KR20180081402A - Bacillus subtilis ds660 strain having antimicrobial activity and uses thereof - Google Patents

Bacillus subtilis ds660 strain having antimicrobial activity and uses thereof Download PDF

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KR20180081402A
KR20180081402A KR1020170002585A KR20170002585A KR20180081402A KR 20180081402 A KR20180081402 A KR 20180081402A KR 1020170002585 A KR1020170002585 A KR 1020170002585A KR 20170002585 A KR20170002585 A KR 20170002585A KR 20180081402 A KR20180081402 A KR 20180081402A
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송홍규
이다솔
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강원대학교산학협력단
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Abstract

The present invention relates to an antimicrobial composition comprising a Bacillus subtilis DS660 (Accession No. KCTC18521P) strain or a culture of the strain as an active ingredient. The strain can usefully be used for antibacterial purposes since the strain can produce surfactant, siderophore, beta-1-3-glucanase and bacterial cell wall lytic enzyme and are excellent in antibacterial activity and stable to temperature and pH change.

Description

항균 활성을 가지는 바실러스 서브틸리스 DS660 균주 및 이의 용도{BACILLUS SUBTILIS DS660 STRAIN HAVING ANTIMICROBIAL ACTIVITY AND USES THEREOF}BACILLUS SUBTILIS DS660 STRAIN HAVING ANTIMICROBIAL ACTIVITY AND USES THEREOF < RTI ID = 0.0 >

본 발명은 항균 활성을 가지는 바실러스 서브틸리스 DS660(기탁번호 KCTC18521P) 균주 및 이의 용도에 관한 것이다.The present invention relates to a strain Bacillus subtilis DS660 (Accession No. KCTC18521P) having antibacterial activity and its use.

바실러스 서브틸리스(Bacillus subtilis)는 흔히 '고초균'으로 불리는 비병원성의 호기성 간균으로, 바실러스 속(Bacillus sp.)에 속하는 그람 양성(gram positive)균이다. 바실러스 서브틸리스는 마른 풀, 토양 등에 존재하며 주모성 편모를 가지고 있어 활발히 운동을 한다. Bacillus subtilis is a non-pathogenic aerobic bacterium commonly referred to as 'Bacillus subtilis', a gram positive bacterium belonging to the genus Bacillus sp. Bacillus subtilis is present in dry grass, soil, etc., and it has active maternal flagella and actively exercises.

바실러스 종은 산업적으로 중요한 아밀레이스(amylase), 프로테이스(protease) 등과 같은 효소를 생산하며, 생물 계면활성제(surfactant), 박테리오신(bacteriocin) 등의 항균물질을 생산하는 것으로 알려져 있다. 항균물질 중에서 생물 계면활성제는 무독성이고, 생분해가 용이하여 환경친화적이기 때문에 환경오염을 감소시킬 수 있다. 또한, 생물 계면활성제는 화장품, 의약품, 식품, 세제, 펄프 및 제지, 원유의 2차 회수, 환경정화 등의 각 분야에서 광범위하게 응용될 수 있다.Bacillus species produces enzymes such as amylase and protease, which are industrially important, and is known to produce antimicrobial substances such as surfactants and bacteriocins. Among the antimicrobial substances, biological surfactants are non-toxic and easily biodegradable, so they can reduce environmental pollution because they are environment-friendly. In addition, biosurfactants can be widely applied in various fields such as cosmetics, medicines, foods, detergents, pulp and paper, secondary recovery of crude oil, and environmental purification.

미생물을 이용한 항균 용도와 관련하여, 대한민국 등록특허 제10-1594446호는 유해 미생물에 대하여 항균 활성을 가지는 바실러스 서브틸리스 RX7 균주를 개시하고 있다(특허문헌 1). 또한, 등록특허 제10-1190901호는 식품 위해 미생물에 대한 항균력이 우수한 바실러스 서브틸리스 CSY191 균주를 개시하고 있으나 주로 대장균, 살모넬라 등의 식중독균에 항균 활성을 보이는 바실러스 서브틸리스 균주를 개시하고 있다(특허문헌 2). Regarding antibacterial use using microorganisms, Korean Patent No. 10-1594446 discloses Bacillus subtilis RX7 strain having antibacterial activity against harmful microorganisms (Patent Document 1). In addition, Patent No. 10-1190901 discloses a Bacillus subtilis CSY191 strain having excellent antimicrobial activity against a food-harmful microorganism, but discloses a Bacillus subtilis strain having antimicrobial activity against food-borne bacteria such as Escherichia coli and Salmonella Patent Document 2).

본 발명자는 토양에서 신규한 바실러스 서브틸리스 균주를 분리하였으며, 상기 균주가 피부 상재균에 대하여 항균 활성이 있음을 확인하였다.The present inventors isolated a novel Bacillus subtilis strain from the soil, and confirmed that the strain has antimicrobial activity against the skin uptake bacteria.

1. 대한민국 등록특허 제10-1594446호1. Korean Patent No. 10-1594446 2. 대한민국 등록특허 제10-1190901호2. Korean Patent No. 10-1190901

1. Novel antilisterial bacteriocin licheniocin 50.2 from Bacillus licheniformis VPS50.2 isolated from soil sample. Journal of Applied Microbiology 2013, 116: 502-510. 1. Novel antilisterial bacteriocin licheniocin 50.2 from Bacillus licheniformis VPS50.2 isolated from soil sample. Journal of Applied Microbiology 2013, 116: 502-510. 2. Bacteriocin (BAC-IB17): Screening, isolation and production from Bacillus subtilis KIBGE IB-17. Pakistan Journal Pharmaceutical Science 2012, 25: 195-201. 2. Bacteriocin (BAC-IB17): Screening, isolation and production from Bacillus subtilis KIBGE IB-17. Pakistan Journal Pharmaceutical Science 2012, 25: 195-201. 3. Characterization of an antibacterial peptide produced by Brevibacterium linens. Journal of Applied Microbiology 2002, 92: 63-70. 3. Characterization of an antibacterial peptide produced by Brevibacterium linens. Journal of Applied Microbiology 2002, 92: 63-70. 4. Comparison of methods to detect biosurfactant production by diverse microorganisms. Journal of Microbiological Methods 2004, 56: 339-347. 4. Comparison of methods to detect biosurfactant production by diverse microorganisms. Journal of Microbiological Methods 2004, 56: 339-347. 5. Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, Microbiological Research, 2004, 159: 73-81. 5. Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens inhibition of Rhizoctonia solani, Microbiological Research, 2004, 159: 73-81. 6. Characterization of a novel bacteriocin produced by Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish. Food Control 2012, 23: 338-344. 6. Characterization of a novel bacteriocin produced by Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish. Food Control 2012, 23: 338-344.

본 발명의 일 양상은 항균 활성을 가지는 바실러스 서브틸리스 DS660(기탁번호 KCTC18521P) 균주를 제공하는 것이다.One aspect of the present invention is to provide a strain of Bacillus subtilis DS660 (Accession No. KCTC18521P) having antibacterial activity.

본 발명의 다른 양상은 상기 균주 또는 균주의 배양물을 포함하는 항균용 조성물을 제공하는 것이다.Another aspect of the present invention is to provide an antimicrobial composition comprising a culture of the strain or strain.

본 발명의 또 다른 양상은 상기 균주의 배양물을 유효성분으로 포함하는 화장료 조성물을 제공하는 것이다.Another aspect of the present invention is to provide a cosmetic composition comprising a culture of the strain as an active ingredient.

본 발명의 일 양상은 항균 활성을 가지는 바실러스 서브틸리스 DS660(기탁번호 KCTC18521P) 균주를 제공한다.One aspect of the present invention provides Bacillus subtilis DS660 (Accession No. KCTC18521P) strain having antibacterial activity.

본 발명의 일 구체예에 따르면 본 발명의 바실러스 서브틸리스 DS660 균주는 토양에서 분리한 미생물로서 16S rDNA 서열분석 및 API 테스트를 통하여 바실러스 서브틸리스에 속하는 신규한 균주인 것을 확인하였다. 따라서, 상기 균주를 한국생명공학연구원 생물자원센터에 기탁하여 KCTC18521P의 기탁번호를 부여받았다.According to one embodiment of the present invention, the Bacillus subtilis DS660 strain of the present invention is a microorganism isolated from soil, and is a novel strain belonging to Bacillus subtilis through 16S rDNA sequence analysis and API test. Therefore, the above strain was deposited with the BRC at the Korea Biotechnology Research Institute and received the deposit number KCTC18521P.

일 구체예에 따르면 본 발명의 바실러스 서브틸리스 DS660 균주는 바실러스 속(genus Bacillus), 스태필로코커스 속(genus Staphylococcus), 아스퍼질러스 속(genus Aspergillus), 슈도모나스 속(genus Pseudomonas), 에스케리치아 속(genus Escherichia) 및 캔디다 속(genus Candida)으로 이루어진 군에서 선택되는 미생물에 대하여 항균 활성을 나타낼 수 있다. 보다 구체적으로는 바실러스 서브틸리스(Bacillus subtilis), 스태필로코커스 아우레우스(Staphylococcus aureus), 아스퍼질러스 나이거(Aspergillus niger), 슈도모나스 에루기노사(Pseudomonas aeruginosa), 에스케리치아 콜라이(Escherichia coli) 및 캔디다 알비칸스(Candida albicans)로 이루어진 군에서 선택되는 미생물에 대하여 항균 활성을 가지는 것이 바람직하다.According to one embodiment of the present invention, the Bacillus subtilis DS660 strain of the present invention is a strain of Bacillus genus Bacillus , genus Staphylococcus , genus Aspergillus , genus Pseudomonas , Genus Escherichia , and genus Candida . ≪ Desc / Clms Page number 2 > More specifically, there can be mentioned Bacillus subtilis , Staphylococcus aureus , Aspergillus niger , Pseudomonas aeruginosa , Escherichia coli, it is preferable that the microorganism has antibacterial activity against a microorganism selected from the group consisting of E. coli and Candida albicans .

