KR100341186B1 - Microencapsulation of Xenorhabdus nematophilus and its preparation and composition - Google Patents

Abstract

The present invention relates to a method for microencapsulation of a Xenorhabdus nematophilus (Xn) microbial agent, a preparation thereof, and a biopesticide composition using the same. In the present invention, microencapsulation of Xn with various polymers (microencapsulation) is provided a microencapsulation method and its preparation for improving the storage stability to the external environment while maintaining the survival of the strain. In addition, the present invention provides a pesticide composition mixed with a Bacillus thuringinesis (Bt) agent in order to increase and sustain the pesticidal effect of the microencapsulated powder Xn agent. The microencapsulation according to the present invention improves the storage stability of Xn, and the mixed composition with BIT exhibits an increased pesticidal effect and more sustained efficacy even for insects which have been low sensitivity to existing biopesticides.

Description

Microencapsulation method of Xenorabdu nematophilus microorganism preparation and its preparation and biopesticide composition using the same {Microencapsulation of Xenorhabdus nematophilus and its preparation and composition}

The present invention relates to a method for microencapsulation of a Xenorhabdus nematophilus (Xn) microbial agent, a preparation thereof, and a biopesticide composition using the same.

Insects that cause serious economic damage to crops are called pests, and pesticides are used to control these pests. Most pesticides in use today are organic synthetic chemical pesticides, which have played an important role in pest control over the last 50 years. However, the overuse and use of chemical pesticides have led to problems such as resistance of pests, major pests of secondary pests, residual pesticides, and environmental destruction. As a result, countries around the world are gradually tightening restrictions on the use of pesticide residue inspection and total spraying regulations for each crop, as well as import and export agricultural products. Negotiations are under way to regulate the prohibition or total production of certain pesticides.

The development of biopesticides has been progressed to complement the chemical pesticides, and in particular, the conversion of biopesticides and the development of microbial pesticides of global pesticide companies have been outstanding recently.

Microbial pesticides use insect pathogenic bacteria, fungi, viruses, and nematodes, which are natural enemies of pests, and can effectively control target pests without harm to humans, livestock, and plants. Insect pathogenic microorganisms suitable for the separation, mass production, and formulation have been commercialized.

Among the microbial insecticides, which have been studied and put into practical use, Bacillus thuringinesis (Bt), an insect pathogenic bacterium, has a strong insecticide resistance and low production cost. However, Beatty has a limited number of pests, has a slower pesticide rate than chemical pesticides, and has recently been reported to be resistant to the outdoors, especially against pests that are resistant to various drugs, such as beech moths. It shows low sensitivity.

Therefore, new biological control factors are needed. Recently, many insect pathogenic nematodes have been studied as biological control factors. Insect pathogenic nematodes are parasitic to insects, which cause host pests to be killed, and have a broad host range and excellent pest detection ability. The insecticidal power of Steinernema carpocapsae among the entomopathogenic nematodes is Xenorhabdus nematophilus (hereinafter referred to as 'Xn'), which is a symbiotic bacteria in the intestine. Proven as a pesticide (Park and Kim, 2000). An important insecticidal mechanism of this bacterium is known to induce immune deterioration of host insects, causing sepsis of blood and kill host within 20 hours after blood penetration.

Xn must be penetrated into insect bodies to achieve its effects. In nature, this is the responsibility of Steinernema carpocapsae , a symbiotic nematode of the bacterium. However, since the symbiotic nematodes are weak in outdoor conditions, there are many problems to be solved in order to use them directly for the control of above-ground pests such as Pabam moths. Despite the high insecticidal properties, biopesticides using Xn have not been put to practical use.

Therefore, conventionally, pesticides using Xenorabdus nematophilus have been used to isolate insecticidal protein toxins from insects, apply them to insects or plants, or process them genetically (Korea Patent Publication No. 10-1998). -701244).

In this regard, a method of applying the cultured Xn microorganism as it has been studied. As a result, a pesticide method using a biopesticide, in particular, a bite, which damages cells and tissues of an insect as a carrier for transporting Xn into an insect body, It has been patented by (Korean Patent Application No. 10-2000-068944).

