KR102655520B1 - Composition for controlling harmful algae containing Kitasatospora sp. as an active ingredient, and use thereof - Google Patents
Composition for controlling harmful algae containing Kitasatospora sp. as an active ingredient, and use thereof Download PDFInfo
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Abstract
본 발명은 키타사토스포라 퍼페오푸스카 CDS15(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 균주에 관한 것으로, 키타사토스포라 퍼페오푸스카 CDS15(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 균주 및 이의 배양액은 우수한 유해 미세조류 저감효과를 나타낸다.The present invention relates to the Kitasatospora purpeofusca CDS15 ( Kitasatospora purpeofusca CDS15; Accession number: KACC81166BP) strain, and the Kitasatospora purpeofusca CDS15 ( Kitasatospora purpeofusca CDS15; Accession number: KACC81166BP) strain and its culture medium are excellent. It shows the effect of reducing harmful microalgae.
Description
본 발명은 키타사토스포라 속(Kitasatospora sp.)을 유효성분으로 포함하는 유해 조류 방제용 살조 조성물 및 이의 용도에 관한 것이다.The present invention relates to an algaic composition for controlling harmful algae containing Kitasatospora sp. as an active ingredient and its use.
최근 우리나라를 비롯한 전 세계의 하천과 호수에서 심한 부영양화로 조류가 대량으로 증식하여 상수 뿐 아니라 산업용수의 수원으로도 위협을 받고 있으며, 이에 따른 피해는 점차 증가하고 있다. 특히 여름철에 빈번히 발생하는 시아노박테리아의 녹조현상은 수질관리의 측면에서 매우 중요하다. 호수에서 가장 흔히 출현하는 시아노박테리아는 마이크로시스티스(Microcystis), 아나베나(Anabaena), 오실레이토리아(Oscillatoria) 등이 알려져 있고, 국내 대부분의 부영양호에서는 마이크로시스티스 에루지노사(Microcystis aeruginosa)가 우점하고 있다(김 등 1999, 이 등 2002, 이 등 2003).Recently, algae has proliferated in large quantities in rivers and lakes around the world, including Korea, due to severe eutrophication, threatening not only water sources but also industrial water sources, and the resulting damage is gradually increasing. In particular, the green algae phenomenon of cyanobacteria, which frequently occurs in summer, is very important in terms of water quality management. The most commonly occurring cyanobacteria in lakes are known to be Microcystis , Anabaena , and Oscillatoria , and Microcystis aeruginosa is found in most eutrophic lakes in Korea. It is dominant (Kim et al. 1999, Lee et al. 2002, Lee et al. 2003).
녹조를 일으키는 미생물은 크게 녹조류와 남조류로 구분되는데, 독성을 분비하는 미생물은 대부분 남조류에 속한다. 남조류의 독성 물질은 수중에 사는 어패류 뿐만 아니라 사람을 포함한 동물이나 곤충들에게도 치명적인 것으로 알려져 있다. 남조류의 일종인 ‘시아노박테리아(cyanobacteria)’가 증가했을 때 발생하는 독성 물질은 특히 어린이와 애완동물에게 치명적인 것으로 보고된바 있으며, 시아노박테리아와 접촉할 경우 안구 충혈, 피부 두드러기, 구토, 설사 등의 증상이 발생할 수 있다. 또한, 녹조류나 남조류에 붙어 포자 상태로 기생하다가 어패류나 곤충, 또는 동물의 체내로 유입되는 감염성 곰팡이에 의해 질병이 유발되기도 한다.Microorganisms that cause green algae are largely divided into green algae and blue-green algae, and most microorganisms that secrete toxicity belong to blue-green algae. Toxic substances in blue-green algae are known to be fatal not only to fish and shellfish living in the water, but also to animals and insects, including humans. Toxic substances produced when 'cyanobacteria', a type of blue-green algae, increase, have been reported to be particularly fatal to children and pets. Contact with cyanobacteria can cause bloodshot eyes, skin hives, vomiting, and diarrhea. Symptoms such as: Additionally, diseases may be caused by infectious fungi that attach to green algae or blue-green algae in the form of spores and then enter the bodies of fish, shellfish, insects, or animals.
