KR20030025023A - Microbial Preparation Using Biodegradating Polymer and Method for Producing Thereof - Google Patents

Abstract

PURPOSE: A method of producing a microorganism preparation by use of a biodegradable polymer and an antagonistic microorganism which is an antagonist against plant pathogens is provided which avoids the destruction of an ecological system and toxic problems caused by use of agrochemicals. CONSTITUTION: A mixed liquid of a microorganism culture and 0.1 to 5.0% by weight of an electrically negative biodegradable polymer solution in a weight ratio of 20:80 to 50:50 is dropped into 0.5 to 3.0% by weight of a metal salt solution to produce beads. Thereafter, the beads are soaked in 0.1 to 3.0% by weight of an electrically positive biodegradable polymer solution and agitated for 30min to 2hr. One or more materials selected from the group consisting of alginate, pectin, carrageenan and polyaspartic acid are used as electrically negative biodegradable polymer materials and one or more materials selected from the group consisting of chitasan, chitosan derivatives and polylysine are used as electrically positive biodegradable polymer materials.

Description

Microbial Preparation Using Biodegradating Polymer and Method for Producing Thereof}

The present invention relates to a microbial preparation using a biodegradable polymer and a method for manufacturing the same. More specifically, the antifungal microorganism is immobilized using a negatively charged biodegradable polymer such as alginate, and the outside thereof is converted into a positively charged biodegradable polymer by electrostatic attraction. It relates to a method for producing a microbial formulation to be formulated by coating.

Until now, organic synthetic pesticides, which have been used since 1902 together with agricultural machinery and chemical fertilizers, have played a key role in solving the food problem of humankind. And water pollution, resulting in lowering of human immunity and decreasing ability to preserve offspring, as well as causing environmental pollution and ecosystem imbalances that threaten humanity as a whole. It is common for countries around the world to end environmentally friendly agriculture, such as green-round already visible and the United Nations Conference on Environment and Development (UNCED) calling for a reduction in the use of chemical pesticides in each country (25% of existing consumption). Korea also promised to reduce the use of chemical pesticides and fertilizers by 50% by 2004. In recent years, biological protection measures to solve food problems with less ecosystem burden have been actively researched, and interest in biopesticides has been increasing as a matter.

The development of microbial pesticides began with pesticides including BT, rather than fungicides. In recent years, the development of fungicides has also become increasingly active. The development of microbial fungicides has given priority to soil diseases that are difficult to control with pesticides. Initially, the microorganisms were treated with useful microorganisms along with the organic matter to be supplied to the soil, but gradually, formulations were developed in the form of hydrates and granules to control major soil diseases such as Rhizoctonia, Pythium, Fusarium, and Verticillium . In addition, microorganisms have been developed that treat useful microorganisms on seeds to control various diseases and promote growth until seeds germinate and grow healthy. However, biopesticides, unlike general chemical pesticides, are often poor in light stability, difficult to guarantee the continuity of effects due to changes in the use environment, and are pointed out as expensive.

In addition, in the case of biopesticides, even if the laboratory effect is seen in the laboratory-level experiments, the development of a suitable formulation that can stably maintain the effect in circulation during commercialization is often insufficient, the drug efficacy is often lowered in actual use.

The present invention is to solve the problems of the prior art as described above, and an object of the present invention is to provide a method for producing a microbial preparation using antagonistic microorganisms antagonistic to biodegradable polymers and pathogenic microorganisms.

One aspect of the present invention for achieving the above object is (a) dropping a mixed solution of the microbial culture medium and 0.1 to 5.0% by weight negatively charged biodegradable polymer solution to 0.5 to 3.0% by weight metal salt solution to prepare the beads; And (b) immersing the beads in 0.1-3.0 wt% positive charge biodegradable polymer solution, stirring for 30 minutes to 2 hours, and coating the beads by electrostatic attraction.

Another aspect of the present invention for achieving the above object relates to a microbial preparation prepared by the above method.

Figure 1a is a photograph showing the surface of the microbial agent before culture;

Figure 1b is a photograph showing the surface change of the microbial agent after culture; And

Figure 2 is a photograph showing the antimicrobial activity of the microbial agent.

Hereinafter, the present invention will be described in more detail.

Preparation of the microbial agent includes the steps of isolating microorganisms antagonistic to the pathogenic microorganism and assaying the antagonism; Selecting a biodegradable polymer (alginate, pectin, carrageenan, chitosan, polylysine, polyaspartic acid, etc.) used for the carrier; Antagonist microorganisms are formulated using biodegradable polymers.

