KR102618322B1 - Novel Lactobacillus strain and composition for preventing, treating, or improving tuberculosis comprising the same - Google Patents
Novel Lactobacillus strain and composition for preventing, treating, or improving tuberculosis comprising the same Download PDFInfo
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- KR102618322B1 KR102618322B1 KR1020210133734A KR20210133734A KR102618322B1 KR 102618322 B1 KR102618322 B1 KR 102618322B1 KR 1020210133734 A KR1020210133734 A KR 1020210133734A KR 20210133734 A KR20210133734 A KR 20210133734A KR 102618322 B1 KR102618322 B1 KR 102618322B1
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- tuberculosis
- strain
- mycobacterium
- lactobacillus
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Classifications
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P31/06—Antibacterial agents for tuberculosis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Abstract
본 발명은 락토바실러스 크리스파투스(Lactobacillus crispatus) 균주에 관한 것으로, 본 발명은 락토바실러스 크리스파투스(Lactobacillus crispatus) 균주를 포함하는 항균용 조성물을 제공한다. 또한, 본 발명은 상기 균주를 포함하는 결핵 질환, 염증성 질환 예방, 치료 및 개선을 위한 약학적 조성물 및 식품 조성물을 제공한다. 보다 구체적으로, 본 발명에 따른 균주는 항균 활성을 가지며, 이에 따라, 마이코박테리움 보다 구체적으로 결핵균에 의해 유발되는 결핵 질환 및 염증성 질환에 대한, 예방, 치료, 및 개선 효과를 가진다.The present invention relates to Lactobacillus crispatus strains, and the present invention provides an antibacterial composition containing Lactobacillus crispatus strains. In addition, the present invention provides pharmaceutical compositions and food compositions for preventing, treating, and improving tuberculosis disease and inflammatory diseases containing the above strains. More specifically, the strain according to the present invention has antibacterial activity, and thus has preventive, therapeutic, and ameliorating effects on tuberculosis disease and inflammatory diseases caused by Mycobacterium tuberculosis, more specifically Mycobacterium tuberculosis.
Description
본 발명은 결핵을 약물의 내성 및 장기간 고용량으로 인한 부작용 없이 치료할 수 있는 신규 균주, 구체적으로 락토바실러스 크리스파투스 균주, 보다 더 구체적으로 락토바실러스 크리스파투스 PMC201 균주 및 이의 용도에 관한 것이다. 본 발명에 따른 균주는 우수한 항결핵 활성을 가지므로 결핵 치료에 이용될 수 있다. 또한, 상기 균주는 세포 독성이 없고, 항염 효과도 존재하여 염증 질환 치료에 이용될 수 있다. The present invention relates to a new strain capable of treating tuberculosis without drug resistance and side effects due to long-term high doses, specifically Lactobacillus crispatus strain, more specifically Lactobacillus crispatus PMC201 strain, and uses thereof. The strain according to the present invention has excellent anti-tuberculosis activity and can be used to treat tuberculosis. In addition, the strain has no cytotoxicity and has an anti-inflammatory effect, so it can be used to treat inflammatory diseases.
결핵은 결핵균(Mycobacterium tuberculosis)에 의해 발생하는 감염병이다. 결핵은 전 세계의 주요 공중 보건문제로 남아있고, 매년 약 180만명의 사망을 유발한다. 다제내성(MDR), 광범위내성(XDR), 완전약제내성(TDR)을 포함한 약제내성 결핵균은 공중보건에 계속 위협이 되고 있다. 항생제 내성 결핵의 경우 기존 항생제의 치료 효과가 낮다. 또한, 약물의 장기간 고용량으로 인한 부작용은 심각한 문제를 일으키고 있다.Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. Tuberculosis remains a major public health problem worldwide, causing approximately 1.8 million deaths each year. Drug-resistant tuberculosis bacteria, including multidrug-resistant (MDR), extensively resistant (XDR), and completely drug-resistant (TDR), continue to pose a threat to public health. In the case of antibiotic-resistant tuberculosis, the treatment effectiveness of existing antibiotics is low. Additionally, side effects caused by long-term, high-dose use of drugs are causing serious problems.
따라서, 이러한 문제점을 극복하기 위해서, 기존의 화합물 기반의 항생제와는 다른 새로운 형태의 항결핵제 개발이 시급한 상황이다. Therefore, in order to overcome these problems, there is an urgent need to develop a new type of anti-tuberculosis drug that is different from existing compound-based antibiotics.
본 발명은 락토바실러스 크리스파투스 균주를 제공하는 것을 목적으로 한다.The purpose of the present invention is to provide Lactobacillus crispatus strains.
본 발명은 또한, 락토바실러스 크리스파투스 균주를 포함하는 항균용 조성물을 제공하는 것을 목적으로 한다. The present invention also aims to provide an antibacterial composition containing Lactobacillus crispatus strains.
본 발명은 또한, 락토바실러스 크리스파투스 균주를 포함하는 결핵 예방, 치료, 또는 개선용 조성물을 제공하는 것을 목적으로 한다. The present invention also aims to provide a composition for preventing, treating, or improving tuberculosis containing Lactobacillus crispatus strains.
본 발명은 또한, 상기 균주를 포함하는 항염증 질환 예방, 치료, 또는 개선용 조성물을 제공하는 것을 목적으로 한다.The present invention also aims to provide a composition for preventing, treating, or improving anti-inflammatory diseases containing the above strain.
본 발명은 락토바실러스 크리스파투스 균주 또는 이의 배양액을 제공한다.The present invention provides a Lactobacillus crispatus strain or a culture medium thereof.
또한, 본 발명은 락토바실러스 크리스파투스(Lactobacillus crispatus) 균주 또는 이의 배양액을 포함하는, 마이크로박테리움 속에 속하는 균에 대한 항균 활성이 있는 항균용 조성물을 제공한다.Additionally, the present invention provides an antibacterial composition having antibacterial activity against bacteria belonging to the genus Microbacterium, including a Lactobacillus crispatus strain or a culture medium thereof.
일 구현예로, 상기 항균용 조성물은 마이코박테리움 압세수스(M. abscessus), 마이코박테리움 아프리카눔(M. africanum), 마이코박테리움 아시아티쿰(M. asiaticum), 마이코박테리움 아비움복합체(M. avium complex (MAC)), 마이코박테리움 보비스(M. bovis), 마이코박테리움 켈로나(M. chelonae), 마이코박테리움 포푸이툼(M. fortuitum), 마이코박테리움 고르도내(M. gordonae), 마이코박테리움 헤모필룸(M. haemophilum), 마이코박테리움 인트라셀룰라레(M. intracellulare), 마이코박테리움 칸사시(M. kansasii), 마이코박테리움 렌티플라붐(M. lentiflavum), 마이코박테리움 레프래(M. leprae), 마이코박테리움 말모엔스(M. malmoense), 마이코박테리움 마리눔(M. marinum), 마이코박테리움 미크로티(M. microti), 마이코박테리움 플라이(M. phlei), 마이코박테리움 스크로풀라세움(M. scrofulaceum), 마이코박테리움 스메그마티스(M. smegmatis), 마이코박테리움 트리플렉스(M. triplex), 마이코박테리움 투베르쿨로시스(M. tuberculosis), 마이코박테리움 울세란스(M. ulcerans), 마이코박테리움 제노피(M. xenopi)로 구성된 군에서 선택된 하나 이상의 균에 대한 항균 활성이 있는, 항균용 조성물이다.In one embodiment, the antibacterial composition is Mycobacterium abscessus ( M. abscessus ), Mycobacterium africanum ( M. africanum ), Mycobacterium asiaticum ( M. asiaticum ), Mycobacterium M. avium complex (MAC), Mycobacterium bovis ( M. bovis ), Mycobacterium chelonae ( M. chelonae ), Mycobacterium fortuitum ( M. fortuitum ), My Cobacterium gordonae ( M. gordonae ), Mycobacterium haemophilum ( M. haemophilum ), Mycobacterium intracellulare ( M. intracellulare ), Mycobacterium kansasii ( M. kansasii ), Mycobacterium kansasii Cobacterium lentiflavum ( M. lentiflavum ), Mycobacterium leprae ( M. leprae ), Mycobacterium malmoense ( M. malmoense ), Mycobacterium marinum ( M. marinum ), Mycobacterium Therium microti ( M. microti ), Mycobacterium fly ( M. phlei ), Mycobacterium scrofulaceum ( M. scrofulaceum ), Mycobacterium smegmatis ( M. smegmatis ), Mycobacterium A group consisting of Terium triplex ( M. triplex ), Mycobacterium tuberculosis ( M. tuberculosis ), Mycobacterium ulcerans ( M. ulcerans ), and Mycobacterium xenopi ( M. xenopi ) It is an antibacterial composition that has antibacterial activity against one or more bacteria selected from.
일 구현예로, 상기 항균용 조성물은 마이코박테리움 투베르쿨로시스에 대한 항균 활성이 있는, 항균용 조성물이다.In one embodiment, the antibacterial composition is an antibacterial composition that has antibacterial activity against Mycobacterium tuberculosis.
일 구현예로, 상기 락토바실러스 크리스파투스(Lactobacillus crispatus) 균주는 KCTC 14673BP의 기탁번호를 갖는 균주이다.In one embodiment, the Lactobacillus crispatus strain is a strain with an accession number of KCTC 14673BP.
일 구현예로, 상기 항균용 조성물은 생체에 이용되는, 항균용 조성물이다.In one embodiment, the antibacterial composition is an antibacterial composition that is used in living organisms.
일 구현예로, 상기 항균용 조성물은 생체 외 환경에 이용되는, 항균용 조성물이다.In one embodiment, the antibacterial composition is an antibacterial composition used in an in vitro environment.
또한, 본 발명은 락토바실러스 크리스파투스(Lactobacillus crispatus) 균주 또는 이의 배양액을 포함하는, 결핵 치료 또는 예방용 약학적 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition for treating or preventing tuberculosis, comprising a Lactobacillus crispatus strain or a culture medium thereof.
일 구현예로, 상기 락토바실러스 크리스파투스(Lactobacillus crispatus) 균주는 KCTC 14673BP의 기탁번호를 갖는 균주이다.In one embodiment, the Lactobacillus crispatus strain is a strain with an accession number of KCTC 14673BP.
또한, 본 발명은 락토바실러스 크리스파투스(Lactobacillus crispatus) 균주 또는 이의 배양액을 포함하는, 결핵 예방 또는 개선용 식품 조성물을 제공한다.Additionally, the present invention provides a food composition for preventing or improving tuberculosis, comprising a Lactobacillus crispatus strain or a culture medium thereof.
일 구현예로, 상기 락토바실러스 크리스파투스(Lactobacillus crispatus) 균주는 KCTC 14673BP의 기탁번호를 갖는 균주이다.In one embodiment, the Lactobacillus crispatus strain is a strain with an accession number of KCTC 14673BP.
일 구현예로, 상기 식품 조성물은 건강 기능 식품 조성물인, 결핵 예방 또는 개선용 식품 조성물이다.In one embodiment, the food composition is a health functional food composition, a food composition for preventing or improving tuberculosis.
본 발명은 락토바실러스 크리스파투스 균주를 제공하며, 이는 항균 활성을 가진다. 보다 구체적으로, 상기 균주는 결핵균에 대한 항균 활성을 가진다. 이에 따라, 상기 균주는 결핵을 치료, 예방, 또는 개선하는데 이용될 수 있다. The present invention provides Lactobacillus crispatus strains, which have antibacterial activity. More specifically, the strain has antibacterial activity against Mycobacterium tuberculosis. Accordingly, the strain can be used to treat, prevent, or improve tuberculosis.
