KR102095200B1 - Novel fungus Penicillium dokdoense and use thereof - Google Patents
Novel fungus Penicillium dokdoense and use thereof Download PDFInfo
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- KR102095200B1 KR102095200B1 KR1020180087265A KR20180087265A KR102095200B1 KR 102095200 B1 KR102095200 B1 KR 102095200B1 KR 1020180087265 A KR1020180087265 A KR 1020180087265A KR 20180087265 A KR20180087265 A KR 20180087265A KR 102095200 B1 KR102095200 B1 KR 102095200B1
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Abstract
본 발명은 신규 진균인 페니실리움 독도엔스(Penicillium dokdoense) 및 이를 활용한 다양한 조성물에 관한 것으로, 산화질소 생성을 억제할 수 있어 염증이나 염증성 질환의 개선, 예방 및 치료에 이용할 수 있다.The present invention is a novel fungus, Penicillium, Penicillium dokdoense ) and various compositions utilizing the same, which can inhibit nitric oxide production, and thus can be used for improvement, prevention and treatment of inflammation or inflammatory diseases.
Description
본 발명은 새로운 페니실리움(Penicillium) 균주인 페니실리움 독도엔스(Penicillium dokdoense) 및 이를 활용한 다양한 기술로 특히 항염증 조성물에 관한 것이다.The present invention relates to a new Penicillium strain, Penicillium dokdoense , and to various anti-inflammatory compositions in particular with various techniques utilizing the same.
상처 발생이나 감염 등에 의해 조직이나 기관이 손상되면 생체 방어기전 중 하나인 염증에 의해 해당 손상 부위를 치유하게 된다. 염증은 만성 염증이나 급성 염증 등 다양한 유형이 존재하고 발생 요인이 매우 다양하며 염증이 지속되는 경우 심각한 질병으로 진행되는 경우도 있다. When a tissue or organ is damaged due to a wound or infection, the damaged area is healed by inflammation, which is one of the biological defense mechanisms. There are various types of inflammation, such as chronic inflammation or acute inflammation, and the occurrence factors are very diverse. In some cases, inflammation continues to be a serious disease.
염증이 발생하면 선천면역 활성을 가지는 대식세포(macrophage)에서 사이토카인, 케모카인, 인터페론, 콜로니 자극인자(colony-stimulating factor), 리소자임, 프로테아제, 성장인자, 에이코사노이드(eicosanoid), 산화질소(nitric oxide, NO) 등 다양한 염증 조절 인자를 생산하게 된다. 대식세포에 생산하는 염증 조절 인자 중 산화질소는 염증을 유발하는 요인들인 박테리아, 바이러스, 암 세포 등에 의한 세포독성이나 세포증식 억제와 관련된 신호전달에 중요한 인자이나 대식세포의 지나친 활성에 의한 과도한 산화질소의 생성은 오히려 다양한 질병을 야기할 수 있는 위험이 있다. When inflammation occurs, cytokines, chemokines, interferons, colony-stimulating factors, lysozymes, proteases, growth factors, eicosanoids, nitric oxides in macrophages with innate immune activity oxide, NO) and various inflammation control factors. Among the inflammation control factors produced in macrophages, nitric oxide is an important factor for signaling related to cytotoxicity or cell proliferation inhibition by bacterial, viral, and cancer cells, which are factors causing inflammation, or excessive nitric oxide due to excessive activity of macrophages Rather, there is a risk of causing various diseases.
염증 조절 인자의 생성을 조절하여 염증을 개선하거나 치료하는 조성물은 식물에서 추출한 천연 화합물이나, 인공 합성 화합물 또는 미생물이 생산하는 성분을 유효성분으로 하는 등 매우 다양하게 연구되고 있다. 예를 들어 한국 공개특허 제10-2016-0060884호에서는 특정 나무의 추출물을 이용한 염증 억제 조성물을 개시하고 있고, 한국 공개특허 제10-2015-0146331호에서는 미생물을 이용한 염증 억제 조성물을 개시하고 있다.Compositions for improving or treating inflammation by regulating the production of inflammatory regulators have been studied in various ways, such as natural compounds extracted from plants, artificial synthetic compounds, or components produced by microorganisms as active ingredients. For example, Korean Patent Publication No. 10-2016-0060884 discloses an anti-inflammatory composition using an extract of a specific tree, and Korean Patent Publication No. 10-2015-0146331 discloses an anti-inflammatory composition using a microorganism.
본 발명은 신규 진균인 페니실리움 독도엔스(Penicillium dokdoense)를 제공하고, 이를 활용하여 항염증 및 염증성 질환을 예방하거나 치료하기 위한 약학적 조성물, 건강기능식품, 화장료 조성물, 사료, 항진균제, 항균제 등을 제공한다.The present invention is a novel fungus, Penicillium, Penicillium dokdoense ), and provides pharmaceutical composition, health functional food, cosmetic composition, feed, antifungal agent, and antibacterial agent to prevent or treat anti-inflammatory and inflammatory diseases using the same.
본 발명은 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)를 제공한다.The present invention is Penicillium dokdoense ) (accession number: KACC 93309p).
본 발명은 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p), 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양액 및 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양 여과액으로 이루어진 군에서 선택되는 하나 이상을 유효성분으로 포함하는 염증 및 염증성 질환의 예방 또는 치료용 약학 조성물을 제공한다. The present invention is Penicillium dokdoense ) (Accession number: KACC 93309p), Penicillium dokdoense ) (Accession No .: KACC 93309p) and Penicillium dokdoense ) (Accession No .: KACC 93309p) provides a pharmaceutical composition for the prevention or treatment of inflammatory and inflammatory diseases comprising at least one selected from the group consisting of culture filtrate.
본 발명은 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p), 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양액 및 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양 여과액으로 이루어진 군에서 선택되는 하나 이상을 유효성분으로 포함하는 항염증용 건강기능식품 조성물을 제공한다.The present invention is Penicillium dokdoense ) (Accession number: KACC 93309p), Penicillium dokdoense ) (Accession No .: KACC 93309p) and Penicillium dokdoense ) (Accession number: KACC 93309p) provides an anti-inflammatory health functional food composition comprising at least one selected from the group consisting of culture filtrate.
본 발명은 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p), 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양액 및 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양 여과액으로 이루어진 군에서 선택되는 하나 이상을 유효성분으로 포함하는 항염증용 화장료 조성물을 제공한다.The present invention is Penicillium dokdoense ) (Accession number: KACC 93309p), Penicillium dokdoense ) (Accession No .: KACC 93309p) and Penicillium dokdoense ) (Accession number: KACC 93309p) provides an anti-inflammatory cosmetic composition comprising at least one selected from the group consisting of culture filtrate.
본 발명은 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p), 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양액 및 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양 여과액으로 이루어진 군에서 선택되는 하나 이상을 유효성분으로 포함하는 항진균 또는 항균용 약학 조성물을 제공한다.The present invention is Penicillium dokdoense ) (Accession number: KACC 93309p), Penicillium dokdoense ) (Accession No .: KACC 93309p) and Penicillium dokdoense ) (Accession number: KACC 93309p) provides an antifungal or antibacterial pharmaceutical composition comprising at least one selected from the group consisting of culture filtrate.
본 발명의 신규 진균인 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p), 이의 배양액 또는 이의 배양 여과액은 산화질소(nitric oxide) 생성을 조절할 수 있어 염증을 효과적으로 치료하고 예방할 수 있을 뿐만 아니라, 단백질 분해 활성을 나타내고, 항진균 활성 및 항균 활성을 가지고 있어 이와 관련된 질병의 치료 및 예방에 활용할 수 있을 것으로 기대된다. Penicillium , a novel fungus of the present invention dokdoense ) (Accession No .: KACC 93309p), its culture fluid or its culture filtrate can control nitric oxide production to effectively treat and prevent inflammation, as well as show proteolytic activity, antifungal activity and antibacterial It is expected to be used for the treatment and prevention of diseases related to this activity.
도 1은 신규 진균인 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1 및 CNUFC-DDS11-2 균주의 계통수(Phylogenetic tree)로 Citrina 섹션 기반의 최대 우도 분석(the maximum likelihood analysis)에 의해 BenA 및 CaM 에 대한 복합 데이터 세트를 사용하여 구성되었고, Penicillium corylophilum을 아웃그룹(outgroup)으로 사용하였다. 노드(node) 수는 1000 복제(replications)의 부트스트랩 값(bootstrap value, 50% 이상)을 나타내고, 새로운 분류군은 파란색으로 표시하였으며 표준균주로 사용된 균주(ex-type)는 굵게 표시하였다.
도 2는 신규 진균인 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1(holotype) 균주의 형태학적 구조를 보여준다(a-d: obverse view, e-h: reverse view). [a 및 e는 yeast extract sucrose agar (YES) 배지에서의 콜로니, b 및 f는 malt extract agar (MEA) 배지에서의 콜로니, c 및 g는 Czapek yeast autolysate agar (CYA) 배지에서의 콜로니, d 및 h는 creatine sucrose agar (CREA) 배지에서의 콜로니, i~l은 윤생 분생자경(Verticillate conidiophore) 및 피알리드(Phialide) 분생포자(conidia)를 보여주고, n는 분생포자(conidia)를 보여준다. 스케일 바(scale bar)는 i는 20㎛, j~l은 10㎛, m은 5㎛, n은 3㎛ 이다.
