium rather than the SDY medium. In addition, it was confirmed that the growth of bacteria and spore formation were excellent in the medium adjusted to pH 5.0 in the medium (Table 2).

In addition, gross differences were observed in the color of pale yellow (original color of the medium) changed to red in SDY medium and GYMK medium, and in spore-forming medium, it was confirmed that red medium was maintained. In ERBM medium, .

In addition, when the GYMK medium and ERBM medium were compared with each other, the number of bacteria was measured as a result of culturing using GYMK medium, and it was confirmed that contamination and maximum spore formation can be confirmed by changing the color of medium, And selected as a medium.

badge pH Step dilution plate method (cfu / ml) The hemocyte counter (spore / ml) Cell mass (g)
SDY 5.0 1.9 × 10 ⁶ 4.0 × 10 ⁶ 4.87 5.7 9.4 × 10 ⁵ 1.4 × 10 ⁶ 4.38
GYMK 5.0 3.1 × 10 ⁷ 5.9 x 10 ⁷ 4.81 6.7 2.3 × 10 ⁶ 1.0 × 10 ⁶ 4.56
ERBM 5.0 4.3 × 10 ⁶ 1.8 x 10 ⁷ 5.24 5.5 1.2 × 10 ⁶ 4.8 × 10 ⁶ 4.24

< Example  2> Identification of the relationship between the amount of spore formation and the contamination of media with changes in medium color

The color of GYMK medium (glucose 2%, yeast extract 0.2%, MgSO _{4 ·} 7H ₂ O 0.005%, K ₂ HPO ₄ 0.05%) was changed when cultured for 6 days. Therefore, the growth of the microorganisms, the degree of spore formation and the degree of contamination according to the color change of the medium were confirmed.

Specifically, 300 ml of GYMK medium was placed in a 500 ml Erlenmeyer flask, and the medium was sterilized at 121 캜 for 15 minutes. 80 쨉 l of a previously prepared spore suspension was inoculated and cultured at 25 캜 at 130 rpm. Then, it was repeated 3 times, and the number of bacteria, the number of spores and the pollution were checked every day from the third culture day to the fifth culture day.

As a result, as shown in Table 3 and Fig. 1, when there was bacterial contamination, the color of the medium did not change regardless of the culture date. However, there was no significant difference in the viable cell count from the day when the color of the medium changed to the next day, but the number of cells of the hemocyte counter increased significantly.

Experimental Method Experiment number One 2 3

Experiment of live cells (cfu / ml)
Three days after culture 1.8 ⁷ 3.1 ⁷ Not measurable
(Because bacteria did not grow due to bacterial contamination, viable counts and hemocyte count tests were not possible) Four days after culture 2.3 ⁷ 3.7 ⁷ After 5 days of culture 2.7 ⁷ 2.7 ⁷
The hemocyte counter (spore / ml)
Three days after culture 9.0 ⁷ 7.6 ⁷ Four days after culture 2.1 ⁸ 1.6 ⁸ After 5 days of culture 1.1 ⁸ 1.3 ⁸

< Example  3> Selection of supplements

Since the surface of mold spores is highly hydrophobic and difficult to disperse in water, a surfactant or the like is used to improve the dispersibility. Therefore, the effect of glycerol and tween 20 (Tween 20), which are easy to use surfactants, on the growth of bacteria was investigated.

Specifically, the adjuvant (glycerol and tween 20) was treated with GYMK medium as a large-scale culture medium, and the medium was sterilized at 21 ° C for 15 minutes. Then, 80 μl of spore suspension was inoculated and cultured at 25 ° C. for 15 days at 130 rpm , Then the viable cell count was measured by the stepwise dilution plate method, and the cell number was measured with a hemocyte counter.

As a result, as shown in Table 4, it was confirmed that glycerol was more stable to the growth of bacteria than tween 20, and in particular, it was confirmed that 2% treatment of glycerol was effective for spore formation without affecting the color change of medium (Table 4).

Supplements % Reputation (cfu / ml) The hemocyte counter (spore / ml)
Glycerol
2 4.0 ×× 10 ⁷ 2.7 × 10 ⁸ 5 6.1 × 10 ⁷ 1.6 x 10 ⁸ 20 7.2 x 10 ⁴ Not verifiable
Tween 20 (Tween 20)
0.1 1.7 × 10 ⁷ 5.2 × 10 ⁷ 0.2 7.7 × 10 ⁶ 2.8 × 0 ⁷ 0.5 4.6 × 10 ⁶ 5.5 × 10 ⁷

Thus, the above-mentioned result shows that Beauveria bassiana , which is an insect pathogenic fungus, can be treated with GYMK medium (glucose 2%, yeast extract 0.2%, MgSO ₄ .7H ₂ O 0.005%, K ₂ HPO ₄ 0.05% , And when the culture was carried out at 25 DEG C and 130 rpm for 5 days, spore formation of the fungus and the growth of the bacterium were remarkable, and the culture conditions were established as the optimum culture conditions and conditions.

Claims (3)

1 to 3% (w / v) glucose, 0.1 to 0.3% (w / v) yeast extract, 0.004 to 0.006% (w / v) MgSO ₄ .7H ₂ O, K ₂ HPO ₄ 0.04 to 0.06% (w / v) baekganggyun liquid medium composition for the improvement of spore formation (Beauveria bassiana) consisting of.
The liquid medium composition for improving spore formation according to claim 1, further comprising 1.5 to 2.5% (v / v) of glycerol.
1) the glucose 1 to 3% (w / v baekganggyun), yeast extract from 0.1 to 0.3% (w / v), MgSO 4 .7H 2 O 0.004 to 0.006% (w / v), K 2 HPO 4 0.04 to 0.06 % (w / v); And
2) culturing the liquid medium of step 1) at pH 4.7 to 5.2 and 22 to 28 ° C at 100 to 150 rpm for 3 to 7 days; Wherein the spore formation is carried out in a culture medium.

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