이때, 상기 균주는 화합물 기반의 약물 치료와 비교하여, 약물 내성 및 장기간 고용량 약물 치료로 인한 부작용이 없는 장점을 가진다. At this time, compared to compound-based drug treatment, the strain has the advantage of no drug resistance and no side effects due to long-term high-dose drug treatment.
도 1은 L. 크리스파투스 균주 PMC201의 게놈 시퀀싱 결과를 나타낸다.
도 1a는 PMC201 균주의 게놈이 CLgenomics 프로그램을 사용하여 시각화된 결과를 나타낸다. 안티센스 및 센스 가닥은 이종 그룹 클러스터(COG) 범주에 따라 색상이 지정되었고, 빨간색의 tRNA와 파란색의 rRNA는 주변에서 중앙으로 표시되었다. 내부 원은 GC 스큐(skew)를 나타낸다. 노란색과 파란색은 각각 양수 값과 음수 값을 나타낸다. GC 함량은 빨간색과 녹색으로 표시되었다.
도 1b는 COG 기능 범주의 상대적 풍부도를 분석한 결과이다.
도 2는 락토바실러스 종의 이용가능한 게놈으로 계산한 OrthoANI 결과를 나타낸다.
도 3은 결핵균에 대한 PMC201의 세포 내 사멸 효과를 나타낸 결과이다.
결핵균에 감염된 대식세포에서 세포내 H37RV(a, b) 및 XDR 균주(c, d)에 대한 PMC201 추출물의 효과는 CFU 분석(a, c) 및 AFB 방법(b, d)으로 확인되었다.
도 4는 브로스 공동 배양 조건에서 PMC201의 항결핵 활성을 나타낸 결과이다.
도 5는 PMC201의 세포독성 결과이다.
도 6은 기니피그에 대한 PMC201의 2주 반복 경구 투여 독성 결과를 나타낸다.
도 7은 인간 장내 미생물 생태계(SHIME) 시뮬레이터를 사용한 장내 미생물군집에 대한 PMC201의 효과를 나타낸다.
도 7a는 Bray-Curtis 기반 베타 세트 유의성 분석 및 주좌표 분석(PCoA)을 통해 평가된 베타 다양성 결과이다. 도 7b는 OTU(Operational Taxonomic Unit) 수에 따른 종 풍부도 및 도 7c는 계통발생적 다양성에 기반한 다양성 지수를 통해 측정된 알파-다양성 결과이다. 두 그룹 간의 평균 분류학적 구성을 도 7d는 문 및 도 7e는 강 수준에서 비교한 결과이다. 도 7f는 PMC201 투여군에서 락토바실러스 속이 유의하게 증가한 결과를 나타낸다. 분류학적 상대 풍부도는 In Wilcoxon rank-sum test를 사용하여 분석되었다.
도 8은 결핵균에 감염된 대식세포에서 아질산염 방출에 대한 PMC201의 효과를 나타낸다.Figure 1 shows the genome sequencing results of L. crispatus strain PMC201.
Figure 1a shows the results of the genome of strain PMC201 visualized using the CLgenomics program. Antisense and sense strands were colored according to cluster of heterogeneous group (COG) categories, with tRNA in red and rRNA in blue from periphery to center. The inner circle represents GC skew. Yellow and blue represent positive and negative values, respectively. GC content was indicated in red and green.
Figure 1b shows the results of analyzing the relative abundance of COG functional categories.
Figure 2 shows OrthoANI results calculated with available genomes of Lactobacillus species.
Figure 3 shows the results showing the intracellular killing effect of PMC201 on Mycobacterium tuberculosis.
The effect of PMC201 extract on intracellular H37RV (a, b) and XDR strains (c, d) in macrophages infected with Mycobacterium tuberculosis was confirmed by CFU analysis (a, c) and AFB method (b, d).
Figure 4 shows the results showing the anti-tuberculosis activity of PMC201 under broth co-culture conditions.
Figure 5 shows the cytotoxicity results of PMC201.
Figure 6 shows the toxicity results of repeated 2-week oral administration of PMC201 in guinea pigs.
Figure 7 shows the effect of PMC201 on the gut microbiome using the Human Intestinal Microbial Ecosystem (SHIME) simulator.
Figure 7a shows the beta diversity results evaluated through Bray-Curtis-based beta set significance analysis and principal coordinate analysis (PCoA). Figure 7b shows the species richness according to the number of OTUs (Operational Taxonomic Units) and Figure 7c shows the alpha-diversity results measured through the diversity index based on phylogenetic diversity. The average taxonomic composition between the two groups is compared at the phylum level in Figure 7d and the class level in Figure 7e. Figure 7f shows the results of a significant increase in the Lactobacillus genus in the PMC201 administered group. Taxonomic relative abundance was analyzed using the In Wilcoxon rank-sum test.
Figure 8 shows the effect of PMC201 on nitrite release from macrophages infected with Mycobacterium tuberculosis.
용어 정의Term Definition
달리 정의되지 않는 한, 본 명세서에서 사용되는 모든 기술적 및 과학적 용어는 본 발명이 속하는 기술분야의 당업자에 의해 통상적으로 이해되는 것과 동일한 의미를 가진다. 본 명세서에서 언급된 모든 간행물, 특허 및 기타 다른 참고문헌은 전체가 참고로 포함된다.Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.
항균antibacterial
본 명세서에서 사용되는 용어 "항균"은 미생물을 죽이거나 미생물의 성장을 억제하는 것을 의미한다. 일 실시예로, 본 명세서에서는 특정 균을 죽이거나 특정 균의 성장을 억제하는 의미로 사용될 수 있다. 구체적인 실시예로, 본 명세서에서는 마이코박테리움(Mycobacterium)속에 속하는 균을 죽이거나 이의 성장을 억제하는 의미로 사용될 수 있다. 보다 더 구체적인 실시예로, 본 명세서에서는 마이코박테리움 압세수스(M. abscessus), 마이코박테리움 아프리카눔(M. africanum), 마이코박테리움 아시아티쿰(M. asiaticum), 마이코박테리움 아비움복합체(M. avium complex (MAC)), 마이코박테리움 보비스(M. bovis), 마이코박테리움 켈로나(M. chelonae), 마이코박테리움 포푸이툼(M. fortuitum), 마이코박테리움 고르도내(M. gordonae), 마이코박테리움 헤모필룸(M. haemophilum), 마이코박테리움 인트라셀룰라레(M. intracellulare), 마이코박테리움 칸사시(M. kansasii), 마이코박테리움 렌티플라붐(M. lentiflavum), 마이코박테리움 레프래(M. leprae), 마이코박테리움 말모엔스(M. malmoense), 마이코박테리움 마리눔(M. marinum), 마이코박테리움 미크로티(M. microti), 마이코박테리움 플라이(M. phlei), 마이코박테리움 스크로풀라세움(M. scrofulaceum), 마이코박테리움 스메그마티스(M. smegmatis), 마이코박테리움 트리플렉스(M. triplex), 마이코박테리움 투베르쿨로시스(M. tuberculosis), 마이코박테리움 울세란스(M. ulcerans), 마이코박테리움 제노피(M. xenopi)로 구성된 군에서 선택된 하나 이상의 균을 죽이거나, 이의 성장을 억제하는 의미로 사용될 수 있다.As used herein, the term “antibacterial” means killing microorganisms or inhibiting their growth. As an example, in this specification, it may be used to mean killing specific bacteria or inhibiting the growth of specific bacteria. As a specific example, in this specification, it may be used to mean killing or inhibiting the growth of bacteria belonging to the genus Mycobacterium. In a more specific embodiment, in the present specification, Mycobacterium abscessus (M. abscessus), Mycobacterium africanum (M. africanum), Mycobacterium asiaticum (M. asiaticum), Mycobacterium M. avium complex (MAC), Mycobacterium bovis (M. bovis), Mycobacterium chelonae (M. chelonae), Mycobacterium fortuitum (M. fortuitum), Mycobacterium bovis (M. chelonae), Mycobacterium fortuitum (M. fortuitum) Cobacterium gordonae (M. gordonae), Mycobacterium haemophilum (M. haemophilum), Mycobacterium intracellulare (M. intracellulare), Mycobacterium kansasii (M. kansasii), mycobacterium Cobacterium lentiflavum (M. lentiflavum), Mycobacterium leprae (M. leprae), Mycobacterium malmoense (M. malmoense), Mycobacterium marinum (M. marinum), Mycobacterium Therium microti (M. microti), Mycobacterium fly (M. phlei), Mycobacterium scrofulaceum (M. scrofulaceum), Mycobacterium smegmatis (M. smegmatis), Mycobacterium A group consisting of M. triplex, M. tuberculosis, M. ulcerans, and M. xenopi It can be used to kill one or more selected bacteria or to inhibit their growth.
이하, 발명의 구체적인 내용을 개시한다.Hereinafter, specific details of the invention are disclosed.
Ⅰ. 균주Ⅰ. strain
1.One. 여성의 질액으로부터 수득 된 균주Strains obtained from female vaginal fluid
본 발명은 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 제공한다. 이때, 상기 균주는 일 실시예로, 여성의 질액으로부터 수득 된 균주일 수 있다. 다만, 이에 제한되지 않는다.The present invention provides a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain. At this time, as an example, the strain may be a strain obtained from a woman's vaginal fluid. However, it is not limited to this.
본 발명의 일 예에서, 락토바실러스 크리스파투스 PMC201 균주는 여성의 질액으로부터 수득되었으며, 신규한 균주로 동정되고, 한국생물자원센터에 KCTC 14673BP의 기탁번호로 기탁되었다.In one example of the present invention, the Lactobacillus crispatus PMC201 strain was obtained from a woman's vaginal fluid, identified as a new strain, and deposited at the Korea Center for Biological Resources under the accession number of KCTC 14673BP.
2.2. 세포 독성이 없는 균주Non-cytotoxic strains
- 생체 및 생체 외 환경에 이용되는 항균용 조성물 - Antibacterial composition used in living and in vitro environments
본 발명은 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 항균용 조성물을 제공한다. 이 때, 상기 균주는 세포 독성이 없으므로, 생체 내 및 생체 외에 이용되는 조성물에 포함될 수 있다.The present invention provides an antibacterial composition containing a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain. At this time, since the strain is not cytotoxic, it can be included in compositions used in vivo and in vitro.
일 실시예로, 상기 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 항균용 조성물은 생체에 투여되는 항균용 조성물 일 수 있다. 이때, 상기 생체는 살아있는 개체를 의미하며, 상기 개체는 인간을 포함한 포유동물을 포함한다. 즉, 본 발명은 일 실시예로, 인간을 포함하는 포유동물 내에 존재하는 원하지 않는 미생물에 대한 항균 활성을 가지는 항균용 조성물을 제공한다. 구체적인 일 실시예로, 본 발명은 인간을 포함하는 포유동물 내에 존재하는 병원성 세균에 대한 항균 활성을 가지는 항균용 조성물을 제공한다.In one embodiment, the antibacterial composition containing the strain belonging to the Lactobacillus crispatus species or the Lactobacillus crispatus PMC201 strain may be an antibacterial composition administered to a living body. At this time, the living organism refers to a living entity, and the entity includes mammals including humans. That is, in one embodiment, the present invention provides an antibacterial composition having antibacterial activity against unwanted microorganisms present in mammals, including humans. In one specific embodiment, the present invention provides an antibacterial composition having antibacterial activity against pathogenic bacteria present in mammals, including humans.