일 구체예에 따르면 본 발명의 바실러스 서브틸리스 DS660 균주는 계면활성제(surfactant)를 생산하는 것일 수 있으며, 바람직하게는 당지질(glycolopid) 구조의 계면활성제를 생산하는 균주일 수 있다.According to one embodiment, the Bacillus subtilis DS660 strain of the present invention may be a strain producing a surfactant, and preferably a strain producing a glycolipid-structured surfactant.

본 명세서의 용어, "계면활성제"는 분자 중에 친수성기 및 친유성기를 모두 포함하는 양친매성 물질을 말하며, 세정력, 분산력, 유화력, 가용화력, 습윤력, 살균력, 기포력 및 침투력이 우수한 물질이다.The term "surfactant" as used herein refers to an amphipathic substance including both a hydrophilic group and a lipophilic group in a molecule, and is a substance excellent in washing power, dispersing power, emulsifying power, solubilizing power, wetting power, sterilizing power, foaming power and penetration power.

일 구체예에 따르면 본 발명의 바실러스 서브틸리스 DS660 균주는 토양 내 미량 존재하는 필수 생리물질인 철 성분을 선택적으로 흡수하여 병원성 미생물의 생장을 억제할 수 있는 시데로포어(siderophore)를 생산하는 것일 수 있다.According to one embodiment, the Bacillus subtilis DS660 strain of the present invention produces siderophore capable of selectively inhibiting the growth of pathogenic microorganisms by selectively absorbing an iron component which is a necessary physiological substance present in a soil in a small amount .

일 구체예에 따르면 본 발명의 바실러스 서브틸리스 DS660 균주는 미생물의 세포벽을 분해할 수 있는 베타-1,3-글루카나제(β-1.3-glucanase)를 생산하는 것일 수 있다.According to one embodiment, the Bacillus subtilis DS660 strain of the present invention can produce beta-1.3-glucanase capable of degrading the cell wall of a microorganism.

일 구체예에 따르면 본 발명의 바실러스 서브틸리스 DS660 균주는 세균 세포벽 용해효소를 생산하는 것일 수 있다.According to one embodiment, the Bacillus subtilis DS660 strain of the present invention may be one which produces a bacterial cell wall lysis enzyme.

일 구체예에 다르면 본 발명의 바실러스 서브틸리스 DS660 균주는 -22 내지 70℃의 온도 범위에서도 항균 활성을 유지하는 것일 수 있으며, 3 내지 12의 넓은 pH 범위에서 항균 활성을 유지하는 것일 수 있다.According to one embodiment, the Bacillus subtilis DS660 strain of the present invention may maintain the antimicrobial activity even in the temperature range of -22 to 70 캜, and may maintain the antimicrobial activity in the wide pH range of 3 to 12.

본 발명의 다른 양상은 바실러스 서브틸리스 DS660(기탁번호 KCTC18521P) 균주, 상기 균주의 배양물, 상기 균주 또는 배양액의 농축물 및 이들의 건조물로 이루어진 군에서 선택된 하나 이상을 포함하는 항균용 조성물을 제공한다.Another aspect of the present invention provides an antimicrobial composition comprising at least one selected from the group consisting of a strain of Bacillus subtilis DS660 (Accession No. KCTC18521P), a culture of the strain, a concentrate of the strain or the culture and a dried product thereof do.

상기 항균용 조성물은 항미생물제를 총칭하는 의미인 항생제와 같은 의미일 수 있고, 항균제, 살균제, 방부, 보존제 또는 제균제와 같은 의미일 수 있으며, 바람직하게는 상기에 언급한 미생물의 발육과 생활 기능을 저지 또는 억제할 수 있는 물질을 의미한다.The antimicrobial composition may have the same meaning as an antibiotic, which is generically referred to as an antimicrobial agent, and may have the same meaning as an antimicrobial agent, a bactericide, an antiseptic, a preservative or a bactericidal agent, ≪ / RTI > or a substance capable of inhibiting or inhibiting < RTI ID =

본 명세서의 용어, "배양물"은 배지에서 일정 기간 동안 배양하여 수득한 균주, 그의 대사물, 여분의 영양분 등을 포함하는 배지, 또는 균주를 배양한 후 균주를 제거한 배양액을 포함하는 개념으로 해석된다.As used herein, the term "culture product" refers to a culture medium obtained by culturing a culture medium for a predetermined period of time, a culture medium containing the same, metabolites thereof, extra nutrients or the like, do.

일 구체예에 따르면 본 발명의 바실러스 서브틸리스 DS660 균주의 배양물은 미생물 배양에 사용되는 배지 중에서 당업자가 목적에 따라 용이하게 선택한 배지를 사용할 수 있으며, 바람직하게는 바실러스균 배양에 사용되는 배지, 보다 바람직하게는 하기 실시예 1에 기재된 NA 또는 LB 배지를 선택하여 사용할 수 있다.According to one embodiment, the culture of the strain Bacillus subtilis DS660 of the present invention can be selected from a medium easily used by a person skilled in the art in a culture medium for microorganism culture, preferably a medium used for Bacillus bacteria culture, More preferably, NA or LB medium described in Example 1 below can be selected and used.

일 구체예에 따르면 상기 NA 또는 LB 배지는 탄소원으로서 소르비톨(sorbitol), 글리세롤(glycerol), 포도당(glucose) 또는 수크로오스(sucroxe)를 포함할 수 있다. 또한, 질소원으로는 황산 암모늄(ammonium sulfate), 트립톤(tryptone), 펩톤(peptone), 효모 추출물(yeast extract), 인산 암모늄(ammonium phosphate), 맥아 추출물(malt extract) 또는 요소(urea)를 포함할 수 있다.According to one embodiment, the NA or LB medium may include sorbitol, glycerol, glucose, or sucroxe as a carbon source. The nitrogen source may also include ammonium sulfate, tryptone, peptone, yeast extract, ammonium phosphate, malt extract or urea. can do.

일 구체예에 따르면 본 발명의 바실러스 서브틸리스 DS660 균주의 배양물은 상기 미생물 배양 배지에 본 발명의 균주를 접종하고, 당업계에 공지된 미생물 배양 방법(예를 들어, 정치배양, 교반배양)에 따라 제조할 수 있다. 상기 바실러스 서브틸리스 DS660 균주 또는 배양액의 농축물 및 이들의 건조물은 당업계에 공지된 미생물 또는 배양액의 농축 또는 건조 방법에 따라 용이하게 제조될 수 있다.According to one embodiment, the culture of the strain Bacillus subtilis DS660 of the present invention can be prepared by inoculating the strain of the present invention into the microorganism culture medium, culturing the microorganism culture method (for example, stationary culture, agitation culture) . ≪ / RTI > The Bacillus subtilis DS660 strain or the concentrate of the culture broth and the dried product thereof can be easily prepared by a method of concentrating or drying the microorganism or culture medium known in the art.

본 발명의 항균용 조성물에서 바실러스 서브틸리스 DS660 균주, 상기 균주의 배양물, 상기 균주 또는 배양액의 농축물 및 이들의 건조물 이외의 성분은 미생물 활성 및 항균 작용을 촉진하는 성분으로써 당업계에 공지된 다양한 성분이 포함될 수 있다.In the antimicrobial composition of the present invention, the components other than the Bacillus subtilis DS660 strain, the culture of the strain, the concentrate of the strain or the culture solution, and the dried components thereof are components that promote microbial activity and antibacterial activity, Various ingredients may be included.

본 발명의 또 다른 양상은 바실러스 서브틸리스 DS660(기탁번호 KCTC18521P) 균주의 배양물, 배양액의 농축물 및 건조물로 이루어진 군에서 선택된 하나 이상을 포함하는 화장료 조성물을 제공한다.Another aspect of the present invention provides a cosmetic composition comprising at least one selected from the group consisting of a culture of a strain of Bacillus subtilis DS660 (Accession No. KCTC18521P), a concentrate of a culture and a dried product.

본 발명의 화장료 조성물은 유효성분으로서 바실러스 서브틸리스 DS660 균주의 배양물, 배양액의 농축물 또는 건조물 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있으며, 예컨대 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다.The cosmetic composition of the present invention may contain, as an active ingredient, components commonly used in cosmetic compositions in addition to the culture of Bacillus subtilis DS660 strain, the concentrate of the culture broth or the dried product, and examples thereof include stabilizers, solubilizers, vitamins, pigments And customary adjuvants such as fragrances, and carriers.