However, even if the problem of transporting the Xn into the insect body in the same way as described above, Xn is weak in storage stability and outdoor conditions, there are still many problems to use directly in the ground pest control. The present invention relates to formulation development to solve this problem.

Several attempts have been made in the development of formulations to reduce the toxicity and risk of conventional pesticides and to improve the safety of the environment and the human body. Among these attempts, microencapsulation, in particular, can control the rate of drug release and sustain the effects, improve drug stability, prevent drug reduction due to exposure to the external environment, masking the odor, and solidifying pesticide substances. It has many advantages that it is easy to apply, and in particular, chemical pesticides have been applied to many chemical synthetic pesticides because they have an advantage of improving safety for humans and the environment and reducing toxicity. Developed by RENNWALT in the United States, methyl parathion, an encapsulated organophosphorus insecticide, was developed as PENNCAP ™. Capsule formulations have been developed.

However, in the case of microbial preparations, the microorganisms are sensitive to temperature, and thus, microorganisms have a problem in that the microorganisms are killed or denatured during microencapsulation. Regardless of the survival of some strains, microencapsulation was attempted when a substance contained in the culture medium was used as a pesticide (e.g., a bite agent), but this was a microencapsulation of a microorganism that microencapsulates the bacteria itself while maintaining survival. It was different from. The present invention relates to a method of microencapsulating a microbial agent, in particular Xenorabdu nematophilus, while maintaining the survival of the strain.

In order for Xenorabdu nematophilus to properly exhibit an insecticidal effect, unlike viti, the strain itself must not be killed and survival must be maintained in the formulation. However, Xn is a symbiotic bacterium in the nematode intestine, which is sensitive to external environment such as ultraviolet rays, temperature, humidity, etc., and thus does not exhibit stability and storage properties, and has no viability when spraying outdoors. As a solution to this problem, it is necessary to develop a formulation capable of protecting Xn from the external environment while maintaining the survival of Xn.

It is an object of the present invention to provide a microencapsulation method and a preparation thereof which microencapsulates Xn with various polymers to improve storage stability to the external environment while maintaining the survival of the strain.

To this end, the present invention provides microencapsulation conditions, namely coating material and spray drying conditions, in particular, the temperature of the nozzle portion and the spray rate of the nozzle, which are advantageous for Xn survival and survival duration.

Microencapsulation method of Xn according to the present invention comprises the steps of emulsifying and dispersing by mixing 1 to 30 parts by weight of Xenorabdus nematophilus (Xn) culture solution to 100 parts by weight of 10 to 50% aqueous solution of the polymer material; Reacting the emulsified and dispersed liquid mixture by an interfacial reaction or an in situ polymerization method to obtain a microcapsule slurry; Spray drying the microcapsule slurry at a condition of a nozzle portion temperature of 50 to 100 ° C. and a spray rate of 1 to 30 ml / min, thereby finally obtaining a microencapsulated powder Xn.

Various synthetic and natural polymers may be used as the polymer material, and preferably, a polymer material selected from the group consisting of starch, gelatin, methacrylic polymer, ethylcellullose and cyclodextrin may be used. .

Still another object of the present invention is to provide a composition in which the microencapsulating agent of Xn is mixed with a Bacillus thuringinesis (Bt) agent in order to increase the insecticidal effect and improve the effect persistence.

When the microencapsulated Xn is sprayed on insects or plants by itself, it cannot be transported to the blood in the insects and thus does not exhibit sufficient insecticidal effect. As a carrier, biopesticides that damage the cells and tissues of insects, especially bites, are used together. This can increase and sustain the pesticidal effect.

The insecticidal effect may vary depending on the composition ratio. Preferably, the mixing ratio of the microencapsulated Xn agent and the beaten agent of the present invention is 1:99 to 50:50.

Other objects and advantages of the invention will be described below, and will be better understood by practice of the invention.

1 is a block diagram showing a manufacturing process of the microcapsules according to the present invention.

2 is a graph showing the stability test results by storage period.

3 is a graph showing the stability test results for each storage temperature.

Figure 4 is a graph showing the sustained insecticidal test results in outdoor conditions.

