KR102296289B1 - Lactobacillus gasseri strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof - Google Patents
Lactobacillus gasseri strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof Download PDFInfo
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- KR102296289B1 KR102296289B1 KR1020200189819A KR20200189819A KR102296289B1 KR 102296289 B1 KR102296289 B1 KR 102296289B1 KR 1020200189819 A KR1020200189819 A KR 1020200189819A KR 20200189819 A KR20200189819 A KR 20200189819A KR 102296289 B1 KR102296289 B1 KR 102296289B1
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Abstract
Description
신규한 미생물, 그의 파쇄물, 배양액, 배양액의 추출물, 소포체 및 이들의 항염증 및/또는 항균 용도에 관한 것이다.It relates to novel microorganisms, lysates thereof, cultures, extracts of cultures, endoplasmic reticulum and their anti-inflammatory and/or antibacterial uses.
마이크로바이옴(Microbiome)은 특정 환경에 존재하고 있는 미생물들과 이들의 유전정보 전체를 말하는 것으로, 단일 생명체의 유전정보 전체를 뜻하는 게놈 (Genome) 들의 집합체를 의미한다. 따라서 인체 마이크로바이옴(Human Microbiome)은 인간 몸체 안팎에 서식하고 있는 미생물들과 그들의 유전정보 전체를 의미한다. Microbiome refers to microorganisms that exist in a specific environment and their entire genetic information, and refers to a collection of genomes, which refers to the entire genetic information of a single organism. Therefore, the human microbiome refers to microorganisms living inside and outside the human body and their entire genetic information.
인간의 몸은 많은 미생물과 공생관계를 이루며 살아가며 특히 장내에는 미생물이 영양분을 섭취하고 체계적인 군집을 형성하기에 최적의 환경으로 가장 많은 미생물이 존재한다. 장내 미생물은 숙주가 지닌 효소만으로는 생성 할 수 없는 영양분을 공급하고 숙주의 대사 및 면역 체계와 깊은 연관을 지니는 한편 과민성대장증후군, 비만, 아토피, 우울증, 류마티스 관절염, 자폐 스펙트럼 장애, 치매 등 다양한 질병의 발생과 관련되어 있다고 보고되고 있다. The human body lives in a symbiotic relationship with many microorganisms, and in particular, the intestine contains the most microorganisms as an optimal environment for the microorganisms to take in nutrients and form a systematic community. Intestinal microorganisms provide nutrients that cannot be produced by the enzymes of the host alone and are closely related to the host's metabolism and immune system. reported to be related to the
최근에는 서구적인 식습관과 무분별한 항생제 사용으로 장내 미생물총(Microbiota)의 불균형이 일어나 장 건강이 악화되고 있으며, 장내미생물과 다양한 질병에 대한 연구로 인해 장내 미생물에 대한 중요성이 부각되고 관심이 대두되고 있다. Recently, due to Western eating habits and reckless use of antibiotics, the intestinal microbiota is imbalanced and intestinal health is deteriorating. .
한편 소포체는 세포가 생성하여 배출하는 20 내지 200 nm 정도의 나노사이즈의 물질로, 세포 간 이동이 자유롭다. 또한 소포체는 막지질, 막단백질, DNA나 RNA 등을 포함하고 이러한 유전물질들이 복합체로 작용하여 세포와 세포 사이에 독성 인자를 전달하고 염증과 면역반응 조절 등의 역할을 한다고 알려져 있다. 단세포 생물에서 다세포 생물에 이르기까지 세포 간 정보교환은 생명현상의 필수적인 과정이며, 최근에는 소포체가 세포 간 정보교환의 매개체로 인식되어 있어 소포를 응용하여 약물 운반체로 활용하는 방법들이 개발되고 있다. On the other hand, the endoplasmic reticulum is a nano-sized material of about 20 to 200 nm produced and discharged by cells, and is free to move between cells. In addition, it is known that the endoplasmic reticulum contains membrane lipids, membrane proteins, DNA or RNA, etc., and these genetic materials act as a complex to deliver toxic factors between cells and regulate inflammation and immune response. From unicellular organisms to multicellular organisms, information exchange between cells is an essential process of life phenomena, and recently, the endoplasmic reticulum is recognized as a medium for information exchange between cells, so methods for using the vesicle as a drug carrier have been developed.
이에, 인간의 장내 유래 신규 미생물, 및 이의 유래의 소포체를 이용한 질병의 개선, 예방, 또는 치료를 위한 물질의 개발이 필요한 실정이다. Accordingly, there is a need for the development of materials for improving, preventing, or treating diseases using novel microorganisms derived from the human gut and endoplasmic reticulum derived therefrom.
일 양상은 기탁번호 KCTC 14298BP로 기탁된, 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 가세리(Lactobacilus gasseri) 균주를 제공하는 것이다. One aspect is to provide a Lactobacillus gasseri ( Lactobacilus gasseri ) strain belonging to the Lactobacillus genus ( Lactobacillus sp. ) deposited with accession number KCTC 14298BP.
다른 양상은 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 또는 배양액을 제공하는 것이다. Another aspect is to provide an endoplasmic reticulum derived from the strain, a lysate of the strain, or a culture solution.
또 다른 양상은 상기 균주, 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 배양액, 또는 이들의 혼합물을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다. Another aspect is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
또 다른 양상은 상기 균주, 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 배양액, 또는 이들의 혼합물을 유효성분으로 포함하는 염증성 질환의 예방 또는 개선용 건강기능식품을 제공하는 것이다. Another aspect is to provide a health functional food for the prevention or improvement of inflammatory diseases comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
또 다른 양상은 상기 균주, 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 배양액, 또는 이들의 혼합물을 유효성분으로 포함하는 장 건강의 개선용 건강기능식품을 제공하는 것이다. Another aspect is to provide a health functional food for improving intestinal health comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
또 다른 양상은 상기 균주, 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 배양액, 또는 이들의 혼합물을 유효성분으로 포함하는 박테리아 감염증의 예방 또는 치료용 약학적 조성물을 제공하는 것이다. Another aspect is to provide a pharmaceutical composition for preventing or treating bacterial infection comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
또 다른 양상은 상기 균주, 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 배양액, 또는 이들의 혼합물을 유효성분으로 포함하는 박테리아 감염증의 예방 또는 개선용 건강기능식품을 제공하는 것이다. Another aspect is to provide a health functional food for preventing or improving bacterial infection comprising the strain, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or a mixture thereof as an active ingredient.
또 다른 양상은 상기 균주, 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 배양액, 또는 이들의 혼합물을 포함하는 화장료 조성물을 제공하는 것이다. Another aspect is to provide a cosmetic composition comprising the strain, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or a mixture thereof.
또 다른 양상은 상기 균주, 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 배양액, 또는 이들의 혼합물을 포함하는 항균용 피부 외용제 조성물을 제공하는 것이다. Another aspect is to provide an antibacterial composition for external application for skin comprising the strain, the endoplasmic reticulum derived from the strain, a lysate of the strain, a culture solution, or a mixture thereof.
일 양상은 락토바실러스 속(Lactobacillus sp.)에 속하는 락토바실러스 가세리(Lactobacilus gasseri) 균주를 제공한다. One aspect provides a Lactobacillus genus ( Lactobacillus sp. ) belonging to the Lactobacillus gasseri (Lactobacilus gasseri) strain.
상기 락토바실러스 가세리 균주는 기탁번호 KCTC 14298BP로 기탁된 균주일 수 있다. The Lactobacillus gasseri strain may be a strain deposited with accession number KCTC 14298BP.
상기 락토바실러스 가세리 균주는 서열번호 1의 16s rRNA를 포함하는 균주일 수 있다. The Lactobacillus gasseri strain may be a strain comprising 16s rRNA of SEQ ID NO: 1.
상기 균주는 항염증 및/또는 항균 활성을 갖는 것일 수 있다. The strain may have anti-inflammatory and/or antibacterial activity.
상기 균주는 염증 유발된 세포에서 산화 질소의 생성을 억제하거나, 염증성 사이토카인(예를 들면, TNF-α 또는 IL-6)의 발현을 억제하거나, 박테리아의 증식(예를 들면, C.difficile)을 억제하는 것일 수 있다. 또한, 상기 균주는 C.difficile에 의해 유도된 염증 인자, 예를 들면, 전염증성 사이토카인(예를 들면, TNF, 또는 CCL2)를 감소시키거나, 항염증성 사이토카인(IL-10)을 증가시키는 것일 수 있다. The strain inhibits the production of nitric oxide in the inflamed cells, inhibits the expression of inflammatory cytokines (eg, TNF-α or IL-6), or inhibits the proliferation of bacteria (eg, C. difficile ) may be inhibiting In addition, the strain reduces inflammatory factors induced by C. difficile, for example, pro-inflammatory cytokines (eg, TNF, or CCL2), or increases anti-inflammatory cytokines (IL-10). it could be
다른 양상은 상기 락토바실러스 가세리(Lactobacilus gasseri) 균주 유래의 소포체, 상기 균주의 파쇄물, 배양액, 배양액의 추출물, 또는 이들의 혼합물을 제공한다. Another aspect provides an endoplasmic reticulum derived from the Lactobacillus gasseri strain, a lysate of the strain, a culture medium, an extract of the culture medium, or a mixture thereof.
