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

Microbial Preparation Using Biodegradating Polymer and Method for Producing Thereof Download PDF

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KR20030025023A
KR20030025023A KR1020010057914A KR20010057914A KR20030025023A KR 20030025023 A KR20030025023 A KR 20030025023A KR 1020010057914 A KR1020010057914 A KR 1020010057914A KR 20010057914 A KR20010057914 A KR 20010057914A KR 20030025023 A KR20030025023 A KR 20030025023A
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biodegradable polymer
solution
beads
microbial
formulation
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KR100531040B1 (en
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김성균
최용복
신중한
김동성
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주식회사 효성
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/08Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
    • A01N25/10Macromolecular compounds
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/26Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10Animals; Substances produced thereby or obtained therefrom

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  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Dentistry (AREA)
  • Plant Pathology (AREA)
  • Pest Control & Pesticides (AREA)
  • Wood Science & Technology (AREA)
  • Agronomy & Crop Science (AREA)
  • Environmental Sciences (AREA)
  • Toxicology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

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

생분해성 고분자를 이용한 미생물제제 및 그의 제조방법 {Microbial Preparation Using Biodegradating Polymer and Method for Producing Thereof}Microbial Preparation Using Biodegradating Polymer and Method for Producing Thereof}

본 발명은 생분해성 고분자를 이용한 미생물제제 및 그의 제조방법에 관한 것으로, 보다 상세하게는 항진균 미생물을 알지네이트 등의 음전하 생분해성 고분자를 이용하여 고정화하고 그 외부를 정전기적 인력에 의하여 양전하 생분해성 고분자로 코팅하여 제형화하는 미생물제제의 제조방법에 관한 것이다.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.

지금까지 인류의 식량문제를 해결하는 데는 농기계, 화학비료와 함께 1902년 이후 사용되어온 유기합성 농약이 그 핵심적인 역할을 해 왔으나, 인구증가에 따른 식량 증산을 위한 대량의 유기합성 농약의 사용은 토양 및 수질오염을 발생시켜 인간의 면역력 저하와 자손보존능력의 감소는 물론, 인류전체를 위협하는 환경오염과 생태계 불균형을 야기시키는 결과를 빚고 있다. 이미 그린라운드(Green-round)가 가시화 되고, 유엔환경개발회의(UNCED)가 각국의 화학농약 사용량 감소(기존 사용량 대비 25%)를 요구하는 등 환경친화농업을 의무화하는 것이 20세기말 세계 각국의 일반적 경향이며, 우리나라 역시 2004년까지 화학농약과 비료의 사용량을 현재의 50% 수준까지 줄이기로 대외적으로 약속한 상태이다. 최근들어 생태계 부담을 보다 적게 하면서, 식량문제를 해결하는 생물학적 보호수단이 활발히 연구되고 있으며, 그 일안으로서 생물농약에 대한 관심이 고조되고 있다.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.

미생물농약의 개발은 살균제보다는 BT제를 비롯한 살충제 분야에서 먼저 시작되었으며 최근들어 살균제의 개발도 점차 활기를 띄기 시작하였다. 미생물 살균제의 개발은 농약으로는 방제가 어려운 토양병이 우선이 되었다. 초기에는 토양에 공급하는 유기물과 함께 유용 미생물을 처리하였으나 점차 수화제, 입제형으로 제형을 개발하여Rhizoctonia, Pythium, Fusarium, Verticillium등 주요 토양병을 방제하였다. 이와 함께 유용 미생물을 종자에 처리하여 종자가 발아하여 건강하게 입모될 때까지 각종 병을 방제하고 생육도 촉진시키는 미생물제제들이 개발되었다. 그러나 생물농약은 일반적인 화학농약과는 달리 광안정성이 미약한 경우가 많고, 사용환경 변화에 따른 효과의 지속성을 보장받기 어렵우며, 고가의 비용이 발생한다는 점이 단점으로 지적되고 있다.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.

또한 생물농약의 경우 실험실 수준 실험에서 큰 효과를 본 경우에도, 상품화시 유통중 효과를 안정적으로 지속시킬 수 있는 적합한 제형 개발이 미비하여 실제사용시에는 약효가 저하되는 경우가 빈번하였다.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.

본 발명은 상기와 같은 종래 기술의 문제점을 해결하기 위한 것으로, 생분해성 고분자와 병원성 미생물에 길항력이 있는 길항 미생물을 사용하여 미생물제제를 제조하는 방법을 제공하는 것을 목적으로 한다.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.