도 3은 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1 배양 여액 농도(50㎕/㎖, 100㎕/㎖)에 따른 대식세포 RAW264.7의 세포 증식을 보여준다.
도 4는 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1 배양 여액에 의한 산화질소(nitric oxide, NO)의 생산 억제 효과를 보여준다.
도 5는 페니실리움 독도엔스(Penicillium dokdoense)(CNUFC-DDS11-1) 배양 여액에 의한 활성 산소 감소 효과를 보여준다(MFI : mean fluorescence intensity).
도 6은 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1 배양 여액에 의한 TNF-α 및 IL-1β의 발현 감소 효과를 보여준다.
도 7은 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1의 효소 활성, 항진균 활성 및 항균 활성을 나타낸다.
A) 탈지유 한천에 페니실리움 독도엔스 CNUFC-DDS11-1의 배양에 의해 형성된 콜로니 주변의 클리어 존, B) 알테나리아 알테나타(Alternaria alternata) EML-BLDF1-4에 대한 페니실리움 독도엔스 CNUFC-DDS11-1의 항진균 활성, 및 C) 스타필로코쿠스 아우레우스( Staphylococcus aureus) ATCC에 대한 페니실리움 독도엔스 CNUFC-DDS11-1의 항균 활성.Figure 1 is a new fungus Penicillium Dokdoens ( Penicillium) dokdoense ) Phylogenetic tree of CNUFC-DDS11-1 and CNUFC-DDS11-2 strains, constructed using a complex data set for BenA and CaM by Citrina section-based the maximum likelihood analysis, Penicillium Corylophilum was used as an outgroup. The number of nodes (nodes) represents the bootstrap value (over 50%) of 1000 replications, the new taxonomic group is indicated in blue, and the strain used as a standard strain (ex-type) is shown in bold.
2 is a new fungus, Penicillium, Penicillium dokdoense ) shows the morphological structure of the CNUFC-DDS11-1 (holotype) strain (ad: obverse view, eh: reverse view). [a and e are colonies in yeast extract sucrose agar (YES) medium, b and f are colonies in malt extract agar (MEA) medium, c and g are colonies in Czapek yeast autolysate agar (CYA) medium, d and h shows colonies in creatine sucrose agar (CREA) medium, i ~ l shows verticillate conidiophore and phialide conidia, and n shows conidia. The scale bar is 20 µm for i, 10 µm for j to l, 5 µm for m, and 3 µm for n.
3 is Penicillium dokdoense ) shows cell proliferation of macrophages RAW264.7 according to CNUFC-DDS11-1 culture filtrate concentrations (50 μl / ml, 100 μl / ml).
4 is Penicillium dokdoense ) CNUFC-DDS11-1 shows the inhibitory effect of nitric oxide (NO) production by the culture filtrate.
5 is Penicillium dokdoense ) (CNUFC-DDS11-1) shows the effect of reducing free radicals by the culture filtrate (MFI: mean fluorescence intensity).
6 is Penicillium dokdoense ) CNUFC-DDS11-1 shows the effect of reducing the expression of TNF-α and IL-1β by the culture filtrate.
7 is Penicillium dokdoense ) It shows the enzyme activity, antifungal activity and antibacterial activity of CNUFC-DDS11-1.
A) Clear zone around colonies formed by culturing Penicillium Dokdoensu CNUFC-DDS11-1 in skim milk agar, B) Alternaria Alternaria alternata ) antifungal activity of penicillium dokone CNUFC-DDS11-1 against EML-BLDF1-4, and C) penicillium dokone CNUFC-DDS11-1 against Staphylococcus aureus ATCC Antibacterial activity.
이하 본 발명에 대하여 구체적으로 상세히 설명하고자 한다.Hereinafter, the present invention will be described in detail.
본 명세서에서 사용되는 용어는 따로 정의하지 않는 경우 해당 분야에서 통상의 지식을 가진 자가 일반적으로 이해하는 내용으로 해석되어야 할 것이다. 본 명세서의 도면 및 실시예는 통상의 기술자가 본 발명을 쉽게 이해하고 실시하기 위한 것으로 도면 및 실시예에서 발명의 요지를 흐릴 수 있는 내용은 생략될 수 있으며, 본 발명이 도면 및 실시예로 한정되는 것은 아니다.Unless otherwise defined, terms used in this specification should be interpreted as contents generally understood by a person having ordinary skill in the relevant field. The drawings and examples in the present specification are for those skilled in the art to easily understand and implement the present invention. In the drawings and examples, contents that may obscure the subject matter of the invention may be omitted, and the present invention is limited to the drawings and examples. It does not work.
본 발명은 신규 진균인 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p) 및 이를 이용한 다양한 용도에 관한 것이다. 특히 본 발명의 페니실리움 독도엔스(Penicillium dokdoense)는 항염증 및 염증성 질환의 예방, 개선 및 치료를 비롯하여, 항진균 또는 항균을 위한 용도에 매우 효과적이다. The present invention is a novel fungus, Penicillium, Penicillium dokdoense ) (accession number: KACC 93309p) and various uses thereof. In particular, Penicillium of the present invention dokdoense ) is very effective for use in antifungal or antibacterial, including prevention, improvement and treatment of anti-inflammatory and inflammatory diseases.
본 발명의 신규 진균인 페니실리움 독도엔스(Penicillium dokdoense)는 한국 동해에 위치한 독도의 동도(동쪽 지역)에서 채취한 토양에서 분리 및 동정하고, 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1 및 CNUFC-DDS11-2로 명명하여, 2018년 7월 23일자로 농진청 미생물은행 (KACC)에 기탁번호 KACC 93309p로 기탁하였다. Penicillium , a novel fungus of the present invention dokdoense ) is separated and identified from soil collected from the East Sea (eastern region) of Dokdo located in the East Sea of Korea, and Penicillium dokdoense ) Named CNUFC-DDS11-1 and CNUFC-DDS11-2, deposited on July 23, 2018 with the Depository No. KACC 93309p to the Microorganism Bank of Korea (KACC).
본 발명의 신규 진균인 페니실리움 독도엔스(Penicillium dokdoense)는 P. terrigenum 및 P. copticola 와 연관성이 높으나, CYA 배지에서 느린 성장을 보이고, MEA 배지에서 빠른 성장을 보여 이들과 구분되는 특징을 가진다. 또한 형태학적 관점에서 페니실리움 독도엔스는 기저경자(Metulae)와 기저경자당 피알리드(Phialide) 수가 P. terrigenum 및 P. copticola 보다 많고, P. terrigenum 및 P. copticola에서 나타나지 않는 분기형(divaricate) 분생자경(Conidiophore)을 생산하였으며, 분생포자(Conidia)가 구형에서 반구형이거나 타원 형태로 이들과 구분되는 특징을 가진다. Penicillium , a novel fungus of the present invention dokdoense ) has a high correlation with P. terrigenum and P. copticola , but it shows slow growth in CYA medium and rapid growth in MEA medium, which is distinct from these. In addition, from the morphological point of view, Penicillium Dokdoen's is the number of P. terrigenum and P. copticola of Metulae and Phialide per base . More, P. terrigenum and P. copticola produced divaricate conidiophores that do not appear, and conidia have distinct features from spherical to hemispherical or elliptic.
본 발명의 페니실리움 독도엔스(Penicillium dokdoense)는 다양한 생물학적 활성 효과를 가지고, 특정 물질의 다량 생산할 수 있어 약학적 조성물, 식품, 화장료, 사료, 항생제 등 다양한 조성물로서 활용될 수 있다. 특히, 상기 페니실리움 독도엔스 및 이로부터 생산되는 물질에는 염증을 억제하거나 조절할 수 있는 기능이 있어 다양한 항염증 조성물과 염증성 질환의 예방, 치료 및 개선에 활용될 수 있다. Penicillium of the present invention dokdoense ) has a variety of biologically active effects, can be produced in large quantities of a specific substance can be utilized as a variety of compositions, such as pharmaceutical compositions, food, cosmetic, feed, antibiotics. In particular, the penicillium doxens and substances produced therefrom have a function to suppress or control inflammation, and thus can be used for prevention, treatment, and improvement of various anti-inflammatory compositions and inflammatory diseases.
본 발명의 일 실시예에 따르면 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)는 균체 자체 뿐만 아니라, 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양액, 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)의 배양액을 여과한 배양 여과액도 다양한 생물학적 활성 효과를 가질 수 있다. According to an embodiment of the present invention, Penicillium dokdoense (Accession No .: KACC 93309p) is not only a cell itself, but also a culture medium of Penicillium dokdoense (Accession No .: KACC 93309p), Penny The culture filtrate obtained by filtering the culture solution of Penicillium dokdoense (Accession No: KACC 93309p) may also have various biological activity effects.