다른 일 실시예로, 상기 락토바실러스 크리스파투스(Lactobacillus crispatus)종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 항균용 조성물은 생체 외 환경에 존재하는 원하지 않는 미생물에 대한 항균 활성을 가지는 항균용 조성물 일 수 있다. 구체적인 일 실시예로, 상기 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 항균용 조성물은 생체 외 환경에 존재하는 병원성 세균에 대한 항균 활성을 가지는 항균용 조성물을 제공한다. 이때, 상기 생체 외 환경은 일 실시예로, 세척이 필요한 물질의 표면을 의미할 수 있다. 이때, 세척은 원하지 않는 미생물을 제거하는 것을 의미할 수 있다. 이때, 상기 제거는 균의 일부를 죽이거나, 균의 증식을 억제하거나, 균의 수를 감소시키는 것을 의미할 수 있다.In another embodiment, the antibacterial composition containing the Lactobacillus crispatus strain or the Lactobacillus crispatus PMC201 strain has antibacterial activity against unwanted microorganisms present in the in vitro environment. It may be an antibacterial composition. In a specific embodiment, the antibacterial composition containing a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain is an antibacterial composition having antibacterial activity against pathogenic bacteria present in an in vitro environment. Provides a composition for At this time, the in vitro environment is an example and may refer to the surface of a material that needs to be cleaned. At this time, washing may mean removing unwanted microorganisms. At this time, the removal may mean killing some of the bacteria, inhibiting the growth of the bacteria, or reducing the number of bacteria.
구체적인 일 실시예로, 상기 물질의 표면은 가구, 식기, 가전 등을 포함하는 생활용품의 표면을 포함할 수 있지만, 이에 제한되지 않고 세척이 필요한 물질의 표면을 모두 포함한다. 즉, 본 발명은 생체 외 환경에 존재하는 병원성 미생물에 대한 항균 활성을 가지는 항균용 조성물을 제공한다.In a specific embodiment, the surface of the material may include the surface of household goods including furniture, dishes, home appliances, etc., but is not limited thereto and includes all surfaces of materials that need to be cleaned. That is, the present invention provides an antibacterial composition having antibacterial activity against pathogenic microorganisms existing in an in vitro environment.
- 생체 및 생체 외 환경에 이용되는 항균 방법 - Antibacterial methods used in in vivo and in vitro environments
본 발명은 또한, 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 항균용 조성물을 이용한 항균 방법을 제공한다.The present invention also provides an antibacterial method using an antibacterial composition containing a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain.
이 때, 일 실시예로, 본 발명은 상기 조성물을 생체에 투여하는 것을 포함하는 항균 방법을 제공한다. 이때, 상기 생체는 살아있는 개체를 의미하며, 상기 개체는 인간을 포함한 포유동물을 포함한다. 즉, 본 발명은 상기 조성물을 인간을 포함하는 포유동물에 투여하는 것을 포함하는 항균 방법을 제공한다.At this time, in one embodiment, the present invention provides an antibacterial method comprising administering the composition to a living body. At this time, the living organism refers to a living entity, and the entity includes mammals including humans. That is, the present invention provides an antibacterial method comprising administering the composition to mammals, including humans.
다른 일 실시예로, 본 발명은 상기 조성물을 생체 외에 처리하는 것을 포함하는 항균 방법을 제공한다. 이때, 생체 외는 일 실시예로, 세척이 필요한 물질의 표면을 의미한다. 즉, 본 발명은 상기 조성물을 세척이 필요한 물질의 표면에 처리하는 항균 방법을 제공한다.In another embodiment, the present invention provides an antibacterial method comprising treating the composition in vitro. At this time, in vitro is an example and refers to the surface of a material that needs to be cleaned. That is, the present invention provides an antibacterial method for treating the surface of a material that needs to be cleaned with the composition.
실시예9 및 도5, 6을 참고하면, 본 발명에서 제공하는 락토바실러스 크리스파투스(Lactobacillus crispatus) PMC201 균주는 3.9Х107 CFU/ml에 해당하는 조건에서 PMC201 세포 추출물 처리는 유의한 세포독성을 나타내지 않았다. 즉, 해당 결과를 참고하여 생체 내 및 생체 외에 이용되는 항균용 조성물 또는 항균 방법에 활용될 수 있다.Referring to Example 9 and Figures 5 and 6, the Lactobacillus crispatus PMC201 strain provided by the present invention showed significant cytotoxicity when treated with PMC201 cell extract under conditions corresponding to 3.9Х10 7 CFU/ml. did not indicate In other words, the results can be referred to and used in antibacterial compositions or antibacterial methods used in vivo and in vitro.
3.3. 조성물에 포함된 균주의 형태Types of strains included in the composition
본 발명은 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주, 및 상기 균주를 포함하는 항균용 조성물, 결핵 및 염증성 질환 치료, 예방, 및 개선용 약학적 조성물 및 식품 조성물을 제공한다. 이 때, 일 실시예로, 상기 조성물은 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 생균, 사균 또는 이의 배양액 형태로 포함할 수 있다. 상기 배양액은 본 발명에서 제공하는 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 배양 원액일 수 있으며, 또는 이의 파쇄물, 원심분리 상층액 또는 펠렛, 농축액, 건조물 등을 포함할 수 있다.The present invention relates to a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain, an antibacterial composition containing the strain, a pharmaceutical composition for treating, preventing, and improving tuberculosis and inflammatory diseases, and A food composition is provided. At this time, in one embodiment, the composition may include a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain in the form of live cells, dead cells, or a culture solution thereof. The culture medium may be a culture solution containing the strain belonging to the Lactobacillus crispatus species or the Lactobacillus crispatus PMC201 strain provided by the present invention, or its lysate, centrifugation supernatant or pellet, or concentrate. , dried matter, etc.
Ⅱ. 균주의 용도Ⅱ. Uses of Strains
1.One. 항균활성Antibacterial activity
- 항균용 조성물 - Antibacterial composition
본 발명에서는 락토바실러스 크리스파투스(Lactobacillus crispatus)종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 항균용 조성물을 제공한다. 일 실시예로, 상기 항균용 조성물은 마이코박테리움(Mycobacterium)속에 속하는 균에 대한 항균활성을 가지는 것인, 항균용 조성물을 제공한다. 구체적인 일 실시예로, 상기 항균용 조성물은 마이코박테리움 압세수스(M. abscessus), 마이코박테리움 아프리카눔(M. africanum), 마이코박테리움 아시아티쿰(M. asiaticum), 마이코박테리움 아비움복합체(M. avium complex (MAC)), 마이코박테리움 보비스(M. bovis), 마이코박테리움 켈로나(M. chelonae), 마이코박테리움 포푸이툼(M. fortuitum), 마이코박테리움 고르도내(M. gordonae), 마이코박테리움 헤모필룸(M. haemophilum), 마이코박테리움 인트라셀룰라레(M. intracellulare), 마이코박테리움 칸사시(M. kansasii), 마이코박테리움 렌티플라붐(M. lentiflavum), 마이코박테리움 레프래(M. leprae), 마이코박테리움 말모엔스(M. malmoense), 마이코박테리움 마리눔(M. marinum), 마이코박테리움 미크로티(M. microti), 마이코박테리움 플라이(M. phlei), 마이코박테리움 스크로풀라세움(M. scrofulaceum), 마이코박테리움 스메그마티스(M. smegmatis), 마이코박테리움 트리플렉스(M. triplex), 마이코박테리움 투베르쿨로시스(M. tuberculosis), 마이코박테리움 울세란스(M. ulcerans), 마이코박테리움 제노피(M. xenopi)로 구성된 군에서 선택된 하나 이상의 균에 대한 항균 활성을 가지는 것인, 항균용 조성물을 제공한다. 보다 더 구체적인 실시예로, 상기 항균용 조성물은 마이코박테리움 투베르쿨로시스(M. tuberculosis)에 대한 항균 활성을 가지는 것인, 항균용 조성물을 제공한다.The present invention provides an antibacterial composition containing a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain. In one embodiment, the antibacterial composition provides an antibacterial composition that has antibacterial activity against bacteria belonging to the genus Mycobacterium . In a specific embodiment, the antibacterial composition is Mycobacterium abscessus ( M. abscessus ), Mycobacterium africanum ( M. africanum ), Mycobacterium asiaticum ( M. asiaticum ), Mycobacterium M. avium complex (MAC), Mycobacterium bovis ( M. bovis ), Mycobacterium chelonae ( M. chelonae ), Mycobacterium fortuitum ( M. fortuitum ), Mycobacterium gordonae ( M. gordonae ), Mycobacterium haemophilum ( M. haemophilum ), Mycobacterium intracellulare ( M. intracellulare ), Mycobacterium kansasii ( M. kansasii ), Mycobacterium lentiflavum ( M. lentiflavum ), Mycobacterium leprae ( M. leprae ), Mycobacterium malmoense ( M. malmoense ), Mycobacterium marinum ( M. marinum ), Mycobacterium Cobacterium microti ( M. microti ), Mycobacterium fly ( M. phlei ), Mycobacterium scrofulaceum ( M. scrofulaceum ), Mycobacterium smegmatis ( M. smegmatis ), My Consisting of Cobacterium triplex ( M. triplex ), Mycobacterium tuberculosis ( M. tuberculosis ), Mycobacterium ulcerans ( M. ulcerans ), and Mycobacterium xenopi ( M. xenopi ) Provided is an antibacterial composition having antibacterial activity against one or more bacteria selected from the group. In a more specific embodiment, the antibacterial composition provides an antibacterial composition having antibacterial activity against Mycobacterium tuberculosis ( M. tuberculosis ).
- 항균 방법 - Antibacterial method
본 발명에서는 락토바실러스 크리스파투스(Lactobacillus crispatus)종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 조성물을 생체에 투여하는 것을 포함하는 항균 방법을 제공한다. 또한, 본 발명에서는 락토바실러스 크리스파투스(Lactobacillus crispatus)종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 조성물을 생체 외 환경에 처리하는 것을 포함하는 항균 방법을 제공한다. 일 실시예로, 상기 항균 방법은 마이코박테리움(Mycobacterium)속에 속하는 균을 제거하는 것인, 항균 방법을 제공한다. 구체적인 일 실시예로, 상기 항균 방법은 마이코박테리움 압세수스(M. abscessus), 마이코박테리움 아프리카눔(M. africanum), 마이코박테리움 아시아티쿰(M. asiaticum), 마이코박테리움 아비움복합체(M. avium complex (MAC)), 마이코박테리움 보비스(M. bovis), 마이코박테리움 켈로나(M. chelonae), 마이코박테리움 포푸이툼(M. fortuitum), 마이코박테리움 고르도내(M. gordonae), 마이코박테리움 헤모필룸(M. haemophilum), 마이코박테리움 인트라셀룰라레(M. intracellulare), 마이코박테리움 칸사시(M. kansasii), 마이코박테리움 렌티플라붐(M. lentiflavum), 마이코박테리움 레프래(M. leprae), 마이코박테리움 말모엔스(M. malmoense), 마이코박테리움 마리눔(M. marinum), 마이코박테리움 미크로티(M. microti), 마이코박테리움 플라이(M. phlei), 마이코박테리움 스크로풀라세움(M. scrofulaceum), 마이코박테리움 스메그마티스(M. smegmatis), 마이코박테리움 트리플렉스(M. triplex), 마이코박테리움 투베르쿨로시스(M. tuberculosis), 마이코박테리움 울세란스(M. ulcerans), 마이코박테리움 제노피(M. xenopi)로 구성된 군에서 선택된 하나 이상의 균을 제거하는 것인, 항균 방법을 제공한다. 보다 더 구체적인 실시예로, 상기 항균 방법은 마이코박테리움 투베르쿨로시스(M. tuberculosis)를 제거하는 것인, 항균 방법을 제공한다.The present invention provides an antibacterial method comprising administering to a living body a composition containing a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain. Additionally, the present invention provides an antibacterial method comprising treating a composition containing a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain in an in vitro environment. In one embodiment, the antibacterial method provides an antibacterial method that removes bacteria belonging to the genus Mycobacterium . In a specific embodiment, the antibacterial method is performed on Mycobacterium abscessus ( M. abscessus ), Mycobacterium africanum ( M. africanum ), Mycobacterium asiaticum ( M. asiaticum ), Mycobacterium M. avium complex (MAC), Mycobacterium bovis ( M. bovis ), Mycobacterium chelonae ( M. chelonae ), Mycobacterium fortuitum ( M. fortuitum ), My Cobacterium gordonae ( M. gordonae ), Mycobacterium haemophilum ( M. haemophilum ), Mycobacterium intracellulare ( M. intracellulare ), Mycobacterium kansasii ( M. kansasii ), Mycobacterium kansasii Cobacterium lentiflavum ( M. lentiflavum ), Mycobacterium leprae ( M. leprae ), Mycobacterium malmoense ( M. malmoense ), Mycobacterium marinum ( M. marinum ), Mycobacterium Therium microti ( M. microti ), Mycobacterium fly ( M. phlei ), Mycobacterium scrofulaceum ( M. scrofulaceum ), Mycobacterium smegmatis ( M. smegmatis ), Mycobacterium A group consisting of Terium triplex ( M. triplex ), Mycobacterium tuberculosis ( M. tuberculosis ), Mycobacterium ulcerans ( M. ulcerans ), and Mycobacterium xenopi ( M. xenopi ) Provides an antibacterial method for removing one or more bacteria selected from. In a more specific embodiment, the antibacterial method provides an antibacterial method for removing Mycobacterium tuberculosis ( M. tuberculosis ).