상기와 같은 목적을 달성하기 위한 본 발명의 한 측면은 (a) 미생물 배양액과 0.1 내지 5.0 중량% 음전하 생분해성 고분자 용액의 혼합액을 0.5~3.0 중량% 금속염 용액에 떨어뜨려 비드를 제조하는 단계; 및 (b) 상기 비드를 0.1∼3.0 중량% 양전하 생분해성 고분자 용액에 침지시켜 30분~2시간동안 교반하여 정전기적 인력에 의하여 코팅하는 단계를 포함하는 미생물 제제의 제형화 방법에 관한 것이다.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.

상기와 같은 목적을 달성하기 위한 본 발명의 다른 측면은 상기의 방법에 의해 제조된 미생물 제제에 관한 것이다.Another aspect of the present invention for achieving the above object relates to a microbial preparation prepared by the above method.

도 1a는 배양전 미생물제제의 표면을 나타낸 사진;Figure 1a is a photograph showing the surface of the microbial agent before culture;

도 1b는 배양후 미생물 제제의 표면변화를 나타낸 사진; 및Figure 1b is a photograph showing the surface change of the microbial agent after culture; And

도 2는 미생물제제의 항균 활성을 나타내는 사진이다.Figure 2 is a photograph showing the antimicrobial activity of the microbial agent.

이하에서 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.

미생물제제의 제조는 병원성 미생물에 길항력이 있는 미생물을 분리하고 길항력을 검정하는 단계; 담체에 이용되는 생분해성 고분자(알지네이트, 펙틴, 카라지난, 키토산, 폴리라이신(polylysine), 폴리아스파르트산(polyaspartic acid) 등)을 선정하는 단계; 생분해성 고분자를 이용하여 길항미생물을 제제화 단계를 거쳐이루어진다.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.

특히 본 발명에 의해 특징적으로 구현되는 생분해성 고분자를 이용하여 길항미생물을 제제화 방법은In particular, a method for formulating antagonism microorganisms using biodegradable polymers characteristically implemented by the present invention is

(a) 알지네이트(alginate), 펙틴(pectin), 카라지난(carrageenan) 또는 폴리아스파르트산(polyaspartic acid)과 같은 음전하 생분해성 고분자의 0.1~5.0 중량% 용액과 길항미생물 배양액의 혼합물을 Ca2+, Cu2+, Zn2+, Ni2+, Co2+, Mn2+, Al2+, Fe2+, 또는 Mg2+을 포함하는 0.5~3.0 중량% 농도의 금속염 용액에 떨어뜨려 비드(bead)를 제조하는 단계; 및(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) 상기 비드를 키토산, 키토산 유도체 또는 폴리라이신(polylysine)과 같은 양전해 생분해성 고분자의 0.1~3.0 중량% 용액에 침지시켜 30분~2시간동안 교반시켜 비드를 정전기적 인력에 의해 코팅하는 단계를 포함하여 이루어진다.(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.

이때 상기 미생물 배양액과 음전하 생분해성 고분자 용액이 20:80 내지 50:50의 무게비로 혼합되는 것이 좋다.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.

상기와 같이 제조된 미생물 제제를 동결건조하여 그대로 사용할 수도 있으나, 효과의 지속성을 높이기 위하여The microbial preparation prepared as described above may be used as it is by lyophilization, in order to increase the durability of the effect

(c) 상기 비드를 50mM 소듐 아세테이트(pH 5.5)로 세척하여 여분의 양전하 생분해성 고분자 물질을 제거하는 단계;(c) washing the beads with 50 mM sodium acetate (pH 5.5) to remove excess positively charged biodegradable polymeric material;

(d) 상기 비드를 0.1 내지 2.0 중량% 음전하 생분해성 고분자 용액에 침지시켜 교반하는 단계; 및(d) immersing the beads in 0.1 to 2.0 wt% negative charge biodegradable polymer solution and stirring; And

(e) 상기 비드를 생리식염수로 세척한 다음 0.1 내지 2.0 중량% 양전하 생분해성 고분자 용액에 침지시켜 교반하여 외막을 형성하는 단계를 추가로 실시하여 그 외부에 생분해성 고분자 막을 좀더 두텁게 형성하는 것에 의해 제조되는 되는 미생물 제제의 효과 지속성을 증대시킬 수 있다.(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.