In particular, a method for formulating antagonism microorganisms using biodegradable polymers characteristically implemented by the present invention is

(a) A mixture of 0.1% to 5.0% by weight of a negatively charged biodegradable polymer, such as alginate, pectin, carrageenan or polyaspartic acid, and a mixture of antagonist cultures with Ca ²⁺ , Beads were dropped in a 0.5 to 3.0 wt% metal salt solution containing Cu ²⁺ , Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Mn ²⁺ , Al ²⁺ , Fe ²⁺ , or Mg ²⁺ . Preparing); And

(b) the beads are immersed in a 0.1 to 3.0% by weight solution of a positively-charged biodegradable polymer such as chitosan, chitosan derivatives or polylysine and stirred for 30 minutes to 2 hours to coat the beads by electrostatic attraction. A step is made.

In this case, the microbial culture solution and the negatively charged biodegradable polymer solution may be mixed at a weight ratio of 20:80 to 50:50.

The microbial preparation prepared as described above may be used as it is by lyophilization, in order to increase the durability of the effect

(c) washing the beads with 50 mM sodium acetate (pH 5.5) to remove excess positively charged biodegradable polymeric material;

(d) immersing the beads in 0.1 to 2.0 wt% negative charge biodegradable polymer solution and stirring; And

(e) washing the beads with physiological saline and then immersing in 0.1 to 2.0 wt% positive charge biodegradable polymer solution to stir to form an outer membrane, thereby forming a thicker biodegradable polymer membrane on the outside thereof. The effect persistence of the microbial agent to be prepared can be increased.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are for the purpose of explanation and the present invention is not limited by the following Examples.

Example 1 Isolation of Antagonist Microorganisms

Soils such as Pyeongtaek, Gyeonggi-do, were collected to isolate the pathogenic microorganisms antagonistic to F. solani . 5 g of the collected soil and 45 ml of physiological saline are mixed and shaken at 30 ° C. for 1 hour, diluted to 10 ^-8 by diluted agar plate method, and 0.1 ml of diluted solution is spread on LB agar medium and incubated in a 30 ° C. thermostat for 3 days. It was. The cultured bacteria were purified and transferred to LB agar medium and stored at 4 ° C.

The isolated microorganisms and retained microorganisms were used to assay the antimicrobial activity. 50 μl of the microbial suspension prepared at the center of PDA medium with a concentration of 0.5% absorbance on each side was inoculated into 50 μl of a 5 mm diameter paper disc, and then incubated for 7 days at 28 ° C. incubator for mycelial growth formed by antagonistic bacteria. Inhibition distance was measured. Among the microorganisms, Baciius subtilis, Burkholderia cepacia, Streptomyces sp. The growth inhibition rate of Pseudomonas sp. Was higher than that of other microorganisms.

Example 2: Antimicrobial Activity According to the Concentration of Chitosan Solution

In order to use chitosan having the highest antimicrobial activity in formulation preparation, the antimicrobial activity of each viscosity was compared. Hydrolysis products of chitosan include hexamer and octamer chitosan oligosaccharides, the main component of which is 0.1% by weight of chitosan with 1.2, 1.5, 2, 3, 10, 18, 32, 50, 100 cps viscosity F. solani , a pathogenic microorganism, was inoculated into PDA agar medium for 5 days in an incubator at 28 ° C, and the growth diameter was measured to determine the growth inhibition rate of each chitosan. From 100 cps to 3 cps, the lower the chitosan viscosity, the higher the antimicrobial activity. The 3 cps chitosan showed a 40% inhibition rate and the same level was maintained up to 1.2 cps.

Example 3 Influence of Concentration of Alginate Solution Used in Formulation

Beads were prepared by mixing a sodium alginate solution having a concentration of 0.1, 0.5, 1.0, 1.5, 2.0, 3.0, and 5.0 wt% with a microbial culture, and dropping it in a 1.5% CaCl ₂ solution. In the case of the alginate solution of 2.0% by weight or more, it was not easy to prepare a formulation due to the high viscosity, and in the case of 1.0% by weight or less, the hardness of the beads was low, making it difficult to fix the microorganism.

Example 4 Influence of Concentration of Pectin Solution Used in Formulation

The formulation was prepared in the same manner except that the alginate was replaced with pectin in Example 3. Less than 1.5% by weight had a problem of low hardness of the formulation.

Example 5 Effect of Concentration of Carrageenan Solution Used in Formulation

The formulation was prepared in the same manner except that the alginate was replaced with carrageenan in Example 3. Formulations were prepared using 0.1-5.0 wt% carrageenan solution, resulting in easy formulation preparation at 2.0-3.0 wt%. At low concentrations, there was a problem of low hardness of the formulation, and high viscosity of the solution had difficulty in formulating.

Example 6 Effect of Concentration of Chitosan Solution Used in Formulation

Beads prepared by dropping a 1.5 wt% alginate solution into a 1.5% CaCl ₂ solution were dipped in 0.1, 0.2, 0.5, 0.7, 1.0 and 2.0 wt% chitosan solutions, respectively, and stirred for 30 minutes to coat the outside of the beads with chitosan. . In solutions of 0.7 wt% or more, the viscosity of the solution is high, making it unsuitable for formulation preparation.