한편, 본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클렌징 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 반드시 한정되는 것은 아니다. 더욱 상세하게는, 유연 화장수, 젤, 수용성 리퀴드, 밀크로션, 크림, 에센스, 스킨토너, 수중유형(oil-in-water) 에멀젼, 유중수형(water-in-oil) 에멀젼, 클렌징 폼, 클렌징 워터, 팩, 바디오일, 바디로션, 수중유형 메이크업베이스, 유중수형 메이크업베이스 및 파운데이션의 제형으로 제조될 수 있다.Meanwhile, the cosmetic composition of the present invention may be prepared in any form conventionally produced in the art, and examples thereof include a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, Containing cleansing oil, powdered foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More particularly, the present invention relates to a cosmetic composition for the preparation of a cosmetic composition, which comprises at least one component selected from the group consisting of a soft lotion, a gel, a water-soluble liquid, a milk lotion, a cream, an essence, a skin toner, an oil-in-water emulsion, , Pack, body oil, body lotion, underwater type make-up base, in-water type make-up base and foundation.

본 발명의 바실러스 서브틸리스 DS660(기탁번호 KCTC18521P) 균주는 계면활성제, 시데로포어, 베타-1,3-글루카나제 및 세균 세포벽 용해효소를 생산하여 항균 활성이 우수하고, 온도 및 pH 변화에 안정적이기 때문에 항균 용도로 유용하게 이용될 수 있다.The strain Bacillus subtilis DS660 (Accession No. KCTC18521P) of the present invention produces a surfactant, cyperophor, beta-l, 3-glucanase and bacterial cell wall lytic enzyme and has excellent antimicrobial activity. And thus can be usefully used for antibacterial use.

도 1은 바실러스 서브틸리스(Bacillus subtilis) DS660 균주의 배양 결과를 나타낸 도이다.
도 2는 6종의 피부 상재균에 대한 B. subtilis DS660 균주의 항균 활성을 측정한 결과를 나타낸 도이다.
도 3은 배양 시간에 따른 B. subtilis DS660 균주의 항균 활성을 측정한 결과를 나타낸 도이다.
도 4는 오일 스프레딩 테스트를 이용하여 B. subtilis DS660 균주의 계면활성제 생산 여부를 확인한 결과를 나타낸 도이다.
도 5는 B. subtilis DS660 균주가 생산하는 계면활성제의 성분을 박층 크로마토그래피로 분석한 결과를 나타낸 도이다.
도 6은 B. subtilis DS660 균주의 배양 시간에 따른 표면장력의 변화를 측정한 결과를 나타낸 도이다.
도 7은 B. subtilis DS660 균주의 배양 상층액 첨가 농도에 따른 표면장력의 변화를 측정한 결과를 나타낸 도이다.
도 8은 B. subtilis DS660 균주의 시데로포어 생산 여부를 확인한 결과를 나타낸 도이다.
도 9는 B. subtilis DS660 균주가 생산하는 시데로포어 농도를 측정한 결과를 나타낸 도이다.
도 10은 B. subtilis DS660 균주의 베타-1,3-글루카나제 생산 여부를 확인한 결과를 나타낸 도이다.
도 11은 B. subtilis DS660 균주가 생산하는 베타-1,3-글루카나제 농도를 측정한 결과를 나타낸 도이다.
도 12는 B. subtilis DS660 균주의 세균 세포벽 용해효소 생산 여부를 확인한 결과를 나타낸 도이다.
도 13은 탄소원 또는 질소원 변화에 따른 B. subtilis DS660 균주의 항균 활성 변화를 측정한 결과를 나타낸 도이다.BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the culture results of Bacillus subtilis strain DS660. FIG.
FIG. 2 is a graph showing the results of measurement of antimicrobial activity of B. subtilis DS660 strain against six types of skin topical bacteria.
FIG. 3 is a graph showing the results of measurement of the antimicrobial activity of B. subtilis DS660 according to culture time. FIG.
FIG. 4 is a graph showing the results of confirming the production of the surfactant of the B. subtilis DS660 strain using the oil spreading test. FIG.
Figure 5 shows the results of thin layer chromatography analysis of the components of the surfactant produced by B. subtilis DS660 strain.
FIG. 6 is a graph showing the results of measurement of the change in surface tension of B. subtilis DS660 according to the incubation time. FIG.
FIG. 7 is a graph showing the results of measurement of the change in surface tension according to the culture supernatant concentration of B. subtilis DS660 strain. FIG.
8 is a diagram showing the results of confirming the production of sea tangle pore of B. subtilis DS660 strain.
FIG. 9 is a graph showing the results of measurement of the concentration of sea urchin produced by B. subtilis DS660 strain. FIG.
10 is a graph showing the results of confirming the production of beta-1,3-glucanase by B. subtilis DS660 strain.
11 is a graph showing the results of measurement of the concentration of beta-1,3-glucanase produced by B. subtilis DS660 strain.
12 is a diagram showing the results of confirming the production of bacterial cell wall lytic enzyme by B. subtilis DS660 strain.
FIG. 13 is a graph showing the results of measurement of the change in antimicrobial activity of B. subtilis DS660 according to carbon source or nitrogen source change. FIG.

이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

실시예Example 1: 항균 활성 균주의 분리 및 동정 1: Isolation and identification of antimicrobial active strains

1-1: 균주의 분리 및 항균 활성 확인1-1: Isolation and Antibacterial Activity of Strain

항균 활성을 나타내는 미생물을 분리하기 위하여 강원도 영월 근처의 식물 근권 토양에서 토양 시료를 채취하였다. 채취한 토양 시료 10 g을 50 ㎖ conical tube에 담은 후 총 부피가 30 ㎖가 되도록 염화나트륨(NaCl 8.5%) 용액을 첨가하여 30분 동안 진탕(shaking)하였다. 이후 염화나트륨(NaCl 8.5%) 용액을 이용하여 시료를 연속 희석(1/103, 1/104)하고, Nutrient agar(peptone 5, beef extract 1.5, yeast extract 1.5, NaCl 5 및 agar 15 g/L; 이하 NA 배지로 표기함) 플레이트 또는 Luria Bertani agar(tryptone 10, yeast extract 5 및 NaCl 10 g/L; 이하 LB 배지로 표기함) 플레이트에 연속 희석한 토양 시료 100 ㎕를 도말하였다. 시료를 도말한 플레이트를 30℃에서 7일 동안 배양하여 성장한 세균 콜로니(colony)를 분리하였고, 분리한 콜로니는 새로운 배지에 계대배양(subculture)하였다.In order to isolate the microorganisms exhibiting antibacterial activity, soil samples were collected from the plant rhizosphere soil near Yeongwol, Kangwon province. 10 g of the collected soil sample was immersed in a conical tube of 50 ml, and a solution of sodium chloride (NaCl 8.5%) was added thereto so as to have a total volume of 30 ml, followed by shaking for 30 minutes. After that, the samples were serially diluted (1/10 3 , 1/10 4 ) with sodium chloride (NaCl 8.5%) solution, and Nutrient agar (peptone 5, beef extract 1.5, yeast extract 1.5, NaCl 5 and agar 15 g / L (Hereinafter referred to as NA medium) plate or Luria Bertani agar (tryptone 10, yeast extract 5 and 10 g / L NaCl, hereinafter referred to as LB medium) plate. Plates on which the samples were stained were cultured at 30 ° C. for 7 days to isolate grown colonies of colonies. Subsequently, the colonies were subcultured to new culture medium.

상기 분리한 콜로니의 항균 활성을 확인하기 위하여 피부 상재균 6종-캔디다 알비칸스(Candida albicans), 바실러스 서브틸리스(Bacillus subtilis), 스태필로코커스 아우레우스(Staphylococcus aureus), 아스퍼질러스 나이거(Aspergillus niger), 슈도모나스 에루기노사(Pseudomonas aeruginosa) 및 에스케리치아 콜라이(Escherichia coli)-을 각각 도말한 배지에 분리한 콜로니의 균주를 획선(streaking)으로 그어 30℃에서 24시간 동안 배양하였다. 24시간 후 콜로니 주변에 생긴 투명대(clear zone)의 크기를 토대로 우수한 항균 활성을 나타내는 균주를 1차로 선별하였다. 선별한 균주 중에서 DS660의 배양 결과를 도 1에 도시하였다.In order to confirm the antimicrobial activity of the isolated colonies, six kinds of skin antibiotics - Candida albicans , Bacillus subtilis , Staphylococcus aureus , (Aspergillus niger), rugi labor Pseudomonas (Pseudomonas aeruginosa and Escherichia coli were each streaked on a streaked culture medium, and the colonies were streaked and cultured at 30 ° C for 24 hours. After 24 hours, strains showing excellent antimicrobial activity were firstly selected based on the size of the clear zone around the colonies. The results of the culture of DS660 among the selected strains are shown in Fig.