이하 본 발명을 실시예를 들어 보다 상세히 설명하나, 하기 실시예는 설명의 목적을 위한 것으로 하기 실시예에 의하여 본 발명이 제한되는 것은 아니다.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.

실시예 1: 길항미생물의 분리Example 1 Isolation of Antagonist Microorganisms

병원성 미생물F. solani에 길항력이 있는 미생물을 분리하기 위하여 경기도 평택 등의 토양을 채취하였다. 채취한 토양 5 g과 생리식염수 45 ml를 혼합하여 30℃에서 1시간 동안 진탕한 후 희석한천 평판법으로 10-8까지 희석한 다음 희석액 0.1 ml를 LB 한천배지에 도말하여 30℃ 항온기에서 3일간 배양하였다. 배양된 세균을 순수분리하여 LB 한천배지로 옮겨 4℃에서 보관하였다.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.

상기 분리 미생물과 보유 미생물을 사용하여 항균성을 검정하였다. PDA배지 중앙에 진균 균사 디스크를 놓고 양쪽에 흡광도 0.5 정도의 농도로 조성된 미생물 현탁액을 직경 5 mm 종이 디스크에 50 ㎕씩 접종한 다음 28℃ 항온기에서 7일동안 배양하면서 길항세균에 의해 형성된 균사 생장억제 거리를 측정하였다. 실험 미생물중Baciius subtilis, Burkholderia cepacia, Streptomycessp.Pseudomonassp.의 생육저해율이 다른 미생물들에 비해 높았다.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.

실시예 2: 키토산용액의 농도에 따른 항균활성Example 2: Antimicrobial Activity According to the Concentration of Chitosan Solution

제형 제조시 가장 항균력이 높은 키토산을 이용하기 위하여 각 점도별 항균력을 비교하였다. 키토산의 가수분해 산물로 헥사머(hexamer)~옥토머(octamer)가 주요성분인 키토산 올리고당과 1.2, 1.5, 2, 3, 10, 18, 32, 50, 100 cps 점도의 키토산 0.1 중량%를 포함하는 PDA한천배지에 병원성 미생물인F. solani를 접종하여 28℃ 배양기에서 5일동안 배양한 다음 생육직경을 측정하여 각 키토산의 생육저해율을 측정하였다. 100 cps부터 3 cps까지는 키토산 점도가 낮아질수록 항균력이 점점 높아져 3 cps의 키토산이 40%의 저해율을 보였으며, 1.2 cps까지는 비슷한 수준을 유지하였다.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.

실시예 3: 제형화시 이용되는 알지네이트용액의 농도의 영향Example 3 Influence of Concentration of Alginate Solution Used in Formulation

0.1, 0.5, 1.0, 1.5, 2.0, 3.0, 5.0 중량%의 농도를 가지는 소듐 알지네이트(sodium alginate)용액과 미생물 배양액을 혼합하여 1.5% CaCl2용액에 떨어뜨려 비드를 제조하였다. 2.0 중량% 이상의 알지네이트 용액의 경우 점도가 높아 제형 제조가 용이하지 않았으며, 1.0 중량% 이하의 경우는 비드의 경도가 낮아 미생물 고정화에 어려움이 있었다.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.

실시예 4: 제형화시 이용되는 펙틴용액의 농도의 영향Example 4 Influence of Concentration of Pectin Solution Used in Formulation

상기 실시예 3에서 알지네이트를 펙틴으로 교체한 것을 제외하고는 동일한 방법으로 제형을 제조하였다. 1.5 중량% 이하에서는 제형의 경도가 낮은 문제점이 있었다.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.

실시예 5: 제형화시 이용되는 카라지난(Carrageenan)용액의 농도의 영향Example 5 Effect of Concentration of Carrageenan Solution Used in Formulation

상기 실시예 3에서 알지네이트를 카라지난(Carrageenan)으로 교체한 것을 제외하고는 동일한 방법으로 제형을 제조하였다. 0.1 내지 5.0 중량% 카라지난 용액을 이용하여 제형을 제조한 결과 2.0 내지 3.0 중량%에서 제형 제조가 용이하였다. 낮은 농도에서는 제형의 경도가 낮은 문제점이 있었으며, 높을 경우 용액의 점도가 높아 제형화 작업에 어려움이 있었다.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.