Cultivation of Xenorabdu nematophilus

About 400 insect pathogenic nematode (Steinernema carpocapsae) infections were localized to the 5th insects. After 10 hours of treatment, the blood of Pabam moth was collected and incubated at 28 ° C. using Xem medium, which is a selective medium of Genodildus nematophilus. The composition of the Xem medium is shown in Table 1 below.

Compound Furtherance Bacto beef extract 3 g Bacto peptone 5 g Bacto agar 15 g Bromothymol blue 0.025 g Triphenyltetrazolium chloride 0.04g Penicillin g 0.0625g Streptomycin 4 g Distilled water 1L

Among colonies formed after 24 hours of cultivation, the colonies having a reddish center and white borders were determined as Xenorhabdus nematophilus , and the colonies were liquid cultured using TSB medium.

The composition of the TSB medium is shown in Table 2 below.

Compound Furtherance Bacto tryptone 17 g Bacto soytone 3 g Bacto dextrose 2.5g Sodium chloride 5 g Dipotassium phosphate 2.5g Distilled water 1L

Microcapsule Preparation of Xn

As a coating material constituting the microcapsules, various synthetic polymers or natural polymers may be used. Examples of polymers that can be used include poly (lactic acid), poly (glycolic acid), and poly (hydroxy acids) such as poly (lactic acid-co-glycolic acid), polyglycolide, polylactide, polylactide-co Glycolide copolymers and mixtures, polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly (ethylene glycol), poly (ethylene oxide) Polyalkylene oxides such as, polyalkylene terephthalates such as poly (ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly (vinyl chloride), polyvinylpyrrolidone , Polysiloxane, poly (vinyl alcohol), poly (vinyl acetate), polystyrene, polyurethane, and alkyl cellulose, hydroxyalkyl cellulose, cellulose Ether, cellulose ester, nitro cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propinate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl Poly (methyl methacrylate), poly (), which is a derivative thereof comprising copolymers derived from cellulose such as cellulose, cellulose triacetate, and cellulose sulfate sodium salt, polymers or copolymers of acrylic acid and methacrylic acid, or esters Ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate) ), Poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate) and poly (octadecyl acrylate), poly (butyl acid), poly ( Valeric acid), and poly (lactide-co-caprolactone), copolymers and mixtures thereof, natural polymers such as starch, gelatin, cellulose and the like. The term 'derivative' as used herein includes polymers having chemical groups such as alkyl, alkylene substitution, addition, hydroxylation, oxidation, and other modifications that may be conventionally made by those skilled in the art.

In the spray drying under the same conditions, the residual moisture content and osmoticity in the capsule are determined by the type of polymer used as the coating material, so the selection of the polymer is an important factor affecting the survival and survival time of the microorganisms.

In the present invention, although not particularly limited, a polymer material such as starch, gelatin, methacrylic polymer, ethyl cellulose (ethylcellullose) and cyclodextrin is preferably used.

Preferably, additives commonly used in polymer polymerization such as NaCl and glutaraldehyde may be added to the polymer to facilitate the action of the polymer and spray drying conditions.

The overall manufacturing process of the microcapsules is shown in FIG. This will be described in detail below.

First, 1-30 parts by weight of an Xn culture solution is mixed with 100 parts by weight of a 10-50% aqueous solution of a high molecular material and emulsified and dispersed. The emulsified and dispersed liquid mixture is reacted by an interfacial reaction and an in situ polymerization method to obtain a microcapsule slurry. At this time, an additive may be preferably added. As the additive, NaCl, glutaraldehyde, or the like may be used.

The obtained microcapsule slurry is spray dried to obtain powdered microencapsulated Xn. At this time, the spray drying condition is to maintain the temperature of the nozzle portion at 50 ~ 100 ℃ and the spray rate of the nozzle is about 1 ~ 30ml / min.

Spray drying technology is widely used in the chemical, food, and pharmaceutical fields, and is advantageous for application to high temperature sensitive materials. In spray drying, the amount of product, the residual moisture content in the capsule, and the osmoticity of the capsule are determined by the temperature of the nozzle portion and the spray rate of the nozzle in addition to the capsule material and additives, which is an important formulation condition. In particular, when the microencapsulated drug is a microorganism as in the present invention, the spray drying condition has an important effect on the survival of the microorganism.