Example 7: Preparation of Bilayer Formulations

The beads prepared in Example 6 were washed with 50 mM sodium acetate (pH 5.5) to remove excess chitosan, and then re-coated with alginate and chitosan on the outside coated with chitosan to increase the stability and persistence of the formulation. Was carried out. If not cleaned enough, excess chitosan and alginate solutions will form a gel that will hinder coating. The washed formulation was immersed in 0.1, 0.5, 1.0, 1.5, 2.0 wt% alginate solution and stirred for 30 minutes. The optimum concentration of the alginate solution was 0.2% by weight, and when the concentration was higher than this, the viscosity of the solution was high, so that the coating work was difficult and the separation of the formulation after the work was difficult. After coating with alginate, the formulation was washed with 0.85% saline solution and then immersed in 0.1, 0.2, 0.5, 0.7, 1.0, 2.0 wt% chitosan solution and stirred for 30 minutes to form a chitosan envelope. At this time, the concentration of the chitosan solution was 0.2, 0.5% by weight was appropriate, and when the concentration was high, the viscosity of the solution was not suitable for coating work.

Example 8 Verification of Preservation and Performance of Microbial Agents

Example 8-1 Microbial Preservation in Formulations

Since the survival of the microorganisms in the formulation is important for a long time, the number of viable microorganisms in the formulation was measured while storing the formulation containing the antagonist microorganisms. The formulations were stored for 0, 2, 4 and 6 months, respectively, and then plated in LB solid medium and incubated at 30 ° C. Stability was measured by comparing the initial microbial number and the number of sacrificed microorganisms. As a result, even after 6 months, the viable cell count in the formulation was stable with little change.

Example 8-2 Release of Microorganisms in Formulations

In order to confirm that the microorganisms immobilized in the formulation were released by decomposing the surrounding chitosan membrane, the formulation was placed on a PDA medium, incubated for 5 days in a 28 ° C. incubator, and the surface of the formulation was observed using an SEM.

As shown in FIGS. 1A and 1B, the chitosan membrane was initially surrounded, but after 5 days, the membrane was decomposed and microorganisms were released to the outside.

Example 8-3 Antimicrobial Activity of Microbial Agents

In order to measure the pathogenic microbial growth inhibitory activity of the formulation containing the antagonist microorganisms, the formulations prepared at both ends of the PDA medium and F. solani disk were placed and incubated for 5 days in a 28 ℃ incubator. The growth radius of pathogenic microorganisms in the control and formulation treatments was measured. As shown in Figure 2 it was observed that the growth of F. solani is inhibited by the microorganisms released from the formulation.

The present invention isolates microorganisms with antagonistic resistance to pathogenic microorganisms and formulates phytopathogen-inhibiting microbial agents by using electrostatic attraction between biodegradable polymers having positive and negative charges, thereby destroying ecosystems and toxicity by using chemical pesticides. In addition to providing a stable fixed carrier against microorganisms, it is very useful for new organic, pesticide and environmentally friendly industries.

Claims (7)

(a) dropping a mixed solution of a microbial culture solution and 0.1 to 5.0 wt% negatively charged biodegradable polymer solution into a 0.5 to 3.0 wt% metal salt solution to prepare beads; And (b) immersing the beads in a 0.1 to 3.0% by weight positively charged biodegradable polymer solution and stirring for 30 minutes to 2 hours to coat by electrostatic attraction. The method of claim 1, (c) washing the beads with 50 mM sodium acetate (pH 5.5) to remove excess positively charged biodegradable polymeric material; (d) immersing the beads in 0.1 to 2.0 wt% negative charge biodegradable polymer solution and stirring; And (e) washing the beads with physiological saline and then immersing in 0.1 to 2.0 wt% positive charge biodegradable polymer solution to stir to form an outer membrane. The method of claim 1, wherein the microbial culture solution and the negatively charged biodegradable polymer solution are mixed at a weight ratio of 20:80 to 50:50. The method of claim 1 or 2, wherein the negatively charged biodegradable polymeric material is at least one selected from the group consisting of alginate, pectin, carrageenan, and polyaspartic acid. A method of formulating a microbial agent, characterized by using a substance. The microbial formulation according to claim 1 or 2, wherein at least one substance selected from the group consisting of chitosan, chitosan derivatives, and polylysine is used as the positively charged biodegradable polymer material. Way. The material of claim 1, wherein Ca ²⁺ , Cu ²⁺ , Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Mn ²⁺ , Al ²⁺ , Fe ^2+, or Mg ²⁺ is used as the metal salt. Formulation method of a microbial agent, characterized in that. Microbial agent prepared by the method of claim 1.