1-2: 균주의 동정(identification)1-2: Identification of the strain

상기 실시예 1-1에서 분리한 DS660 균주의 16S rDNA 서열(서열번호 1)을 마크로젠에 분석 의뢰한 결과 바실러스 서브틸리스와 99%의 상동성을 나타내는 것을 확인하였다. 또한, 제조사(Biomerieux, 프랑스)의 지시에 따라 API test(API 50CH, 20E)를 실시하여 DS660 균주의 생리생화학적 특성을 확인하였으며 그 결과를 하기 표 1에 기재하였다. 상기 서열분석 및 API test 결과를 종합하여 분리한 DS660 균주를 최종적으로 바실러스 서브틸리스 DS660(이하, DS660으로 표기함)으로 동정하였다. 동정한 바실러스 서브틸리스 DS660 균주를 2016년 12월 2일 한국생명공학연구원 생물자원센터에 기탁하여 미생물 기탁번호 KCTC18521P를 부여받았다.The 16S rDNA sequence (SEQ ID NO: 1) of the strain DS660 isolated in Example 1-1 was analyzed by Macrogen and showed 99% homology with Bacillus subtilis. In addition, an API test (API 50CH, 20E) was conducted according to the instructions of the manufacturer (Biomerieux, France) to confirm the physiological and biochemical characteristics of the strain DS660. The DS660 strain isolated from the sequence analysis and API test results was finally identified as Bacillus subtilis DS660 (hereinafter referred to as DS660). The identified Bacillus subtilis strain DS660 was deposited on December 2, 2016 at the Korea Research Institute of Bioscience and Biotechnology, and received the microorganism deposit number KCTC18521P.

Figure pat00001
Figure pat00001

실시예Example 2: 분리한 DS660 균주의 항균 활성 확인 2: Identification of the antimicrobial activity of isolated DS660 strain

2-1: 피부 2-1: Skin 상재균에In the supernatant 대한 생장 억제 효과 확인 Confirmation of growth inhibition effect

LB 액체배지에서 선배양(30℃, 160 rpm)한 DS660 균주를 새로운 액체배지에 2x108 CFU/㎖ 농도로 개체수를 보정하여 접종하고, 48시간 동안 배양하였다. 배양이 끝난 후 배양액을 4℃, 12000 g에서 20분 동안 원심분리하고, 배양 상층액을 수거하였다. DS660 균주와 별도로 피부 상재균 5종을 2x108 CFU/㎖ 농도로 개체수를 보정하여 NA 배지에 도말하였으며, A. niger는 변형시킨 PDA(potato dextrose agar; potato starch 2 g, dextrose 10 g, beef extract 1.5 g, peptone 2.5 g) 배지에 도말하였다. 이후 배지에 10 ㎜ 직경의 웰(well)을 뚫고, 각 웰에 DS660 균주 배양 상층액 100 ㎕를 첨가한 후 24 내지 48시간 동안 배양하였으며 배양이 끝난 후 생성된 투명대의 직경을 측정하였다.The strain DS660, which was predominantly in LB liquid medium (30 ° C, 160 rpm), was inoculated with fresh liquid medium at a concentration of 2 × 10 8 CFU / ml and cultured for 48 hours. After the culture was completed, the culture was centrifuged at 12,000 g for 20 minutes at 4 ° C, and the culture supernatant was collected. Five different types of skin fungi were isolated on a NA medium supplemented with 2 × 10 8 CFU / ㎖, separately from the DS660 strain. A. niger was cultured on a modified PDA (potato dextrose agar; potato starch 2 g, dextrose 10 g, beef extract 1.5 g, peptone 2.5 g). Then, a 10-mm-diameter well was inserted into the culture medium. 100 μl of the culture supernatant of the strain DS660 was added to each well, followed by culturing for 24 to 48 hours. The diameter of the generated zygote after the cultivation was measured.

그 결과, 하기 표 2 및 도 2에 나타난 것과 같이 DS660 균주가 6종의 피부 상재균의 생장을 억제하는 것을 확인할 수 있었으며, 특히 S. aureus , A. niger 및 P. aeruginosa에 대한 항균 활성이 우수한 것을 알 수 있었다.As a result, as shown in Table 2 and FIG. 2, it was confirmed that the strain DS660 inhibited the growth of six types of skin topical bacterium. Especially, the antimicrobial activity against S. aureus , A. niger and P. aeruginosa was excellent .

Figure pat00002
Figure pat00002

상기 실험 결과는 본 발명의 DS660 균주가 비특허문헌 1에 개시된 Bacillus licheniformis VPS50.2와 비교하여 넓은 범위의 그람 양성 및 그람 음성 세균과 곰팡이, 효모의 생장을 억제할 수 있음을 의미한다. 또한, 비특허문헌 2에 개시된 Bacillus subtilis KIBGE IB-17 균주와 비교하면, KIBGE IB-17 균주는 S. aureus ATCC 6538, P. aeruginosa 및 E. coli에 대한 생장 억제 직경이 0, 0 및 18 ㎜이기 때문에 본 발명의 DS660 균주가 항균 활성이 현저히 우수한 것을 알 수 있다.The above experimental results indicate that the strain DS660 of the present invention can inhibit the growth of a wide range of gram-positive and gram-negative bacteria, fungi and yeast as compared with Bacillus licheniformis VPS50.2 disclosed in Non-patent Document 1. [ Compared with the Bacillus subtilis KIBGE IB-17 strain disclosed in the non-patent document 2, the KIBGE IB-17 strain had growth inhibition diameters of 0, 0 and 18 mm against S. aureus ATCC 6538, P. aeruginosa and E. coli , It can be seen that the strain DS660 of the present invention has remarkably excellent antibacterial activity.

2-2: DS660 균주의 배양시간에 따른 항균 활성 확인2-2: Identification of antimicrobial activity of DS660 strain by incubation time

LB 액체배지에서 선배양(30℃, 160 rpm)한 DS660 균주를 새로운 액체배지에 2x108 CFU/㎖ 농도로 개체수를 보정하여 접종하고, 48시간 동안 배양하였다. 배양이 끝난 후 배양액을 4℃, 12000 g에서 20분 동안 원심분리하고, 배양 상층액을 수거하였다. 수거한 배양 상층액은 PBS를 이용하여 2배씩 연속 희석하여 실험에 이용하였다. DS660 균주와 별도로 지시 균주(indicator strain)로 이용할 S. aureus를 2x108 CFU/㎖ 농도로 개체수를 보정하여 NA 배지에 도말한 뒤 배지에 6 ㎜ 직경의 웰을 뚫었다. 상기 웰에 2배씩 연속 희석한 DS660 균주 배양 상층액 20 ㎕를 처리하고, 24시간 동안 배양하였다. 24시간 후 투명대가 나타난 희석배수를 통해 항균 활성을 평가하였으며 활성 단위 AU/㎖는 희석배수x50으로 표현하였다.The strain DS660, which was predominantly in LB liquid medium (30 ° C, 160 rpm), was inoculated with fresh liquid medium at a concentration of 2 × 10 8 CFU / ml and cultured for 48 hours. After the culture was completed, the culture was centrifuged at 12,000 g for 20 minutes at 4 ° C, and the culture supernatant was collected. The culture supernatants collected were successively diluted 2-fold with PBS and used for the experiment. S. aureus used as an indicator strain separately from DS660 strain was plated on NA medium at a concentration of 2 x 10 8 CFU / ml, and wells of 6 mm diameter were punched in the medium. 20 [mu] l of the culture supernatant of DS660 strain, which was serially diluted 2-fold, was treated in the wells and cultured for 24 hours. After 24 hours, the antimicrobial activity was evaluated by dilution drainage showing the zona pellucida. The activity unit AU / ml was expressed as dilution factor x50.

그 결과, 도 3에 나타난 것과 같이 배양 시간 의존적으로 DS660 균주의 항균 활성은 증가하였으며 배양 1일차에 최대활성인 5333±923 AU/㎖을 나타내었다. 도 3의 그래프에서 ▲는 OD600에서 측정한 흡광도를 의미하며, □는 항균 활성 측정 결과를 의미한다.As a result, as shown in FIG. 3, the antimicrobial activity of the strain DS660 was increased depending on the culture time, and the maximum activity was 5333 ± 923 AU / ml on the first day of culture. In the graph of FIG. 3,? Represents the absorbance measured at OD 600 , and? Represents the results of the antimicrobial activity measurement.

본 발명의 DS660 균주와 비특허문헌 3에 개시된 Brevibacterium linens 균주의 항균 활성을 비교하면, 상기 B. linens 균주는 최대 항균 활성이 400 AU/㎖이고, DS660 균주는 5333±923 AU/㎖이기 때문에 DS660 균주의 항균 활성이 현저히 우수한 것을 알 수 있다.Comparing the antimicrobial activity of the strain DS660 of the present invention and the strain Brevibacterium linens disclosed in the non-patent document 3, the maximum antimicrobial activity of the B. linens strain was 400 AU / ml, and the strain DS660 was 5333 ± 923 AU / The antimicrobial activity of the strain is remarkably excellent.

실시예Example 3: 생물 계면활성제 확인 3: Identification of biological surfactants

3-1: 오일 3-1: Oil 스프레딩Spreading 테스트(oil spreading test)를 이용한 계면활성제 생산 여부 확인 Confirmation of surfactant production using oil spreading test

DS660 균주를 2 내지 3일 동안 배양한 후 원심분리(3,400 rpm, 40분, 4℃)하여 배양 상층액을 회수하였다. 유리 페트리 디쉬(petri dish)에 증류수 50 ㎖를 담은 후 크루드 오일(crude oil) 20 ㎕를 떨어뜨리고, DS660 균주의 배양 상층액 10 ㎕를 가한 뒤 생기는 투명대의 크기를 측정하였다.The DS660 strain was cultured for 2 to 3 days and centrifuged (3,400 rpm, 40 minutes, 4 ° C) to recover the culture supernatant. In a glass petri dish, 50 ml of distilled water was added, 20 ㎕ of crude oil was dropped, and 10 쨉 l of the culture supernatant of the strain DS660 was added.