If the temperature of the nozzle portion is high, the amount of the product increases but adversely affects the survival of the microorganisms and the residual moisture content of the capsule. In addition, the spray rate of the nozzle affects the amount of product and the residual moisture content of the capsule, the faster the spray rate is reduced the product amount and the higher the residual moisture content of the capsule.

The microencapsulated quildine Xn powder of the present invention prepared as described above greatly improves the storage stability. However, when it is sprayed on insects or plants alone, it is not transported to the blood in the insects and thus does not exhibit sufficient insecticidal effect.

Accordingly, the present invention provides a pesticidal composition in which the Xn agent of the present invention is mixed with a biopesticide that damages cells and tissues of an insect, in particular, in order to increase and sustain an insecticidal effect.

The pesticidal effect of the composition may vary depending on the composition ratio. Preferably, the mixing ratio of the microencapsulated Xn agent and the beaten agent of the present invention is 1:99 to 50:50.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited by the following examples, and those of ordinary skill in the art to which the present invention pertains should be within the equivalent scope of the technical concept of the present invention and the claims to be described below. Of course, various modifications and variations are possible.

Example 1

Biodegradable starch was used as a microencapsulating coating material of Xn. 10 g of Xn culture solution was added to 100 g of 30% aqueous biodegradable starch (Suncap ™, Samyang) and stirred for 10 minutes at 7000 rpm with a homogenizer to prepare a microcapsule slurry. This slurry liquid was sprayed using a Mini Spray Dryer B-191, Buchi co. At a nozzle temperature of 80 ° C. and a spray rate of 8 to 9 ml / min. Got.

Example 2

Except for using gelatin as a coating material, Xn was microencapsulated and spray dried in the same manner as in Example 1 to obtain microencapsulated Xn in powder form.

Example 3

Xn was microencapsulated and spray dried in the same manner as in Example 1, except that a methacrylic polymer was used as the encapsulating material, thereby obtaining powdered microencapsulated Xn.

Example 4

For comparative experiments, the microencapsulated Xn culture medium was sprayed under the same spray drying conditions as in Examples 1 to 3 (the nozzle part temperature was 8 and the spraying speed of the nozzle was 8-9 ml / min). Xn was obtained.

Example 5

A commercial insecticide composition was prepared by mixing a commercially available beetase (Cactar Granular Watering Agent ™, Advance Industries) and the microencapsulated powder Xn agent of the present invention obtained in Example 3 in a ratio of 99: 1.

Example 6

A mixed insecticide composition was prepared in the same manner as in Example 5, except that the VAT agent and the Xn agent were mixed at a ratio of 90:10.

Example 7

A mixed insecticide composition was prepared in the same manner as in Example 5, except that the BTT agent and the Xn agent were mixed at a ratio of 50:50.

Example 8

Storage Stability Test of Microencapsulated Xn

(1) How to measure the concentration of Xn

1) Step 1: A suspension of the Xn powder obtained in the above Examples and Comparative Examples was prepared by the following method.

Examples 1, 2 and 4: 0.5 g of the microencapsulated powder obtained in Examples 1, 2 and 4 was added to 4.5 ml of sterilized water, and the mixture was shaken sufficiently until the capsule powder was completely dissolved to prepare a suspension solution.

Example 3: A suspension was prepared by adding 0.5 g of the microencapsulated powder obtained in Example 3 to 4.5 ml of 15% alcohol and shaking sufficiently until the capsule powder was completely dissolved.

Xn broth: 0.5 ml of unencapsulated Xn broth was added to 19.5 ml of sterilized water, and shaken sufficiently to prepare a suspension.

2) Step 2: Dilute the suspension prepared above by the 10-step dilution method.

3) Step 3: 100 μl of the diluent is evenly spread on the NA plate medium and incubated in a culture chamber at 30 ° C. to measure the number of colonies to measure the concentration of Xn.