상기 균주에 대해서는 상기한 바와 같다. The strain is as described above.
본 명세서에서 용어 "소포체(vesicle)"는 세포에서 분비되어 세포 외 공간으로 방출된 입자를 의미하는 것으로서, 엑소좀(exosome), 엑토좀(ectosome), 마이크로소낭(microvesicle), 마이크로입자(microparticle), 엑소좀 유사 소포체 (exosome like vesicle) 등의 다수의 상이한 종을 포함할 수 있다. 세포밖 소포체는 분비하는 기원 세포(공여 세포)의 상태를 반영할 수 있으며, 어떤 세포에서 분비되었는가에 따라 다양한 생물학적 활성을 나타내고, 세포들 사이에 유전 물질과 단백질을 옮기면서 세포 간 상호작용에 중요한 역할을 할 수 있다. 또한, 상기 소포체를 포함하는 세포 유래 물질들은 질병을 일으키거나 또는 면역세포를 자극하여 질병에 대항하게 하며, 미생물의 대사과정을 통해 사람이 소화시키지 못하는 물질들을 분해하여 흡수할 수 있도록 도와주는 효과가 있다. 상기 소포체는 막 구조 소포체로 내부와 외부가 구분되며, 세포의 세포막 지질(plasma membrane lipid)과 세포막 단백질(plasma membrane protein), 핵산(nucleic acid), 및 세포질 성분 등을 가지고 있고, 원래 세포보다 크기가 작은 것일 수 있다.As used herein, the term “vesicle” refers to particles secreted from cells and released into the extracellular space, and includes exosomes, ectosomes, microvesicles, and microparticles. , exosome-like vesicles, and the like. The extracellular ER can reflect the state of the secreting cell of origin (donor cell), exhibit various biological activities depending on which cell it is secreted from, and play an important role in cell-to-cell interactions by transferring genetic material and proteins between cells can do. In addition, the cell-derived substances including the endoplasmic reticulum cause disease or stimulate immune cells to fight disease, and have the effect of helping to break down and absorb substances that humans cannot digest through the metabolic process of microorganisms. have. The endoplasmic reticulum is a membrane-structured endoplasmic reticulum, which is divided into inside and outside, and has plasma membrane lipid and plasma membrane protein, nucleic acid, and cytoplasmic components of the cell, and is larger than the original cell. may be small.
일 구체예에 있어서, 상기 소포체는 락토바실러스 가세리 균주의 배양액의 세포 파쇄물로부터 분리된 것일 수 있다.In one embodiment, the endoplasmic reticulum may be isolated from the cell lysate of the culture solution of the Lactobacillus gasseri strain.
일 구체예에 있어서, 상기 세포 외 소포체는 10 nm 내지 400 nm의 직경을 갖는 것일 수 있다. 예를 들어, 10 nm 내지 400 nm, 10 nm 내지 350 nm, 10 nm 내지 300 nm, 10 nm 내지 250 nm 일 수 있다. In one embodiment, the extracellular vesicles may have a diameter of 10 nm to 400 nm. For example, it may be 10 nm to 400 nm, 10 nm to 350 nm, 10 nm to 300 nm, or 10 nm to 250 nm.
본 명세서에서 용어"배양액"은 "배양 상층액", "조건 배양액" 또는 "조정 배지"와 호환적으로 사용될 수 있고, 락토바실러스 가세리가 시험관 내에서 성장 및 생존할 수 있도록 영양분을 공급할 수 있는 배지에 상기 균주를 일정기간 배양하여 얻는 상기 균주, 이의 대사물, 여분의 영양분 등을 포함하는 전체 배지를 의미할 수 있다. 또한, 상기 배양액은 균주를 배양하여 얻은 균체 배양액에서 균체를 제거한 배양액을 의미할 수 있다. 한편, 상기 배양액 중 균체를 제거한 액체를 "상등액"이라고도 하며, 배양액을 일정시간 가만히 두어 하층에 가라앉은 부분을 제외한 상층의 액체만을 취하거나, 여과를 통해 균체를 제거하거나, 배양액을 원심분리하여 하부의 침전을 제거하고 상부의 액체만을 취하여 획득할 수 있다. 상기 "균체"는 본 발명의 균주 자체를 의미하는 것으로 피부 샘플 등으로부터 분리하여 선별한 균주 자체 또는 상기 균주를 배양하여 배양액으로부터 분리한 균주를 포함한다. 상기 균체는 배양액을 원심분리하여 하층에 가라앉은 부분을 취하여 획득할 수 있고, 또는 중력에 의해 배양액의 하층으로 가라앉으므로 일정 시간동안 가만히 두었다가 상부의 액체를 제거함으로써 획득할 수 있다.As used herein, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and a medium capable of supplying nutrients so that Lactobacillus gasseri can grow and survive in vitro. to the strain obtained by culturing the strain for a certain period of time, its metabolites, and may mean the entire medium including extra nutrients. In addition, the culture solution may refer to a culture solution obtained by culturing the strain in which the cells are removed from the culture solution. On the other hand, the liquid from which the cells have been removed from the culture solution is also called a “supernatant”, and the culture solution is left for a certain period of time to take only the liquid from the upper layer except for the part that has sunk to the lower layer, remove the cells through filtration, or centrifuge the culture solution to the lower part It can be obtained by removing the precipitation of and taking only the liquid at the top. The "cell" of the present invention refers to the strain itself of the present invention, and includes the strain isolated and selected from a skin sample, or the strain isolated from the culture solution by culturing the strain. The cells can be obtained by centrifuging the culture solution to take the part that has sunk to the lower layer, or by removing the liquid from the upper layer after leaving it still for a certain period of time because it sinks into the lower layer of the culturing solution by gravity.
상기 배양액은 균주를 배양하여 수득된 배양액 자체, 그의 농축물, 또는 동결건조물 또는 배양액로부터 균주를 제거하여 수득된 배양 상층액, 그의 농축물 또는 동결건조물을 포함할 수 있다. The culture medium may include the culture medium itself obtained by culturing the strain, its concentrate, or a lyophilisate or a culture supernatant obtained by removing the strain from the culture medium, its concentrate or lyophilisate.
상기 배양액은 락토바실러스 가세리를 적절한 배지(예를 들면, R2A 배지 또는 TSA 배지) 에서 10 ℃초과 또는 40 ℃미만 중 어느 온도에서 일정 시간, 예를 들면, 4 내지 50시간 동안 배양하여 수득된 것일 수 있다. The culture medium is Lactobacillus gasseri in an appropriate medium (eg, R2A medium or TSA medium) at any temperature above 10 °C or below 40 °C for a certain time, for example, 4 to 50 hours. can
일 구체예에서, 균주의 배양 상층액은 균주 배양액을 원심분리나 여과시켜 균주를 제거하는 단계에 의해 수득될 수 있다.In one embodiment, the culture supernatant of the strain may be obtained by centrifuging or filtering the strain culture to remove the strain.
다른 구체예에서, 농축물은 상기 균주 배양액 자체, 또는 상기 배양액을 원심분리나 필터를 이용하여 여과한 후 수득된 상층액을 농축하는 단계에 의해 수득될 수 있다. In another embodiment, the concentrate may be obtained by concentrating the supernatant obtained after filtering the strain culture solution itself, or the culture solution using centrifugation or a filter.
상기 락토바실러스 가세리를 배양하기 위한 배양용 배지 및 배양 조건은 통상의 지식을 가진 자가 적절하게 선택하거나 변형하여 이용할 수 있다.The culture medium and culture conditions for culturing the Lactobacillus gasseri may be appropriately selected or modified by those of ordinary skill in the art.
본 명세서에서 용어 "파쇄액"은 균주 자체를 화학적 또는 물리적 힘에 의하여 균주의 세포벽을 파쇄하여 얻은 산물을 의미할 수 있다.As used herein, the term “lysate” may refer to a product obtained by disrupting the cell wall of the strain itself by chemical or physical force.