이에 본 발명은 유해 미세조류인 마이크로시스티스 에루지노사(Microcystis aeruginosa)에 뛰어난 방제효과를 보이는 키타사토스포라 속 균주를 확인하여 본 발명을 완성하였다.Accordingly, the present invention was completed by identifying a strain of the genus Kitasatospora that shows excellent control effects against the harmful microalgae Microcystis aeruginosa .
본 발명은 부영양화에 의한 녹조 발생을 방지 또는 제어하기 위한 키타사토스포라 속(Kitasatospora sp.) 신규 균주를 제공하는 것을 목적으로 한다.The purpose of the present invention is to provide a new strain of Kitasatospora sp. for preventing or controlling the occurrence of green algae due to eutrophication.
상기한 바와 같은 기술적 과제를 달성하기 위해서 본 발명은, 유해 조류에 대한 살조 활성을 가지는, 키타사토스포라 퍼페오푸스카 CDS15(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 균주를 제공한다.In order to achieve the technical problems described above, the present invention provides a strain of Kitasatospora purpeofusca CDS15 ( Kitasatospora purpeofusca CDS15; Accession number: KACC81166BP), which has algicidal activity against harmful algae.
본 발명의 일 실시예에 있어서, 상기 유해 조류는 마이크로시스티스 속(Microcystis spp.), 아나베나 속(Anabaena spp.) 및 오실레이토리아 속(Oscillatoria spp.)으로 이루어진 군에서 선택되는 어느 하나 이상인 것을 특징으로 할 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the harmful algae is at least one selected from the group consisting of Microcystis spp., Anabaena spp., and Oscillatoria spp. It may be characterized, but is not limited to this.
또한, 본 발명은 상기 키타사토스포라 퍼페오푸스카 CDS15(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 균주 또는 이의 배양액을 유효성분으로 포함하는, 유해 조류 방제용 살조 조성물을 제공한다.In addition, the present invention provides an algae composition for controlling harmful algae, comprising the Kitasatospora purpeofusca CDS15 (Accession number: KACC81166BP) strain or its culture medium as an active ingredient.
또한, 상기 유해 조류 방제용 살조 조성물을 녹조가 발생한 하천, 호소 또는 습지에 방제하는 단계를 포함하는, 유해 조류 살조 방법을 제공한다.In addition, a method for controlling harmful algae is provided, comprising the step of controlling the algae composition for controlling harmful algae in a river, lake, or wetland where green algae occurs.
본 발명의 일 실시예에 있어서, 상기 유해 조류는 마이크로시스티스 속 (Microcystis spp.), 아나베나 속 (Anabaena spp.) 및 오실레이토리아 속 (Oscillatoria spp.)으로 이루어진 군에서 선택되는 어느 하나 이상인 것을 특징으로 할 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the harmful algae is at least one selected from the group consisting of Microcystis spp., Anabaena spp., and Oscillatoria spp. It may be characterized, but is not limited to this.
본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 및 이의 배양액은 우수한 유해 미세조류 저감효과를 가지므로 유해 조류 방제용 살조 조성물로 사용될 수 있다.The 0508CDS15 strain ( Kitasatospora purpeofusca CDS15; Accession number: KACC81166BP) of the present invention and its culture medium have excellent harmful microalgae reduction effects and can be used as an algae composition for controlling harmful algae.
도 1 내지 9는 55종의 균주에 대한 살조 활성을 나타낸 도이다.
도 10은 본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP)의 계통수를 나타낸 도이다.
도 11은 본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP)와 Kitasatospora purpeofusca 표준균주의 살조활성을 비교한 도이다.
도 12는 본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 처리에 따른 Microcystis aeruginosa의 live cell 변화 추이를 나타낸 도이다.