(2) Stability test by storage period

The unencapsulated Xn cultures used in Examples 1 to 4 and Comparative Examples were each placed at room temperature, and the concentration of Xn was measured by the method of (1) at 15 days intervals to evaluate the stability according to the storage period. The results are shown in FIG. As shown in FIG. 2, Examples 1 to 4 which microencapsulate Xn than Xn culture showed better storage stability, and in particular, Example 3 showed the highest storage stability.

(3) Stability test by storage temperature

The unencapsulated Xn culture solution and the microencapsulated powder of Example 3 were stored at 4 ° C., 20 ° C. and 40 ° C., respectively. Evaluated. The results are shown in FIG. As shown in FIG. 3, Xn of Example 3 microencapsulated than the Xn culture medium had higher storage stability, and the lower the storage temperature, the higher the storage stability.

Example 9

Insecticidal test indoors

For the insecticidal effect test, the aqueous solutions of the pesticides of Examples 5 to 7 were prepared at concentrations of 2000, 1000, 500, 250, and 125 ppm, and for comparison, the Xn preparation alone and the commercially available vitiagent were used for comparison. ™, Advance Industries) alone produced an aqueous solution in the same manner. Cabbage leaf slices (diameter 30 mm) were immersed in the prepared aqueous solution for 0.5 to 1 minute, dried in a hood, and placed in a petri dish (50 mm) covered with filter paper and inoculated with 10 spodoptera exigua. After 72 hours, the number of insects was examined, and the insecticide concentrations were determined by using the half lethal concentration (LC ₅₀ value). The results are shown in Table 2 below.

process LC ₅₀ value (ppm) Xn alone 1257.6 (1150.1 to 1365.1) 쎈 alone 516.4 (456.1-576.5) Example 5 245.5 (170.4-321.0) Example 6 153.9 (104.2 to 203.6) Example 7 372.2 (274.7--469.7)

As can be seen from Table 2, the mixed composition of Examples 5 to 7 was found to have better insecticidal properties than Xn alone or beetle alone. In particular, the case of Example 6 was found to have the highest insecticide, from which it can be seen that the mixing ratio of the bitty agent and microencapsulated Xn affects the insecticide.

Example 10

Insecticidal test under outdoor conditions

The cabbages cultivated for about 60 days for the sustained insecticidal test were sprayed with 1000 ppm each of commercially available VITIZE (Cactar Granular Watering Agent ™, Advance Industry) alone, Xn alone obtained in Example 3, and the composition of Example 6, respectively. After 5, 10, 15 and 20 days, the cabbage leaves were cut and examined for insecticide indoors. The result is shown in FIG. 4. It can be seen from FIG. 4 that the insecticidal properties of these mixed compositions last longer than those of Xn alone or Beatty alone.

Since the microencapsulated Xn agent according to the present invention protects the strain from the external environment to maintain survival for a longer time and improve storage stability, it is easy to utilize Xn, which has poor viability when spraying, and has not properly exhibited an insecticidal effect. In addition, the biopesticide composition in which the microencapsulated Xn agent and the bite agent according to the present invention are mixed, the insecticidal effect is increased and sustained efficacy even for insects that have low sensitivity to the conventional biopesticides.

Claims (5)

Emulsifying and dispersing by mixing 1-30 parts by weight of a Genorabdus nematophilus (Xn) culture solution with 100 parts by weight of a 10-50% aqueous solution of a polymer material; Reacting the emulsified and dispersed liquid mixture by an interfacial reaction or an in situ polymerization method to obtain a microcapsule slurry; And spray-drying the microcapsule slurry under conditions of a temperature of 50 to 100 ° C. of the nozzle portion and a spray rate of 1 to 30 ml / min of the nozzle to powderize the microcapsule slurry. The method of claim 1, wherein the polymer material is selected from the group consisting of starch, gelatin, methacrylic polymer, ethyl cellulose, and cyclodextrin. A pesticide comprising the microencapsulated powder Xn prepared by the method of claim 1 as an active ingredient. A pesticide composition comprising a microencapsulated powder Xn and a Bacillus thuringinesis agent prepared by the method of claim 1. 5. The insecticide composition according to claim 4, wherein the Xn agent and the bitty agent are mixed at a ratio of 1:99 to 50:50.