KR100835689B1 - Method for Enhancing the Thermal Tolerance of Entomopathogenic Fungal Spores, blastospores and enzymes - Google Patents

Abstract

The present invention relates to a method for maintaining the activity of spores and enzymes produced by an insect pathogenic fungus, and more particularly, to a method for maintaining the activity of spores and enzymes produced by an insect pathogenic fungus by adding a powder obtained by adsorbing an enzyme derived from an insect pathogenic fungus spore powder or an insect pathogenic fungus with a mineral adsorbent, And preserving the spores and enzymes produced by the insect pathogenic fungus.

The method of maintaining the activity of the spores and enzymes produced by the insect pathogenic fungus according to the present invention was evaluated to be a method for effectively maintaining the activity of the spores and enzymes produced by the insect pathogenic fungus usable as the material of the microbial pesticide, It is expected that it can be effectively applied to mass production and formulation of highly active microbial pesticides.

Insect pathogenicity, fungi, spores, enzymes, active maintenance

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for maintaining the activity of spores and enzymes produced by insect pathogenic fungi,

1 is Beauveria for the practice of this invention sp. DBB2507 is a graph showing the thermal stability of the spore in water. (Left: Spore, Right: Apoja)

Figure 2 is a graphical representation of a Beauveria < RTI ID = 0.0 > sp. DBB2507 is a graph showing the stability of spores by oil type. (Left: room temperature condition, right: heat treatment condition)

Figure 3 is a graphical representation of a < RTI ID = 0.0 > Beauveria & sp. DBB2507 is a graph showing the thermal stability of the spores in the heat treatment time in oil phase. (Left: Spore, Right: Apoja)

Figure 4 is a graphical representation of the < RTI ID = 0.0 > Beauveria & sp. This graph shows the thermal stability of chitinase produced by DBB2507.

Figure 5 is a graphical representation of the < RTI ID = 0.0 > Beauveria & sp. This is a graph showing the yield of chitinase produced by DBB2507 during centrifugation.

Figure 6 is a graphical representation of the < RTI ID = 0.0 > Beauveria & sp. This graph shows the adsorption capacity of enzyme adsorbents for chitinase produced by DBB2507.

Figure 7 is a graphical representation of the < RTI ID = 0.0 > Beauveria & sp. FIG. 4 is a graph showing the thermal stability of the enzyme-adsorbed powder against chitinase produced by DBB2507 in oil phase. FIG.

The present invention relates to a method for maintaining the activity of spores and enzymes produced by an entomopathogenic fungi usable as a material for microbial pesticides.

Recently, problems such as increased tolerance of insects due to excessive use of chemical insecticides, ecosystem confusion, and concern about adverse effects on human body have been raised. As an alternative, interest in the development of microbial pesticides using insect pathogenic microorganisms in the natural world is increasing.

Biological pesticides including microorganisms can be said to be biologic agents used to effectively control pests such as insects, mites, nematodes, and various plant pathogens or weeds, which directly use microorganisms or use microorganism-containing preparations to harm crops .

Recently, the role of chitinase in fungi, bacteria, and plants has been clarified. As a result, interest in chitinase has been increasingly concerned with the ecological interaction between chitinase and biocontrol.

Biological pesticides, including insect pathogenic fungi, are less toxic and less vulnerable than organic synthetic pesticides, have less impact on ecosystems, and do not exhibit tolerance to or resistance to drugs. However, since the activity of the enzyme produced by fungal spores or fungi during commercialization is not maintained for a long time, the disadvantage of short circulation period is being questioned.

It is an object of the present invention to provide a method for effectively maintaining the activity of spores and enzymes produced by an insect pathogenic fungus usable as a material for microbial pesticides.

In the present invention, spores generally tend to be less active due to moisture. In order to prevent the spores from being exposed to the maximum moisture, the storage medium for oil is examined to improve the stability. In the case of chitinase, In order to confirm the tendency of the activity to decrease when dissolved in water, and to reduce the contact with water, the enzyme dissolved in the water was first pulverized and stored in the selected oil to improve the stability.

The present invention relates to a method for maintaining the activity of spores and enzymes produced by insect pathogenic fungi, and more particularly, to a method for maintaining the activity of spores and enzymes produced by insect pathogenic fungi, The present invention also relates to a method for maintaining the activity of spores and enzymes produced by an insect pathogenic fungus.