그 결과, 도 4에 나타난 것과 같이 배양 상층액의 첨가에 의하여 투명대가 형성되는 것을 확인할 수 있었으며, 이를 통하여 DS660 균주가 계면활성제를 생산하는 것을 알 수 있었다. As a result, it was confirmed that a zona pellucida was formed by the addition of the culture supernatant as shown in FIG. 4, and it was found that the DS660 strain produced a surfactant.

또한, 하기 표 3에 개시한 것과 같이 DS660 균주 배양 상층액은 5.1 cm의 직경으로 크루드 오일을 퍼트렸으며, 이를 토대로 표면장력을 측정한 결과 증류수의 표면장력인 75 mN/m를 32.3 mN/m로 현저하게 낮추는 효과가 있는 것을 확인하였다.In addition, as shown in Table 3 below, the culture supernatant of DS660 strain was spread on crude oil with a diameter of 5.1 cm. As a result of measurement of the surface tension thereof, the surface tension of distilled water was 75 mN / m to 32.3 mN / m As shown in Fig.

Figure pat00003
Figure pat00003

비특허문헌 4에 개시된 미생물과 DS660 균주를 비교하면, 비특허문헌 4에 개시된 미생물은 최대 3 ㎝ 직경으로 크루드 오일을 퍼뜨렸으나 DS660 균주는 5 ㎝ 이상의 직경으로 오일을 퍼뜨리기 때문에 계면활성제 생산이 현저히 우수한 것을 알 수 있으며, 표면장력을 감소시키는 효능 또한 우수한 것을 알 수 있다.When comparing the microorganism disclosed in Non-Patent Document 4 with the strain DS660, the microorganism disclosed in Non-Patent Document 4 spreads crude oil at a maximum diameter of 3 cm. However, because the DS660 strain spreads oil at a diameter of 5 cm or more, And the effect of reducing the surface tension is also excellent.

3-2: 생물 계면활성제 추출 및 분석3-2: Biological Surfactant Extraction and Analysis

DS660 균주를 2 내지 3일 동안 배양한 후 원심분리(3,400 rpm, 40분, 4℃)하여 배양 상층액을 회수하였다. 회수한 배양 상층액을 2 M 염산(HCl)을 이용하여 pH2로 적정하고, 동량의 에틸 아세테이트(ethyl acetate)를 첨가한 후 30분 동안 진탕(shaking; 300 rpm, 30분)하였다. 진탕이 끝난 후 상층액을 다시 원심분리(3,400 rpm, 1분, 4℃)하여 에틸 아세테이트만 회수하고, 회수한 용액을 감압 및 증발시킨 후, 남은 시료를 소량의 에틸 아세테이트로 용해시켜 계면활성제 시료를 수득하였다.The DS660 strain was cultured for 2 to 3 days and centrifuged (3,400 rpm, 40 minutes, 4 ° C) to recover the culture supernatant. The recovered culture supernatant was titrated to pH 2 with 2 M hydrochloric acid (HCl), and the same amount of ethyl acetate was added thereto, followed by shaking (300 rpm, 30 minutes) for 30 minutes. After shaking, the supernatant was centrifuged again (3,400 rpm, 1 min, 4 ° C) to recover only ethyl acetate. The recovered solution was reduced in pressure and evaporated, and the remaining sample was dissolved in a small amount of ethyl acetate to obtain a surfactant sample ≪ / RTI >

배지 추출물을 대조군으로 하여, 수득한 계면활성제 시료를 박층 크로마토그래피(Thin Layer Chromatography, TLC)로 분석하였다. 구체적으로, TLC 플레이트(silicagel 60)에 계면활성제 시료를 점적하고, TLC 플레이트를 이동상 (chloroform/methanol/glacial acetic acid = 65:15:2)에 위치시킨 후 전개하였다. 전개가 끝난 후 UV를 이용하여 밴드를 관찰하였고, 1% 닌하이드린(ninhydrin), 브로모티몰 블루(bromothymol blue) 및 오르시놀(orcinol)로 염색하여 계면활성제의 성분을 조사하였다.As a control, the obtained extracts of the medium were analyzed by thin layer chromatography (TLC). Specifically, a surfactant sample was loaded onto a TLC plate (silicagel 60) and the TLC plate was placed in a mobile phase (chloroform / methanol / glacial acetic acid = 65: 15: 2) and developed. After the development, the band was observed using UV and the components of the surfactant were examined by staining with 1% ninhydrin, bromothymol blue and orcinol.

그 결과, 도 5에 나타난 것과 같이 DS660 균주가 생산하는 계면활성제는 오르시놀에 의해 염색이 되는 것을 확인하여 당지질(glycolipid) 구조를 가지는 것을 알 수 있었다.As a result, as shown in FIG. 5, it was confirmed that the surfactant produced by the strain DS660 had a glycolipid structure by confirming staining by orcinol.

3-3: DS660 배양 시간에 따른 배양 3-3: Culture according to DS660 culture time 상층액의Supernatant 표면장력 측정 Surface tension measurement

DS660 균주를 2 내지 3일 동안 배양한 후 OD600을 1로 보정하여 새로운 배지에 10% 농도로 접종하고, 24시간을 주기로 배양액을 회수한 후 원심분리(3,400 rpm, 40분)하여 배양 상층액을 수득하였다. 수득한 배양 상층액의 표면장력은 Du Nouy Ring method를 이용하는 표면장력 측정기(조선 계측기, 대한민국)로 측정하였다.After incubation for 2 to 3 days the DS660 strains inoculated at 10% concentration in fresh medium to correct the OD 600 of 1, and after recovering the culture period for 24 hours and centrifuged (3,400 rpm, 40 minutes) the culture supernatant ≪ / RTI > The surface tension of the obtained culture supernatant was measured by a surface tension meter (Shipbuilding instrument, Korea) using the Du Nouy Ring method.

그 결과, 도 6에 나타난 것과 같이 DS660 균주는 배양 시간에 관계없이 거의 일정한 수준의 표면장력을 유지하는 것을 알 수 있었다.As a result, as shown in FIG. 6, it was found that the DS660 strain maintained almost constant surface tension regardless of the incubation time.

3-4: 임계 미셀 농도(Critical Micelle Concentration, 3-4: Critical Micelle Concentration, CMCCMC )측정 )Measure

DS660 균주를 2 내지 3일 동안 LB 액체배지에서 배양한 후 원심분리(3,400 rpm, 40분, 4℃)하여 배양 상층액을 회수하였다. 유리 페트리 디쉬에 증류수 50 ㎖를 담은 후 크루드 오일(crude oil) 20 ㎕를 떨어뜨리고, 회수한 배양 상층액의 첨가 정도를 변화시키면서 표면장력의 변화를 측정하였다.The DS660 strain was cultured in LB liquid medium for 2 to 3 days and centrifuged (3,400 rpm, 40 minutes, 4 ° C) to recover the culture supernatant. After transferring 50 ml of distilled water into the glass petri dish, 20 ㎕ of crude oil was dropped, and the change of the surface tension was measured while changing the degree of addition of the recovered culture supernatant.

그 결과, 도 7에 나타난 것과 같이 DS660 균주의 CMC는 1 내지 2%인 것을 확인할 수 있었다.As a result, it was confirmed that CMC of DS660 strain was 1 to 2% as shown in Fig.

실시예Example 4:  4: 시데로포어Sideropore (( siderophoresiderophore ) 확인) Confirm

4-1: DS660 균주의 4-1: expression of DS660 strain 시데로포어Sideropore 생산 여부 확인 Confirm production

CAS 한천 배지(60 ㎎ CAS(Chrome Azurol S), 1 mM FeCl3, 10 mM HCl 10 ㎖, 72.9 ㎎ HDTAB, 16 g agar, 30.2 g pipes 및 pH 6.8) 플레이트를 제작하고, 6 ㎜ 직경의 웰을 뚫었다. 웰에 DS660 균주 배양 상층액 70 ㎕를 첨가하고, 플레이트를 30℃ 암조건에서 48시간 동안 배양하여 웰 주변에 노란색 환의 형성 유무로 시데로포어 생산 여부를 확인하였다.Plates were prepared with CAS agar medium (60 ㎎ CAS (Chrome Azurol S), 1 mM FeCl 3 , 10 mM HCl 10 ml, 72.9 mg HDTAB, 16 g agar, 30.2 g pipes and pH 6.8) Pierced. 70 [mu] l of the culture supernatant of the strain DS660 was added to the wells, and the plate was cultured at 30 [deg.] C for 48 hours to confirm the production of cytidophores with or without formation of yellow rings around the wells.

그 결과, 도 8에 나타난 것과 같이 DS660 균주를 첨가한 웰 주변에 노란색 환이 크게 형성된 것을 확인하여 DS660 균주가 시데로포어를 생산하는 것을 알 수 있었다.As a result, as shown in FIG. 8, it was confirmed that a yellow circle was formed largely around the well to which the DS660 strain was added, indicating that the DS660 strain produced sideropore.