이때, 상기 제거는 균의 일부를 죽이거나, 균의 증식을 억제하거나, 균의 수를 감소시키는 것을 의미할 수 있다.At this time, the removal may mean killing some of the bacteria, inhibiting the growth of the bacteria, or reducing the number of bacteria.
실시예8 및 도3,4를 참고하면, 본 발명에서 제공하는 균주는 항마이코박테리움 활성을 가지는 것을 확인할 수 있다. 이에 따라, 상기 균주는 항균용 조성물 또는 항균 방법에 이용될 수 있다.Referring to Example 8 and Figures 3 and 4, it can be confirmed that the strain provided in the present invention has anti-mycobacterium activity. Accordingly, the strain can be used in an antibacterial composition or antibacterial method.
2.2. 약학적 조성물pharmaceutical composition
1)One) 질환 치료 또는 예방용 약학적 조성물Pharmaceutical composition for treating or preventing disease
본 발명에서는 락토바실러스 크리스파투스(Lactobacillus crispatus)종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 질환 치료 또는 예방용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for treating or preventing diseases containing a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain.
본 명세서에서 사용되는 용어 "예방"이란 본 발명의 조성물의 투여에 의해 질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미하며, "치료"란 본 발명의 조성물을 투여함으로써 질환에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "prevention" refers to all actions that suppress or delay the onset of a disease by administering the composition of the present invention, and "treatment" refers to the improvement of disease symptoms by administering the composition of the present invention. It means any action that becomes or changes beneficially.
본 발명에서 제공하는 약학 조성물은 담체를 더 포함할 수 있으며, 본 발명의 약학 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences(19th ed., 1995)에 상세히 기재되어 있다.The pharmaceutical composition provided by the present invention may further include a carrier, and pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, such as lactose, dextrose, sucrose, sorbitol, Mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, Includes, but is not limited to, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
본 발명에 따른 상기 약학 조성물을 제제화할 경우, 당해 분야에서 통상적으로 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 하나 이상 혼합하여 제조할 수 있다.When formulating the pharmaceutical composition according to the present invention, it can be prepared by mixing one or more diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants commonly used in the field.
일 실시예로, 경구투여를 위한 고형 제제에는 정제, 환자, 산제, 과립제, 캡슐제, 트로키제 등이 포함되며, 이러한 고형 제제는 본 발명의 균주 또는 이의 배양액에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로스(sucrose) 또는 락토오스(lactose) 또는 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 다른 일 실시예로, 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면, 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제, 동결건조제제, 좌제 등이 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.In one embodiment, solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc., and such solid preparations include at least one excipient in the strain of the present invention or its culture medium, for example, It is prepared by mixing starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc are also used. In another embodiment, liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents and sweeteners are used. , fragrances, preservatives, etc. may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.
이때, 상기 약학적 조성물에 있어서, 상기 질환은 결핵 질환, 염증성 질환을 포함한다.At this time, in the pharmaceutical composition, the disease includes tuberculosis disease and inflammatory disease.
- 결핵질환 - Tuberculosis disease
상기 결핵 질환은 결핵균에 의해 전염되는 감염성 질환을 의미한다. 상기 결핵 질환은 일 실시예로, 폐결핵, 소화기계 결핵, 비뇨생식계 결핵, 골관절 결핵, 중추신경계 결핵을 모두 포함한다. 즉, 본 발명에서 개시하는 결핵 질환 예방 및 치료용 약학적 조성물은 일 실시예로, 폐결핵, 소화기계 결핵, 비뇨생식계 결핵, 골관절 결핵, 중추신경계 결핵으로 구성된 군에서 선택된 하나 이상의 질환 예방 및 치료용 약학적 조성물을 의미할 수 있다.The tuberculosis disease refers to an infectious disease transmitted by tuberculosis bacteria. In one embodiment, the tuberculosis disease includes pulmonary tuberculosis, digestive tuberculosis, genitourinary tuberculosis, bone and joint tuberculosis, and central nervous system tuberculosis. That is, the pharmaceutical composition for preventing and treating tuberculosis disease disclosed in the present invention is an embodiment, and is used for preventing and treating one or more diseases selected from the group consisting of pulmonary tuberculosis, digestive tuberculosis, urogenital tuberculosis, bone and joint tuberculosis, and central nervous system tuberculosis. It may refer to a pharmaceutical composition.
실시예 8 및 도3, 4를 참고하면, 본 발명에서 제공하는 균주는 항결핵 활성을 가지므로, 결핵질환을 치료하는데 이용될 수 있다.Referring to Example 8 and Figures 3 and 4, the strain provided in the present invention has anti-tuberculosis activity and can be used to treat tuberculosis disease.
- 염증성 질환 - Inflammatory disease
상기 염증성 질환은 심내막염, 심낭염, 심근염, 구내염, 간염, 담낭염, 담도염, 식도염, 대장염, 위염, 장염, 충수염, 췌장염, 기관지염, 갑상선염, 난소염, 방광염, 요도염, 전립선염, 질염, 골수염, 관절염 등의 염증성 질환을 포함하나, 이에 제한되지 않고, 당업계에 염증성 질환으로 알려진 모든 염증성 질환을 포함한다.The inflammatory diseases include endocarditis, pericarditis, myocarditis, stomatitis, hepatitis, cholecystitis, cholangitis, esophagitis, colitis, gastritis, enteritis, appendicitis, pancreatitis, bronchitis, thyroiditis, oophoritis, cystitis, urethritis, prostatitis, vaginitis, osteomyelitis, arthritis, etc. Including, but not limited to, inflammatory diseases, and includes all inflammatory diseases known in the art as inflammatory diseases.
실시예11 및 도8을 참고하면, 본 발명에서 제공하는 락토바실러스 크리스파투스(Lactobacillus crispatus) PMC201 균주가 항염증 활성을 가지는 것을 확인할 수 있다. 이에 따라, 본 명세서에 개시되는 락토바실러스 크리스파투스(Lactobacillus crispatus) PMC201 균주는 항염증 용도로 이용될 수 있다. Referring to Example 11 and Figure 8, it can be confirmed that the Lactobacillus crispatus PMC201 strain provided by the present invention has anti-inflammatory activity. Accordingly, the Lactobacillus crispatus PMC201 strain disclosed herein can be used for anti-inflammatory purposes.
2)2) 질환 치료 방법How to treat the disease
본 발명에서는 락토바실러스 크리스파투스(Lactobacillus crispatus)종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 조성물을 개체에 투여하는 것을 포함하는 질환 치료 방법을 제공한다. 이때, 상기 개체는 인간을 포함하는 포유동물을 의미할 수 있다. 이때, 상기 질환은 위에 기재한 결핵 질환, 염증성 질환에 관한 내용을 모두 포함한다. The present invention provides a method of treating a disease comprising administering to an individual a composition containing a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain. At this time, the entity may mean a mammal, including humans. At this time, the disease includes all of the tuberculosis disease and inflammatory disease described above.
본 발명에서 제공하는 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The composition provided in the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and the dosage is determined by the patient's condition and weight, and the severity of the disease. It varies depending on the degree, drug form, administration route and time, but can be appropriately selected by a person skilled in the art.
본 발명에 따른 조성물은 약학적으로 유효한 양으로 투여된다. 상기 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the present invention is administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the patient's disease, the activity of the drug, and the It may depend on factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
구체적으로, 본 발명에 따른 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 ㎏당 0.1 내지 100 mg, 바람직하게는 0.5 내지 10 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and weight, and is generally administered at 0.1 to 100 mg, preferably 0.5 to 10 mg, per 1 kg of body weight every day or every other day, or 1 It can be administered in divided doses 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.
3.3. 식품 조성물food composition
본 발명에서는 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 포함되는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 질환 예방 또는 개선용 식품 조성물을 제공한다. 이때, 상기 질환은 위에 기재한 결핵 질환, 염증성 질환에 관한 내용을 모두 포함한다.The present invention provides a food composition for preventing or improving diseases containing a strain included in the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain. At this time, the disease includes all of the tuberculosis disease and inflammatory disease described above.
이때, 상기 식품 조성물은 건강 기능 식품 조성물을 포함하며, 상기 건강 기능 식품은 상기 락토바실러스 크리스파투스(Lactobacillus crispatus) 종에 속하는 균주 또는 락토바실러스 크리스파투스 PMC201 균주를 포함하는 것이라면 특별히 제한하지 않으나, 바람직하게는 건강기능 식품 조성물 전체 중량 중 상기 균주를 0.01 ~ 20 중량%로 포함할 수 있다.At this time, the food composition includes a health functional food composition, and the health functional food is not particularly limited as long as it contains a strain belonging to the Lactobacillus crispatus species or a Lactobacillus crispatus PMC201 strain, Preferably, the above-mentioned strain may be included in an amount of 0.01 to 20% by weight based on the total weight of the health functional food composition.
상기 건강기능 식품의 종류에는 통상적으로 제조 및/또는 판매되는 것이라면 특별히 제한하지 않는다. 예를 들면, 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료 및 비타민 복합제 등이 있으며, 환제, 분말, 과립, 침제, 정제, 캡슐 또는 음료인 형태로 사용할 수 있고 통상적인 의미에서의 건강기능 식품을 모두 포함한다.There are no particular restrictions on the types of health functional foods as long as they are commonly manufactured and/or sold. For example, meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. It can be used in the form of pills, powders, granules, precipitates, tablets, capsules or beverages, and includes all health functional foods in the conventional sense.
본 발명에서 건강 음료 조성물은 상술한 바와 같은 균주를 포함하는 것 외에는 액체성분에는 특별한 제한은 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.In the present invention, the health drink composition has no particular restrictions on the liquid ingredients other than containing the strains described above, and may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary drinks.
상기 천연 탄수화물의 예로는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린; 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제 (타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.Examples of the natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; polysaccharides such as dextrins, cyclodextrins; These are common sugars such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. there is.
상술한 추가 성분 이외에 본 발명의 건강기능 식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 포함할 수 있다.In addition to the additional ingredients described above, the health functional food of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and additives (cheese, chocolate, etc.), pectic acid and its It may include salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.
그 밖에 본 발명의 건강기능 식품들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다.In addition, the health functional foods of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination.
이하, 실시예를 통해 본 발명을 상세히 설명하고자 한다.Hereinafter, the present invention will be described in detail through examples.
이때 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들의 실시예에 의해 제한되지 않는다는 것은 본 발명이 속한 기술분야에서 통상의 지식을 가진 자에 있어 자명하다.At this time, the examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples.