본 명세서에서 용어 "배양액 추출물"은 상기 배양액 또는 그의 농축액로부터 추출한 것을 의미하며, 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이들 조정제물 또는 정제물, 이를 분획한 분획물을 포함할 수 있다. As used herein, the term "culture solution extract" means extraction from the culture solution or its concentrate, and may include an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these prepared or purified products, and a fraction obtained by fractionating it. can
또 다른 양상은 락토바실러스 가세리(Lactobacilus gasseri), 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 배양액, 또는 배양액의 추출물의 질병 개선, 예방 또는 치료 용도를 제공한다. Another aspect is Lactobacillus gasseri ( Lactobacillus gasseri ), the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture solution, or the disease improvement, prevention or treatment use of the extract of the culture solution.
본 명세서에서 용어 "치료 (treat)"는 자연 치유에 비하여 단축된 시간에 염증 또는 박테리아 감염증 등이 치유되는 것을 의미할 수 있다. 상기 치료는 염증 또는 박테리아 감염증의 개선 및/또는 완화를 포함할 수 있다. 또한, 상기 치료는 염증 또는 박테리아 감염증으로부터 유발되는 증상의 치유 및/또는 회복을 의미할 수 있다.As used herein, the term “treat” may mean that inflammation or bacterial infection is cured in a shorter time compared to natural healing. The treatment may include amelioration and/or alleviation of inflammation or bacterial infection. In addition, the treatment may refer to healing and/or recovery of symptoms resulting from inflammation or bacterial infection.
상기 균주의 용도는 염증성 질환의 예방, 개선, 또는 치료(항 염증 활성), 박테리아 감염증의 예방, 개선, 또는 치료(항균 활성), 또는 장 건강의 예방 또는 개선을 포함할 수 있다. The use of the strain may include preventing, ameliorating, or treating an inflammatory disease (anti-inflammatory activity), preventing, ameliorating, or treating a bacterial infection (antibacterial activity), or preventing or improving intestinal health.
상기 염증성 질환은 소화기계(위장관 등)의 염증, 안 내 염증, 구강 내 염증, 폐를 포함하는 호흡계의 염증, 피부의 염증, 심장혈관계 내 염증, 뇌의 염증, 귀 내 염증 등을 포함할 수 있다. The inflammatory disease may include inflammation of the digestive system (gastrointestinal tract, etc.), intraocular inflammation, oral inflammation, respiratory system inflammation including lung, skin inflammation, cardiovascular inflammation, brain inflammation, and ear inflammation. have.
더욱 상세하게는 상기 염증성 질환은 염증성 장 질환(inflammatory bowel diseases, IBD); 과민성 대장증후군(irritable bowel syndrome); 베체트 병(Behcet's disease); 장염(enteritis), 크론병(Crohn's disease); 궤양성 대장염(ulcerative colitis); 혈관염(vasculitis); 점막염(mucositis); 구내염(stomatitis); 임플란트 주위염(peri-implantitis); 치주염(periodontitis); 치수염(pulpitis); 치은염(gingivitis); 폐렴; 피부염(dermatitis); 아토피 피부염(atopic dermatitis); 접촉성 피부염(contact dermatitis); CREST 증후군; 포진성 피부염(dermatitis herpetiformis); 피부근염(dermatomyositis); 전신성 공피증(systemic scleroderma); 결절성 홍반(erythema nodosum); 헤노흐-쇤라인 자반병(Henoch-Schonlein purpura); 화농성 한선염(Hidradenitis suppurativa); 편평태선(Lichen planus); 마지드 증후군(Majeed syndrome); 슈니츨러 증후군(Schnitzler syndrome); 건선(psoriasis); 습진(eczema); 여드름(acne); 구강염(mouth ulcers); 포도막염(uveitis); 인두염(pharyngitis); 편도염(tonsillitis); 중이염을 포함하는 이염(otitis); 관절염(psoriatic arthritis); 활액막염(synovitis); 수막염(meningitis); 뇌염(encephalitis); 비커스테프 뇌염(Bickerstaff's encephalitis) 뇌척수염(encephalomyelitis); 척추염(spondylitis); 골수염(osteomyelitis); 길리안 바레 증후군(Guillain-barre syndrome); 척수염(myelitis); 시속신경수염(neuromyelitis optica); 방광염(cystitis); 감염 또는 상처 부위에 급성 염증; 신염(nephritis); 및 사구체신염(glomerulonephritis)로 이루어진 군으로부터 선택된 어느 하나인 것일 수 있다. More specifically, the inflammatory disease is inflammatory bowel disease (IBD); irritable bowel syndrome; Behcet's disease; enteritis, Crohn's disease; ulcerative colitis; vasculitis; mucositis; stomatitis; peri-implantitis; periodontitis; pulpitis; gingivitis; Pneumonia; dermatitis; atopic dermatitis; contact dermatitis; CREST syndrome; dermatitis herpetiformis; dermatomyositis; systemic scleroderma; erythema nodosum; Henoch-Schonlein purpura; Hidradenitis suppurativa; lichen planus; Majeed syndrome; Schnitzler syndrome; psoriasis; eczema; acne; mouth ulcers; uveitis; pharyngitis; tonsillitis; otitis, including otitis media; arthritis (psoriatic arthritis); synovitis; meningitis; encephalitis; Bickerstaff's encephalitis encephalomyelitis; spondylitis; osteomyelitis; Guillain-barre syndrome; myelitis; neuromyelitis optica; cystitis; Acute inflammation at the site of an infection or wound; nephritis; And it may be any one selected from the group consisting of glomerulonephritis.
또한, 상기 장 건강의 개선은 장내 유익증식 및 유해균 억제에의 도움, 면역을 조절하여 장 건강에의 도움, 또는 배변활동 원할에의 도움인 것일 수 있다. In addition, the improvement of the intestinal health may be a help in intestinal beneficial growth and suppression of harmful bacteria, a help in intestinal health by regulating immunity, or a help in smooth bowel movements.
용어 "항균제"는 본원에서 사용될 때, (i) 박테리아의 성장을 억제, 감소 또는 방지하거나; (ii) 박테리아가 대상에서 감염을 발생시키는 능력을 억제 또는 감소시키거나; 또는 (iii) 박테리아가 환경에서 증식하거나 감염성을 유지하는 능력을 억제 또는 감소시키는 것이 가능한 물질을 가리킨다. The term “antibacterial agent,” as used herein, refers to (i) inhibiting, reducing or preventing the growth of bacteria; (ii) inhibit or reduce the ability of the bacteria to cause infection in a subject; or (iii) a substance capable of inhibiting or reducing the ability of a bacterium to multiply or maintain infectivity in the environment.
상기 박테리아 감염증의 예시는 그람 양성균 또는 그람 음성균에 의한 감염증을 포함할 수 있다. 상세하게는, 상기 박테리아 감염증은 클로스트리디움(Clostridium), 헬리코박터(Helicobactor), 에스케리키아(Escherichia), 살모넬라(Salmonella), 스타필로코커스(Staphylococcus), 스트렙토코커스(Streptococcus), 해모필루스(Haemophilus), 클레브시엘라(Klebsiella), 모락셀라(Moraxella), 엔테로박터(Enterobacter), 프로테우스(Proteus), 세라티아(Serratia), 슈도모나스(Pseudomonas), 아시네토박터(Acinetobacter), 시트로박터(Citrobacter), 스테노프로포모나스(Stenotrophomonas), 박테로이드(Bacteroides), 프레보텔라(Prevotella), 푸소박테리움(Fusobacterium) 속에 속하는 박테리라에 의한 감염증을 포함할 수 있다. 더욱 상세하게는 상기 박테리아 감염증은 클로스트리디움 디피실 감염증(CDI), 또는 클로스트리디움 디피실 관련 질환(CDAD: Clostridium difficile associated disease), 예를 들면, 클로스티리디움 디피실 관련 설사(Clostridium difficile associated diarrhea)를 포함할 수 있다.Examples of the bacterial infection may include an infection caused by gram-positive bacteria or gram-negative bacteria. Specifically, the bacterial infection is Clostridium (Clostridium), Helicobacter (Helicobactor), Escherichia (Escherichia), Salmonella (Salmonella), Staphylococcus (Staphylococcus), Streptococcus (Streptococcus), by a brush loose (Haemophilus ), Klebsiella (Klebsiella), morak Cellar (Moraxella), Enterobacter bakteo (Enterobacter), Proteus (Proteus), Serratia marcescens (Serratia), Pseudomonas (Pseudomonas), ahsine Sat bakteo (Acinetobacter), bakteo (Citrobacter a sheet ), Stenotrophomonas ), Bacteroides ), Prevotella ( Prevotella ), Fusobacterium ( Fusobacterium ) It may include an infection caused by bacteria belonging to the genus. More specifically, the bacterial infection is a Clostridium difficile infection (CDI), or a Clostridium difficile associated disease (CDAD), for example, Clostridium difficile associated diarrhea. diarrhea) may be included.