도 13은 본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 처리에 따른 Microcystis aeruginosa의 Chlorophyll a 농도 변화 추이를 나타낸 도이다.
도 14는 본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 처리에 따른 Microcystis aeruginosa에 대한 살조 활성을 확인한 도이다.
도 15는 본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 접종에 따른 활성산소 함량을 나타낸 도이다.
도 16은 본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 접종에 따른 MDA 함량을 나타낸 도이다.
도 17은 본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 접종에 따른 SOD 활성을 나타낸 도이다.
도 18은 본 발명의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 접종에 따른 CAT 활성을 나타낸 도이다.Figures 1 to 9 are diagrams showing algicidal activity against 55 strains.
Figure 10 is a diagram showing the phylogenetic tree of strain 0508CDS15 ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP) of the present invention.
Figure 11 is a diagram comparing the algicidal activity of strain 0508CDS15 of the present invention ( Kitaspora purpeofusca CDS15; accession number: KACC81166BP) and the standard strain of Kitasatospora purpeofusca .
Figure 12 is a diagram showing the change trend of live cells of Microcystis aeruginosa according to treatment with water extract of culture medium of strain 0508CDS15 ( Kitaspora purpeofusca CDS15; Accession number: KACC81166BP) of the present invention.
Figure 13 is a diagram showing the change in Chlorophyll a concentration of Microcystis aeruginosa according to treatment with the culture water extract of the 0508CDS15 strain ( Kitaspora purpeofusca CDS15; Accession number: KACC81166BP) of the present invention.
Figure 14 is a diagram confirming the algicidal activity against Microcystis aeruginosa according to treatment with the culture water extract of the 0508CDS15 strain ( Kitaspora purpeofusca CDS15; Accession number: KACC81166BP) of the present invention.
Figure 15 is a diagram showing the active oxygen content according to the inoculation of the culture water extract of the 0508CDS15 strain ( Kitaspora purpeofusca CDS15; Accession number: KACC81166BP) of the present invention.
Figure 16 is a diagram showing the MDA content according to inoculation with the culture water extract of the 0508CDS15 strain ( Kitaspora purpeofusca CDS15; Accession number: KACC81166BP) of the present invention.
Figure 17 is a diagram showing the SOD activity according to inoculation with the culture water extract of the 0508CDS15 strain ( Kitaspora purpeofusca CDS15; Accession number: KACC81166BP) of the present invention.
Figure 18 is a diagram showing CAT activity according to inoculation with culture water extract of the 0508CDS15 strain ( Kitaspora purpeofusca CDS15; Accession number: KACC81166BP) of the present invention.
본 발명은 유해 조류에 대한 살조 활성을 가지는, 키타사토스포라 퍼페오푸스카 CDS15(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 균주를 제공한다.The present invention provides a strain of Kitasatospora purpeofusca CDS15 ( Kitasatospora purpeofusca CDS15; Accession number: KACC81166BP), which has algicidal activity against harmful algae.
또한, 본 발명은 키타사토스포라 퍼페오푸스카 CDS15(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 균주 또는 이의 배양액을 유효성분으로 포함하는 유해 조류 방제용 살조 조성물을 제공한다.In addition, the present invention provides an algae composition for controlling harmful algae comprising Kitasatospora purpeofusca CDS15 (Accession number: KACC81166BP) strain or its culture medium as an active ingredient.
본 발명에 있어서, 상기 유해 조류는 마이크로시스티스 속(Microcystis spp.), 아나베나 속(Anabaena spp.) 및 오실레이토리아 속(Oscillatoria spp.)으로 이루어진 군에서 선택되는 어느 하나 이상인 것일 수 있다.In the present invention, the harmful algae may be any one or more selected from the group consisting of Microcystis spp., Anabaena spp., and Oscillatoria spp.
또한, 본 발명은 상기 유해 조류 방제용 살조 조성물을 녹조가 발생한 하천, 호소 또는 습지에 방제하는 단계를 포함하는 유해 조류 살조 방법을 제공한다.In addition, the present invention provides a method for controlling harmful algae, comprising the step of controlling the algae composition for controlling harmful algae in a river, lake, or wetland where green algae occurs.