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The vegetable oil of the present invention may be selected from soybean oil, olive oil, cotton seed oil or corn oil, preferably olive oil, Can be selected.

In addition, the insect pathogenic fungus-derived enzyme-adsorbing powder of the present invention can be obtained by lyophilizing a pellet formed by adding an enzyme adsorbent to a centrifugal supernatant of a fungal strain culture. The drying process is not limited to lyophilization, and it will be obvious that a person skilled in the art can employ a known drying process if necessary. The enzyme adsorbent of the present invention may preferably be kaolin.

The insect pathogenic fungi-derived enzyme of the present invention may be chitinase, but is not limited thereto.

On the other hand, in the present invention, a new insect pathogenic fungus Beauveria sp. DBB2507 strain (Accession No .: KFCC 11378) was used to test the method for maintaining the activity of the spores and enzymes derived from the fungus

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the structure of the present invention will be described in more detail with reference to preferred embodiments. However, these embodiments are only for illustrating the present invention specifically, and the scope of the present invention is not limited by these embodiments. The scope should be limited only by what is stated in the claims.

Example 1: Maintaining the activity of spores produced by insect pathogenic fungi

1-1. Spore Thermal stability  Confirmation test

Beauveria sp. DBB2507 strains (KFCC 11378) were cultured on SDA + Y agar (Saubraud dextrose medium + 0.5% weight yeast extract) medium and SDA + Y broth liquid culture medium to produce spores and blastospores, respectively. And then lyophilized and used for the test. Spore and apo-sporosate suspension was stored in a 50 ° C incubator for 60 minutes and 120 minutes, and then 10 μl was suspended in SDA + Y agar as a suspension. After incubation at 28 ℃ for 12 hours, spores germinated in 100 spores were investigated to investigate the spore germination rate.

Both spores and blastospores produced by insect pathogenic fungi were significantly lower in spore germination rate of about 10% after one hour at 50 캜 heat treatment conditions (Fig. 1).

1-2. Stability of spore oil at room temperature condition Thermal stability  Comparative test

Beauveria sp. DBB2507 The strain was cultured in SDA + Y agar for 3 weeks. The spores were harvested with a brush and lyophilized to prepare a spores powder to be used for the test. The spore powder is mixed with various kinds of oils such as soybean oil, olive oil, cotton seed oil, corn oil, mineral oil and methyl oleate. And stored for 120 minutes at room temperature (25 ° C) and heat treatment conditions (50 ° C incubator), and then 10 μl was suspended in SDA + Y agar as a suspension. After incubation at 28 ℃ for 12 hours, spores germinated in 100 spores were investigated and the germination rate of spores was compared.

The stability of spores present in oil phase at room temperature showed a spore germination rate of about 90% similar to that of untreated soybean oil, olive oil, cotton seed oil and corn oil (FIG. 2). In addition, at 50 ℃, the spore germination rate in olive oil was similar to that of non-heat treated germination (Fig. 2). Therefore, it was confirmed that the thermal stability was improved when the spore was stored in olive oil rather than water.

1-3. Spore Oil-phase  Heat treatment time Thermal stability  Confirmation test

Beauveria sp. DBB2507 strain was cultured on SDA + Y agar for 3 weeks, and spores were harvested with a brush and lyophilized to prepare spores to be used for the test. Spore powder was mixed in olive oil and stored for 60 minutes and 120 minutes in a heat treatment condition (50 ° C incubator), and 10 μl of the suspension was dispensed in SDA + Y agar. After incubation at 28 ℃ for 12 hours, spores germinated in 100 spores were investigated and the germination rate of spores was compared.

As a result, as shown in Fig. 3, it was confirmed that both the spores as well as the blastospores were stable to heat in the olive oil condition (Fig. 3).

<Example 2> Method for maintaining the activity of an enzyme produced by an insect pathogenic fungus

2-1. Enzymatic Thermal stability  Confirmation test

Beauveria sp. DBB2507 strain was cultured in SDA + Y broth for 3 days and centrifuged at 15,000 rpm for 5 minutes to prepare supernatant. The supernatant was stored in a 50 ° C incubator for 60 minutes and 120 minutes, and the activity of the chitinase was examined. The activity of chitinase was determined by mixing 100 μl of the enzyme solution, 100 μl of p-nitrophenyl β-DN-acetylglucosaminide (PNG) and 300 μl of 0.1M citrate-phosphate buffer, followed by reaction at 37 ° C for 1 hour. The absorbance of nitrophenol (405nm) was measured and examined.