상기 조성물은 조성물 총 중량에 대하여 0.00001 중량% 내지 80 중량%, 예를 들면, 0.00001 중량% 내지 60 중량%, 0.00001 중량% 내지 40 중량%, 0.00001 중량% 내지 30 중량%, 0.00001 중량% 내지 20 중량%, 0.00001 중량% 내지 10 중량%, 0.00001 중량% 내지 5 중량%, 0.05 중량% 내지 60 중량%, 0.05 중량% 내지 40 중량%, 0.05 중량% 내지 30 중량%, 0.05 중량% 내지 20 중량%, 0.05 중량% 내지 10 중량%, 0.05 중량% 내지 5 중량%, 0.1 중량% 내지 60 중량%, 0.1 중량% 내지 40 중량%, 0.1 중량% 내지 30 중량%, 0.1 중량% 내지 20 중량%, 0.1 중량% 내지 10 중량%, 또는 0.1 중량% 내지 5 중량%의 균주, 이의 파쇄액, 배양액, 또는 이의 배양액의 추출물을 포함할 수 있다.The composition comprises 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, based on the total weight of the composition %, 0.00001% to 10% by weight, 0.00001% to 5% by weight, 0.05% to 60% by weight, 0.05% to 40% by weight, 0.05% to 30% by weight, 0.05% to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % to 10% by weight, or 0.1% to 5% by weight of a strain, a lysate thereof, a culture solution, or an extract of a culture solution thereof.
용어, "유효성분으로 포함"은 상기에서 언급한 효과를 나타낼 수 있는 정도로 본 명세서의 균주, 상기 균주 유래의 소포체, 상기 균주의 파쇄액, 배양액, 또는 이의 배양액의 추출물이 첨가되는 것을 의미하고, 약물전달 및 안정화 등을 위하여 다양한 성분을 부성분으로 첨가하여 다양한 형태로 포뮬레이션 (formulation)되는 것을 포함하는 의미이다.The term, "included as an active ingredient" means that the strain of the present specification, the endoplasmic reticulum derived from the strain, the lysate of the strain, the culture medium, or the extract of the culture medium is added to the extent that it can exhibit the above-mentioned effects, For drug delivery and stabilization, it is meant to include formulations in various forms by adding various components as sub-components.
다른 구체예에 있어서, 상기 조성물은 약학적 조성물일 수 있다. In another embodiment, the composition may be a pharmaceutical composition.
상기 약학적 조성물은 약제학적으로 허용가능한 희석제 또는 담체를 추가적으로 포함할 수 있다. 상기 희석제는 유당, 옥수수 전분, 대두유, 미정질 셀룰로오스, 또는 만니톨, 활택제로는 스테아린산 마그네슘, 탈크, 또는 그 조합일 수 있다. 상기 담체는 부형제, 붕해제, 결합제, 활택제, 또는 그 조합일 수 있다. 상기 부형제는 미결정 셀룰로오즈, 유당, 저치환도 히드록시셀룰로오즈, 또는 그 조합일 수 있다. 상기 붕해제는 카르복시메틸셀룰로오스 칼슘, 전분글리콜산 나트륨, 무수인산일수소 칼슘, 또는 그 조합일 수 있다. 상기 결합제는 폴리비닐피롤리돈, 저치환도 히드록시프로필셀룰로오즈, 히드록시프로필셀룰로오즈, 또는 그 조합일 수 있다. 상기 활택제는 스테아린산 마그네슘, 이산화규소, 탈크, 또는 그 조합일 수 있다.The pharmaceutical composition may further include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be carboxymethyl cellulose calcium, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.
상기 약학적 조성물은 경구 또는 비경구 투여 제형으로 제형화될 수 있다. 경구 투여 제형은 과립제, 산제, 액제, 정제, 캅셀제, 건조시럽제, 또는 그 조합일 수 있다. 비경구 투여 제형은 주사제일 수 있다.The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or a combination thereof. The parenteral dosage form may be an injection.
상기 조성물은 건강기능식품 조성물을 일 수 있다. The composition may be a health functional food composition.
상기 건강기능식품 조성물은 상기 균주 또는 이의 배양액 단독 또는 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 명세서의 조성물은 원료에 대하여 15 중량부 이하의 양으로 첨가될 수 있다. 상기 건강기능식품의 종류에는 특별한 제한은 없다. 건강기능식품의 종류 중 음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 건강식품 조성물은 또한 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제, 또는 그 조합을 함유할 수 있다. 상기 건강기능식품 조성물은 또한, 천연 과일쥬스, 과일쥬스 음료, 야채 음료의 제조를 위한 과육, 또는 그 조합을 함유할 수 있다.The health functional food composition may be used alone or in combination with other foods or food ingredients of the strain or its culture, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment). In general, in the production of food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of the health functional food. Among the types of health functional food, the beverage composition may contain various flavoring agents or natural carbohydrates as additional components, like a conventional beverage. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health food composition may also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages the carbonation agent used, or a combination thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverage, fruit flesh for the production of vegetable beverage, or a combination thereof.
상기 조성물은 화장료 조성물일 수 있다. The composition may be a cosmetic composition.
상기 화장료 조성물은 예를 들면, 유연화장수, 영양화장수, 마사지크림, 영양크림, 에센스, 팩, 젤, 앰플 또는 피부 점착 타입의 화장료 제형을 갖는 것일 수 있다.The cosmetic composition may have a cosmetic formulation of, for example, a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule, or a skin adhesion type.
상기 화장료 조성물에 포함되는 성분은 유효성분으로서 상기 조성물 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함할 수 있으며, 예를 들면, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제 및 담체를 포함할 수 있다.Components included in the cosmetic composition may include components commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances. may include.
또한, 상기 조성물은 피부외용제용 조성물일 수 있다. In addition, the composition may be a composition for external application to the skin.
본 명세서에서, 상기 피부외용제는 크림, 겔, 연고, 피부 유화제, 피부 현탁액, 경피전달성 패치, 약물 함유 붕대, 로션, 또는 그 조합일 수 있다. 상기 피부외용제는 통상 화장품이나 의약품 등의 피부외용제에 사용되는 성분, 예를 들면 수성성분, 유성성분, 분말성분, 알코올류, 보습제, 증점제, 자외선흡수제, 미백제, 방부제, 산화방지제, 계면활성제, 향료, 색제, 각종 피부 영양제, 또는 이들의 조합과 필요에 따라서 적절하게 배합될 수 있다. 상기 피부외용제는, 에데트산이나트륨, 에데트산삼나트륨, 시트르산나트륨, 폴리인산나트륨, 메타인산나트륨, 글루콘산 등의 금속봉쇄제, 카페인, 탄닌, 벨라파밀, 감초추출물, 글라블리딘, 칼린의 과실의 열수추출물, 각종생약, 아세트산토코페롤, 글리틸리틴산, 트라넥삼산 및 그 유도체 또는 그 염등의 약제, 비타민 C, 아스코르브산인산마그네슘, 아스코르브산글루코시드, 알부틴, 코지산, 글루코스, 프룩토스, 트레할로스 등의 당류등도 적절하게 배합할 수 있다.In the present specification, the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component normally used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed. The external preparation for skin includes metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extract, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.
또한, 다른 양상은 유효한 양의 상기한 조성물을 그를 필요로 하는 개체에 처리 또는 투여하는 단계를 포함하는 개체의 상태를 예방, 개선, 또는 치료하는 방법을 제공한다. Another aspect also provides a method of preventing, ameliorating, or treating a condition in a subject comprising treating or administering to the subject in need thereof an effective amount of the composition described above.
상기 개체의 상태는 염증과 관련된 상태, 또는 박테리아 감염과 관련된 상태일 수 있다. The subject's condition may be a condition associated with inflammation or a condition associated with a bacterial infection.
투여는 당업계에 알려진 방법에 의하여 투여될 수 있다. 투여는 예를 들면, 정맥내, 근육내, 경구, 경피 (transdermal), 점막, 코안 (intranasal), 기관내 (intratracheal) 또는 피하 투여와 같은 경로로, 임의의 수단에 의하여 개체로 직접적으로 투여될 수 있다. 상기 투여는 전신적으로 또는 국부적으로 투여될 수 있다.Administration may be administered by methods known in the art. Administration can be administered directly to a subject by any means, for example, by routes such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. can The administration may be systemically or locally.
상기 개체는 포유동물, 예를 들면, 사람, 소, 말, 돼지, 개, 양, 염소, 또는 고양이일 수 있다. 상기 개체는 염증과 관련된 상태, 또는 박테리아 감염과 관련된 상태의 개선 효과를 필요로 하는 개체일 수 있다.The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject in need of improvement of a condition associated with inflammation or a condition associated with bacterial infection.