본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시 예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 “포함”한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.The terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when it is said that a part “includes” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 '%'는 별도의 언급이 없는 경우, 고체/고체는 (w/w) %, 고체/액체는 (w/v) %, 그리고 액체/액체는 (v/v) %이다.Throughout this specification, '%' used to indicate the concentration of a specific substance means (w/w) % for solid/solid, (w/v) % for solid/liquid, and Liquid/Liquid is (v/v) %.
이하, 실시예 및 실험예를 통하여 본 발명을 보다 자세히 설명한다. 다만, 하기 실시예 및 실험예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다.Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the following examples and experimental examples are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description is omitted. It can be done, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.
<실험예 1> 살조 활성을 나타내는 균주 분리 및 동정<Experimental Example 1> Isolation and identification of strains exhibiting algicidal activity
1-1. 살조 활성을 나타내는 균주 확인1-1. Confirmation of strains showing algicidal activity
충청북도 옥천군 대청호 지류 추동, 추소리 담수로부터 총 55종의 균주를 분리하였으며, 55종의 배양상청을 대상으로 유해 미세조류인 Microcystis aeruginosa(이하, M.aeruginosa)에 대한 살조 활성을 측정하였다. 고체배지 상의 55종의 균주 군락을 24 well plate를 사용하여 각 well 당 2 ㎖ TBS, R2A 및 PPB 액체배지에 접종한 후, 25 ℃에서 7일간 100 rpm으로 진탕배양 하였다. 배양 후의 배양액을 원심 분리 및 필터링(syringe filter; pore size 0.2 um, ADVANTEC) 하여 살조 실험의 시료로 이용하였다. 균주 배양액을 종 농도 5%로 접종하여 M.aeruginosa에 대한 살조 활성을 확인하였다. M.aeruginosa는 1.0 × 107 cells/㎖, 96 well culture plate 200 ㎍/㎖ 스케일로 살조 활성 실험을 진행하였으며, 접종일로부터 4일째 살조 활성을 측정하였다. 살조활정 측정방법은 plate 리더기를 사용한 흡광도 측정법(OD680) 또는, Fluorescein Diacetate(2 ㎍/㎖) 염색 후, Flowcytometer 분석기(Guava flowcytometer, Luminex)를 이용하여 live cell의 수를 측정하였다. A total of 55 strains were isolated from the Chudong and Chusori freshwater tributaries of Daecheong Lake in Okcheon-gun, Chungcheongbuk-do, and the algicidal activity against the harmful microalgae Microcystis aeruginosa (hereinafter referred to as M. aeruginosa ) was measured on 55 types of culture supernatants. Colonies of 55 strains on solid media were inoculated into 2 ml of TBS, R2A, and PPB liquid media per well using a 24 well plate, and then cultured with shaking at 100 rpm for 7 days at 25°C. After culturing, the culture medium was centrifuged and filtered (syringe filter; pore size 0.2 um, ADVANTEC) and used as a sample for the algaecide experiment. Algicidal activity against M. aeruginosa was confirmed by inoculating the strain culture at a species concentration of 5%. M. aeruginosa was tested for algicidal activity on a scale of 1.0 Algicidal activity was measured by absorbance measurement (OD680) using a plate reader, or by staining with Fluorescein Diacetate (2 ㎍/ml) and measuring the number of live cells using a flowcytometer analyzer (Guava flowcytometer, Luminex).