실시예Example
1.One. 인간 장 및 질 유래 프로바이오틱스의 제조Preparation of human intestinal and vaginal derived probiotics
여성 질액 샘플링은 순천향대학교 천안병원(eIRB) 윤리위원회 산하 순천향대학교 천안병원 산부인과에서 시행되었다(IRB No. 2019-10-017-005). 면봉을 사용하여 건강한 폐경 전 여성(연령: 18-50세)의 전체 질벽과 자궁경부를 채취했다. 시료를 식염수에 희석하여 MRS agar(BD Difco, USA)에 도말한 후 37℃에서 2일간 배양하였다. 다음날, de Man, Rogosa and Sharp(MRS) 한천(Goldstein et al., 2015)에 대한 락토바실러스 집락 특성(보통 백색 및 점액성 집락)에 기초하여 단일 집락으로부터 순수 배양을 수행하고 37℃에서 2일 배양하였다. 이후 순수한 단일 콜로니를 MRS 브로쓰(BD Difco, USA)에 접종하고 진탕 배양기(BioFree, 한국)에서 37℃에서 밤새 배양하였다. 이어서, OD600nm에서 휴대용 VIS 분광광도계(DR 1900, Hatch, USA)를 사용하여 프로바이오틱 배양물의 성장을 확인하였다. 그런 다음 15% 글리세롤에서 프렙하고 식별을 위해 회사(Biofact, 한국)에 16S rRNA 시퀀싱을 위해 보냈다. 인간 장 유래 프로바이오틱스는 한국 장내 미생물군유전체은행(KGMB, Korea)에서 입수했다.Female vaginal fluid sampling was conducted at the Department of Obstetrics and Gynecology at Soonchunhyang University Cheonan Hospital under the Ethics Committee of Soonchunhyang University Cheonan Hospital (eIRB) (IRB No. 2019-10-017-005). The entire vaginal wall and cervix of healthy premenopausal women (age: 18–50 years) were sampled using cotton swabs. The sample was diluted in saline solution, spread on MRS agar (BD Difco, USA), and cultured at 37°C for 2 days. The next day, pure cultures were performed from single colonies based on Lactobacillus colony characteristics (usually white and mucoid colonies) on de Man, Rogosa and Sharp (MRS) agar (Goldstein et al., 2015) and incubated at 37°C for 2 days. Cultured. Then, pure single colonies were inoculated into MRS broth (BD Difco, USA) and cultured overnight at 37°C in a shaking incubator (BioFree, Korea). Subsequently, the growth of the probiotic culture was confirmed using a portable VIS spectrophotometer (DR 1900, Hatch, USA) at OD 600 nm . They were then prepared in 15% glycerol and sent for 16S rRNA sequencing to our company (Biofact, Korea) for identification. Human gut-derived probiotics were obtained from the Korean Gut Microbiome Bank (KGMB, Korea).
2.2. 시험 후보 프로바이오틱스의 세포 추출물 제조Preparation of cell extracts of test candidate probiotics
16s rRNA 시퀀싱 결과를 기반으로, 가능한 모든 후보 프로바이오틱스는 스톡 배양에서 얻어지고 이전에서 언급한 조건에 따라 MRS 브로스(0.1% 접종)에서 배양되었다. 다음 날 세균 증식을 확인한 후 원심분리기(Combi R515, Hanil Scientific, Inc. Korea)를 이용하여 1000 xg에서 10분간 원심분리하였다. 펠렛을 멸균 증류수로 세척하였다. 원심분리와 세척과정을 3회 반복하여 배양액과 기타 오염물질을 적절히 제거하였다. 마지막으로, 펠렛을 0.85% 염화나트륨 용액에 재현탁하여 대략 2Х1010 CFU/ml의 농도가 되도록 하였다. 다음으로, 현탁액을 bead-beating(FastPrep-24 5G, MP Biomedicals, USA)을 통한 기계적 용해를 위해 0.1 mm의 저밀도 zirconia/silica 비드를 함유하는 용해 매트릭스 B 튜브(MP Biomedicals, USA)에 무균적으로 옮겼다. 눈에 보이는 입자가 없는 상층액을 항결핵 스크리닝을 위한 세포 추출물로 수집했다.Based on the 16s rRNA sequencing results, all possible candidate probiotics were obtained from stock cultures and cultured in MRS broth (0.1% inoculum) according to the previously mentioned conditions. The next day, after confirming bacterial growth, the cells were centrifuged at 1000 xg for 10 minutes using a centrifuge (Combi R515, Hanil Scientific, Inc. Korea). The pellet was washed with sterile distilled water. The centrifugation and washing process was repeated three times to properly remove culture medium and other contaminants. Finally, the pellet was resuspended in 0.85% sodium chloride solution to a concentration of approximately 2Х10 10 CFU/ml. Next, the suspension was aseptically transferred into lysis matrix B tubes (MP Biomedicals, USA) containing 0.1 mm low-density zirconia/silica beads for mechanical lysis via bead-beating (FastPrep-24 5G, MP Biomedicals, USA). moved. The supernatant without visible particles was collected as a cell extract for anti-tuberculosis screening.
3.3. M. tuberculosis 및 배양 조건M. tuberculosis and culture conditions
M. tuberculosis H37Rv(ATCC 27294)는 American Type Culture Collection(ATCC, USA)에서 구입하였고, M. tuberculosis의 임상적으로 분리된 광범위 약제내성(XDR) 균주(KMRC 00203-00197)는 한국 마이코박테리움 자원센터(KMRC) (충북, 한국)에서 구입하였다. M. tuberculosis은 10% OADC(올레산-알부민-덱스트로스-카탈라제)(BD Difco, USA) 및 0.5% Tween 80(Sigma-Aldrich, USA)이 보충된 Middlebrook 7H9 브로쓰(BD Difco, USA)에서 배양되었고, 37℃의 쉐이킹 인큐베이터에서 배양되었다. M. tuberculosis을 이용한 실험은 순천향대학교 동물생물안전 3등급 연구실(ABSL-3, KDCA-20-3-04)에서 진행하였다.M. tuberculosis H37Rv (ATCC 27294) was purchased from the American Type Culture Collection (ATCC, USA), and a clinically isolated extensively drug-resistant (XDR) strain of M. tuberculosis (KMRC 00203-00197) was obtained from Korean Mycobacterium. It was purchased from the Resource Center (KMRC) (Chungbuk, Korea). M. tuberculosis was cultured in Middlebrook 7H9 broth (BD Difco, USA) supplemented with 10% OADC (oleic acid-albumin-dextrose-catalase) (BD Difco, USA) and 0.5% Tween 80 (Sigma-Aldrich, USA). and was cultured in a shaking incubator at 37°C. Experiments using M. tuberculosis were conducted at Soonchunhyang University Animal Biosafety Level 3 Laboratory (ABSL-3, KDCA-20-3-04).
4.4. 상업용 약물commercial drugs
이소니아지드(INH), 스트렙토마이신(STR), 리팜피신(RIF), 피라지나메이드(PZA) 및 에탐부톨(EMB)은 Sigma-Aldrich(St. Louis, MO, USA)에서 구입하였다. Isoniazid (INH), streptomycin (STR), rifampicin (RIF), pyrazinamade (PZA), and ethambutol (EMB) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
5.5. 세포주 및 배양 조건Cell lines and culture conditions
Murine raw 264.7 대식세포 세포주(KCLB 40071)는 한국 세포주 은행(KCLB, Seoul, Korea)에서 구입하여 37℃, 5% CO2의 가습 환경에서 10% 소태아혈청(FBS) (Gibco, USA) 및 1% 항생제(100U/ml의 페니실린 및 100μg/ml의 스트렙토마이신)(HyClone, USA)이 보충된 Dulbecco's Modified Eagle Medium(DMEM, Gibco, USA)에서 유지하였다. Murine raw 264.7 macrophage cell line (KCLB 40071) was purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and incubated with 10% fetal bovine serum (FBS) (Gibco, USA) and 1 ml in a humidified environment of 5% CO 2 at 37°C. % were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) supplemented with antibiotics (100 U/ml of penicillin and 100 μg/ml of streptomycin) (HyClone, USA).
6.6. 항결핵 효과가 있는 프로바이오틱스의 분리Isolation of probiotics with anti-tuberculosis effect
트립판 블루 분석 기반 세포독성은 인간의 장과 질에서 추출한 30여종의 프로바이오틱스를 이용하여 측정하였다(표 1). Trypan blue assay-based cytotoxicity was measured using 30 types of probiotics extracted from human intestines and vagina (Table 1).
a. 프로바이오틱스 0.25% 농도에서 독성을 나타내지 않음.b. 프로바이오틱스 0.5% 농도에서 독성을 나타내지 않음.a. Probiotics do not exhibit toxicity at a concentration of 0.25%.b. Not toxic at 0.5% probiotic concentration.
KGMB, Korean Gut Microbiome Bank.KGMB, Korean Gut Microbiome Bank.
SCH hospital, Soonchunhyang University Hospital.SCH hospital, Soonchunhyang University Hospital.
0.1% 접종 조건에서 갓 배양한 프로바이오틱 배양 배지(OD600nm = 1.0, 30ml)를 0.85% NaCl 용액으로 3회 세척하여 최종적으로 1ml가 되도록 하여 대식세포에 0.5%와 0.25% 농도로 처리하였다. L. crispatus, L. paragasseri, L. animalis, L. brevis, L. crispatus, 및 L. sakei 는 0.25% 농도에서 독성을 나타내지 않았다. L. paragasseri 및 L. crispatus 는 0.5%에서도 독성이 없었다. 이 중, 질 유래 L. crispatus는 세포 내 항마이코박테리아 활성 분석을 통해 항결핵 효과가 있는 것으로 확인되었다.Probiotic culture medium (OD600nm = 1.0, 30ml) freshly cultured under 0.1% inoculation conditions was washed three times with 0.85% NaCl solution to make a final volume of 1ml, and treated with macrophages at 0.5% and 0.25% concentrations. L. crispatus , L. paragasseri , L. animalis , L. brevis , L. crispatus , and L. sakei did not show toxicity at a concentration of 0.25%. L. paragasseri and L. crispatus were not toxic even at 0.5%. Among these, vaginal-derived L. crispatus was confirmed to have an anti-tuberculosis effect through analysis of intracellular anti-mycobacterial activity.
7.7. 균주 분석Strain analysis
7-1.7-1. 16S rRNA 유전자 기반 메타게놈 분석16S rRNA gene-based metagenomic analysis
제조업체의 지침에 따라 QIAamp DNA Mini Kit(Qiagen, Germany)를 사용하여 각 샘플에서 총 DNA를 추출하였다. 추출 후 dsDNA HS Assay Kit(Invitrogen, USA)를 사용하여 Qubit Fluorometer로 DNA를 정량화하였다. 또한 아가로스겔 전기영동을 하여 ChemiDoc(BioRad, USA)로 품질을 확인하였다. 다음으로, 우리는 V4 영역에 대한 범용 프라이머를 사용하여 16S rRNA 유전자의 V4 초가변 영역을 증폭하였다. (Forward primer: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 및 Reverse primer: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). Agencourt AMPure XP(Beckman Coulter, USA) 비드를 PCR 산물을 정제하기 위해 모든 PCR 단계 후에 사용하였다. Qubit dsDNA HS Assay Kit(Thermo Fisher Scientific, USA)를 사용하여 PCR 산물을 정량화하였다. Nextera XT DNA Library Prep Kit(Illumina, USA)를 사용하여 Metagenomic 라이브러리를 준비하였다. 다음으로 샘플을 정규화하고 풀링하고 PhiX control v3(Illumina, USA)와 혼합하고 Illumina iSeq 100 플랫폼(Illumina, USA)을 사용하여 시퀀싱하였다. 마지막으로 시료 내 전체 미생물 조성을 나타내는 염기서열 데이터를 Ezbiocloud 소프트웨어(Chunlab, Korea)를 사용하여 분석하였다. Total DNA was extracted from each sample using the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. After extraction, DNA was quantified with a Qubit Fluorometer using the dsDNA HS Assay Kit (Invitrogen, USA). Additionally, agarose gel electrophoresis was performed and the quality was confirmed using ChemiDoc (BioRad, USA). Next, we amplified the V4 hypervariable region of the 16S rRNA gene using universal primers for the V4 region. (Forward primer: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and Reverse primer: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). Agencourt AMPure XP (Beckman Coulter, USA) beads were used after all PCR steps to purify PCR products. PCR products were quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). Metagenomic libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina, USA). Next, samples were normalized, pooled, mixed with PhiX control v3 (Illumina, USA), and sequenced using the Illumina iSeq 100 platform (Illumina, USA). Finally, sequence data representing the overall microbial composition in the sample were analyzed using Ezbiocloud software (Chunlab, Korea).