상기 투여는 일 구체예에 따른 조성물을 개체당 일당 0.00001 mg 내지 1,000 mg, 예를 들면, 0.00001 mg 내지 500 mg, 0.00001 mg 내지 100 mg, 0.00001 mg 내지 50 mg, 0.00001 mg 내지 25 mg, 1 mg 내지 1,000 mg, 1 mg 내지 500 mg, 1 mg 내지 100 mg, 1 mg 내지 50 mg, 1 mg 내지 25 mg, 5mg 내지 1,000 mg, 5 mg 내지 500 mg, 5 mg 내지 100 mg, 5 mg 내지 50 mg, 5 mg 내지 25 mg, 10mg 내지 1,000 mg, 10 mg 내지 500 mg, 10 mg 내지 100 mg, 10 mg 내지 50 mg, 또는 10 mg 내지 25 mg을 투여하는 것일 수 있다. 다만, 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있고, 당업자라면 이러한 요인들을 고려하여 투여량을 적절히 조절할 수 있다. 투여 횟수는 1일 1회 또는 임상적으로 용인 가능한 부작용의 범위 내에서 2회 이상이 가능하고, 투여 부위에 대해서도 1개소 또는 2개소 이상에 투여할 수 있으며, 매일 또는 2 내지 5일 간격으로 총 투여 일수는 한번 치료 시 1일에서 30일까지 투여될 수 있다. 필요한 경우, 적정 시기 이후에 동일한 치료를 반복할 수 있다. 인간 이외의 동물에 대해서도, kg당 인간과 동일한 투여량으로 하거나, 또는 예를 들면 목적의 동물과 인간과의 기관(심장 등)의 용적비(예를 들면, 평균값) 등으로 상기의 투여량을 환산한 양을 투여할 수 있다.The administration is 0.00001 mg to 1,000 mg per subject per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity, and those skilled in the art The dosage may be appropriately adjusted in consideration of these factors. The number of administration can be once a day or twice or more within the range of clinically acceptable side effects, and it can be administered to one or two or more places for the administration site, and total daily or at intervals of 2 to 5 days. The number of days of administration may range from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For animals other than humans, the dose is the same as that of humans per kg, or the above dose is converted, for example, by the volume ratio (for example, the average value) of the target animal and the organ (heart, etc.) One dose can be administered.
일 양상에 따른 신규 균주 및 이의 유래의 소포체에 의하면, 염증 관련 상태, 또는 박테리아 감염의 예방, 개선, 또는 치료에 유용하게 사용될 수 있는 효과가 있다. According to the novel strain and its derived endoplasmic reticulum according to an aspect, there is an effect that can be usefully used for prevention, improvement, or treatment of inflammation-related conditions, or bacterial infections.
도 1은 일 구체예에 따른 균주의 소포체의 세포 처리에 따른 산화질소의 생성량을 나타낸 그래프이다; N: 음성 대조군, P: 무처리 대조군, EV: 실시예 2의 소포체.
도 2는 일 구체예에 따른 균주의 소포체의 세포 처리에 염증성 사이토카인(TNF-α 및 IL-6)의 단백질 발현양을 나타낸 그래프이다; N: 음성 대조군, P: 무처리 대조군, EV: 실시예 2의 소포체.
도 3은 일 구체예에 따른 균주의 소포체의 세포 처리에 항염증성 사이토카인(IL-10)의 단백질 발현양을 나타낸 그래프이다; N: 음성 대조군, P: 무처리 대조군, EV: 실시예 2의 소포체.
도 4는 일 구체예에 따른 균주의 소포체의 C.difficile에의 처리에 따른 C.difficile의 배양율을 나타낸 그래프이다.
도 5는 일 구체예에 따른 균주의 소포체의 세포 독성 결과를 나타낸 그래프이다; N: 음성 대조군, P: 무처리 대조군, EV: 실시예 2의 소포체.
도 6은 일 구체예에 따른 균주와 표준균주의 소포체의 세포독성 결과를 나타낸 그래프이다; CdEV: 클로스트리디움 디피실 소포체, BBH008: 실시예 1 균주의 소포체, Type strain: 락토바실러스 가세리 표준 균주의 소포체.
도 7은 일 구체예에 따른 균주의 소포체의 클로스트리디움 디피실에 의해 유도된 염증의 감소 활성을 나타낸 그래프이다; CdEV: 클로스트리디움 디피실 소포체, BBH008: 실시예 1 균주의 소포체, Type strain: 락토바실러스 가세리 표준 균주의 소포체. 1 is a graph showing the amount of nitric oxide production according to the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
Figure 2 is a graph showing the protein expression level of inflammatory cytokines (TNF-α and IL-6) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
Figure 3 is a graph showing the protein expression level of the anti-inflammatory cytokine (IL-10) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
Figure 4 is a graph showing the culture rate of C. difficile according to the treatment with C. difficile of the endoplasmic reticulum of the strain according to one embodiment.
5 is a graph showing the cytotoxicity results of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
6 is a graph showing the cytotoxicity results of the endoplasmic reticulum of the strain and the standard strain according to one embodiment; CdEV: Clostridium difficile ER, BBH008: ER of Example 1 strain, Type strain: ER of Lactobacillus gasseri standard strain.
7 is a graph showing the activity of reducing inflammation induced by Clostridium difficile of the endoplasmic reticulum of the strain according to one embodiment; CdEV: Clostridium difficile ER, BBH008: ER of Example 1 strain, Type strain: ER of Lactobacillus gasseri standard strain.
이하 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.
실시예 1. 균주의 분리 및 동정Example 1. Isolation and identification of strains
사람의 분변으로부터 균주를 분리 및 동정하기 위해 다음과 같이 수행하였다. 먼저, 인체로부터 수집한 분변 1g과 인산완충식염수(phosphate buffer saline, Dongin biotech) 10ml을 균질화 하였다. 그런 뒤 소화되지 않은 음식 및 작은 입자 물질 등을 제거하기 위하여 cell strainer(100um, SPL)로 여과하여 분변 용액을 제조하였다.In order to isolate and identify the strain from human feces, it was performed as follows. First, 1 g of feces collected from the human body and 10 ml of phosphate buffer saline (Dongin biotech) were homogenized. Then, a fecal solution was prepared by filtration with a cell strainer (100um, SPL) to remove undigested food and small particles.
상기 미생물을 포함하는 조성물로부터 5% sheep blood을 함유하는 TSA 배지(Tryptic soy agar, Green Tech)에 연속희석(Serial dilution)하여 Plate에 Spreading하였고 37 ℃에 2일간 배양하는 과정으로 균을 선별하였다. 배양이 완료된 집락에 대해 PCR 증폭을 수행하였고, 분리 배양된 미생물 집락 중 결정된 16S rRNA부위의 염기서열을 미국 국립생물정보센터(NCBI, National Center for Biotechnology Information) 홈페이지에서 제공되는 BLAST 프로그램으로 등록된 다른 균주들과 비교 분석 하였다. 비교 분석 결과 상동성 98%의 Lactobacilus gasseri BBH 008을 분리하였다. 선별된 Lactobacilus gasseri BBH 008 균주를 2020년 9월 3일자로 한국생물자원센터에 기탁하여 기탁번호 KCTC 14298BP를 부여 받았고, Lactobacilus gasseri BBH 008 균주는 서열번호 1(complementary DNA)의 16s rRNA 서열을 갖는다.The composition containing the microorganism was serially diluted in TSA medium (Tryptic soy agar, Green Tech) containing 5% sheep blood, spread on a plate, and the bacteria were selected by incubating at 37 ° C. for 2 days. PCR amplification was performed on the cultured colonies, and the nucleotide sequence of the 16S rRNA region determined among the isolated and cultured microbial colonies was transferred to another registered BLAST program provided on the website of the National Center for Biotechnology Information (NCBI). The strains were compared and analyzed. As a result of comparative analysis, Lactobacilus gasseri BBH 008 having 98% homology was isolated. The selected Lactobacilus gasseri BBH 008 strain was deposited with the Korea Center for Biological Resources on September 3, 2020 and was given accession number KCTC 14298BP, and the Lactobacilus gasseri BBH 008 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).
실시예 2. 소포체의 분리 Example 2. Isolation of the endoplasmic reticulum
상기 실시예에서 분리된 균주의 소포체를 분리하였다. The endoplasmic reticulum of the strain isolated in the above example was isolated.