도 1 내지 도 4에 균주배양액(TBS)의 살조 활성 결과를, 도 5 내지 도 8에 균주배양액(R2A)의 살조 활성 결과를, 도 9에 균주배양액(PPB)의 살조 활성 결과를 나타내었다. 도 4의 0508SA2와 도 6의 0508CDS15에서 85% 이상의 살조 활성이 나타났다. 이하, 살조활성이 가장 높은 것으로 확인된 0508CDS15 균주를 분리 및 동정하였다.Figures 1 to 4 show the algicidal activity results of the strain culture medium (TBS), Figures 5 to 8 show the algicidal activity results of the strain culture medium (R2A), and Figure 9 show the algicidal activity results of the strain culture medium (PPB). Algicidal activity of more than 85% was observed in 0508SA2 in Figure 4 and 0508CDS15 in Figure 6. Hereinafter, strain 0508CDS15, which was confirmed to have the highest algicidal activity, was isolated and identified.
1-2. 0508CDS15 균주 분리 및 동정1-2. Isolation and identification of strain 0508CDS15
0508CDS15 균주는 충청북도 옥천군 대청호 지류 추동의 강가에서 채수한 담수로부터 분리되었다. 채수한 담수 2 L를 3.0 ㎛의 Membrane filter(Advantec 社)를 이용하여 여과한 뒤 미생물 및 현탁물을 채집하고, 20 ㎖의 여과된 담수액에 현탁시켜 약 100배의 담수 농축액을 제조하였다. 농축액에 화학적(cycloheximide) 및 물리적 처리(heat chock 65℃, 15분)를 한 뒤 ISP4 배지에 100 ㎕ 도말하여 25 ℃에서 7일간 배양하였다. 배양 후, 배지상에 나타나는 군락을 새로운 ISP4 배지로 순수 분리하였다. 분리된 균주를 포함하는 plate를 (주)마크로젠에 의뢰하여 16S rRNA gene을 기반으로 균동정을 수행한 결과, 0508CDS15 균주는 Kitasatospora purpeofusca에 similarity 99.86 % (1453/1486, NCBI)로 나타났다. 최종 분리된 균주는 Kitasatospora purpeofusca CDS15(수탁번호: KACC81166BP)이다. 도 10에 0508CDS15 균주의 분자생물학적 계통수를 나타내었다.Strain 0508CDS15 was isolated from fresh water collected from the riverside of Chudong, a tributary of Daecheong Lake, Okcheon-gun, Chungcheongbuk-do. After filtering 2 L of collected fresh water using a 3.0 ㎛ membrane filter (Advantec), microorganisms and suspensions were collected and suspended in 20 ml of filtered fresh water to prepare a fresh water concentrate about 100 times larger. After chemical (cycloheximide) and physical treatment (heat chock at 65°C, 15 minutes) of the concentrate, 100 μl was spread on ISP4 medium and cultured at 25°C for 7 days. After culturing, the colonies appearing on the medium were separated into new ISP4 medium. The plate containing the isolated strain was commissioned by Macrogen Co., Ltd. to perform fungal identification based on the 16S rRNA gene. As a result, strain 0508CDS15 was found to have a similarity of 99.86% (1453/1486, NCBI) to Kitasatospora purpeofusca . The final isolated strain was Kitasatospora purpeofusca CDS15 (accession number: KACC81166BP). Figure 10 shows the molecular biological phylogenetic tree of strain 0508CDS15.
1-3. 0508CDS15 균주의 살조 활성 확인1-3. Confirmation of algicidal activity of strain 0508CDS15
Kitasatospora purpeofusca의 표준균주를 이용하여 M.aeruginosa에 대한 살조 활성을 확인하였다(Control: 1% DMSO 또는 5% R2A배지, CHL: Chlroramphenicol 200 ㎍/㎖, 배양상청은 각각 5 %(v/v) 처리).Algicidal activity against M. aeruginosa was confirmed using the standard strain of Kitasatospora purpeofusca (Control: 1% DMSO or 5% R2A medium, CHL: Chlroramphenicol 200 ㎍/㎖, culture supernatant treated with 5% (v/v) each. ).