7-2.7-2. 16s rRNA 유전자 시퀀싱 기반 식별16s rRNA gene sequencing-based identification
항결핵 효과가 있는 여성 질 유래 균주에 대해 16s rRNA 유전자 염기서열 분석을 수행하였다(표 2). 16s rRNA gene sequence analysis was performed on female vaginal strains with anti-tuberculosis effects (Table 2).
NCBI, National Center for Biotechnology Information.NCBI(National Center for Biotechnology Information) 데이터베이스의 L. crispatus, L. amylovorus 및 L. kitasatonis의 해당 서열과 99% 이상의 서열 동일성을 공유하였다.It shared more than 99% sequence identity with the corresponding sequences of L. crispatus, L. amylovorus, and L. kitasatonis in the National Center for Biotechnology Information (NCBI) database.
7-3.7-3. 전체 게놈 시퀀싱Whole genome sequencing
균주 레벨에서 후보 프로바이오틱스를 식별하기 위해, 미생물을 식별, 비교 및 분류하기 위한 고해상도를 갖는 강력한 방법인 전체 게놈 시퀀싱(WGS) 분석을 수행하였다(Gilchrist et al., 2015). 시퀀싱을 위해, 37℃에서 24시간 동안 MRS 브로스에서 프로바이오틱스를 배양하였다. 배양하는 동안, 기하급수적 성장 단계에 대해 주기적으로 배양 조건을 확인하고 배양액을 원심분리하여 펠렛을 얻었다. 마지막으로 WGS를 위해 상업용 DNA 시퀀싱 서비스(Chunlab, Inc., Seoul, Korea)에 박테리아 펠렛을 보냈다. PacBio 시퀀싱 데이터는 HGAP2 프로토콜(Pacific Biosciences, USA)을 사용하여 PacBio SMRT Analysis 2.3.0으로 수집되었다. PacBio 시퀀싱 데이터의 결과 contig는 Circlator 1.4.0(Sanger Institute, UK)을 사용하여 추가로 원형화되었다. 시퀀스 리드 및 어셈블리는 NCBI 데이터베이스(BioProject: PRJNA735892)에 보관된다. 예측 코딩 시퀀스(CDS)는 이종 그룹 클러스터(COG) 기능 분류에 따라 그룹화되었다(Tatusov et al., 2000). 분류학적 목적을 위해 Orthologous Average nucleotide identity(OrthoANI) 방법을 사용하여 평균 뉴클레오티드 동일성을 계산하였다(Lee et al., 2016).To identify candidate probiotics at the strain level, whole genome sequencing (WGS) analysis was performed, a powerful method with high resolution to identify, compare, and classify microorganisms (Gilchrist et al., 2015). For sequencing, probiotics were cultured in MRS broth for 24 hours at 37°C. During the culture, the culture conditions were periodically checked for exponential growth phase, and the culture was centrifuged to obtain a pellet. Finally, the bacterial pellet was sent to a commercial DNA sequencing service (Chunlab, Inc., Seoul, Korea) for WGS. PacBio sequencing data were collected with PacBio SMRT Analysis 2.3.0 using the HGAP2 protocol (Pacific Biosciences, USA). The resulting contigs from PacBio sequencing data were further circularized using Circlator 1.4.0 (Sanger Institute, UK). Sequence reads and assemblies are deposited in the NCBI database (BioProject: PRJNA735892). Predicted coding sequences (CDS) were grouped according to the cluster of heterogeneous groups (COG) functional classification (Tatusov et al., 2000). For taxonomic purposes, average nucleotide identity was calculated using the Orthologous Average nucleotide identity (OrthoANI) method (Lee et al., 2016).
7-4.7-4. 전체 게놈 특성 및 비교 분석Whole genome characterization and comparative analysis
항결핵 효과가 있는 균주에 대해 전체 게놈 시퀀싱 분석을 수행하였다(도 1). 평균 GC 함량이 37.3%인 2,391,600bp의 원형 염색체를 생성하였다(도 1a). 그것은 2,031개의 단백질을 인코딩하는 2,428개의 CDS를 가지고 있었다. 이 단백질은 COG 기능 분류에 따라 그룹화되었다(도 1b). 생물학적 기능은 1,458개의 예측된 단백질에 대해 정의될 수 있는 반면 573개의 CDS는 다른 유기체에서 알려지지 않은 기능을 가진 보존된 단백질과 상동성을 가졌다. 나머지 397개의 가상 단백질은 데이터베이스에 있는 알려진 단백질과 일치하지 않았다. 또한 71개의 tRNA와 15개의 rRNA 유전자가 예측되었다. Whole genome sequencing analysis was performed on strains with anti-tuberculosis effects (Figure 1). A circular chromosome of 2,391,600 bp with an average GC content of 37.3% was generated (Figure 1a). It had 2,428 CDSs encoding 2,031 proteins. These proteins were grouped according to COG functional classification (Figure 1B). Biological functions could be defined for 1,458 predicted proteins, while 573 CDSs had homology to conserved proteins with unknown functions in other organisms. The remaining 397 hypothetical proteins did not match any known proteins in the database. Additionally, 71 tRNA and 15 rRNA genes were predicted.
7-5.7-5. 이종 평균 뉴클레오티드 동일성Heterogeneous average nucleotide identity
OrthoANI 분석은 높은 처리량의 게놈 시퀀싱 데이터를 사용하여 수행되었다(도 2). 이 연구에서 확인된 분리주의 게놈 서열은 공개적으로 이용 가능한 L. 크리스파투스의 B4, C25 및 1D 균주와 각각 97.22%, 96.93% 및 96.79% 서열 유사성을 공유하였다. 그러나 L. crispatus 이외의 종과 90% 미만의 게놈 서열 유사성을 공유하였다. 따라서 항결핵 효과가 있는 균주는 L. 크리스파투스임을 확인하였다.OrthoANI analysis was performed using high-throughput genome sequencing data (Figure 2). The genome sequence of the isolate identified in this study shared 97.22%, 96.93%, and 96.79% sequence similarity with publicly available B4, C25, and 1D strains of L. crispatus, respectively. However, it shared less than 90% genome sequence similarity with species other than L. crispatus. Therefore, it was confirmed that the strain with anti-tuberculosis effect was L. crispatus.
7-6.7-6. L. 크리스파투스 균주의 염색체 특성 비교Comparison of chromosomal characteristics of L. crispatus strains
항결핵 효과가 있는 L. crispatus의 유전적 특성을 같은 종의 다른 균주와 비교하였다(표 3). The genetic characteristics of L. crispatus, which has anti-tuberculosis effects, were compared with other strains of the same species (Table 3).
B4, https://www.ncbi.nlm.nih.gov/assembly/GCF_013456995.1/; C25 (Rezvani et al., 2016); ST1 (Ojala et al., 2010); ST1 (Edelman et al., 2012); 1D, https://www.ncbi.nlm.nih.gov/assembly/GCF_013487905.1/; NCK971, https://www.ncbi.nlm.nih.gov/assembly/GCF_008694205.1/.이 균주를 L. 크리스파투스의 B4, C25, ST1, 1D 및 NCK971 균주와 비교했을 때, 소스, 게놈 크기(bp), G+C 함량(%), 예측 CDS, rRNA 수 및 tRNA 수는 서로 달랐다. 따라서 이 L. 크리스파투스 PMC201은 기존 균주와 다른 신규 균주임을 알 수 있었다.B4, https://www.ncbi.nlm.nih.gov/assembly/GCF_013456995.1/; C25 (Rezvani et al., 2016); ST1 (Ojala et al., 2010); ST1 (Edelman et al., 2012); 1D, https://www.ncbi.nlm.nih.gov/assembly/GCF_013487905.1/; NCK971, https://www.ncbi.nlm.nih.gov/assembly/GCF_008694205.1/. When comparing this strain to strains B4, C25, ST1, 1D and NCK971 of L. crispatus, source, genome Size (bp), G+C content (%), predicted CDS, rRNA number, and tRNA number were different. Therefore, it was found that this L. crispatus PMC201 was a new strain different from the existing strain.
8.8. 항균 활성 실험Antibacterial activity test
8-1.8-1. AFB 염색에 의한 세포 내 항마이코박테리움 활성Intracellular antimycobacterial activity by AFB staining
대식세포를 세포 배양 슬라이드(SPL life science, Korea)에 5Х10 cells/ml의 밀도로 접종하고 70-80% confluence에 도달할 때까지 37℃, 5% CO2에서 밤새 배양하였다. 이어서, 세포 단층을 세포당 10:1 간균의 감염 다중도(MOI)로 2시간 동안 M. tuberculosis에 노출시켜 세포가 간균을 흡수할 수 있도록 하였다. 다음으로 세포를 PBS(phosphate-buffer saline)로 3회 세척하여 남아있는 세포 외 간균을 제거하였다. 그 후, 감염된 세포를 다른 항생제와 함께 시험 후보 프로바이오틱스의 세포 추출물의 소정 농도의 존재 하에 약물 없는(drug-free) DMEM 배지에서 3일 동안 배양하였다. 시험 물질이 없는 상태에서 감염된 세포를 용매 DMSO로 양성 대조군으로 사용하였다. 처리 3일 후 세포를 세척하고 AFB(acid fast bacilli) 염색을 한 후 광학현미경(AX10, Carl Zeiss, Germany)으로 100배 확대 관찰하였다. 이때 carbol fuchsin과 methylene blue를 사용하여 각각 M. tuberculosis와 대식세포를 염색하였다. Macrophages were seeded on cell culture slides (SPL life science, Korea) at a density of 5Х10 cells/ml and cultured overnight at 37°C and 5% CO 2 until 70-80% confluence was reached. Cell monolayers were then exposed to M. tuberculosis for 2 hours at a multiplicity of infection (MOI) of 10:1 bacilli per cell to allow cells to take up the bacilli. Next, the cells were washed three times with PBS (phosphate-buffer saline) to remove remaining extracellular bacilli. Thereafter, the infected cells were cultured in drug-free DMEM medium for 3 days in the presence of predetermined concentrations of cell extracts of test candidate probiotics along with other antibiotics. Infected cells in the absence of test substances were used as a positive control with the solvent DMSO. After 3 days of treatment, the cells were washed, stained with AFB (acid fast bacilli), and observed at 100x magnification using an optical microscope (AX10, Carl Zeiss, Germany). At this time, carbol fuchsin and methylene blue were used to stain M. tuberculosis and macrophages, respectively.