구체적으로, 소포체를 제조하기 위해, 상기 분리된 균주를 MRS broth(De Man, Rogosa, Sharpe)에서 37 ℃, 혐기 조건에서 3일간 배양하였다. 이후에 배양액을 3000 RPM으로 20분 동안 원심분리 한 뒤 다시 3000 RPM으로 40분 동안 원심분리하여 균의 잔해를 제거하였다. 이후에, 0.45um 필터로 여과 한 뒤 다시 0.22um 필터로 여과하고 필터(Millipore Pellicon XL50, Biomax-100 filter)를 이용한 TFF 시스템(Tangential Flow Filtration System, Green Tech)을 사용하여 100 kda 이상의 물질을 농축하였고 상기 분리된 균주의 소포체를 분리하였다. Specifically, in order to prepare the endoplasmic reticulum, the isolated strain was cultured in MRS broth (De Man, Rogosa, Sharpe) at 37 ° C., anaerobic conditions for 3 days. Thereafter, the culture medium was centrifuged at 3000 RPM for 20 minutes and then centrifuged again at 3000 RPM for 40 minutes to remove bacterial debris. After that, it was filtered with a 0.45um filter and then filtered again with a 0.22um filter, and a TFF system (Tangential Flow Filtration System, Green Tech) using a filter (Millipore Pellicon XL50, Biomax-100 filter) was used to concentrate the material over 100 kda. and the endoplasmic reticulum of the isolated strain was isolated.
실험예 1. 항염증 활성 분석 Experimental Example 1. Analysis of anti-inflammatory activity
상기 실시예 2.에서 분리된 균주의 소포체의 항염증 활성을 분석하였다. The anti-inflammatory activity of the endoplasmic reticulum of the strain isolated in Example 2. was analyzed.
먼저, 항염증 활성을 평가하기 위해, 산화 질소(NO) 생성량 감소를 측정하였다. 마우스 대식세포 Raw264.7 세포를 20% 소태아혈청 (FBS: fetal bovine serum), 1% 항생제 (100U/mL 페니실린 및 100㎍/mL 스트렙토마이신)를 포함하는 RPMI1640 배양액으로 5% CO2 존재 하에서 37 ℃로 배양하였다. 이후에, 상기 Raw 264.7 세포를 48 웰 플레이트에 5Х104세포/웰의 농도로 250μL씩 분주하고, CO2 배양기에서 37 ℃및 24 시간 동안 배양하였다. 웰 상층액을 버리고 염증 유발을 위해 lipopolysaccharide(LPS) 10ug/ml가 첨가된 배지를 분주한 후 4 시간 동안 추가 배양하였다. LPS가 들어있는 상층액을 버리고 상기 소포체를 0.01 내지 100 ug/ml의 농도로 배지에 첨가하여 처리한 다음 37 ℃에서 16시간 배양하였다. 이후에, 웰 상층액 중 50μL와 Griess 시약 50 μL를 섞어서 실온에서 10분간 반응시킨 후 플레이트 리더기로 570 nm에서 흡광도 측정하여 산화 질소의 생성량을 측정하였고, 그 결과는 도 1에 나타내었다.First, in order to evaluate the anti-inflammatory activity, a decrease in nitric oxide (NO) production was measured. Mouse macrophage Raw264.7 cells were treated with RPMI1640 culture medium containing 20% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) in the presence of 5% CO 2 37 Incubated at ℃. Thereafter, the Raw 264.7 cells were aliquoted at a concentration of 5Х10 4 cells/well in a 48-well plate by 250 μL, and incubated at 37° C. and 24 hours in a CO 2 incubator. The well supernatant was discarded and a medium supplemented with lipopolysaccharide (LPS) 10ug/ml was dispensed to induce inflammation, followed by further incubation for 4 hours. The supernatant containing LPS was discarded and the endoplasmic reticulum was added to the medium at a concentration of 0.01 to 100 ug/ml and treated, and then incubated at 37° C. for 16 hours. Thereafter, 50 μL of the well supernatant and 50 μL of Griess reagent were mixed and reacted at room temperature for 10 minutes, and then absorbance was measured at 570 nm with a plate reader to measure the amount of nitric oxide produced, and the results are shown in FIG. 1 .
도 1은 일 구체예에 따른 균주의 소포체의 세포 처리에 따른 산화질소의 생성량을 나타낸 그래프이다; N: 음성 대조군, P: 무처리 대조군, EV: 실시예 2의 소포체.1 is a graph showing the amount of nitric oxide production according to the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
도 1에 나타낸 바와 같이, 일 구체예에 따른 균주의 소포체는 염증 유발된 세포의 NO 생성량을 유의하게 감소시킨 것을 알 수 있었다. As shown in FIG. 1 , it was found that the endoplasmic reticulum of the strain according to one embodiment significantly reduced the amount of NO production in the cells induced by inflammation.
다음으로, 상기 균주의 소포체의 염증성 사이토카인 억제 활성을 측정하였다. 구체적으로, 상기와 동일한 방법으로 LPS 처리된 Raw264.7 세포에 상기 소포체를 0.01 내지 100 ug/ml의 농도로 처리한 다음 37 ℃에서 48시간 배양하였다. 이후에, 상기 세포의 TNF, IL-6 및 IL-10의 단백질 발현을 ELISA kit (eBioscience, 미국)를 이용하여 제조사의 지시에 따라 540nm에서 흡광도를 측정하였고, 그 결과를 각각 도 2 및 도 3에 나타내었다. Next, the inflammatory cytokine inhibitory activity of the endoplasmic reticulum of the strain was measured. Specifically, the LPS-treated Raw264.7 cells in the same manner as above were treated with the endoplasmic reticulum at a concentration of 0.01 to 100 ug/ml, and then cultured at 37° C. for 48 hours. Thereafter, the protein expression of TNF, IL-6 and IL-10 of the cells was measured for absorbance at 540 nm using an ELISA kit (eBioscience, USA) according to the manufacturer's instructions, and the results are shown in FIGS. 2 and 3, respectively. shown in
도 2는 일 구체예에 따른 균주의 소포체의 세포 처리에 염증성 사이토카인(TNF 및 IL-6)의 단백질 발현양을 나타낸 그래프이다; N: 음성 대조군, P: 무처리 대조군, EV: 실시예 2의 소포체.Figure 2 is a graph showing the protein expression level of inflammatory cytokines (TNF and IL-6) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
도 3은 일 구체예에 따른 균주의 소포체의 세포 처리에 항염증성 사이토카인(IL-10)의 단백질 발현양을 나타낸 그래프이다; N: 음성 대조군, P: 무처리 대조군, EV: 실시예 2의 소포체.Figure 3 is a graph showing the protein expression level of the anti-inflammatory cytokine (IL-10) in the cell treatment of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
도 2 및 도 3에 나타낸 바와 같이, 일 구체예에 따른 균주의 소포체는 염증성 사이토카인을 무처리 대조군 대비 현저하게 감소시키고, 항염증성 사이토카인을 무처리 대조군 대비 현저하게 증가시킴을 알 수 있었다. As shown in Figures 2 and 3, the endoplasmic reticulum of the strain according to one embodiment significantly reduced inflammatory cytokines compared to the untreated control group, and it was found that the anti-inflammatory cytokines significantly increased compared to the untreated control group.
이상의 결과는 일 구체예에 따른 균주가 염증성 질환, 특히 염증성 장질환 또는 과민성대장증후군의 예방, 개선, 또는 치료에 유용하게 사용될 수 있음을 의미한다. The above results mean that the strain according to one embodiment can be usefully used for the prevention, improvement, or treatment of inflammatory diseases, particularly inflammatory bowel disease or irritable bowel syndrome.
실험예 2. 항균 활성 분석 Experimental Example 2. Antibacterial activity analysis
상기 실시예 1.에서 분리된 균주의 항균 활성을 분석하였다.The antibacterial activity of the strain isolated in Example 1. was analyzed.