250ml 배양플라스크에 150ml BG-11 배지를넣고 M.aeruginosa 를 OD680=0.2 배양액 10%로 접종하였다. 배양일차 11~17일차의 log Phase의 세포를 원심분리하여 배양액을 제거한 뒤, 다시 BG-11 배지를 첨가하여 OD680=0.2 로 suspension하였다. 준비된 조류 플라스크에 OD680=0.2의 배양액을 10ml 접종하였다. 접종한 플라스크는 4000 lux, 20도, 빛:암흑 타임코스 14H:10으로 광배양기에서 하루 pre 인큐베이션하였다. Kitasatospora purpeofusca CDS15(수탁번호: KACC81166BP) 및 Tpye strain을 R2A배지 50ml 플라스크에 접종한 뒤, 일주일간 shaking(50 rpm), 25 ℃ 조건에서 배양한 후, 균주의 배양상청을 무균상태로 회수하여 M.aeruginosar의 플라스크에 접종하였다. 배양상청 접종 4일후 200 ㎕씩(n=3) 샘플링하여 Flowcytometer 분석기(Guava flowcytometer, Luminex)로 셀수를 계수 (cells/ml) 및 셀 활성을 분석하였다.150ml BG-11 medium was added to a 250ml culture flask, and M. aeruginosa was inoculated with 10% of the OD680=0.2 culture medium. Cells in log phase on days 11 to 17 of culture were centrifuged to remove the culture medium, and BG-11 medium was added again to suspend them at OD680=0.2. 10ml of culture medium with OD680=0.2 was inoculated into the prepared algal flask. The inoculated flask was pre-incubated for one day in a photoincubator at 4000 lux, 20 degrees, light:dark time course 14H:10. Kitasatospora purpeofusca CDS15 (accession number: KACC81166BP) and Tpye strain were inoculated into a 50ml flask of R2A medium, shaken (50 rpm) for a week, and cultured at 25°C. Then, the culture supernatant of the strain was recovered in sterile condition and M. aeruginosar was inoculated into the flask. Four days after inoculation of the culture supernatant, 200 ㎕ each (n=3) was sampled and the number of cells (cells/ml) and cell activity were analyzed using a flowcytometer analyzer (Guava flowcytometer, Luminex).
도 11에서 보듯이, 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 이외의 표준균주에서는 살조 활성이 나타나지 않았다(*p≥0.001).As shown in Figure 11, standard strains other than strain 0508CDS15 ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP) did not show algicidal activity (*p≥0.001).
<실험예 2> 균주 배양액 추출물의 살조 활성 확인<Experimental Example 2> Confirmation of algicidal activity of strain culture extract
고체배지 상의 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 군락을 200 ㎖ R2A 액체배지에 접종한 후, 25 ℃에서 8일간 200 rpm으로 진탕배양 하였다. 배양 후의 배양액과 동일한 양의 1-buOH으로 3번 추출한 후 물(aqueous)층과 부탄올(1-buOH)층으로 분별 깔대기를 이용하여 분리하였다. evaporator를 이용하여 물(aqueous)층으로부터 약 150 mg, 부탄올(1-buOH)층으로부터 20 mg의 residue가 추출되었다.A colony of strain 0508CDS15 ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP) on solid medium was inoculated into 200 ml R2A liquid medium and cultured with shaking at 200 rpm for 8 days at 25°C. It was extracted three times with the same amount of 1-buOH as the culture medium after culturing, and then separated into an aqueous layer and a butanol (1-buOH) layer using a separatory funnel. About 150 mg of residue was extracted from the aqueous layer and 20 mg of residue from the butanol (1-buOH) layer using an evaporator.