4-2: DS660 균주의 4-2: expression of DS660 strain 시데로포어Sideropore 생산량 확인 Check production

DS660 균주를 2 내지 3일 동안 배양한 후 OD600을 1로 보정하여 새로운 배지에 10% 농도로 접종하고, 24시간을 주기로 15 ㎖의 배양액을 회수한 후 원심분리(3,400 rpm, 40분)하여 배양 상층액을 수득하였다. 수득한 배양 상층액의 pH를 1 M 염산을 이용하여 pH 2.9로 보정하고, 배양 상층액과 동량의 에틸 아세테이트를 첨가하여 300 rpm에서 30분 동안 extraction shaker(Recipro shaker RS-1, Jeio Tech)를 이용하여 1회 추출하였다. 추출한 용액을 원심분리(3400xg, 15분, 4℃)하여 상층의 에틸 아세테이트층 5 ㎖과 Hathway 반응 용액(0.1 M FeCl3 1 ㎖, 0.1 M potassium ferricyanide 1 ㎖, 0.1 L 증류수) 5 ㎖을 30분 동안 반응시켰다. 반응이 끝난 후 분광광도계를 이용하여 OD700에서 흡광도를 측정하였으며, 표준곡선은 디히드록시 벤조익산(dihydroxy benzoic acid)을 이용하여 정량하였다.After the second incubation for 1-3 days DS660 strains were inoculated at 10% concentration in fresh medium to correct the OD 600 of 1, and then recovering the culture medium of 15 ㎖-clock cycle by centrifugation (3,400 rpm, 40 minutes) A culture supernatant was obtained. The pH of the obtained culture supernatant was adjusted to pH 2.9 using 1 M hydrochloric acid, and an extraction shaker (Recipro shaker RS-1, Jeio Tech) was added for 30 minutes at 300 rpm by adding the same amount of ethyl acetate as the culture supernatant And extracted once. 5 ml of the ethyl acetate layer of the upper layer and 5 ml of Hathway reaction solution (1 ml of 0.1 M FeCl 3, 1 ml of 0.1 M potassium ferricyanide, 0.1 L of distilled water) were centrifuged at 3400 xg for 15 minutes at 4 ° C for 30 minutes Lt; / RTI > After the reaction, the absorbance was measured at OD 700 using a spectrophotometer, and the standard curve was quantified using dihydroxy benzoic acid.

그 결과, 도 9에 나타난 것과 같이 DS660 균주는 배양 2일차에 57±8 umol/㎖ 농도의 시데로포어를 생산하여 시데로포어 생산량이 우수한 것을 확인할 수 있었다. 도 9에서 X는 OD600에서 측정한 흡광도를 나타내고, ◆는 DS660 균주가 생산하는 시데로포어의 생산량을 의미한다.As a result, as shown in FIG. 9, the DS660 strain produced a sideropore at a concentration of 57 8 umol / ml on the second day of culture, indicating that sideropore production was excellent. In Figure 9, X represents the absorbance measured at OD 600 , and ◆ represents the yield of the siderophores produced by the strain DS660.

비특허문헌 5에 개시된 Pseudomonas fluorescens와 비교하면 P. fluorescens의 시데로포어 최대 생산량은 13 umol/㎖로 DS660 균주의 시데로포어 생산량이 약 4.3배 높은 것을 알 수 있다.Non-Patent Document 5: Pseudomonas In comparison with P. fluorescens pores up to the production of side fluorescens it can be seen that the high spore production by about 4.3 times the side of the strain DS660 with 13 umol / ㎖.

실시예Example 5: 베타-1,3- 5: beta-1,3- 글루카나제Glucanase (β-1.3-(β-1,3- glucanaseglucanase ) 확인) Confirm

5-1: 베타-1,3-5-1: Beta- 글루카나제Glucanase 생산 여부 확인 Confirm production

Peptone-bouillon-yeast agar(1% peptone, 1% beef extract, 2% baker's yeast, 2% agar 및 pH 7) 플레이트에 DS660 균주를 획선으로 도말하여 배양하고, 투명대(clear zon)의 형성 여부로 효소 생성 여부를 판단하였다.The strain DS660 was streaked on a plate with Peptone-bouillon-yeast agar (1% peptone, 1% beef extract, 2% baker's yeast, 2% agar and pH 7) It was judged whether or not it was generated.

그 결과, 도 10에 나타난 것과 같이 DS660 균주를 도말한 곳에는 투명대가 형성된 것을 확인할 수 있었으며, 이를 통하여 DS660 균주가 베타-1,3-글루카나제를 생산하는 것을 알 수 있었다.As a result, as shown in FIG. 10, it was confirmed that a zona pellucida was formed in the place where the strain DS660 was stained, and it was found that the DS660 strain produced beta -1,3-glucanase.

5-2:5-2: 베타-1,3-Beta-1,3- 글루카나제Glucanase 활성 확인 Verify Active

DS660 균주를 배지(5 g glucose, 2 g peptone, 2 g laminarin, 1 g K2HPO4, 0.5 g MgSO4 및 0.5 g NaCl)에 2 내지 3일 동안 배양한 후 OD600을 1로 보정하여 새로운 배지에 10% 농도로 접종하였다. 24시간 주기로 배양액 1 ㎖를 수거하여 원심분리(12000 g, 20분, 4℃)하고, 배양 상층액만 취한 후 상층액 0.25 ㎖, 0.1 M 인산 버퍼(pH 5.5) 500 ㎕ 및 0.2% 라미나린(laminarin) 500 ㎕을 각각 혼합하였다. 혼합액을 2시간 동안 40℃ 항온수조에서 반응시키고, 반응액 100 ㎕를 DNS(3,5-dinitrosalicylic acid 10 g/L, sodium hydroxide 0.3 M 및 rochelle salt 250 g/L) 용액 200 ㎕와 90에서 10분 동안 추가로 반응시킨 후 냉각시켜 반응을 종료하였다. 반응이 끝난 후 반응액의 흡광도를 575 ㎚에서 측정하고, 대조군으로 PBS를 이용하여 흡광도 차이를 조사하고, 포도당(glucose)으로 표준곡선을 작성하여 정량하였다. 1 유닛(unit)은 1분 동안 1 ㎛의 N-아세틸-D-글루코사민(N-acetyl-D-glucosamine, NAG)을 생성하는 효소량으로 정의하였고, 브래드포드(Bradford) 방법으로 시료의 단백질을 정량하였으며, DNS 결과와 단백질 정량 결과를 조합하여 효소 생성량을 조사하였다. 표준곡선은 소혈청 알부민(bovin serum albumin; BSA)을 이용하였다.During the DS660 strains medium (5 g glucose, 2 g peptone , 2 g laminarin, 1 g K 2 HPO 4, 0.5 g MgSO 4 and 0.5 g NaCl) 2 to 3 days after culturing to correct the OD 600 of 1 new The medium was inoculated at 10% concentration. The culture supernatant was collected by centrifugation (12000 g, 20 minutes, 4 ° C) at a 24-hour cycle, and the culture supernatant was collected. Then, the supernatant was diluted with 0.25 ml of 0.1 M phosphate buffer (pH 5.5) laminarin) were mixed, respectively. The reaction mixture was reacted in a constant temperature water bath at 40 ° C for 2 hours and 200 μl of a solution of DNS (3,5-dinitrosalicylic acid 10 g / L, sodium hydroxide 0.3 M and rochelle salt 250 g / L) Lt; / RTI > for one minute and then cooled to terminate the reaction. After the reaction, the absorbance of the reaction solution was measured at 575 nm, the absorbance difference was examined using PBS as a control group, and a standard curve was prepared using glucose as a control. One unit was defined as the amount of enzyme that produced 1 占 퐉 N-acetyl-D-glucosamine (NAG) for 1 minute, and the protein of the sample was quantified by the Bradford method The enzyme production was investigated by combining the results of DNS and protein quantification. The standard curve was bovine serum albumin (BSA).

그 결과, 도 11에 나타난 것과 같이 DS660 균주는 배양 24시간에 169.2±9.9 nmol/min/mg protein의 효소를 생산하고, 배양 시간이 증가함에 따라 효소 활성이 감소하는 것을 확인할 수 있었다.As a result, as shown in FIG. 11, the strain DS660 produced 169.2 ± 9.9 nmol / min / mg of protein at 24 hours of culture and decreased enzyme activity with increasing incubation time.

비특허문헌 5에 개시된 P. fluorescens와 비교하면, P. fluorescens의 베타-1,3-글루카나제 생산량은 150 nmol/min/mg protein로 DS660 균주의 베타-1,3-글루카나제 생산량이 약 20% 정도 높은 것을 알 수 있다.Compared with P. fluorescens disclosed in Non-Patent Document 5, the yield of β-1,3-glucanase of P. fluorescens was 150 nmol / min / mg protein and the yield of β-1,3- Which is about 20% higher.

실시예Example 6: 세균 세포벽 용해효소 확인 6: Identification of bacterial cell wall lytic enzyme

3종의 피부 상재균-B. subtilis, P. aeruginosa 및 E. coli-의 배양액을 원심분리(3,400 rpm, 15분, 4℃)하여 세균 펠렛(pellet)을 수거하였다. 수거한 세균 펠렛을 121℃에서 15분 동안 멸균하고 건조시킨 후, 0.2% 농도로 첨가하여 NA 배지를 제작하였다. NA 배지에 DS660 균주를 획선으로 도말하여 30에서 배양한 후 콜로니 주변에 생긴 투명대를 토대로 세균 세포벽 용해효소 생산 여부를 판단하였다.Bacterial pellets were harvested by centrifugation (3,400 rpm, 15 min, 4 ° C) of the cultures of three subcutaneous skin subfamilies - B. subtilis , P. aeruginosa and E. coli . The collected bacterial pellets were sterilized at 121 DEG C for 15 minutes, dried and added at a concentration of 0.2% to prepare NA medium. The strain DS660 was streaked on NA medium and cultured at 30, and the production of bacterial cell wall lytic enzyme was judged based on the zona around the colony.