실시예 6: 제형화시 이용되는 키토산용액의 농도의 영향Example 6 Effect of Concentration of Chitosan Solution Used in Formulation

1.5 중량% 알지네이트 용액을 1.5% CaCl2용액에 떨어뜨려 제조한 비드를 각각 0.1, 0.2, 0.5, 0.7, 1.0, 2.0 중량% 키토산 용액에 침지시킨 다음 30분동안 교반하여 비드 외부를 키토산으로 코팅하였다. 0.7 중량% 이상의 용액에서는 용액의 점도가 높아 제형 제조에 부적절하였다.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.

실시예 7: 이중층 제형의 제조Example 7: Preparation of Bilayer Formulations

실시예 6에서 제조된 비드를 50mM 소듐 아세테이트(pH 5.5)로 세척하여 여분의 키토산을 제거한 다음, 제형의 잔효성 및 지속성을 증대시키기 위하여 키토산으로 코팅되어 있는 외부에 알지네이트와 키토산을 이용하여 다시 코팅을 실시하였다. 세척을 충분히 하지 않을 경우 여분의 키토산 용액과 알지네이트 용액이 젤을 형성하여 코팅작업을 저해한다. 세척한 제형을 0.1, 0.5, 1.0, 1.5, 2.0 중량%의 알지네이트 용액에 침지시켜 30분동안 교반하였다. 알지네이트 용액의 최적농도는 0.2 중량%였으며, 농도가 이보다 높을 경우 용액의 점도가 높아 코팅작업이 어려울뿐 아니라 작업후 제형 분리가 어려운 문제점이 있었다. 알지네이트로 코팅 후 0.85% 생리식염수로 제형을 세척한 다음 0.1, 0.2, 0.5, 0.7, 1.0, 2.0 중량% 키토산 용액에 침지시켜 30분동안 교반하여 키토산 외막을 형성하였다. 이때 키토산 용액의 농도는 0.2, 0.5 중량%가 적당하였으며, 농도가 높아질 경우 용액의 점도가 높아져 코팅작업에 부적절하였다.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.

실시예 8: 미생물제제의 보존성 및 성능 검증Example 8 Verification of Preservation and Performance of Microbial Agents

실시예 8-1: 제형 내의 미생물 보존성Example 8-1 Microbial Preservation in Formulations

제형 내의 미생물 생존이 장기간 유지되는 것이 중요하므로 길항미생물이 들어있는 제형을 보관하면서 제형 내의 생존 미생물의 수를 측정하였다. 각각 0, 2, 4, 6개월동안 제형을 보관한 다음 LB 고체배지에 도말하여 30℃에서 배양하였다. 초기 미생물 수와 희생된 미생물 수를 비교하여 안정성을 측정하였다. 그 결과 6개월이 경과한 후에도 제형 내의 생균수는 거의 변화없이 안정하였다.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.

실시예 8-2: 제형 내 미생물의 방출Example 8-2 Release of Microorganisms in Formulations

제형 내에 고정화된 미생물들이 외부를 싸고 있는 키토산 막을 분해시켜 방출되는지 확인하기 위하여 제형을 PDA 배지 위에 올려놓고 28℃ 배양기에서 5일동안 배양한 다음 SEM을 이용하여 제형의 표면을 관찰하였다.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.

도 1a 및 도 1b에서 나타난 바와 같이, 초기에는 키토산 막이 둘러싸고 있지만, 5일이 경과한 후에는 막이 분해되어 미생물들이 외부로 방출되고 있음을 관찰할 수 있었다.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.

실시예 8-3: 미생물제제의 항균 활성Example 8-3 Antimicrobial Activity of Microbial Agents

길항미생물을 포함하는 제형의 병원성 미생물 생육억제 활성을 측정하기 위하여 PDA 배지의 양쪽 끝에 제조한 제형과F. solani디스크를 올려놓고 28℃ 배양기에서 5일동안 배양하였다. 대조구와 제형 처리구의 병원성 미생물의 생육반경을 측정하였다. 도 2에서 나타난 바와 같이 제형으로부터 방출된 미생물에 의하여F. solani의 생육이 저해되는 것을 관찰할 수 있었다.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.