또한 페니실리움 독도엔스 및 이의 배양액, 또는 배양 여과액에는 당류, 단백질을 분해할 수 있는 효소 활성을 가지고 있으며, 이러한 효소 활성을 통해 일예로, 셀룰로오스, 헤미셀룰로오스, 전분 및 단백질 등을 분해할 수 있으나, 효소 활성의 범위가 이에 한정하는 것은 아니다.In addition, penicillium doxens and its culture broth or culture filtrate has enzyme activity capable of decomposing sugars and proteins. For example, cellulose, hemicellulose, starch and protein can be decomposed through such enzyme activity. However, the scope of enzyme activity is not limited thereto.
상기 페니실리움 독도엔스 및 이의 배양액, 또는 배양 여과액에 포함된 효소 중 단백질 분해 활성(proteolytic activity)을 갖는 효소는 치즈 숙성을 위한 식품 산업, 단백질 가수 분해물 생산 및 빵의 제조 등에 사용되기 때문에 당업계에서 중요한 효소 활성으로 간주되고 있다. 페니실리움 spp.의 여러 종류의 효소에 대한 연구가 현재까지 이루어져 왔으나, 단백질 분해 효소에 대한 연구가 보고된 경우는 거의 없다.The enzyme having proteolytic activity among the enzymes contained in the penicillium doxens and its culture medium, or the culture filtrate, is used in the food industry for cheese ripening, protein hydrolyzate production, and bread production. It is considered an important enzyme activity in the industry. Studies on various types of enzymes of Penicillium spp. Have been conducted to date, but few studies have been reported on proteolytic enzymes.
본 발명의 신규 진균인 페니실리움 독도엔스(Penicillium dokdoense)는 항진균 또는 항균 활성을 가지고 있어, 약학적 조성물, 식품, 화장료, 사료, 항생제 등 다양한 조성물로서 활용될 수 있다. Penicillium , a novel fungus of the present invention dokdoense ) has antifungal or antibacterial activity, and can be used as various compositions such as pharmaceutical compositions, food, cosmetics, feed, and antibiotics.
본 발명의 일 실시예에 의하면, 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1은 단백질에 대해 분해 활성을 나타내는 것을 새롭게 확인하였으며, 시트리나 섹션(section Citrina)에 속하는 새로운 페니실리움 종인 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1은 효소 생산에 대한 가능성을 제시하였다.According to an embodiment of the present invention, Penicillium dokdoense CNUFC-DDS11-1 was newly confirmed to exhibit decomposition activity against proteins, and is a new penicillium species belonging to the section Citrina. Penicillium dokdoense ) CNUFC-DDS11-1 suggested the potential for enzyme production.
본 발명의 일 실시예에 따른 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1는 ALT, AOH, ATX-1, AME의 4가지 미코톡신(mycotoxin)을 생산하는 알테나리아 알테나타(Alternaria alternata ) EML-BLDF1-4에 대해 항진균 활성(antifungal activity)가 있음을 확인하였다. Penicillium according to an embodiment of the present invention dokdoense ) CNUFC-DDS11-1 is an Alternaria that produces four mycotoxins, ALT, AOH, ATX-1, and AME. alternata ) EML-BLDF1-4 was confirmed to have antifungal activity.
본 발명의 일 실시예에 따른 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1의 유기용매 추출물은 병원균인 스타필로코쿠스 아우레우스(Staphylococcus aureus) ATCC에 대해 항균 활성이 있음을 확인하였다. Penicillium according to an embodiment of the present invention dokdoense ) It was confirmed that the organic solvent extract of CNUFC-DDS11-1 has antibacterial activity against the pathogen Staphylococcus aureus ATCC.
배양액 및 배양액의 여과액 외에도 배양액의 동결건조 분말, 배양 여과액의 동결건조 분말 또는 동결건조 분말을 용해한 용액을 여러 분야에서 활용할 수 있다. In addition to the culture medium and the filtrate of the culture medium, a lyophilized powder of the culture medium, a lyophilized powder of the culture filtrate, or a solution in which the lyophilized powder is dissolved may be used in various fields.
본 발명에서 배양액은 균체를 배지에 배양하여 균체가 생성한 물질, 균체 자체 및 배지를 포함할 수 있다. In the present invention, the culture medium may include a substance produced by the cell, the cell itself, and the medium by culturing the cell in the medium.
본 발명에서 배양 여과액은 배양액을 원심분리하여 얻은 상등액, 상층액을 여과하여 얻은 액체를 포함할 수 있다. In the present invention, the culture filtrate may include a supernatant obtained by centrifuging the culture solution, and a liquid obtained by filtering the supernatant.
본 발명의 다양한 조성물, 식품 등의 제조 방법은 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)을 배양 배지에 배양하는 단계 및 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)을 배양하여 얻은 배양액을 수득하여 제형화 하는 단계를 포함할 수 있다. Various compositions, methods of manufacture of food of the present invention penny room Solarium islets Enschede (Penicillium dokdoense) (Accession No: KACC 93309p) the culturing in the culture medium and Penny room Solarium islets Enschede (Penicillium dokdoense ) (Accession number: KACC 93309p) may be obtained by culturing to obtain a culture medium and may be formulated.
본 발명의 조성물의 제조 방법은 배양액을 원심분리하여 층분리가 일어난 배양액의 상등액을 얻어 상등액을 제형화하는 단계를 더 포함할 수 있다.The method for preparing a composition of the present invention may further include the step of formulating the supernatant by centrifuging the culture medium to obtain a supernatant of the culture medium in which the layer separation has occurred.
본 발명의 조성물의 제조 방법은 배양액을 원심분리하여 층분리가 일어난 배양액의 상등액을 여과하여 얻은 배양 여과액을 제형화하는 단계를 더 포함할 수 있다. The method for preparing the composition of the present invention may further include the step of formulating a culture filtrate obtained by filtering the supernatant of the culture medium in which the layer separation has occurred by centrifuging the culture medium.
본 발명에서 페니실리움 독도엔스(Penicillium dokdoense)(기탁번호: KACC 93309p)을 배양하기 위한 배지는 MEA(Blakeslee's malt extract agar) 배지, YES(yeast extract sucrose agar) 배지, CREA(Czapek yeast autolysate agar) 배지 및 PDA(potato dextrose agar) 배지에서 선택되는 배지일 수 있으나 이에 제한되는 것은 아니다. 본 발명의 실시예에 따르면 바람직하게는 MEA 배지이고, CYA 및 YES 배지에서는 성장이 느릴 수 있다. Penicillium in the present invention ( Penicillium The medium for cultivating dokdoense (accession number: KACC 93309p) is in MEA (Blakeslee's malt extract agar) medium, YES (yeast extract sucrose agar) medium, CREA (Czapek yeast autolysate agar) medium, and PDA (potato dextrose agar) medium. The medium may be selected, but is not limited thereto. According to an embodiment of the present invention is preferably MEA medium, growth may be slow in CYA and YES medium.
본 발명에 따른 조성물은 염증성 사이토카인, 활성산소종(reactive oxygen species, ROS) 및 산화질소(Nitric oxide, NO)의 생성 억제 및 조절에 특히 효과적일 수 있다. 구체적으로 본 발명에 따르면 페니실리움 독도엔스, 이의 배양액 혹은 이의 배양액의 여액(filtrate)은 염증에 의해 발현이 증가하는 사이토카인인 TNF-α 및 IL-1β을 효과적으로 억제할 수 있어 지나친 염증의 발생과 이로부터 진행될 수 있는 여러 질병을 효과적으로 치료 및 예방할 수 있다. 또한 본 발명에 따르면 페니실리움 독도엔스, 이의 배양액 혹은 이의 배양액의 여액(filtrate)은 산화질소와 활성산소종을 효과적으로 억제하고 이들의 생산을 조절할 수 있어 염증성 반응 저해에 특히 효과적일 수 있고, 나아가 다양한 염증성 질환의 예방과 치료도 가능할 수 있다. The composition according to the present invention may be particularly effective in inhibiting and regulating the production of inflammatory cytokines, reactive oxygen species (ROS) and nitric oxide (NO). Specifically, according to the present invention, penicillium dokone, its culture medium or its filtrate can effectively suppress TNF-α and IL-1β, cytokines whose expression is increased by inflammation, resulting in excessive inflammation. And it can effectively treat and prevent various diseases that can progress from it. In addition, according to the present invention, penicillium doxens, a culture solution thereof, or a filtrate thereof can effectively inhibit nitric oxide and reactive oxygen species and control their production, and thus may be particularly effective in inhibiting inflammatory reactions. Prevention and treatment of various inflammatory diseases may also be possible.
본 발명에서 염증성 질환은 산화질소와 같은 염증성 사이토카인과 관련된 염증성 질환이라면 특별히 제한되는 것은 아니다. 바람직하게는 아토피, 천식, 위궤양, 위염, 간염, 크론병(crohn's disease), 방광염, 신장염, 류마티스, 관절염, 폐렴, 염증성 알레르기, 결막염, 비염, 중이염, 인후염, 통풍, 건염증, 근육염증, 급성 염증 질환 및 만성 염증 질환으로 이루어지는 군으로부터 선택되는 어느 하나일 수 있으나, 이에 제한되는 것은 아니다. In the present invention, the inflammatory disease is not particularly limited as long as it is an inflammatory disease associated with inflammatory cytokines such as nitric oxide. Preferably atopy, asthma, gastric ulcer, gastritis, hepatitis, crohn's disease, cystitis, nephritis, rheumatism, arthritis, pneumonia, inflammatory allergies, conjunctivitis, rhinitis, otitis media, sore throat, gout, tendinitis, myositis, acute It may be any one selected from the group consisting of inflammatory diseases and chronic inflammatory diseases, but is not limited thereto.