그 결과, 도 12에 나타난 것과 같이 DS660 균주 주변에 투명대가 현저하게 형성된 것을 확인하여 DS660 균주가 세포벽 용해효소를 생산하는 것을 알 수 있었다. 도 12에서 (a)는 B. subtilis, (b)는 P. aeruginosa이고, (c)는 E. coli이다.As a result, it was confirmed that a zona pellucida was formed remarkably around the strain DS660 as shown in Fig. 12, and it was found that the strain DS660 produced a cell wall lytic enzyme. 12 (a) is B. subtilis , (b) is P. aeruginosa , and (c) is E. coli .

실시예Example 7: DS660 균주의 특성 확인 7: Characterization of strain DS660

7-1: 최적 7-1: Optimal 탄소원Carbon source 확인 Confirm

효모 추출물(Yeast extract)이 첨가된 배지(10 g/L)에 5종류의 탄소원(sorbitol, D-mannitol, glycerol, sucrose 및 glucose)을 20 g/L로 각각 첨가하여 배지를 제작하고, DS660 균주를 선배양하였다. 선배양한 배양액을 OD600에서 1로 보정하여 새로운 배지에 접종하고, 48시간 뒤에 배양액을 원심분리(12000 g, 15분, 4℃)하여 배양 상층액을 수거하였다. 지시 균주인 스태필로코커스 아우레우스를 2x108 CFU/㎖로 도말한 NA 배지에 10 ㎚ 직경의 웰을 뚫고, DS660 균주 배양 상층액을 첨가하여 30℃에서 24시간 동안 배양한 후, 형성된 투명대의 크기를 측정하였다.Five kinds of carbon sources (sorbitol, D-mannitol, glycerol, sucrose and glucose) were added at 20 g / L to a medium containing yeast extract (10 g / L) Respectively. Senior amount of the culture liquid to the calibrated to 1 OD 600 inoculated in fresh medium, and the culture broth was centrifuged after 48 hours (12000 g, 15 bun, 4 ℃) was collected culture supernatant. Wells having a diameter of 10 nm were punched into NA medium spiked with 2 x 10 8 CFU / ml of Staphylococcus aureus as an indicator strain, and the culture supernatant of strain DS660 was added and cultured at 30 ° C for 24 hours. The size was measured.

그 결과, 도 13에 나타난 것과 같이 탄소원으로 수크로오스(sucrose)를 이용하였을 때 항균 활성이 가장 우수한 것을 확인할 수 있었다.As a result, when sucrose was used as a carbon source as shown in FIG. 13, it was confirmed that the antimicrobial activity was the most excellent.

7-2: 최적 질소원 확인7-2: Identification of the optimum nitrogen source

상기 실시예 7-1과 동일한 방법을 이용하여 DS660 균주의 최적 질소원을 확인하였다. 다만, 포도당이 첨가된 배지(10 g/L)에 질소원 7가지(ammonium phosphate, tryptone, urea, Peptone, yeast extract, malt extract, ammonium sulfate)를 각각 10 g/L로 첨가한 배지를 이용하였다.The optimum nitrogen source of the strain DS660 was confirmed using the same method as in Example 7-1. However, the medium supplemented with glucose (10 g / L) and 7 kinds of nitrogen sources (ammonium phosphate, tryptone, urea, peptone, yeast extract, malt extract and ammonium sulfate) was added at 10 g / L.

그 결과, 도 13에 나타난 것과 같이 질소원으로 효모 추출물을 이용하였을 때 형성된 투명대의 크기가 가장 큰 것을 확인할 수 있었다.As a result, it was confirmed that the size of the zona pellucida formed when the yeast extract was used as the nitrogen source as shown in Fig. 13 was the largest.

7-3: 온도 안정성 확인 7-3: Confirmation of temperature stability

DS660 균주를 48시간 동안 배양한 후 원심분리(3,400 rpm, 40분, 4℃)하여 배양 상층액을 회수하고, 회수한 배양 상층액에서 2 ㎖씩 취하여 다양한 온도(-22, 4, 25, 50, 60, 70, 80, 90, 100℃에서 30분 및 121℃에서 15분)에서 방치하였다. 이후 스태필로코커스 아우레우스를 2x108 CFU/㎖로 도말한 NA 배지에 10 ㎚ 직경의 웰을 뚫고, DS660 균주 배양 상층액을 첨가하여 30℃에서 24시간 동안 배양한 후, 형성된 투명대의 크기를 측정하였다.The culture supernatant was recovered by centrifugation (3,400 rpm, 40 min, 4 ° C) after culturing the strain of strain DS660 for 48 hours. 2 ml of the culture supernatant was taken at various temperatures (-22, , 60, 70, 80, 90, 30 minutes at 100 占 폚 and 15 minutes at 121 占 폚). Then, wells having a diameter of 10 nm were drilled in NA medium spiked with Staphylococcus aureus at 2 x 10 8 CFU / ml, and the culture supernatant of strain DS660 was added thereto. After culturing at 30 ° C for 24 hours, Respectively.

그 결과, 표 4에 나타난 것과 같이 DS660 균주가 생산하는 항균 물질은 -22 내지 70℃의 온도 범위에서 항균 활성이 남아 있는 것을 확인할 수 있었다.As a result, as shown in Table 4, it was confirmed that the antimicrobial substance produced by the strain DS660 remained in the temperature range of -22 to 70 캜.

Figure pat00004
Figure pat00004

7-4: pH에 대한 안정성 확인7-4: Confirm stability against pH

DS660 균주를 48시간 동안 배양한 후 원심분리(3,400 rpm, 40분, 4℃)하여 배양 상층액을 회수하고, 회수한 배양 상층액에서 2 ㎖씩 취하여 2 M 염산 및 수산화나트륨(NaOH)을 이용하여 pH 3 내지 12 범위로 적정한 뒤 30분 동안 방치하였다. 이후 적정한 배양 상층액의 pH를 기존의 배양 상층액이 가지고 있던 pH로 재적정하고, 스태필로코커스 아우레우스(2x108 CFU/㎖)를 도말한 NA 배지에 10 ㎚ 직경의 웰을 뚫은 후, 상기 재적정한 배양 상층액을 첨가하여 30℃에서 24시간 동안 배양한 후, 형성된 투명대의 크기를 측정하였다.The culture supernatant was recovered by centrifugation (3,400 rpm, 40 minutes, 4 ° C) after culturing the strain of strain DS660 for 48 hours. Take 2 ml of the supernatant from the culture supernatant and use 2 M hydrochloric acid and sodium hydroxide And the pH was adjusted to the range of 3 to 12, followed by allowing to stand for 30 minutes. Thereafter, the pH of the culture supernatant was adjusted to the pH of the existing culture supernatant, and wells having a diameter of 10 nm were punched in NA medium stained with Staphylococcus aureus (2 x 10 8 CFU / ml) The re-titrated culture supernatant was added and cultured at 30 占 폚 for 24 hours, and the size of the formed zona pellucida was measured.

그 결과, 표 5에 나타난 것과 같이 DS660 균주가 생산하는 항균 물질은 pH 3 내지 12의 넓은 범위에서 항균 활성을 유지하는 것을 확인할 수 있었다.As a result, as shown in Table 5, it was confirmed that the antimicrobial substance produced by the strain DS660 maintained the antimicrobial activity over a wide range of pH 3 to 12. [

Figure pat00005
Figure pat00005

비특허문헌 6에 개시된 Lactobacillus sakei LSJ618 균주와 DS660 균주의 항균 활성을 비교하면, LSJ618 균주는 pH 10 내지 12 범위에서 항균 활성을 잃어버리지만 DS660 균주는 어느 pH 범위에서도 최소 50% 이상의 항균 활성을 유지하는 것을 알 수 있으며, DS660 균주가 생산하는 항균 물질이 안정한 것을 알 수 있다. Lactobacillus sakei disclosed in Non-Patent Document 6 Comparing the antimicrobial activity of LSJ618 strain and DS660 strain, LSJ618 strain lost its antimicrobial activity in the range of pH 10 to 12. However, DS660 strain maintained antimicrobial activity at least 50% in any pH range. DS660 strain It can be seen that the antimicrobial substance produced by the present invention is stable.

7-5: 계면활성제에 대한 안정성 확인7-5: Confirm stability for surfactant

DS660 균주를 48시간 동안 배양한 후 원심분리(3,400 rpm, 40분, 4℃)하여 배양 상층액을 회수하고, 회수한 배양 상층액에서 2 ㎖씩 취하여 1% 농도로 계면활성제(urea, tween 20, tween 80 및 triton X-100)를 각각 처리하였다. 계면활성제 처리가 끝난 후 스태필로코커스 아우레우스(2x108 CFU/㎖)를 도말한 NA 배지에 10 ㎚ 직경의 웰을 뚫은 후, 배양 상층액을 첨가하여 30℃에서 24시간 동안 배양한 후, 형성된 투명대의 크기를 측정하였다.The culture supernatant was recovered by centrifugation (3,400 rpm, 40 min, 4 ° C) after culturing the strain of DS660 for 48 hours. Take 2 ml of the supernatant from the recovered culture supernatant and add the surfactant (urea, tween 20 , tween 80 and triton X-100), respectively. After the surfactant treatment, wells with a diameter of 10 nm were punched into NA medium spiked with Staphylococcus aureus (2 x 10 8 CFU / ml), and then the culture supernatant was added and cultured at 30 ° C for 24 hours, The size of the formed transparent bar was measured.