균주 배양액 물 추출물 20 mg/㎖(DMSO), 균주 배양액 부탄올 추출물 15 mg/㎖(DMSO)를 각각 50, 100, 200㎍/㎖로 접종하여 M.aeruginosa에 대한 살조 활성을 확인하였다. M.aeruginosa는 1.0 × 107 cells/㎖, culture plask 30 ㎖ 스케일로 살조 활성 실험을 진행하였으며, 접종일로부터 2일마다 8일째까지 매일 살조 활성을 측정하였다. Fluorescein Diacetate(2 ㎍/㎖) 염색 후, Flowcytometer 분석기(Guava flowcytometer, Luminex)를 이용하여 live cell의 수를 측정하였으며, 동일 배양 측정일의 Chlorophyll a(이하, Chl-a) 농도를 측정하였다.Algicidal activity against M. aeruginosa was confirmed by inoculating 20 mg/ml of strain culture water extract (DMSO) and 15 mg/ml of strain culture butanol extract (DMSO) at 50, 100, and 200 μg/ml, respectively. M. aeruginosa was tested for algicidal activity at 1.0 After staining with Fluorescein Diacetate (2 ㎍/ml), the number of live cells was measured using a flowcytometer analyzer (Guava flowcytometer, Luminex), and the concentration of Chlorophyll a (hereinafter referred to as Chl-a) on the same culture measurement day was measured.
살조 활성은 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물에서 나타났다. 도 12에 M.aeruginosa의 live cell 수를, 도 13에 M.aeruginosa의 Chl-a 농도 변화를 나타내었다. 배양일수 6일째부터 높은 살조 활성을 나타내었으며 추출물의 접종 농도가 높을수록 강한 살조 활성을 나타내었다. 배양일수에 따른 Chl-a 농도도 추출물의 접종 농도가 높을수록 감소하였으며, control 과의 차이는 배양일수 8일차에 가장 크게 나타났다.Algicidal activity was shown in the culture water extract of strain 0508CDS15 ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP). Figure 12 shows the number of live cells of M. aeruginosa , and Figure 13 shows the change in Chl-a concentration of M. aeruginosa . High algicidal activity was shown from the 6th day of culture, and the higher the inoculum concentration of the extract, the stronger the algicidal activity. Chl-a concentration according to the number of days of culture also decreased as the inoculum concentration of the extract increased, and the difference with the control was greatest on the 8th day of culture.
또한, 균주 배양액 물 추출물을 15, 75, 150 및 300 ㎍/㎖로 접종하여 M.aeruginosa에 대한 살조 활성을 확인하였다. 도 14에 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물의 살조 활성을 나타내었다. In addition, the algicidal activity against M. aeruginosa was confirmed by inoculating the strain culture water extract at 15, 75, 150, and 300 ㎍/㎖. Figure 14 shows the algicidal activity of the culture water extract of strain 0508CDS15 ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP).
<실험예 3> 활성산소, 지질과산화, 항산화 효소 측정<Experimental Example 3> Measurement of active oxygen, lipid peroxidation, and antioxidant enzymes
상기 실험예 2에서 배양일수 별로 1 ㎖의 미세조류 배양액을 회수하여 활성산소(Reactive oxygen species, ROS), 지질과산화(Malondialdehyde, MDA), 항산화효소(Superoxide Dismutase, SOD 및 Catalase, CAT)를 측정하였다. 동시에 단위 protein 당 물질양을 측정하기 위해 Total protein을 추출하여 물질 정량에 이용하였다. ROS (Reactive Oxygen Species Detection Reagents CM-H2DCFDA, Invitorgen) MDA (TBARS Assay kit STA-330, CELL BIOLABS, INC), SOD (Fish SOD ELISA kit, MyBioSource CORP), CAT (ARG82219 Catalase Activity Assay Kit, arigobio) 및 Total protein (Quick Start™ Bradford Kit1, BIO-RAD)은 각각의 키트를 이용하여 측정하였다.In Experimental Example 2, 1 ml of microalgae culture medium was recovered for each number of days of culture, and reactive oxygen species (ROS), lipid peroxidation (Malondialdehyde, MDA), and antioxidant enzymes (Superoxide Dismutase, SOD and Catalase, CAT) were measured. . At the same time, to measure the amount of substance per unit protein, total protein was extracted and used for substance quantification. ROS (Reactive Oxygen Species Detection Reagents CM-H2DCFDA, Invitorgen) MDA (TBARS Assay kit STA-330, CELL BIOLABS, INC), SOD (Fish SOD ELISA kit, MyBioSource CORP), CAT (ARG82219 Catalase Activity Assay Kit, arigobio) and Total protein (Quick Start™ Bradford Kit1, BIO-RAD) was measured using each kit.