본 발명에 따른 조성물 중 건강기능식품이나 식품첨가물을 제조하는 경우 그 형태나 유형은 특별히 제한되지 않으며 항염 활성을 증가시킬 수 있는 타 성분과 혼합하여 제조할 수도 있다. 식품의 종류에도 특별히 제한은 없고, 예를 들어 드링크제, 비타민제, 각종 음료수, 가공 육류, 가공 소세지, 가공 면류, 가공 유지류, 사탕, 껌 등 통상적으로 제조 및 판매되는 식품류를 포함할 수 있다. When preparing a dietary supplement or a food additive in the composition according to the present invention, its form or type is not particularly limited and may be prepared by mixing with other ingredients capable of increasing anti-inflammatory activity. There is no particular limitation on the type of food, and for example, it may include foods commonly produced and sold, such as drinks, vitamins, various beverages, processed meats, processed sausages, processed noodles, processed fats and oils, candy and gum.
바람직하게는 드링크제나 기타 음료 형태 조성물로 제조하는 것이 좋고, 감미료와 같은 다양한 향미제 등을 추가 성분으로서 함유하여 섭취에 도움을 줄 수 있다. 단당, 과당, 포도당 등 다양한 맛을 탄수화물류를 포함할 수 있고, 솔비톨이나 자일리톨과 같은 당알콜도 포함할 수 있다. 감미제의 예로는 천연 감미제뿐만 아니라 사카린과 같은 합성 감미제 등도 사용할 수 있다. Preferably, it is good to prepare it as a drink agent or other beverage form composition, and it may contain various flavoring agents such as sweeteners as additional ingredients to help intake. Various flavors such as monosaccharides, fructose, and glucose may include carbohydrates and sugar alcohols such as sorbitol and xylitol. Examples of sweeteners include not only natural sweeteners, but also synthetic sweeteners such as saccharin.
본 발명에 따른 조성물 중 약학 조성물을 제조하는 경우 그 형태나 유형은 특별히 제한되지 않으며 투여 또는 섭취 방식에 따라 다양한 제형으로 제조할 수 있다. 또한 약학 조성물의 제조시 해당 분야에서 통상적으로 사용하는 부형제, 담체 혹은 희석제도 적절히 더 포함할 수 있다. 제형의 예로는 정제, 시럽, 캡슐, 산제 등의 경구형태 외에도 외용제나 주사용제 등 다양한 제형이 가능할 수 있다. 담체나 부형제 등의 예로는 솔비톨, 자일리톨이나 말티올과 같은 당알코올류, 단당류, 다당류, 젤라틴, 물, 알코올, 전분, 식용가능한 고무류, 광물유 등을 포함한 다양한 화합물 및 이들의 혼합물을 사용할 수 있다. 제제화시 일반적으로 사용하는 습윤제, 계면활성제, 부형제, 충진제나 결합제 등의 종류도 특별히 제한되지 않는다.In the case of preparing the pharmaceutical composition of the composition according to the present invention, its form or type is not particularly limited and may be prepared in various dosage forms depending on the method of administration or intake. In addition, when preparing pharmaceutical compositions, excipients, carriers or diluents commonly used in the field may be further included. As an example of the formulation, various formulations such as an external preparation or an injection may be possible in addition to oral forms such as tablets, syrups, capsules, and powders. Examples of carriers or excipients include various compounds including sugar alcohols such as sorbitol, xylitol or maltiol, monosaccharides, polysaccharides, gelatin, water, alcohol, starch, edible rubbers, mineral oils, and mixtures thereof. The type of wetting agents, surfactants, excipients, fillers or binders generally used in formulation is also not particularly limited.
이하에서 본 발명에 대한 실시예를 통해 보다 구체적으로 설명하며, 본 발명이 하기 실시예에 의해 제한되는 것은 아니다. Hereinafter, the present invention will be described in more detail through examples, and the present invention is not limited by the following examples.
진균의 동정Identification of fungi
실시예 1-1. 새로운 진균의 동정을 위한 샘플링 및 분리Example 1-1. Sampling and separation for identification of new fungi
한국의 동해(the East Sea of Korea)에 위치한 독도(Dokdo Island)의 동도(Dongdo)에서 깊이 10~15cm 부근의 토양 샘플을 채취하였다. 채취한 샘플을 멸균된 50㎖ 원추형 튜브에 옮기고 4℃에서 보관하였다. 곰팡이 균은 연속 희석 평판법(serial dilution plating method)으로 분리 하였다. 구체적으로, 샘플인 흙 1g을 멸균 증류수 9㎖와 혼합하고 실온에서 15 분간 흔들어 섞은 다음 10-1에서 10-4의 농도까지 단계별로 희석하였다. 각 희석액 0.1㎖를 감자 포도당 한천 배지(PDA 배지)(39g potato dextrose agar [Becton, Dickinson, and Co., Sparks, MD, USA])에 옮겨 25℃에서 3 ~ 7 일 동안 배양하였다. 순수한 배양체를 분리하기 위해, 다양한 형태의 개별 콜로니를 PDA 플레이트로 옮겼다. 순수분리된 균주는 한국 광주 전남대학교 균주 수집처(Chonnam National University Fungal Collection, CNUFC)에서 -80℃의 20 % 글리세롤에 보관하였고 CNUFC-DDS11-1 및 CNUFC-DDS11-2으로 명명하여 사용하였다.A soil sample of 10 to 15 cm in depth was collected from Dongdo on Dokdo Island located in the East Sea of Korea. The collected sample was transferred to a sterilized 50 ml conical tube and stored at 4 ° C. Fungi were separated by a serial dilution plating method. Specifically, 1 g of sample soil was mixed with 9 ml of sterile distilled water, shaken for 15 minutes at room temperature, and then diluted stepwise from 10 -1 to 10 -4 . 0.1 ml of each dilution was transferred to potato glucose agar medium (PDA medium) (39 g potato dextrose agar [Becton, Dickinson, and Co., Sparks, MD, USA]) and incubated at 25 ° C. for 3 to 7 days. To isolate pure cultures, individual colonies of various types were transferred to PDA plates. The purely isolated strain was stored in 20% glycerol at -80 ° C at the Chonnam National University Fungal Collection (CNUFC) in Gwangju, Korea, and used as CNUFC-DDS11-1 and CNUFC-DDS11-2.
실시예 1-2. DNA 추출, PCR 및 시퀀싱(sequencing)Example 1-2. DNA extraction, PCR and sequencing
분리한 균의 균사체(mycelia)로부터 Solgent Genomic DNA prep kit(Solgent Co. Ltd., 대전, 한국)를 이용하여 직접 게놈 DNA를 추출 하였다. 프라이머 쌍 ITS1/ITS4(White et al, 1990), Bt2a/Bt2b(Glass & Donaldson 1995) 및 Cmd5/Cmd6(Samson et al, 2014)을 사용하여 rDNA ITS(The internal transcribed spacer)S1-5.8S-ITS2 영역, 베타 튜블린(beta tubulin)을 암호화(encoding)하는 BenA 부분 유전자 및 칼모듈린(calmodulin)을 암호화하는 CaM 부분 유전자를 증폭 및 확인하여 이용하였다([표 1]).Genomic DNA was directly extracted from the isolated mycelia of mycelia using Solgent Genomic DNA prep kit (Solgent Co. Ltd., Daejeon, Korea). The internal transcribed spacer (rDNA ITS) using primer pairs ITS1 / ITS4 (White et al, 1990), Bt2a / Bt2b (Glass & Donaldson 1995) and Cmd5 / Cmd6 (Samson et al, 2014) S1-5.8S-ITS2 The region, BenA partial gene encoding beta tubulin and the CaM partial gene encoding calmodulin were amplified and used (Table 1).
각각의 PCR 증폭 혼합물(총 부피, 20 ㎕)은 2 ㎕의 균주 DNA 주형(10 ng), 각각의 프라이머 (5pM/㎕) 1.5㎕; Taq DNA 중합 효소, dNTP, 완충액 및 트래킹 염료(tracking dye)를 함유한 Accupower®PCR premix(Bioneer Corp., 대전, 한국) 1㎕ 및 멸균수 14㎕를 포함하였다. PCR 생성물을 Accuprep®PCR 정제 키트 (Bioneer)로 제조사의 지시에 따라 정제하였다. DNA 시퀀싱은 ABI 3700 automated DNA sequencer(Applied Biosystems Inc., Foster City, CA, USA)를 이용하여 수행 하였다.Each PCR amplification mixture (total volume, 20 μl) contained 2 μl of strain DNA template (10 ng), 1.5 μl of each primer (5 pM / μl); 1 μl of Accupower®PCR premix (Bioneer Corp., Daejeon, Korea) and 14 μl of sterile water containing Taq DNA polymerase, dNTP, buffer and tracking dye. The PCR product was purified with Accuprep® PCR purification kit (Bioneer) according to the manufacturer's instructions. DNA sequencing was performed using an ABI 3700 automated DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA).