본 발명은 병원성 미생물에 대해 길항력이 있는 미생물을 분리하고 양전하, 음전하를 가지는 생분해성 고분자 물질간의 정전기적 인력을 이용하여 식물병원균 억제 미생물제제를 제형화함으로써 화학농약 사용에 따른 생태계 파괴와 독성 문제를 해결할 수 있을 뿐만 아니라 미생물에 대해 안정한 고정담체를 제공함으로써 신규한 유기농업, 농약산업 및 환경친화 산업상 매우 유용하다.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) 미생물 배양액과 0.1 내지 5.0 중량% 음전하 생분해성 고분자 용액의 혼합액을 0.5~3.0 중량% 금속염 용액에 떨어뜨려 비드를 제조하는 단계; 및(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) 상기 비드를 0.1∼3.0 중량% 양전하 생분해성 고분자 용액에 침지시켜 30분~2시간동안 교반하여 정전기적 인력에 의하여 코팅하는 단계를 포함하는 미생물 제제의 제형화 방법.(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. 제 1항에 있어서,The method of claim 1, (c) 상기 비드를 50mM 소듐 아세테이트(pH 5.5)로 세척하여 여분의 양전하 생분해성 고분자 물질을 제거하는 단계;(c) washing the beads with 50 mM sodium acetate (pH 5.5) to remove excess positively charged biodegradable polymeric material; (d) 상기 비드를 0.1 내지 2.0 중량% 음전하 생분해성 고분자 용액에 침지시켜 교반하는 단계; 및(d) immersing the beads in 0.1 to 2.0 wt% negative charge biodegradable polymer solution and stirring; And (e) 상기 비드를 생리식염수로 세척한 다음 0.1 내지 2.0 중량% 양전하 생분해성 고분자 용액에 침지시켜 교반하여 외막을 형성하는 단계를 추가로 포함하는 것을 특징으로 하는 미생물 제제의 제형화 방법.(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. 제 1항에 있어서, 상기 미생물 배양액과 음전하 생분해성 고분자 용액이 20:80 내지 50:50의 무게비로 혼합되는 것을 특징으로 하는 미생물 제제의 제형화 방법.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. 제 1항 또는 제 2항에 있어서, 상기 음전하 생분해성 고분자 물질로 알지네이트(alginate), 펙틴(pectin), 카라지난(carrageenan), 및 폴리아스파르트산(polyaspartic acid)으로 구성된 그룹으로부터 선택되는 1종 이상의 물질을 사용하는 것을 특징으로 하는 미생물제제의 제형화 방법.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. 제 1항 또는 제 2항에 있어서, 상기 양전하 생분해성 고분자 물질로 키토산, 키토산 유도체, 및 폴리라이신(polylysine)으로 구성된 그룹으로부터 선택되는 1종 이상의 물질을 사용하는 것을 특징으로 하는 미생물제제의 제형화 방법.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. 제 1항에 있어서, 상기 금속염으로 Ca2+, Cu2+, Zn2+, Ni2+, Co2+, Mn2+, Al2+, Fe2+또는 Mg2+를 발생시키는 물질을 사용하는 것을 특징으로 하는 미생물제제의 제형화 방법.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. 제 1항의 방법에 의해 제조된 미생물 제제.Microbial agent prepared by the method of claim 1.
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US8119780B2 (en) 2006-06-02 2012-02-21 Synedgen, Inc. Chitosan-derivative compounds and methods of controlling microbial populations
KR20230111895A (en) 2022-01-19 2023-07-26 한국과학기술연구원 Polymer-microorganism composites and fabrication method thereof

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US8119780B2 (en) 2006-06-02 2012-02-21 Synedgen, Inc. Chitosan-derivative compounds and methods of controlling microbial populations
US8658775B2 (en) 2006-06-02 2014-02-25 Shenda Baker Chitosan-derivative compounds and methods of controlling microbial populations
US9029351B2 (en) 2006-06-02 2015-05-12 Synedgen, Inc. Chitosan-derivative compounds and methods of controlling microbial populations
US9732164B2 (en) 2006-06-02 2017-08-15 Synedgen, Inc. Chitosan-derivative compounds and methods of controlling microbial populations
US10494451B2 (en) 2006-06-02 2019-12-03 Synedgen, Inc. Chitosan-derivative compounds and methods of controlling microbial populations
KR100864399B1 (en) 2007-06-20 2008-10-20 경상대학교산학협력단 A method for capsulating useful agricultural culture using alginate shell bead having improved drought resistance viability of useful agricultural culture
KR20230111895A (en) 2022-01-19 2023-07-26 한국과학기술연구원 Polymer-microorganism composites and fabrication method thereof

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