8-2.8-2. CFU 분석에 의한 세포내 항마이코박테리움 활성 Intracellular antimycobacterium activity by CFU assay
대식세포를 96-웰 플레이트에 5 x 105개 세포/ml의 밀도로 시딩하고 밤새 성장시켰다. 그런 다음 그들은 10:1의 MOI로 M. tuberculosis에 감염되었고 세포에 의해 M. tuberculosis bacilli가 흡수될 수 있도록 2시간 동안 5% CO2와 함께 37℃에서 배양되었다. 이어서, 세포를 이전에서 설명한 대로 세척하고 미리 결정된 농도의 프로바이오틱스가 존재하는 200㎕의 약물 없는 DMEM 배지에서 3일 동안 배양하였다. 시험 물질의 존재 없이 감염된 세포를 용매 DMSO로 양성 대조군으로 사용하였다. 3일 후, 감염된 세포를 멸균 증류수를 사용하여 용해하고, 연속적으로 희석된 용해물을 Middlebrook 7H10 한천 플레이트에 플레이팅하여 생존 가능한 간균의 수를 결정하였다. Macrophages were seeded in 96-well plates at a density of 5 × 10 5 cells/ml and grown overnight. Then they were infected with M. tuberculosis at an MOI of 10:1 and incubated at 37°C with 5% CO 2 for 2 hours to allow uptake of M. tuberculosis bacilli by the cells. Cells were then washed as described previously and cultured for 3 days in 200 μl of drug-free DMEM medium in the presence of predetermined concentrations of probiotics. Cells infected without the presence of test substances were used as a positive control with the solvent DMSO. After 3 days, infected cells were lysed using sterile distilled water, and serially diluted lysates were plated on Middlebrook 7H10 agar plates to determine the number of viable bacilli.
8-3.8-3. PMC201의 세포내 항마이코박테리움 활성Intracellular antimycobacterial activity of PMC201
H37Rv 균주에 대한 PMC201의 세포내 항결핵 효과를 CFU(도 3a) 및 AFB 방법(도 3b)으로 시험하였다. 7.8Х107, 3.9Х107, 1.95Х107, 및 0.97Х107 CFU/ml에 해당하는 조건에서 PMC201 추출물 처리 후 미처리군과 비교하였을 때, H37Rv 균주의 각각 97.0%, 95.8%, 95.4% 및 94.4%가 유의하게 억제되었다. 함께 테스트된 0.39 및 0.78 μg/ml의 RIF, INH, EMB 및 PZA도 H37Rv 균주를 유의하게 억제하였다(도 3a). AFB 방법으로, H37Rv 균주의 염색 정도는 3.9Х107, 1.95Х107, 및 0.95Х107 CFU/ml에 해당하는 PMC201 추출물을 처리하였을 때 무약물 시료에 비해 더 낮았다(감염 3일 후)(도 3b). 특히, 3.9Х107 CFU/ml에 해당하는 PMC201 추출물 처리 후 H37Rv 염색 수준은 감염 직후(감염 후 0일) H37Rv 염색 수준보다 낮았다. PMC201 추출물의 XDR 균주의 세포내 억제능은 CFU(도 3c)와 AFB 방법(도 3d)으로 측정하였다. 3.9Х107, 1.95Х107, 및 0.97Х107 CFU/ml에 해당하는 조건에서 PMC201 추출물 처리 후, XDR 균주는 무약물 샘플과 비교하여 각각 87.5%, 57.8%, 및 50% 유의하게 감소하였다(도 3c). 그러나, INH는 5 μg/ml에서도 XDR 균주를 유의하게 억제하지 않았다. 이러한 결과는 AFB 염색 방법으로 얻은 결과와 유사하였다(도 3d). XDR 균주에 대한 PMC201과 INH의 시너지 효과는 CFU 방법(도 3e) 및 AFB 방법(도 3f)에 의해 확인되었다. 5㎍/ml의 INH를 단독 처리에 사용한 경우, INH는 XDR 균주를 억제하지 않았다. 그러나, 3.9Х107 및 1.95Х107 CFU/ml에 해당하는 PMC201 처리에 사용했을 때, XDR 균주는 유의하게 감소하였다.The intracellular anti-tuberculosis effect of PMC201 against H37Rv strain was tested by CFU (Figure 3a) and AFB methods (Figure 3b). When compared to the untreated group after treatment with PMC201 extract under conditions corresponding to 7.8Х10 7 , 3.9Х10 7 , 1.95Х10 7 , and 0.97Х10 7 CFU/ml, 97.0%, 95.8%, 95.4%, and 94.4% of the H37Rv strain, respectively. was significantly suppressed. RIF, INH, EMB, and PZA tested together at 0.39 and 0.78 μg/ml also significantly inhibited the H37Rv strain ( Fig. 3A ). By the AFB method, the degree of staining of the H37Rv strain was lower when treated with PMC201 extracts corresponding to 3.9Х10 7 , 1.95Х10 7 , and 0.95Х10 7 CFU/ml compared to drug-free samples (3 days after infection) (Fig. 3b) ). In particular, the level of H37Rv staining after treatment with PMC201 extract, corresponding to 3.9Х10 7 CFU/ml, was lower than the level of H37Rv staining immediately after infection (day 0 after infection). The intracellular inhibitory ability of the PMC201 extract against the XDR strain was measured by CFU (Figure 3c) and AFB methods (Figure 3d). After treatment with PMC201 extract under conditions corresponding to 3.9Х10 7 , 1.95Х10 7 , and 0.97Х10 7 CFU/ml, the 3c). However, INH did not significantly inhibit the XDR strain even at 5 μg/ml. These results were similar to those obtained with the AFB staining method (Figure 3d). The synergistic effect of PMC201 and INH against the XDR strain was confirmed by the CFU method (Figure 3e) and the AFB method (Figure 3f). When 5 μg/ml INH was used for treatment alone, INH did not inhibit the XDR strain. However, when used for PMC201 treatment, corresponding to 3.9Х10 7 and 1.95Х10 7 CFU/ml, the XDR strain was significantly reduced.
8-4.8-4. 공동 배양 분석Co-culture assay
프로바이오틱스의 항-마이코박테리움 사멸 활성은 프로바이오틱스(4.5Х107 CFU/ml)와 M. tuberculosis(1Х105 or 1Х106 CFU/ml)의 배양액을 혼합한 공배양법을 통해 조사하였다. MRS 및 7H9 브로쓰의 부피는 각각 10% 및 90%였다. 이어서, 공동 배양 브로스를 2주 동안 37℃의 진탕 인큐베이터에서 인큐베이션하였다. 살아있는 간균의 수는 인큐베이션 후 0, 3, 7 및 14일에 Middlebrook 7H10 한천 플레이트에 연속 희석된 브로스 배양액을 플레이팅하여 결정하였다.The anti-mycobacterium killing activity of probiotics was investigated through a co-culture method mixing probiotics (4.5Х10 7 CFU/ml) and M. tuberculosis (1Х10 5 or 1Х10 6 CFU/ml) culture medium. The volumes of MRS and 7H9 broth were 10% and 90%, respectively. The co-culture broth was then incubated in a shaking incubator at 37°C for 2 weeks. The number of viable bacilli was determined by plating serially diluted broth cultures on Middlebrook 7H10 agar plates at 0, 3, 7, and 14 days after incubation.
8-5.8-5. 브로쓰 동시배양 조건에서 PMC201의 항결핵 활성Anti-tuberculosis activity of PMC201 under broth co-culture conditions.
PMC201 (1Х10 CFU/ml) 은 브로스 공동 배양 분석에서 H37Rv의 생존 세포 수를 감소시키는 효과를 나타냈다 (도 4). 시험 시작 시, 1Х106 CFU/ml에서 H37Rv는 PMC201 처리 후 3, 7, 및 14일에 각각 26.8%, 99.9%, 및 99.9% 감소하였다(각각 2.2Х104 CFU/ml, 1.1Х105 CFU/ml, 3.3Х105 CFU/ml으로) 반면 대조군은 각각 1Х1010 CFU/ml, 1Х1010 CFU/ml, 4.5Х105 CFU/ml으로 증가하였다. H37Rv of 1Х105 CFU/ml 밀도의 H37Rv는 PMC201 처리 후 3, 7, 및 14일에 각각 0%, 99.98%, 및 99.9%감소하였다(각각 9.9Х103 CFU/ml, 2.2Х103 CFU/ml, 및 2.2Х103 CFU/ml으로). 그러나, 대조군의 경우에는 각각 2.2Х103 CFU/ml, 1.3Х107 CFU/ml, 및 2.2Х108 CFU/ml으로 증가하였다.PMC201 (1Х10 CFU/ml) showed an effect in reducing the viable cell number of H37Rv in broth co-culture assay (Figure 4). At 1Х10 6 CFU/ml at the start of the test, H37Rv was reduced by 26.8%, 99.9%, and 99.9% at 3, 7, and 14 days after PMC201 treatment (2.2Х10 4 CFU/ml and 1.1Х10 5 CFU/ml, respectively). , 3.3Х10 5 CFU/ml), while the control group increased to 1Х10 10 CFU/ml, 1Х10 10 CFU/ml, and 4.5Х10 5 CFU/ml, respectively. H37Rv of 1Х10 5 CFU/ml Density of H37Rv was reduced by 0%, 99.98%, and 99.9%, respectively, at 3, 7, and 14 days after PMC201 treatment (9.9Х10 3 CFU/ml, 2.2Х10 3 CFU/ml, respectively) and 2.2Х10 3 CFU/ml). However, in the control group, it increased to 2.2Х10 3 CFU/ml, 1.3Х10 7 CFU/ml, and 2.2Х10 8 CFU/ml, respectively.
9.9. 세포 독성 측정Cytotoxicity measurements
9-1.9-1. 세포독성cytotoxicity
프로바이오틱스 처리 후 264.7 세포의 생존력을 결정하기 위해 공개된 방법(Cantatore et al., 2013)에 약간의 수정을 가하여 트립판 블루 분석을 수행하였다. 간단히 말해서, 대식세포 raw 세포(1.5 x 105 cells/ml)를 2-well 세포 배양 슬라이드(SPL life science, Korea)에 접종하고 37℃, 5% CO2로 24시간 동안 인큐베이션하였다. 인큐베이션 후, 세포를 PBS로 세척하고, 상이한 농도의 프로바이오틱스로 처리하고, 24시간 동안 인큐베이션하였다. 이후 PBS로 세포를 세척하고 트립판 블루(Gibco, USA)로 염색한 후, 혈구계산기(Marienfeld, Germany)를 이용하여 광학현미경으로 계수하였다. To determine the viability of 264.7 cells after probiotic treatment, a trypan blue assay was performed using a published method (Cantatore et al., 2013) with some modifications. Briefly, macrophage raw cells ( 1.5 After incubation, cells were washed with PBS, treated with different concentrations of probiotics, and incubated for 24 hours. Afterwards, cells were washed with PBS, stained with trypan blue (Gibco, USA), and counted under an optical microscope using a hemocytometer (Marienfeld, Germany).