[표 1] [Table 1] PCRPCR 증폭 및 시퀀싱에 사용한 Used for amplification and sequencing 프라이머primer
실시예Example 1-3. 계통발생분석(Phylogenetic analysis) 1-3. Phylogenetic analysis
시퀀싱 결과와 GenBank 데이터베이스에서 얻은 서열 데이터([표 2] 참조)를 Clustal_X v.1.83(Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research: 4876-82) 및 Bioedit v. 5.0.9.1 소프트웨어(Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic. Acids Symp Ser 1999;41:95-8)를 사용하여 분석하였다. 계통 발생 분석은 MEGA 6 소프트웨어(Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729)를 사용하여 수행되었다. Clustal_X v.1.83 (Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment) for sequencing results and sequence data obtained from the GenBank database (see [Table 2]) aided by quality analysis tools.Nucleic Acids Research: 4876-82) and Bioedit v. 5.0.9.1 software (Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98 / NT.Nucleic.Acids Symp Ser 1999; 41: 95-8). Phylogenetic analysis was performed using MEGA 6 software (Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729).
CNUFC-DDS11-1, CNUFC-DDS11-2 및 Citrina 섹션(the section Citrina) 관련 종을 포함하는 계통 발생 수는 최대 우도 분석(the maximum likelihood analysis)에 의해 BenA 및 CaM 에 대한 복합 데이터 세트를 사용하여 구성되었고, Penicillium corylophilum 서열이 아웃 그룹으로 선정되었다. Phylogenetic counts, including CNUFC-DDS11-1, CNUFC-DDS11-2, and Citrina section related species, were determined using a complex data set for BenA and CaM by the maximum likelihood analysis. Constituent, Penicillium corylophilum The sequence was selected out group.
각각의 균주에 대한 Basic Local Alignment Search Tool(BLASTn)(NCBI) 검색에 의해 서열 일치율을 얻었다. 내부 브랜치의 신뢰성은 1000 부트 스트랩 복제(1000 bootstrap replicates)를 사용하는 p-거리 대체 모델을 사용하여 평가하였다.Sequence matching rates were obtained by Basic Local Alignment Search Tool (BLASTn) (NCBI) search for each strain. The reliability of the internal branch was evaluated using a p-distance alternative model using 1000 bootstrap replicates.
(Strain no.)Collection no.
(Strain no.)
실시예Example 1-4. 형태학적 분석 1-4. Morphological analysis
자세한 형태 연구를 위해 분리한 균주를 블레이크슬리(Blakeslee)의 맥아 추출물 한천(MEA; Blakeslee, 1915), yeast extract sucrose 한천(YES; Frisvad, 1981), Czapek yeast autolysate agar(CYA; Pitt, 1979) 및 크레아틴 자당 한천(CREA; Frisvad 1981)에서 배양하였다. 플레이트(각 배지에 대해 3 복제물)를 3-점(three-point)에 접종하였다. 7일간 배양한 후 암실에서 5℃10℃, 15℃, 20℃25℃30℃, 35℃ 및 37℃에서 Rayner의 컬러 차트(the color charts of Rayner(1970))를 사용하여 성장을 확인하였다. 샘플을 락토페놀용액(Junsei Chemical Co. Ltd., Tokyo, Japan)에 마운팅하고 DIC 광학 장치(Olympus, Tokyo, Japan)가 장착된 Olympus BX51 현미경으로 관찰 하였다.The strains isolated for detailed morphology studies were Blakeslee's malt extract agar (MEA; Blakeslee, 1915), yeast extract sucrose agar (YES; Frisvad, 1981), Czapek yeast autolysate agar (CYA; Pitt, 1979), and Cultured in creatine sucrose agar (CREA; Frisvad 1981). Plates (3 replicates for each medium) were inoculated at three-point. After incubation for 7 days, growth was confirmed using Rayner's color charts (the color charts of Rayner (1970)) at 5 ° C, 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C, and 37 ° C in the dark. The sample was mounted on a lactophenol solution (Junsei Chemical Co. Ltd., Tokyo, Japan) and observed with an Olympus BX51 microscope equipped with a DIC optical device (Olympus, Tokyo, Japan).
미세 균질 구조는 주사 전자 현미경(SEM)(Hitachi S4700; Hitachi, Tokyo, Japan)로 관찰하였다. 분리 균주를 0.05M 인산 완충액(pH 7.2) 중 2.5% 파라 포름알데히드 - 글루타르알데히드 paraformaldehyde-glutaraldehyde)에 2시간 동안 고정시킨 후, 카코딜염산(cacodylate) 버퍼(Junsei Chemical Co. Ltd.)로 세척 하였다. 카코딜염산에 1 시간 동안 희석한 1% 오스뮴테트록시드(Electron Microscopy Sciences, Hatfield, PA, USA)에 시료를 고정시킴으로써 세포막을 보존 하였다. 샘플을 다시 카코딜염산 버퍼로 세척하고, 에탄올(graded ethanol) (Emsure, Darmstadt, Germany) 및 이소아밀아세테이트(isoamyl acetate)(Junsei Chemical Co. Ltd.)로 탈수시키고 흄 후드(fume hood)에서 건조시켰다. 마지막으로, 샘플을 금으로 스퍼터 코팅하고 한국기초과학연구소에서 Hitachi S4700 FE-SEM(field emission scanning electron microscope)을 사용하여 관찰 하였다.The micro-homogenous structure was observed with a scanning electron microscope (SEM) (Hitachi S4700; Hitachi, Tokyo, Japan). The isolated strain was fixed in 2.5% paraformaldehyde-glutaraldehyde in 0.05M phosphate buffer (pH 7.2) for 2 hours, and then washed with cacodylate buffer (Junsei Chemical Co. Ltd.) Did. Cell membranes were preserved by immobilizing the sample in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) diluted with chacodil hydrochloric acid for 1 hour. The sample was washed again with a carcodil hydrochloric acid buffer, dehydrated with ethanol (graded ethanol) (Emsure, Darmstadt, Germany) and isoamyl acetate (Junsei Chemical Co. Ltd.) and dried in a fume hood Ordered. Finally, the samples were sputter coated with gold and observed using a Hitachi S4700 field emission scanning electron microscope (FE-SEM) at the Korea Basic Science Institute.
실시예Example 1-5. 계통발생분석 결과(Phylogenetic analysis) 1-5. Phylogenetic analysis result
신규 균주의 관련 종 계통 발생 관계를 분석은 신규 균주 및 연관된 종간의 ITS, BenA 및 CaM 유전자에서 나타나는 계통발생 관계 분석을 통해 진행하였다. ITS rDNA 서열을 GeneBank에서 추출한 관련 종과 비교한 서열 분석에 결과에 따라 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1 및 CNUFC-DDS11-2로 NCBI 데이터베이스에 기탁하고, 이를 이용하여 분석하였다([표 2] 참조). 분석 수행은 ITS (서열번호 1), BenA (서열번호 2) 및 CaM (서열번호 3) 유전자를 이용하였다. Analysis of the related strain phylogenetic relationship of the new strain was conducted through analysis of phylogenetic relationship in the ITS, BenA, and CaM genes between the new strain and related species. According to the results of s rDNA sequence comparison with the related species extracted from GeneBank, Penicillium dokdoense ) Deposited in the NCBI database with CNUFC-DDS11-1 and CNUFC-DDS11-2, and analyzed using this (see [Table 2]). Analysis was performed using ITS (SEQ ID NO: 1), BenA (SEQ ID NO: 2) and CaM (SEQ ID NO: 3) genes.
CNUFC-DDS11-1 및 CNUFC-DDS11-2 균주의 ITS rDNA 서열을 GenBank에서 추출한 관련 종과 비교했을 때, BLASTn 검색에 의한 서열 분석 결과 CNUFC-DDS11-1 및 CNUFC-DDS11-2가 Penicillium sp . E3(GenBank 접근번호 FJ471589)와 98.4% (548/557 bp)로 가장 근접한 연관성을 보였다. When ITS rDNA sequences of CNUFC-DDS11-1 and CNUFC-DDS11-2 strains were compared with related species extracted from GenBank, sequencing results by BLASTn search showed that CNUFC-DDS11-1 and CNUFC-DDS11-2 were Penicillium sp . E3 (GenBank accession number FJ471589) and 98.4% (548/557 bp) showed the closest association.
P. terrigenum MUT 5749 및 P. terrigenum CBS 127345의 BenA 서열은 분리균의 BenA와 각각 96.9% (444/458 bp) 및 96.8% (429/443 bp) 의 서열 상동성을 보였다. P. terrigenum DTO 9D4 및 JM439의 CaM 서열은 분리균의 CaM과 각각 97.1% (505/520 bp) 및 96.4% (396/411 bp)의 서열 상동성을 보였다. BenA sequences of P. terrigenum MUT 5749 and P. terrigenum CBS 127345 showed sequence homology of 96.9% (444/458 bp) and 96.8% (429/443 bp) with BenA of isolates, respectively. The CaM sequences of P. terrigenum DTO 9D4 and JM439 showed sequence homology with 97.1% (505/520 bp) and 96.4% (396/411 bp) of CaM of isolates, respectively.