그 결과, 표 6에 나타난 것과 같이 계면활성제의 첨가 유무와 관계없이 DS660 균주가 생산하는 항균 물질은 항균 활성을 유지하는 것을 확인할 수 있었다.As a result, as shown in Table 6, it was confirmed that the antimicrobial substance produced by the strain DS660 maintains the antimicrobial activity regardless of whether or not the surfactant is added.

Figure pat00006
Figure pat00006

이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

한국생명공학연구원Korea Biotechnology Research Institute KCTC18521PKCTC18521P 2016120220161202

<110> Kangwon University-Industry Cooperation Foundation <120> BACILLUS SUBTILIS DS660 STRAIN HAVING ANTIMICROBIAL ACTIVITY AND USES THEREOF <130> PN160347 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1553 <212> DNA <213> Bacillus subtilis <400> 1 gtacaaagcc cccgttagag tttgaatcat ggctcaggac gaacgctggc ggcgtgccta 60 atacatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt 120 gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat 180 accggatggt tgtttgaacc gcatggttca gacataaaag gtggcttcgg ctaccactta 240 cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg 300 cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 360 cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 420 tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgccgttc 480 aaatagggcg gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 540 gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca 600 ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 660 tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag 720 agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag 780 cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 840 tgctaagtgt tagggggttt ccgcctcctt agtgctgcag ctaacgcatt aagcactccg 900 cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg 960 gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 1020 tgacaatcct agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg 1080 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct 1140 tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg 1200 tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1260 acagaacaaa gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt 1320 cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc 1380 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt 1440 gtaacacccg aagtcggtga ggtaaccttt taggagccag ccgccgaagg tgggacagat 1500 gattggggtg aagtcgtaac aggtaaaccg taaatttact tcccttcctc cat 1553 <110> Kangwon University-Industry Cooperation Foundation <120> BACILLUS SUBTILIS DS660 STRAIN HAVING ANTIMICROBIAL ACTIVITY AND          USES THEREOF <130> PN160347 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1553 <212> DNA <213> Bacillus subtilis <400> 1 gtacaaagcc cccgttagag tttgaatcat ggctcaggac gaacgctggc ggcgtgccta 60 atacatgcaa gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt 120 gagtaacacg tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat 180 accggatggt tgtttgaacc gcatggttc gacataaaag gtggcttcgg ctaccactta 240 cagatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg 300 cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc cagactccta 360 cgggaggcag cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg 420 tgagtgatga aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgccgttc 480 aaatagggcg gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca 540 gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca 600 ggcggtttct taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac 660 tggggaactt gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag 720 agatgtggag gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag 780 cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag 840 tgctaagtgt tagggggttt ccgcctcctt agtgctgcag ctaacgcatt aagcactccg 900 cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg 960 gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 1020 tgacaatcct agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg 1080 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct 1140 tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg 1200 tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1260 acagaacaaa gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt 1320 cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc 1380 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt 1440 gtaacacccg aagtcggtga ggtaaccttt taggagccag ccgccgaagg tgggacagat 1500 gattggggtg aagtcgtaac aggtaaaccg taaatttact tcccttcctc cat 1553

Claims (8)

항균 활성을 가지는 바실러스 서브틸리스 DS660(기탁번호 KCTC18521P) 균주.
Bacillus subtilis DS660 (Accession No. KCTC18521P) having antibacterial activity.
제1항에 있어서, 상기 바실러스 서브틸리스 DS660 균주는 바실러스 속(genus Bacillus), 스태필로코커스 속(genus Staphylococcus), 아스퍼질러스 속(genus Aspergillus), 슈도모나스 속(genus Pseudomonas), 에스케리치아 속(genus Escherichia) 및 캔디다 속(genus Candida)으로 이루어진 군에서 선택되는 미생물에 대하여 항균 활성을 갖는 것을 특징으로 하는 균주.
The method according to claim 1, wherein the Bacillus subtilis DS660 strain is selected from the group consisting of genus Bacillus , genus Staphylococcus , genus Aspergillus , genus Pseudomonas , wherein the strain has an antimicrobial activity against a microorganism selected from the group consisting of genus Escherichia and genus Candida .
제1항에 있어서, 상기 바실러스 서브틸리스 DS660 균주는 계면활성제를 생산하는 것을 특징으로 하는 균주.
The strain according to claim 1, wherein the Bacillus subtilis DS660 strain produces a surfactant.
제1항에 있어서, 상기 바실러스 서브틸리스 DS660 균주는 시데로포어(siderophore)를 생산하는 것을 특징으로 하는 균주.
The strain according to claim 1, wherein the strain Bacillus subtilis DS660 produces siderophore.
제1항에 있어서, 상기 바실러스 서브틸리스 DS660 균주는 베타-1,3-글루카나제(β-1.3-glucanase)를 생산하는 것을 특징으로 하는 균주.
The strain according to claim 1, wherein the strain Bacillus subtilis DS660 produces beta-1,3-glucanase.
제1항에 있어서, 상기 바실러스 서브틸리스 DS660 균주는 세균 세포벽 용해효소를 생산하는 것을 특징으로 하는 균주.
The strain according to claim 1, wherein the strain Bacillus subtilis DS660 produces a bacterial cell wall lytic enzyme.
바실러스 서브틸리스 DS660(기탁번호 KCTC18521P) 균주, 상기 균주의 배양물, 상기 균주 또는 배양액의 농축물 및 이들의 건조물로 이루어진 군에서 선택된 하나 이상을 포함하는 항균용 조성물.
A strain of Bacillus subtilis DS660 (Accession No. KCTC18521P), a culture of the strain, a concentrate of the strain or the culture, and a dried product thereof.
바실러스 서브틸리스 DS660(기탁번호 KCTC18521P) 균주의 배양물, 배양액의 농축물 및 건조물로 이루어진 군에서 선택된 하나 이상을 포함하는 화장료 조성물.A culture of Bacillus subtilis DS660 (Accession No. KCTC18521P), a concentrate of the culture broth, and a dried product.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113181220A (en) * 2021-05-18 2021-07-30 郭云 Bacillus subtilis spray and preparation method thereof
CN116426403A (en) * 2022-11-04 2023-07-14 武汉悦呼吸科技有限公司 Bacillus subtilis with antibacterial and mite-inhibiting activities and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101190901B1 (en) 2010-06-14 2012-10-12 몽고식품 주식회사 Bacillus subtilis CSY191 strain and probiotics containing the strain
KR101594446B1 (en) 2013-04-29 2016-02-17 단국대학교 천안캠퍼스 산학협력단 Bacillus subtilis RX7 strain having antimicrobial activity against harmful microorganism and uses thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200927764A (en) * 2007-09-30 2009-07-01 Umo Inc Co Ltd Peptide having antimicrobial and/or biosurfactant property
SE535660C2 (en) * 2011-02-02 2012-10-30 Lifting or carrying device intended for recreational craft
CN102550607B (en) * 2011-11-01 2014-05-28 江苏省农业科学院 Bacillus subtilis PTS-394 for preventing and controlling soil-borne diseases of solanaceous fruit vegetables and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101190901B1 (en) 2010-06-14 2012-10-12 몽고식품 주식회사 Bacillus subtilis CSY191 strain and probiotics containing the strain
KR101594446B1 (en) 2013-04-29 2016-02-17 단국대학교 천안캠퍼스 산학협력단 Bacillus subtilis RX7 strain having antimicrobial activity against harmful microorganism and uses thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
1. Novel antilisterial bacteriocin licheniocin 50.2 from Bacillus licheniformis VPS50.2 isolated from soil sample. Journal of Applied Microbiology 2013, 116: 502-510.
2. Bacteriocin (BAC-IB17): Screening, isolation and production from Bacillus subtilis KIBGE IB-17. Pakistan Journal Pharmaceutical Science 2012, 25: 195-201.
3. Characterization of an antibacterial peptide produced by Brevibacterium linens. Journal of Applied Microbiology 2002, 92: 63-70.
4. Comparison of methods to detect biosurfactant production by diverse microorganisms. Journal of Microbiological Methods 2004, 56: 339-347.
5. Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, Microbiological Research, 2004, 159: 73-81.
6. Characterization of a novel bacteriocin produced by Lactobacillus sakei LSJ618 isolated from traditional Chinese fermented radish. Food Control 2012, 23: 338-344.

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113181220A (en) * 2021-05-18 2021-07-30 郭云 Bacillus subtilis spray and preparation method thereof
CN116426403A (en) * 2022-11-04 2023-07-14 武汉悦呼吸科技有限公司 Bacillus subtilis with antibacterial and mite-inhibiting activities and application thereof
CN116426403B (en) * 2022-11-04 2023-10-03 武汉悦呼吸科技有限公司 Bacillus subtilis with antibacterial and mite-inhibiting activities and application thereof

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