구체적으로, 실시예 2의 균주의 소포체의 클로스트리디움 디피실(Clostridium difficile)에 대한 항균 활성을 분석하기 위해, 상기 실시예 1의 균주와 C.difficile을 5ml BHI 브로스(Brain Heart Infusion, Eco cell)에 전 배양 하고 분광광도계를 사용하여 OD(600nm) 0.5로 맞추었다. 그 후, 균주 배양액을 30ml BHI broth에 10% 비율로 접종한 뒤 37 ℃에서 48시간 동안 혐기적 조건으로 배양하였다. 배양한 균주를 4000RPM으로 30분 동안 원심분리 하여 펠렛과 상층액을 분리한 뒤 Specifically, in order to analyze the antibacterial activity against Clostridium difficile of the endoplasmic reticulum of the strain of Example 2, the strain of Example 1 and C. difficile 5ml BHI broth (Brain Heart Infusion, Eco cell) ) and adjusted to OD (600 nm) 0.5 using a spectrophotometer. After that, the strain culture medium was inoculated in 30ml BHI broth at a rate of 10% and then cultured anaerobically at 37°C for 48 hours. After centrifuging the cultured strain at 4000RPM for 30 minutes to separate the pellet and the supernatant,
배양 상층액을 4000으로 30분 동안 원심분리하여 소포체를 분리하였다. 분리한 상층액을 원심분리 튜브(Amicon centrifuge tubes)를 이용하여 농축하였고, 3 X 106 cfu/ml의 C.difficile에 90%의 비율로 접종한 후, BHI 브로스 배지에서 1일 동안 혐기적 조건에서 배양하였다. 이후에 C.difficile의 배양율을 콜로니 형성 단위 계산법(Colony forming unit calculation)의 방법으로 측정하였고, 그 결과를 도 5에 나타내었다.The culture supernatant was centrifuged at 4000 for 30 minutes to separate the endoplasmic reticulum. The separated supernatant was concentrated using centrifuge tubes (Amicon centrifuge tubes), and inoculated to 3 X 10 6 cfu/ml C. difficile at a ratio of 90%, anaerobic conditions for 1 day in BHI broth medium cultured in Thereafter, the culture rate of C. difficile was measured by the method of colony forming unit calculation, and the results are shown in FIG. 5 .
도 4는 일 구체예에 따른 균주의 소포체의 C.difficile에의 처리에 따른 C.difficile의 배양율을 나타낸 그래프이다. Figure 4 is a graph showing the culture rate of C. difficile according to the treatment with C. difficile of the endoplasmic reticulum of the strain according to one embodiment.
도 4에 나타낸 바와 같이, 일 구체예에 따른 균주의 소포체는 C.difficile의 배양율을 현저하게 감소시키는 것을 알 수 있었다. As shown in Figure 4, the endoplasmic reticulum of the strain according to one embodiment was found to significantly reduce the culture rate of C. difficile.
이상의 결과는 일 구체예에 따른 균주의 소포체가 박테리아, 예를 들면, 그람 음성균에 대한 항균 활성을 가짐을 의미한다. 특히, 이러한 결과는 일 구체예에 따른 균주의 소포체는 클로스트리디움 디피실 감염증(Clostridium difficile infection, CDI), 또는 그로 인해 나타나는 과민성 대장증후군의 예방, 개선, 또는 치료에 유용하게 사용될 수 있음을 의미한다. The above result means that the endoplasmic reticulum of the strain according to one embodiment has antibacterial activity against bacteria, for example, Gram-negative bacteria. In particular, these results indicate that the endoplasmic reticulum of the strain according to one embodiment can be usefully used for the prevention, improvement, or treatment of Clostridium difficile infection (CDI), or irritable bowel syndrome resulting therefrom. do.
실험예 3. 세포 독성 실험 Experimental Example 3. Cytotoxicity test
상기 실시예 2.에서 분리된 균주의 소포체의 세포 독성 실험을 위해 PreMix WST-1 Cell Proliferation Assay System을 이용하였다. PreMix WST-1 Cell Proliferation Assay System was used for the cytotoxicity test of the endoplasmic reticulum of the strain isolated in Example 2.
구체적으로, Raw264.7 세포를 48 웰 플레이트에 5Х104세포/웰의 농도로 250μL씩 분주하고, CO2 배양기에서 37 ℃ 및 24 시간 동안 배양하였다. 웰 상층액을 버리고 염증 유발을 위해 lipopolysaccharide(LPS) 10ug/ml가 첨가된 배지를 분주한 후 4 시간 동안 추가 배양하였다. LPS가 들어있는 상층액을 버리고 상기 소포체를 0.01 내지 100 ug/ml의 농도로 배지에 첨가하여 처리한 다음 37 ℃에서 16시간 배양하였다. 이후에, WST-1 용액을 각 웰에 25㎕씩 처리하고 4시간 동안 반응시켰다. 4시간 후, 살아있는 세포의 미토콘드리아에 존재하는 숙신산 탈수소효소 (succinate dehydrogenase)에 의하여 생성된 수용성 포르마잔 (formazan)의 농도를 560 nm에서 흡광도를 측정하여 세포 생존율을 측정하였고, 그 결과를 도 5에 나타내었다. Specifically, Raw264.7 cells were aliquoted at a concentration of 5Х10 4 cells/well in a 48-well plate by 250 μL, and incubated at 37° C. and 24 hours in a CO 2 incubator. The well supernatant was discarded and a medium supplemented with lipopolysaccharide (LPS) 10ug/ml was dispensed to induce inflammation, followed by further incubation for 4 hours. The supernatant containing LPS was discarded and the endoplasmic reticulum was added to the medium at a concentration of 0.01 to 100 ug/ml and treated, and then incubated at 37° C. for 16 hours. Thereafter, 25 μl of WST-1 solution was treated in each well and reacted for 4 hours. After 4 hours, the concentration of water-soluble formazan produced by succinate dehydrogenase present in mitochondria of living cells was measured at 560 nm to measure cell viability, and the results are shown in FIG. indicated.
도 5는 일 구체예에 따른 균주의 소포체의 세포 독성 결과를 나타낸 그래프이다; N: 음성 대조군, P: 무처리 대조군, EV: 실시예 2의 소포체.5 is a graph showing the cytotoxicity results of the endoplasmic reticulum of the strain according to one embodiment; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.
도 5에 나타낸 바와 같이, 일 구체예에 따른 균주의 소포체는 100 ug/ml의 농도에서 세포 독성이 전혀 관찰되지 않음을 알 수 있었다. As shown in FIG. 5, it was found that no cytotoxicity was observed in the endoplasmic reticulum of the strain according to one embodiment at a concentration of 100 ug/ml.
실험예 4. 클로스트리디움 디피실에 의해 유도된 염증의 감소 활성 분석Experimental Example 4. Analysis of the activity of reducing inflammation induced by Clostridium difficile
상기 실시예 1에서 분리된 균주의 클로스트리디움 디피실에 의해 유도된 전염증성 사이토카인의 감소 효과를 확인하였다. The effect of reducing the pro-inflammatory cytokines induced by Clostridium difficile of the strain isolated in Example 1 was confirmed.
먼저, 상기 실험예 1과 동일한 방법으로 마우스 대식세포 Raw264.7 세포에 클로스트리디움 디피실의 소포체(CdEV)를 100gu/ml의 양으로 처리한 다음 37 ℃의 조건에서 4시간 동안 배양하였다. 이후에, 락토바실러스 가세리의 표준 균주(ATCC33323)와 실시예 1의 균주의 소포체를 0.01ug/ml의 양으로 처리한 후, 37 ℃의 조건에서 16시간 동안 배양하였고, 세포 생존율을 상기 실험예 2와 동일한 방법으로 측정하여 그 결과를 도 6에 나타내었다.First, in the same manner as in Experimental Example 1, Clostridium difficile endoplasmic reticulum (CdEV) was treated in mouse macrophage Raw264.7 cells in an amount of 100gu/ml, and then cultured at 37°C for 4 hours. After that, the standard strain of Lactobacillus gasseri (ATCC33323) and the endoplasmic reticulum of the strain of Example 1 were treated in an amount of 0.01ug/ml, and cultured at 37°C for 16 hours, and the cell viability was measured in Experimental Example 2 Measured in the same manner as shown in FIG. 6 .
또한, 상기 세포에서 사이토카인인 TNF 및 CCL2의 상대적 농도를 ELISA kit (BD bioscience, 미국)를 이용하여 제조사의 지시에 따라 540nm에서 흡광도를 측정하였고, 그 결과를 도 7에 나타내었다. In addition, the relative concentrations of the cytokines TNF and CCL2 in the cells were measured for absorbance at 540 nm using an ELISA kit (BD bioscience, USA) according to the manufacturer's instructions, and the results are shown in FIG. 7 .
도 6은 일 구체예에 따른 균주의 소포체의 세포독성 결과를 나타낸 그래프이다; CdEV: 클로스트리디움 디피실 소포체, BBH008: 실시예 1 균주의 소포체, Type strain: 락토바실러스 가세리 표준 균주의 소포체. 6 is a graph showing the cytotoxicity results of the endoplasmic reticulum of the strain according to one embodiment; CdEV: Clostridium difficile ER, BBH008: ER of Example 1 strain, Type strain: ER of Lactobacillus gasseri standard strain.
도 7은 일 구체예에 따른 균주의 소포체 클로스트리디움 디피실에 의해 유도된 염증의 감소 활성을 나타낸 그래프이다; CdEV: 클로스트리디움 디피실 소포체, BBH008: 실시예 1 균주의 소포체, Type strain: 락토바실러스 가세리 표준 균주의 소포체. 7 is a graph showing the reduction activity of inflammation induced by the endoplasmic reticulum Clostridium difficile of the strain according to one embodiment; CdEV: Clostridium difficile ER, BBH008: ER of Example 1 strain, Type strain: ER of Lactobacillus gasseri standard strain.