계통수(phylogenetic trees)는 CNUFC-DDS11-1 및 CNUFC-DDS11-2의 BenA 및 CaM과 GenBank에서 추출한 Citrina 섹션에 속하는 관련 종(도 1)에 대한 데이터 세트를 통해 추론되었다. 계통 발생 분석은 Citrina 섹션에 분리한 진균을 두었고, 분리 진균은 별도의 클레드(clade)를 형성하여 분리 진균이 별개의 Penicillium 종이라는 것을 보여 주었다.Phylogenetic trees were deduced from the data set for the relevant species (FIG. 1) belonging to the Citrina section extracted from BenA and CaM and GenBank of CNUFC-DDS11-1 and CNUFC-DDS11-2. Phylogenetic analysis put the isolated fungi in the Citrina section, and the isolated fungi formed separate clades to separate the isolated fungi from the Penicillium. Showed that it was paper.
신규 균주는 CYA 배지에서 느린 성장을 하여 P. terrigenum 및 P. copticola와 구분되었고, P. terrigenum는 YES 및 CYA에서 빠른 성장을 나타냈다. New strains by slower growth in CYA medium was separated from P. terrigenum and P. copticola, P. terrigenum showed a rapid growth in YES and CYA.
기저경자(Metulae)와 기저경자당 피알리드 수는 P. terrigenum 및 P. copticola에서 관찰된 것보다 훨씬 많았다. 또한 신규 균주는 P. terrigenum 및 P. copticola에 없는 이중 윤생상(divaricate) 분생자경을 생산하였다. 신규 균주의 분생포자 형태는 구형에서 반구형 또는 타원으로 타원 형태인 P. terrigenum와 구분되었다. The number of fialides per basal etiology (Metulae) and basal erythema was much higher than that observed in P. terrigenum and P. copticola . In addition, the new strain produced a double divaricate conidia that is not in P. terrigenum and P. copticola . The conidia form of the new strain was distinguished from the elliptical form P. terrigenum from spherical to hemispherical or elliptical.
신규 균주와 P. terrigenum 및 P. copticola의 특성 비교는 아래 [표 3]과 같이 정리할 수 있었다. Comparison of the characteristics of the new strain and P. terrigenum and P. copticola could be summarized as in [Table 3] below.
Ampuliform, 3 - 9 per metula, 6.7 - 11.5 × 2.0 - 3.5 μm
새로운 진균을 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1 및 CNUFC-DDS11-2로 명명하고 이 중 페니실리움 독도엔스(Penicillium dokdoense) CNUFC-DDS11-1을 농진청 미생물은행 (KACC)에 기탁하였다. Penicillium, a new fungus dokdoense ) Named CNUFC-DDS11-1 and CNUFC-DDS11-2, of which Penicillium dokdoense ) CNUFC-DDS11-1 was deposited with the Agribusiness Administration Microbial Bank (KACC).
2-1. 신규2-1. new 페니실리움Penicillium 독도엔스Dokdo Nance (( PenicilliumPenicillium dokdoensedokdoense ) ) CNUFCCNUFC -- DDS11DDS11 -1의 항염증 효과 확인 Confirm anti-inflammatory effect of -1
신규 페니실리움 독도엔스의 항염증 효과 확인은 내독소 물질인 LPS(lipopolysaccharide)에 의해 자극되어 활성화된 대식세포를 통해 확인하였다. 항염증 효과 확인은 산화질소(nitric oxide, NO), 활성 산소, TNF-α 및 IL-1β를 측정하여 확인하였다. Confirmation of the anti-inflammatory effect of the new penicillium doxens was confirmed by macrophages stimulated and activated by the endotoxin substance LPS (lipopolysaccharide). The anti-inflammatory effect was confirmed by measuring nitric oxide (NO), free radicals, TNF-α and IL-1β.
Dulbecco's modified Eagle's medium (DMEM), antimyotic-antibiotics, fetal bovine serum (FBS), and lipopolysaccharide (LPS), MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) 는 Invitrogen-Gibco (New York, USA)에서 구입하여 사용하였고 나머지 다른 화학물질은 Merck, Sigma 또는 Junsei에서 구입하여 사용하였다. Dulbecco's modified Eagle's medium (DMEM), antimyotic-antibiotics, fetal bovine serum (FBS), and lipopolysaccharide (LPS), MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 -(4-sulfophenyl) -2H-tetrazolium) was purchased from Invitrogen-Gibco (New York, USA) and the rest of the chemicals were purchased from Merck, Sigma or Junsei.
대식세포인 RAW 264.7 세포를 96 웰-플레이트(96 well-plate)에 2 × 104 cells/well로 씨딩(seeding)하고, 37℃에서 24시간 5% CO의 습한 대기 조건에서 인큐베이션 하였다. 24시간 후 배지 50μL 및 MTS와 PMS를 포함한[혼합비 20:1 (MTS:PMS)]한 진단시약 50μL를 혼합하였다. 상층액(supernatant) 100㎕를 96 웰-플레이트로 3회 옮기고 형성된 포르 마잔의 흡광도를 마이크로 플레이트 리더기(microplate reader)로 490nm에서 측정하였다.Macrophage RAW 264.7 cells were seeded in 96 well-plates at 2 × 10 4 cells / well and incubated at 37 ° C. in humid atmosphere at 5% CO for 24 hours. After 24 hours, 50 μL of the medium and 50 μL of the diagnostic reagents containing MTS and PMS [mix ratio 20: 1 (MTS: PMS)] were mixed. 100 μl of the supernatant was transferred three times to a 96-well plate and the absorbance of the formed formazan was measured at 490 nm with a microplate reader.
산화질소(nitric oxide, NO)는 Griess 시약 시스템을 이용하여 아질산염(nitrite)의 수준을 분석하여 측정하였다. 구체적으로는 다음과 같다. RAW 264.7 세포를 2 × 105 cells/well 밀도로 12 웰-플레이트에 씨딩하고 LPS(500ng/㎖)으로 24시간 동안 자극한 후, 96- 웰 플레이트에서 같은 부피의 Griess 시약으로 세포 배양 상등액 30㎕를 실온에서 10분 동안 배양하였다. Griess 시약 시스템에 따라 아질산염 표준 물질(0.1M 아질산 나트륨)을 준비하였고, 흡광도는 마이크로 플레이트 리더기를 사용하여 540nm에서 측정하였다. 3회 반복으로 2번 실험을 진행하였다. Nitric oxide (NO) was measured by analyzing the level of nitrite using the Griess reagent system. Specifically, it is as follows. RAW 264.7 cells were seeded in 12 well-plates at a density of 2 × 10 5 cells / well and stimulated with LPS (500 ng / ml) for 24 hours, followed by 30 μl of cell culture supernatant with the same volume of Griess reagent in a 96-well plate. Was incubated at room temperature for 10 minutes. A nitrite standard (0.1M sodium nitrite) was prepared according to the Griess reagent system, and the absorbance was measured at 540 nm using a microplate reader. Two experiments were conducted in three replicates.
활성산소(reactive oxygen species, ROS)는 DCFDA(2′ 7′diacetate)를 사용하여 측정하였다. 구체적으로 RAW 264.7 세포를 2 × 105 cells/well 밀도로 12 웰-플레이트에 씨딩하고 LPS(500ng/㎖)으로 24시간 동안 자극한 후, 배지를 제거하고 세포를 암실에서 30분간 10μM DCFDA 처리하고 37℃에서 배양하였다. 이어서 세포를 세척하고 FACs 버퍼에 재현탁(re-suspension)한 후 FACs Calibur 유세포 분석기로 분석하였다. Reactive oxygen species (ROS) were measured using DCFDA (2 ′ 7′diacetate). Specifically, RAW 264.7 cells were seeded in 12 well-plates at a density of 2 × 10 5 cells / well and stimulated with LPS (500 ng / ml) for 24 hours, the medium was removed, and the cells were treated with 10 μM DCFDA for 30 minutes in the dark. Cultured at 37 ° C. Cells were then washed, re-suspensioned in FACs buffer and analyzed with a FACs Calibur flow cytometer.
TNF-α 및 IL-1β 측정은 TRISOL 시약을 사용하여 총 RNA를 분리하고, 분리한 RNA를 이용하여 cDNA를 합성한 후 RT-PCR(real time polymerase chain reaction)을 수행하여 측정하였다. RT-PCR에 사용한 프라이머는 아래 [표 4]와 같다. TNF-α and IL-1β were measured by isolating total RNA using TRISOL reagent, and synthesizing cDNA using the isolated RNA, followed by RT-PCR (real time polymerase chain reaction). Primers used for RT-PCR are shown in [Table 4] below.
[표 4][Table 4]
모든 측정값은 3회 반복 측정 평균 ± 표준오차로 나타내었다. 통계 분석은 Tukey 's test에 의한 ANOVA(analysis of variance)로 수행하였다. All measurements are expressed as the mean of 3 repeat measurements ± standard error. Statistical analysis was performed by ANOVA (analysis of variance) by Tukey's test.