As a result, insect pathogenic fungi ( Beauveria sp. DBB2507) showed a decrease from initial 4.2 unit / hr to 0.34 unit / hr after 2 hours at 50 ° C heat treatment (Fig. 4).

2-2. Examination of enzyme yield in centrifugation process

Beauveria sp. DBB2507 strain was cultured in SDA + Y broth for 3 days and centrifuged at 15,000 rpm for 5 minutes to separate the supernatant and the pellet. The pellet was again calibrated to the concentration of the culture medium before centrifugation with 0.1 M citrate-phosphate buffer. The chitinase activity of the supernatant and the centrifugal pallet suspension was measured.

In order to harvest chitinase produced by liquid culture, the centrifugation process is required, but the harvest efficiency by precipitating the enzyme present in the aqueous solution state only through the centrifugation process is as low as 4.5% (0.3 / 6.7) (Fig. 5).

2-3. Adsorbent selection test to improve efficiency of centrifugation of enzyme

Beauveria sp. DBB2507 strain was cultured in SDA + Y broth for 3 days and centrifuged at 15,000 rpm for 5 minutes to prepare supernatant. The enzyme adsorbents such as silicagel, cellulose, pyrophilite, skim milk, bentonite, celite, kaoline and polyvinyl alcohol were added to the supernatant at a concentration of 0.5% (w / v) Followed by centrifugation for one minute to obtain a pellet adsorbed by the enzyme. The enzyme adsorption rates of adsorbents were investigated by comparing chitinase activities of supernatant and enzyme adsorbed pellet.

In order to increase the harvest efficiency of chitinase, the enzyme adsorption rate was confirmed by using minerals and polysaccharides. Among the candidate compounds, kaoline treatment showed the highest chitinase activity of 11.1 unit / hr in the sediment and relatively low chitinase activity of 0.2 unit / hr in the sediment filtrate. The enzyme adsorption rate of kaoline was about 90.2% (Fig. 6).

2-4. Of the powder adsorbed by the enzyme Oil-phase Thermal stability  Confirmation test

The pellet adsorbed on the enzyme obtained above was lyophilized. The enzyme-adsorbed powder was mixed with olive oil, a vegetable oil, and then heat-treated for 60 minutes and 120 minutes in an incubator at 50 ° C. The chitinase activity of the enzyme - adsorbed powder was compared with the water - loaded sample to confirm the stability in the oil phase.

As a result, the thermal stability of kaoline powder adsorbed on chitinase was found to be 8 ~ 10 times higher than that of liquid form, and it was 1.25 times higher than that of powder (Fig. 7).

As described above, the method for maintaining the activity of spores and enzymes produced by the insect pathogenic fungus according to the present invention is a method capable of effectively maintaining the activity of spores and enzymes produced by the insect pathogenic fungus usable as the material of the microbial pesticide Respectively.

In addition, it is expected that the method of maintaining the activity of spores and enzymes produced by the insect pathogenic fungus according to the present invention can be effectively applied to the formulation of microbial pesticides with high controlling activity.

Claims (6)

A chitinase adsorption powder derived from a spore powder or an insect pathogenic fungus produced by an insect pathogenic fungus of the genus Beauveria sp. Is put into vegetable oil, mixed and stored, and then the insect pathogenic fungus is produced Spores and enzymes. The method according to claim 1, wherein the vegetable oil is at least one selected from the group consisting of soybean oil, olive oil, cotton seed oil and corn oil. A method for maintaining the activity of spores and enzymes produced by the method. delete The chitinase adsorbent powder of claim 1, wherein the chitinase adsorbent powder derived from the insect pathogenic fungus is obtained by lyophilizing a pellet formed by adding an enzyme adsorbent to a centrifugal supernatant of a fungal strain culture. Methods for maintaining the activity of spores and enzymes produced by pathogenic fungi. 5. The method according to claim 4, wherein the enzyme adsorbent is at least one selected from the group consisting of kaolin, silicagel, pyrophilite, and bentonite. The spore and enzyme produced by the insect pathogenic fungus / RTI &gt; The method of claim 1, wherein the insect pathogenic fungus is selected from the group consisting of Beauveria sp. DBB2507 strain (KFCC 11378). The method for maintaining the activity of spores and enzymes produced by an insect pathogenic fungus.

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