도 6에 나타낸 바와 같이, 일 구체예에 따른 균주와 표준균주의 소포체 모두에서 세포독성은 확인되지 않았다. As shown in Figure 6, cytotoxicity was not confirmed in both the endoplasmic reticulum of the strain and the standard strain according to one embodiment.
또한, 도 7에 나타낸 바와 같이, 일 구체예에 따른 균주는 전염증성 사이토카인 TNF 및 CCL2을 표준 균주 대비 클로스트리디움 디피실의 소포체에 의해 증가한 사이토카인의 양을 유의하게 감소시켰음을 알 수 있었다.In addition, as shown in FIG. 7, the strain according to one embodiment significantly reduced the amount of cytokines increased by the endoplasmic reticulum of Clostridium difficile compared to the standard strain for pro-inflammatory cytokines TNF and CCL2. .
이러한 결과는 일 구체예에 따른 균주 및/또는 그의 유래의 소포체는 현저한 항염 활성을 가져, 클로스트리디움 디피실 감염증(Clostridium difficile infection, CDI), 또는 그로 인해 나타나는 과민성 대장증후군의 예방, 개선, 또는 치료에 유용하게 사용될 수 있음을 의미한다. These results show that the strain and / or the endoplasmic reticulum derived therefrom according to one embodiment has significant anti-inflammatory activity, Clostridium difficile infection (CDI), or prevention, improvement, or irritable bowel syndrome resulting therefrom It means that it can be usefully used for treatment.
<110> BioBankHealing <120> Lactobacilus gasseri strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof <130> PN202445 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1489 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus gasseri <400> 1 cctaatcatc tgtcctacct tagacggctg actcctataa aggttatccc accggctttg 60 ggtgttacag actctcatgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac 120 cgcggcgtgc tgatccgcga ttactagcga ttccagcttc gtgtaggcga gttgcagcct 180 acagtccgaa ctgagaacgg ctttcagaga tccgcttgcc ttcgcaggtt cgcttctcgt 240 tgtaccgtcc attgtagcac gtgtgtagcc caggtcataa ggggcatgat gacttgacgt 300 catccccacc ttcctccggt ttgtcaccgg cagtctcatt agagtgccca acttaatgat 360 ggcaactaat gacaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga 420 gctgacgaca gccatgcacc acctgtctca gcgtccccga agggaactcc taatctctta 480 ggtttgcact ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca 540 tgctccaccg cttgtgcggg cccccgtcaa ttcctttgag tttcaacctt gcggtcgtac 600 tccccaggcg gagtgcttaa tgcgttagct gcagcactga gaggcggaaa cctcccaaca 660 cttagcactc atcgtttacg gcatggacta ccagggtatc taatcctgtt cgctacccat 720 gctttcgagc ctcagcgtca gttgcagacc agagagccgc cttcgccact ggtgttcttc 780 catatatcta cgcattccac cgttacacat ggagttccac tctcctcttc tgcactcaag 840 ttcaacagtt tctgatgcaa ttctccggtt gagccgaagg ctttcacatc agacttattg 900 aaccgcctgc actcgcttta cgcccaataa atccggacaa cgcttgccac ctacgtatta 960 ccgcggctgc tggcacgtag ttagccgtga ctttctaagt aattaccgtc aaataaaggc 1020 cagttactac ctctatcttt cttcactacc aacagagctt tacgagccga aacccttctt 1080 cactcacgcg gcgttgctcc atcagacttg cgtccattgt ggaagattcc ctactgctgc 1140 ctcccgtagg agtttgggcc gtgtctcagt cccaatgtgg ccgatcagtc tctcaactcg 1200 gctatgcatc attgccttgg taagccgtta ccttaccaac tagctaatgc accgcaggtc 1260 catccaagag tgatagcaga accatctttt aaactctaga catgcgtcta gtgttgttat 1320 ccggtattag catctgtttc caggtgttat cccagtctct tgggcaggtt acccacgtgt 1380 tactcacccg tccgccgctc gcttgtatct agtttcattt ggtgcaagca ccaaattcat 1440 ctaggcaagc tcgctcgact tgcatgtatt aggcacgccg ccagcgttc 1489 <110> BioBankHealing <120> Lactobacilus gasseri strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of them <130> PN202445 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1489 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus gasseri <400> 1 cctaatcatc tgtcctacct tagacggctg actcctataa aggttatccc accggctttg 60 ggtgttacag actctcatgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac 120 cgcggcgtgc tgatccgcga ttactagcga ttccagcttc gtgtaggcga gttgcagcct 180 acagtccgaa ctgagaacgg ctttcagaga tccgcttgcc ttcgcaggtt cgcttctcgt 240 tgtaccgtcc attgtagcac gtgtgtagcc caggtcataa ggggcatgat gacttgacgt 300 catccccacc ttcctccggt ttgtcaccgg cagtctcatt agagtgccca acttaatgat 360 ggcaactaat gacaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga 420 gctgacgaca gccatgcacc acctgtctca gcgtccccga agggaactcc taatctctta 480 ggtttgcact ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca 540 tgctccaccg cttgtgcggg cccccgtcaa ttcctttgag tttcaacctt gcggtcgtac 600 tccccaggcg gagtgcttaa tgcgttagct gcagcactga gaggcggaaa cctcccaaca 660 cttagcactc atcgtttacg gcatggacta ccagggtatc taatcctgtt cgctacccat 720 gctttcgagc ctcagcgtca gttgcagacc agagagccgc cttcgccact ggtgttcttc 780 catatatcta cgcattccac cgttacacat ggagttccac tctcctcttc tgcactcaag 840 ttcaacagtt tctgatgcaa ttctccggtt gagccgaagg ctttcacatc agacttattg 900 aaccgcctgc actcgcttta cgcccaataa atccggacaa cgcttgccac ctacgtatta 960 ccgcggctgc tggcacgtag ttagccgtga ctttctaagt aattaccgtc aaataaaggc 1020 cagttactac ctctatcttt cttcactacc aacagagctt tacgagccga aacccttctt 1080 cactcacgcg gcgttgctcc atcagacttg cgtccattgt ggaagattcc ctactgctgc 1140 ctcccgtagg agtttgggcc gtgtctcagt cccaatgtgg ccgatcagtc tctcaactcg 1200 gctatgcatc attgccttgg taagccgtta ccttaccaac tagctaatgc accgcaggtc 1260 catccaagag tgatagcaga accatctttt aaactctaga catgcgtcta gtgttgttat 1320 ccggtattag catctgtttc caggtgttat cccagtctct tgggcaggtt acccacgtgt 1380 tactcacccg tccgccgctc gcttgtatct agtttcattt ggtgcaagca ccaaattcat 1440 ctaggcaagc tcgctcgact tgcatgtatt aggcacgccg ccagcgttc 1489
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KR20170039637A (en) * | 2015-04-16 | 2017-04-11 | 주식회사 고바이오랩 | Lactobacilli having inhibitory effect against pathogenic microorganisms in vagina |
KR20170049216A (en) * | 2015-10-28 | 2017-05-10 | 고려대학교 산학협력단 | Novel Lactobacillus gasseri and Uses Thereof |
WO2018115361A1 (en) * | 2016-12-21 | 2018-06-28 | Agriculture And Food Development Authority (Teagasc) | Human lactobacilli strains |
KR102063554B1 (en) | 2018-09-12 | 2020-01-09 | (주)성운파마코피아 | Lactobacillus gasseri swpm102 which has antifungal activity or antibacterial activity |
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KR20170039637A (en) * | 2015-04-16 | 2017-04-11 | 주식회사 고바이오랩 | Lactobacilli having inhibitory effect against pathogenic microorganisms in vagina |
KR20170049216A (en) * | 2015-10-28 | 2017-05-10 | 고려대학교 산학협력단 | Novel Lactobacillus gasseri and Uses Thereof |
WO2018115361A1 (en) * | 2016-12-21 | 2018-06-28 | Agriculture And Food Development Authority (Teagasc) | Human lactobacilli strains |
KR102063554B1 (en) | 2018-09-12 | 2020-01-09 | (주)성운파마코피아 | Lactobacillus gasseri swpm102 which has antifungal activity or antibacterial activity |
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WO2022146096A1 (en) * | 2020-12-31 | 2022-07-07 | 주식회사 바이오뱅크힐링 | Lactobacillus sp. strain, bifidobacterium sp. strain, or ruminococcus sp. strain, and vesicles derived therefrom, and anti-inflammatory and antibacterial uses thereof |
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