대식세포를 이용한 페니실리움 독도엔스의 항염증 효과 확인 결과 중 세포 독성에 있어서, 페니실리움 독도엔스는 50μg/mL(배양 상등액 기준)에서 대식세포 RAW 264.7에 대한 세포 독성은 나타나지 않았고 100μg/mL에서 대식세포 RAW 264.7에 대해 약간의 세포 독성이 나타났다(도 3). Among the results of confirming the anti-inflammatory effect of penicillium dokone using macrophages, in terms of cytotoxicity, penicillium dokone showed no cytotoxicity against macrophage RAW 264.7 at 50 μg / mL (based on culture supernatant) and 100 μg / mL Showed a slight cytotoxicity against macrophage RAW 264.7 (Fig. 3).
페니실리움 독도엔스의 배양 여액을 LPS 자극된 대식세포 RAW 264.7에 처리시 아질산염의 수준은 7.5 μM로 나타나 NO 생성을 억제하는 활성을 확인할 수 있었다(도 4). When treating the culture filtrate of Penicillium Dokdoen's with LPS-stimulated macrophages RAW 264.7, the level of nitrite was 7.5 μM, which confirmed the activity of inhibiting NO production (FIG. 4).
페니실리움 독도엔스의 배양 여액을 LPS 자극된 대식세포 RAW 264.7에 처리시 ROS의 생성을 현저히 감소시킬 수 있음을 확인할 수 있었다(도 5).When the culture filtrate of Penicillium Dokdoen's was treated with LPS-stimulated macrophage RAW 264.7, it was confirmed that the production of ROS was significantly reduced (FIG. 5).
페니실리움 독도엔스의 배양 여액을 LPS 자극된 대식세포 RAW 264.7에 처리시 LPS 단독 처리시 유의적으로 증가한 TNF-α 및 IL-1β의 mRNA 발현이 현저히 감소함을 확인할 수 있었다(도 6).It was confirmed that the mRNA expression of TNF-α and IL-1β significantly increased when LPS alone was treated when the culture filtrate of Penicillium Dokdoen's was treated with LPS-stimulated macrophage RAW 264.7 (FIG. 6).
2-2. 신규2-2. new 페니실리움Penicillium 독도엔스Dokdo Nance (( PenicilliumPenicillium dokdoensedokdoense ) ) CNUFCCNUFC -- DDS11DDS11 -1로부터 활성 화합물의 추출Extraction of active compound from -1
본 발명의 페니실리움 독도엔스 CNUFC-DDS11을 25℃의 PDB(potato dextrose broth)에서 10일간 배양하였다. 배양 후, 배양 배지를 Whatman 여과지 No. 2로 여과하였다. 배양 여액을 실온에서 에틸아세테이트(EtOAc)의 부피인 150 mL로 3회 추출하였다. 에틸아세테이트 층은 회전 증발기에서 40℃에서 농축하여 에틸아세테이트 용매를 제거하였다. 유기 추출물을 증발시켜 0.04g을 수득하였다.Penicillium dokdoensu CNUFC-DDS11 of the present invention was cultured in a potato dextrose broth (PDB) at 25 ° C for 10 days. After incubation, the culture medium was replaced with Whatman filter paper No. Filtered by 2. The culture filtrate was extracted three times at room temperature with 150 mL of the volume of ethyl acetate (EtOAc). The ethyl acetate layer was concentrated at 40 ° C. in a rotary evaporator to remove the ethyl acetate solvent. Evaporation of the organic extract gave 0.04 g.
2-3. 신규2-3. new 페니실리움Penicillium 독도엔스Dokdo Nance (( PenicilliumPenicillium dokdoensedokdoense ) ) CNUFCCNUFC -- DDS11DDS11 -1의 단백질 분해활성(-1 proteolytic activity ( proteolyticproteolytic activity) activity)
2% 한천(agar)을 함유한 한천 플레이트에 본 발명의 페니실리움 독도엔스 CNUFC-DDS11-1 균주를 접종하고, 2% 탈지유를 보충하였다. 플레이트는 25℃에서 4일(3-5일)간 배양하였다.The agar plate containing 2% agar (agar) was inoculated with the Penicillium Dokdoens CNUFC-DDS11-1 strain of the present invention, and supplemented with 2% skim milk. Plates were incubated at 25 ° C for 4 days (3-5 days).
신규 페니실리움 독도엔스의 단백질 분해활성에 대한 스크리닝 결과를 도 7A에 도시하였다. 이로부터, 본 발명의 페니실리움 독도엔스는 24시간 배양 후 단백질 분해 효소를 생산하였으며, 이는 콜로니를 둘러싸고 있는 클리어 존(clear zone)으로부터 확인할 수 있었다.The screening results for proteolytic activity of the new Penicillium dokdoence are shown in Figure 7A. From this, the penicillium doxens of the present invention produced proteolytic enzymes after incubation for 24 hours, which was confirmed from a clear zone surrounding the colony.
2-4. 신규2-4. new 페니실리움Penicillium 독도엔스Dokdo Nance (( PenicilliumPenicillium dokdoensedokdoense ) ) CNUFCCNUFC -- DDS11DDS11 -1의 항진균 활성(-1 antifungal activity ( antifungalantifungal activity) activity)
본 발명의 페니실리움 독도엔스 CNUFC-DDS11-1의 항진균 활성은 PDA(potato dextrose agar) 배지에서 이중 배양법(dual culture method)으로 측정하였다.The antifungal activity of Penicillium Dokdoen's CNUFC-DDS11-1 of the present invention was measured by a dual culture method in PDA (potato dextrose agar) medium.
곰팡이 병원균인 알테나리아 알테나타(Alternaria alternata) EML-BLDF1-4의 균사체 플러그(mycelial plug)를 PDA 플레이트의 중앙에 위치하고, 본 발명의 페니실리움 독도엔스 CNUFC-DDS11-1 배양균은 상기 병원성 진균으로부터 2.5 cm 떨어진 곳에 플레이팅하였다. Alternaria , a fungal pathogen alternata ) The mycelial plug of EML-BLDF1-4 was located in the center of the PDA plate, and the Penicillium Dokdoen's CNUFC-DDS11-1 culture of the present invention was plated 2.5 cm from the pathogenic fungus.
모든 플레이트는 25℃의 암실에서 6일(5-7일)간 배양하고, 각 실험은 3회씩 반복하였다.All plates were cultured for 6 days (5-7 days) in a dark room at 25 ° C, and each experiment was repeated three times.
이후, 병원균과 페니실리움 독도엔스 CNUFC-DDS11-1 균주 사이의 저해 영역(inhibition zone)의 폭을 밀리미터 단위로 측정하여, 항진균 활성으로 해석하였다.Subsequently, the width of the inhibition zone between the pathogen and the Penicillium Dokdoensu CNUFC-DDS11-1 strain was measured in millimeters and analyzed as antifungal activity.
신규 페니실리움 독도엔스의 항진균 활성에 대한 스크리닝 결과를 도 7B에 도시하였다.The screening results for the antifungal activity of the new Penicillium dokdoensu are shown in Figure 7B.
이로부터, 본 발명의 페니실리움 독도엔스는 진균성 병원균인 알테나리아 알테나타(Alternaria alternata) EML-BLDF1-4의 균사 성장을 억제하는 항진균 활성이 있는 것을 확인할 수 있었다.From this, it was confirmed that the penicillium dokone of the present invention has an antifungal activity that inhibits mycelial growth of the fungal pathogen Alternaria alternata EML-BLDF1-4.
2-5. 신규2-5. new 페니실리움Penicillium 독도엔스Dokdo Nance (( PenicilliumPenicillium dokdoensedokdoense ) ) CNUFCCNUFC -- DDS11DDS11 -1의 항균 활성(antimicrobial activity)-1 antimicrobial activity
본 발명의 페니실리움 독도엔스 CNUFC-DDS11-1의 항균 활성은 병원균인 스타필로코쿠스 아우레우스(Staphylococcus aureus) ATCC에 대한 페이퍼 디스크(paper disc) 방법으로 측정하였다.The antibacterial activity of Penicillium Dokdoen's CNUFC-DDS11-1 of the present invention was measured by the paper disc method for Staphylococcus aureus ATCC, a pathogen.
상기 병원균의 신선한 배양액을 LB(luria-bertani) 한천 배지의 표면에 접종하고, 멸균된 직경 6 mm의 페이퍼 디스크에는 아세톤에 용해된 화합물의 시험 농도인 0.10 mg/disc (0.05~0.25 mg/disc)가 로딩되었다.A fresh culture solution of the pathogen was inoculated on the surface of a LB (luria-bertani) agar medium, and 0.10 mg / disc (0.05 to 0.25 mg / disc), which is a test concentration of the compound dissolved in acetone, was sterilized on a paper disk having a diameter of 6 mm. Was loaded.
상기와 같이 접종된 플레이트를 25℃에서 24 시간 배양하였고, 상기 실험은 3회 반복 수행하였으며, 페이퍼 디스크 주변의 성장 저해 영역(growth inhibition zone)을 측정하여 기록하였다.The inoculated plate was cultured at 25 ° C. for 24 hours, the experiment was repeated 3 times, and the growth inhibition zone around the paper disc was measured and recorded.
신규 페니실리움 독도엔스의 항균 활성에 대한 스크리닝 결과를 도 7B에 도시하였다.The screening results for the antibacterial activity of the new Penicillium dokdoensu is shown in Figure 7B.