도 15에 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 접종에 따른 활성산소 함량을 나타내었다. 활성산소는 접종 2일째에 증가하였으나, 4일째부터 감소하였으며, 접종 농도가 높을수록 더욱 감소하는 것으로 나타났다. Figure 15 shows the active oxygen content according to the inoculation of the culture water extract of strain 0508CDS15 ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP). Active oxygen increased on the 2nd day of inoculation, but decreased from the 4th day, and decreased further as the inoculation concentration increased.
도 16에 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 접종에 따른 MDA 함량을 나타내었다. 접종 2일째에는 ROS에 의한 세포손상으로 인해 MDA 함량이 증가하였으나, 접종 4일째에는 수치가 다시 감소하였다. Figure 16 shows the MDA content according to inoculation with culture water extract of strain 0508CDS15 ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP). On the 2nd day of inoculation, the MDA content increased due to cell damage caused by ROS, but on the 4th day of inoculation, the level decreased again.
도 17에 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 접종에 따른 SOD 활성을, 도 18에 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물 접종에 따른 CAT 활성을 나타내었다. SOD와 CAT 활성은 접종일수에 따라 증가하는 양상을 보였다. SOD와 CAT 활성은 ROS를 단계적으로 제거하는 것으로 나타났다.Figure 17 shows SOD activity following inoculation with culture water extract of strain 0508CDS15 ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP), and Figure 18 shows CAT activity following inoculation of culture water extract of strain 0508CDS15 ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP). It was. SOD and CAT activities showed an increase according to the number of days of vaccination. SOD and CAT activities were shown to eliminate ROS step by step.
즉, 0508CDS15 균주(Kitasatospora purpeofusca CDS15; 수탁번호: KACC81166BP) 배양액 물 추출물은 M.aeruginosa의 성장을 억제하였으며, 세포의 방어기작으로 나타나는 항산화활성 관련 contents 및 효소활성으로 보아, 배양일수 2일째 활성산소에 의한 세포손상으로 인한 MDA가 추출물의 처리농도에 비례하여 나타났으며, 활성산소를 단계적으로 제거하는 SOD 및 CAT가 배양일수 및 처리농도에 비례하게 나타났다. 종합적으로 고려할 때 0508CSD15균주 추출물은 M.aeruginosa의 성장을 억제하는 기능을 가지며 그 작용기작은 세포기능 저하에 따른 ROS 발생과 항산화활성 방어기작으로 나타났다.In other words, the water extract of the 0508CDS15 strain ( Kitasatospora purpeofusca CDS15; accession number: KACC81166BP) culture medium inhibited the growth of M. aeruginosa , and judging from the antioxidant activity-related contents and enzyme activity shown as a cellular defense mechanism, it was exposed to active oxygen on the second day of culture. MDA due to cell damage appeared in proportion to the treatment concentration of the extract, and SOD and CAT, which gradually remove active oxygen, appeared in proportion to the number of days of culture and treatment concentration. When comprehensively considered, the extract of strain 0508CSD15 has the function of inhibiting the growth of M. aeruginosa , and its mechanism of action appears to be ROS generation due to decreased cell function and antioxidant activity defense mechanism.
Claims (5)
Kitasatospora purpeofusca CDS15 ( Kitasatospora purpeofusca CDS15; Accession number: KACC81166BP) strain, which has algicidal activity against Microcystis aeruginosa .
An algicidal composition for controlling Microcystis aeruginosa , comprising the Kitasatospora purpeofusca CDS15 ( Kitasatospora purpeofusca CDS15; Accession number: KACC81166BP) strain of claim 1 or its culture medium as an active ingredient.
A method of killing Microcystis aeruginosa , comprising the step of controlling the composition of claim 3 in a river, lake or wetland where green algae has occurred.
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