이로부터, 본 발명의 페니실리움 독도엔스의 에틸 아세테이트 추출물은 인간으로부터 질병을 일으키는 그람 양성균인 포도상 구균(Staphylocuccus aureus) ATCC에 대한 성장을 억제하는 항균 활성이 있는 것을 확인할 수 있었다.From this, ethyl acetate extracts of Staphylococcus aureus Gram-positive bacteria that cause human disease from Penny room Solarium islets Enschede of the present invention (Staphylocuccus aureus ) It was confirmed that there is an antibacterial activity that inhibits growth against ATCC.
<110> University Industry Liaison Office Of Chonnam National University <120> Novel fungus Penicillium dokdoense and use thereof <130> P18030120190 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 576 <212> DNA <213> Artificial Sequence <220> <223> MG906868 Penicillium dokdoense CNUFC-DDS11-1 sp. nov. [ITS region] <400> 1 aggatctata ccgcagtaga gagacctctg ggtccagcct cccacccgtg ttttccgaac 60 cctgttgctt cggcgggccc gcctcacggc cgccgggggg cttctgcccc cgggcccgcg 120 cccgccgaag acacctgtga acgctgtctg aagttgcagt ctgagaaact agctaaatta 180 gttaaaactt tcaacaacgg atctcttggt tccggcatcg atgaagaacg cagcgaaatg 240 cgataactaa tgtgaattgc agaattcagt gaatcatcga gtctttgaac gcacattgcg 300 ccctctggta ttccggaggg catgcctgtc cgagcgtcat tgctgccctc aagcacggct 360 tgtgtgttgg gcccccgccc ccccccgcac cgggggggcg ggcccgaaag gcagcggcgg 420 caccgcgtcc ggtcctcgag cgtatggggc ttcgtcaccc gctcttgtag gcccggccgg 480 cgccagccga ccccccctca atctattttt tcaggttgac ctcggatcag gtagggatac 540 ccgctgaact taagcatatc ataagccggc aggaaa 576 <210> 2 <211> 452 <212> DNA <213> Artificial Sequence <220> <223> MH243037 Penicillium dokdoense CNUFC-DDS11-1 sp. nov. (BenA gene [encoding beta tubulin]) <400> 2 ggtgcgttgc tgcatcttga tcatcattgt tggatttacg gggcaatata ctgattaatt 60 tcacaggcaa accattgctg gcgagcacgg ccttgatggc gatggacagt gagttcttct 120 cgacaaagtt ttgatttcca agaatggcgg tctgatattt ttgggcagct acaacggtac 180 ttccgacctc cagctggagc gcatgaacgt ctacttcaac cacgtaagtc aacccaacaa 240 tatcaattgc acttgtgaca tcgactctaa ttgatttgct ctcttttttg tcaataggct 300 tccggtgaca agtatgttcc ccgcgctgtt ctggtcgatc tggagcccgg taccatggac 360 gctgtccgtg ccggtccctt cggcaagctt ttccgtcccg acaacttcgt cttcggccag 420 tctggtgctg gtaacaactg ggccaagggt ca 452 <210> 3 <211> 517 <212> DNA <213> Artificial Sequence <220> <223> MH243031 Penicillium dokdoense CNUFC-DDS11-1 sp. nov. (CaM gene [encoding calmodulin]) <400> 3 agcgtaggtg atcattatag gctgacgggt tatttgtgat cgacaggaca aggatggcga 60 tggtgagtgc agtcgtgtct aaacaaatcg ggtctctttc acgggcggca tttctattgc 120 tgcgattggg gctccgtcct tccctcttta aacgcagcta atggatatgt gatcaatagg 180 acaaattacc accaaggagc tcggcaccgt catgcgctcc ctcggccaga acccctccga 240 gtccgagctg caggatatga tcaacgaggt tgacgccgat aacaatggca ccattgattt 300 ccctggtacg atacccgcaa cctcagatcc tcttcctttc ttcccccatc ctccatgctg 360 cctcccacga taggatcaat attgacatgc gcctcacaga gttcttgacc atgatggccc 420 gtaagatgaa ggataccgac tctgaggagg agatccgtga ggcattcaag gttttcgacc 480 gcgacaacaa cggcttcatt tccgccgctg agctgcg 517 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Forward <400> 4 cccttattga cctcaactac atggt 25 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Reverse <400> 5 gaggggccat ccacagtctt ctg 23 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Forward <400> 6 catcttctca aaattcgagt gacaa 25 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Reverse <400> 7 tgggagtaga caaggtacaa ccc 23 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta Forward <400> 8 caaccaacaa gtgatattct ccatg 25 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta Reverse <400> 9 gatccacact ctccagctgc a 21 <110> University Industry Liaison Office Of Chonnam National University <120> Novel fungus Penicillium dokdoense and use thereof <130> P18030120190 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 576 <212> DNA <213> Artificial Sequence <220> <223> MG906868 Penicillium dokdoense CNUFC-DDS11-1 sp. nov. [ITS region] <400> 1 aggatctata ccgcagtaga gagacctctg ggtccagcct cccacccgtg ttttccgaac 60 cctgttgctt cggcgggccc gcctcacggc cgccgggggg cttctgcccc cgggcccgcg 120 cccgccgaag acacctgtga acgctgtctg aagttgcagt ctgagaaact agctaaatta 180 gttaaaactt tcaacaacgg atctcttggt tccggcatcg atgaagaacg cagcgaaatg 240 cgataactaa tgtgaattgc agaattcagt gaatcatcga gtctttgaac gcacattgcg 300 ccctctggta ttccggaggg catgcctgtc cgagcgtcat tgctgccctc aagcacggct 360 tgtgtgttgg gcccccgccc ccccccgcac cgggggggcg ggcccgaaag gcagcggcgg 420 caccgcgtcc ggtcctcgag cgtatggggc ttcgtcaccc gctcttgtag gcccggccgg 480 cgccagccga ccccccctca atctattttt tcaggttgac ctcggatcag gtagggatac 540 ccgctgaact taagcatatc ataagccggc aggaaa 576 <210> 2 <211> 452 <212> DNA <213> Artificial Sequence <220> <223> MH243037 Penicillium dokdoense CNUFC-DDS11-1 sp. nov. (BenA gene [encoding beta tubulin]) <400> 2 ggtgcgttgc tgcatcttga tcatcattgt tggatttacg gggcaatata ctgattaatt 60 tcacaggcaa accattgctg gcgagcacgg ccttgatggc gatggacagt gagttcttct 120 cgacaaagtt ttgatttcca agaatggcgg tctgatattt ttgggcagct acaacggtac 180 ttccgacctc cagctggagc gcatgaacgt ctacttcaac cacgtaagtc aacccaacaa 240 tatcaattgc acttgtgaca tcgactctaa ttgatttgct ctcttttttg tcaataggct 300 tccggtgaca agtatgttcc ccgcgctgtt ctggtcgatc tggagcccgg taccatggac 360 gctgtccgtg ccggtccctt cggcaagctt ttccgtcccg acaacttcgt cttcggccag 420 tctggtgctg gtaacaactg ggccaagggt ca 452 <210> 3 <211> 517 <212> DNA <213> Artificial Sequence <220> <223> MH243031 Penicillium dokdoense CNUFC-DDS11-1 sp. nov. (CaM gene [encoding calmodulin]) <400> 3 agcgtaggtg atcattatag gctgacgggt tatttgtgat cgacaggaca aggatggcga 60 tggtgagtgc agtcgtgtct aaacaaatcg ggtctctttc acgggcggca tttctattgc 120 tgcgattggg gctccgtcct tccctcttta aacgcagcta atggatatgt gatcaatagg 180 acaaattacc accaaggagc tcggcaccgt catgcgctcc ctcggccaga acccctccga 240 gtccgagctg caggatatga tcaacgaggt tgacgccgat aacaatggca ccattgattt 300 ccctggtacg atacccgcaa cctcagatcc tcttcctttc ttcccccatc ctccatgctg 360 cctcccacga taggatcaat attgacatgc gcctcacaga gttcttgacc atgatggccc 420 gtaagatgaa ggataccgac tctgaggagg agatccgtga ggcattcaag gttttcgacc 480 gcgacaacaa cggcttcatt tccgccgctg agctgcg 517 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Forward <400> 4 cccttattga cctcaactac atggt 25 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Reverse <400> 5 gaggggccat ccacagtctt ctg 23 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Forward <400> 6 catcttctca aaattcgagt gacaa 25 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Reverse <400> 7 tgggagtaga caaggtacaa ccc 23 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta Forward <400> 8 caaccaacaa gtgatattct ccatg 25 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta Reverse <400> 9 gatccacact ctccagctgc a 21
Claims (6)
상기 페니실리움 독도엔스 CNUFC-DDS11-1(기탁번호: KACC 93309p)는 산화질소(NO) 생성을 억제하는 것을 특징으로 하는 페니실리움 독도엔스 CNUFC-DDS11-1(기탁번호: KACC 93309p).According to claim 1,
The penicillium dokdoensu CNUFC-DDS11-1 (accession number: KACC 93309p) is a penicillium dokdoensu CNUFC-DDS11-1 (accession number: KACC 93309p) characterized by inhibiting the production of nitrogen oxide (NO).
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