9-2.9-2. PMC201의 세포독성Cytotoxicity of PMC201
대식세포 Raw 264.7 세포주에서 다양한 농도의 PMC201 추출물의 세포독성을 평가하였다(도 5). 세포 생존력은 트립판 블루 및 혈구계 직접 세포 계수를 사용하여 정량적으로 결정되었다(도 5a). 3.9Х107 CFU/ml에 해당하는 조건에서 PMC201 세포 추출물 처리는 유의한 세포독성을 나타내지 않았다. 그러나, 7.8Х107 CFU/ml 및 15.6Х107 CFU/ml의 농도에서는 세포 생존율이 현저히 감소하였다(각각, 52.2% 및 63.3%로). PMC201을 3.9Х107 CFU/ml로 처리한 후 광학 현미경하에서 Raw 264.7세포(메틸렌 블루로 염색한 후)의 형태에는 큰 변화가 없었다 (도 5b). 그러나, Raw 264.7 세포에 7.8Х107 CFU/ml의 PMC201을 처리했을 때 약간의 형태학적 변화가 관찰되었다. 이들 세포는 15.6Х107 및 31.2Х107 CFU/ml의 PMC201을 처리했을 때 파열 및 분리되었다.The cytotoxicity of various concentrations of PMC201 extract was evaluated in the macrophage Raw 264.7 cell line (Figure 5). Cell viability was quantitatively determined using trypan blue and hemocytometer direct cell counting (Figure 5A). Treatment with PMC201 cell extract under conditions corresponding to 3.9Х10 7 CFU/ml did not show significant cytotoxicity. However, at concentrations of 7.8Х10 7 CFU/ml and 15.6Х10 7 CFU/ml, cell viability was significantly reduced (to 52.2% and 63.3%, respectively). After treatment with PMC201 at 3.9Х10 7 CFU/ml, there was no significant change in the morphology of Raw 264.7 cells (after staining with methylene blue) under a light microscope (Fig. 5b). However, when Raw 264.7 cells were treated with 7.8Х10 7 CFU/ml of PMC201, some morphological changes were observed. These cells ruptured and detached when treated with 15.6Х10 7 and 31.2Х10 7 CFU/ml of PMC201.
9-3.9-3. 기니피그에서 프로바이오틱스의 2주간 반복 경구투여독성 평가 Toxicity evaluation of repeated 2-week oral administration of probiotics in guinea pigs
프로바이오틱스의 급성 독성은 체중이 1000g에서 1200g인 성체 수컷 기니피그에서 2주 동안 평가되었다. 이 동물들은 20~25℃ 범위의 온도와 30%~70%의 상대습도에서 12시간 명암 주기의 케이지에 수용 및 유지되었다. 이 동물들을 두 그룹으로 나누었다. 한 그룹은 대조군으로 하고, 다른 그룹은 프로바이오틱스를 경구 투여하였다. 요약하면, 프로바이오틱스를 37℃의 MRS 브로스에서 밤새 배양하고, 멸균 증류수로 세척하고, 재현탁하고, 2주 동안 주 5일, 1일 1회 경구 투여(2 x 109 CFU/체중 kg)하였다. 대조군은 프로바이오틱스 처리군과 동일한 조건에서 멸균 식염수만 투여하였다. 동물은 전체 치료 기간 동안 임상 징후, 사망률 및 체중에 대해 관찰되었다. 본 실험은 식품의약품안전처에서 고시한 의약품 안전성 평가시험지침(고시 제2015-82호)에 따라 순천향대학교 ABSL-2 시설(LML 20-591)에서 수행되었다. 동물 연구 프로토콜은 순천향 기관 동물 관리 및 사용 위원회(IACUC)의 검토 및 승인을 받았다(승인 번호: SCH21-0017).Acute toxicity of probiotics was evaluated in adult male guinea pigs weighing 1000 to 1200 g over a period of 2 weeks. These animals were housed and maintained in cages with a 12-h light/dark cycle at a temperature ranging from 20 to 25°C and a relative humidity of 30% to 70%. These animals were divided into two groups. One group served as a control group, and the other group was orally administered probiotics. Briefly, probiotics were cultured overnight in MRS broth at 37°C, washed with sterile distilled water, resuspended, and administered orally (2 x 109 CFU/kg body weight) once daily, 5 days a week for 2 weeks. The control group was administered only sterile saline solution under the same conditions as the probiotic treatment group. Animals were observed for clinical signs, mortality, and body weight throughout the entire treatment period. This experiment was conducted at Soonchunhyang University's ABSL-2 facility (LML 20-591) in accordance with the Pharmaceutical Safety Evaluation Test Guidelines (Notice No. 2015-82) announced by the Ministry of Food and Drug Safety. The animal study protocol was reviewed and approved by the Soonchunhyang Institutional Animal Care and Use Committee (IACUC) (approval number: SCH21-0017).
9-4.9-4. PMC201의 2주 반복 경구 투여 독성Toxicity of repeated 2-week oral administration of PMC201
PMC201은 2주 동안 2Х109 CFU/kg의 용량으로 기니피그에 반복적으로 경구 투여되었다(도 6 및 표 4). 일반적인 외모, 체중, 임상 징후 및 사망률을 2주 동안 관찰하였다. PMC201의 경구 투여는 유의한 체중 감소를 일으키지 않았다(도 6). 두 그룹 사이의 임상 관찰에는 유의한 차이가 없었다(표 4).PMC201 was repeatedly administered orally to guinea pigs at a dose of 2Х10 9 CFU/kg for 2 weeks (Figure 6 and Table 4). General appearance, body weight, clinical signs and mortality were observed for 2 weeks. Oral administration of PMC201 did not cause significant body weight loss (Figure 6). There were no significant differences in clinical observations between the two groups (Table 4).
(CFU/kg)Volume
(CFU/kg)
in fecal volumeDecrease
in fecal volume
in fecal volumeDecrease
in fecal volume
NOA, No observable abnormality.NOA, No observable abnormality.
10.10. 장내 미생물에 미치는 영향 측정 실험Experiment measuring effects on intestinal microorganisms
10-1.10-1. 인간 장내 미생물 생태계 시뮬레이션Human gut microbial ecosystem simulation
인간 장내 미생물 생태계(SHIME) 모델의 트윈 시뮬레이터(Pro Digest, 벨기에)를 사용하여 제조업체의 지침에 따라 후보 프로바이오틱스를 처리하는 동안 주어진 대변 샘플의 미생물 활동 및 조성의 변화를 조사하였다. SHIME 시스템은 pH, 온도, 접종원 및 체류 시간과 같은 환경 조건을 제어하여 생체 내 장 미생물 생태계(위, 소장, 상행 결장, 횡단 및 하행 결장)를 모방할 수 있는 10개의 이중 잭 용기로 구성되었다 (Molly et al., 1993; Molly et al., 1994; Sivieri et al., 2013). 간단히 말해서, 이 연구 이전 6개월 동안 항생제 치료 이력이 없는 건강한 성인 남성 지원자의 대변 샘플을 트윈 SHIME의 결장 혈관에 접종하였다. 환원제로서 티오글리콜산나트륨 1g/L를 함유하는 멸균 인산염 완충액(0.1mol/L, pH 7.0) 100ml로 희석하고 균질화하여 샘플을 제조하였다. 접종물은 탄수화물 기반 배지에서 2주의 기간 동안 안정화되었고 결장 혈관의 특정 환경 조건에 적응하도록 허용되었다. 그 후, 하나의 SHIME 세트에서 2주 동안 매일 2 x 109 CFU/ml의 프로바이오틱 배양 배지 2 ml를 위 구획에 첨가하였다. 프로바이오틱스는 대조군으로 설정된 다른 SHIME에서 사용되지 않았다. 결장 반응기에서 샘플을 수집하였다. 인간의 대변 샘플은 순천향대학교 부천병원 소화기내과에서 제공하였다. 분변시료 SHIME 실험법은 순천향대학교 부천병원 윤리위원회의 심사를 거쳐 승인되었다(IRB No. 2021-03-017-012).The Twin Simulator of the Human Intestinal Microbial Ecosystem (SHIME) Model (Pro Digest, Belgium) was used to investigate changes in microbial activity and composition of a given stool sample during treatment with candidate probiotics according to the manufacturer's instructions. The SHIME system consisted of 10 double jack vessels that could mimic the in vivo gut microbial ecosystem (stomach, small intestine, ascending colon, transverse and descending colon) by controlling environmental conditions such as pH, temperature, inoculum and residence time ( Molly et al., 1993; Molly et al., 1994; Sivieri et al., 2013). Briefly, stool samples from healthy adult male volunteers with no history of antibiotic treatment in the 6 months prior to this study were inoculated into the colonic vessels of twin SHIME. Samples were prepared by diluting with 100 ml of sterile phosphate buffer (0.1 mol/L, pH 7.0) containing 1 g/L of sodium thioglycolate as a reducing agent and homogenizing. The inoculum was stabilized for a period of 2 weeks in carbohydrate-based medium and allowed to adapt to the specific environmental conditions of the colonic blood vessels. Afterwards, 2 ml of probiotic culture medium at 2 x 109 CFU/ml was added to the gastric compartment every day for 2 weeks in one SHIME set. Probiotics were not used in other SHIMEs set as controls. Samples were collected from the colon reactor. Human stool samples were provided by the Department of Gastroenterology at Soonchunhyang University Bucheon Hospital. The fecal sample SHIME experimental method was approved after review by the Ethics Committee of Soonchunhyang University Bucheon Hospital (IRB No. 2021-03-017-012).
10-2.10-2. 장내 미생물군집에 대한 PMC201의 효과 평가Evaluation of the effect of PMC201 on the intestinal microbiome
PMC201 투여가 장내 미생물군유전체에 미치는 영향은 인공 장내 미생물군집 시뮬레이터인 SHIME를 이용하여 평가하였다(도 7). PMC201을 처리한 그룹과 PMC201을 처리하지 않은 그룹 사이의 세균 군집에는 유의한 차이가 없었다(도 7a). PMC201은 종 풍부도(도 7b) 또는 다양성 지수(도 7c)에서도 유의한 차이를 유도하지 않았다. 문(Phylum) 수준에서, 방선균(Actinobacteria)의 존재비는 대조군에 비해 PMC201 처리군에서 4.2%에서 3.2%(p<0.05)로 감소하였고, 후벽균(Firmicutes)은 49.3%에서 39.9%로 감소(p<0.01)하였다. 그러나 의간균류(Bacteroidetes)의 풍부함은 40.3%에서 50.5%로 증가했다(p<0.001). 프로테오박테리아(Proteobacteria)의 풍부함에는 큰 차이가 없었다(도 7d). 강(class) 수준에서 대조군과 비교하여 방선균(Actinobacteria), 네가티비콕쿠스강(Negativicutes) 및 클로스트리디움강(Clostridia)의 존재비는 각각 4.0%에서 3.0%로(p<0.05), 21.3%에서 14.7%로(p<0.05), 27.2%에서 24.5%(p<0.01)로 감소했다. 그러나 박테로이디아(Bacteroidia)의 존재비는 대조군에 비해 40.3%에서 50.5%로 증가하였다(p<0.001). 반면, 감마프로테오박테리아와 델타프로테오박테리아의 존재비는 크게 변하지 않았다(도 7e). 특히, PMC201 처리군에서 Lactobacillus 속의 존재비는 대조군에 비해 중앙값을 기준으로 0.01%에서 0.07%로 유의하게 증가하였다(p<0.01)(도 7f).The effect of PMC201 administration on the intestinal microbiome was evaluated using SHIME, an artificial intestinal microbiome simulator (Figure 7). There was no significant difference in the bacterial community between the PMC201-treated group and the PMC201-untreated group (Figure 7a). PMC201 did not induce significant differences in species richness (Figure 7b) or diversity index (Figure 7c). At the phylum level, the abundance of Actinobacteria decreased from 4.2% to 3.2% (p<0.05) in the PMC201 treatment group compared to the control group, and Firmicutes decreased from 49.3% to 39.9% (p <0.01). However, the abundance of Bacteroidetes increased from 40.3% to 50.5% (p<0.001). There was no significant difference in the abundance of Proteobacteria (Figure 7d). At the class level, compared to the control group, the abundance of Actinobacteria, Negativicutes, and Clostridia increased from 4.0% to 3.0% (p<0.05) and 21.3%, respectively. It decreased from 14.7% (p<0.05) and from 27.2% to 24.5% (p<0.01). However, the abundance of Bacteroidia increased from 40.3% to 50.5% compared to the control group (p<0.001). On the other hand, the abundance ratio of Gammaproteobacteria and Deltaproteobacteria did not change significantly (Figure 7e). In particular, the abundance of the Lactobacillus genus in the PMC201 treatment group significantly increased from 0.01% to 0.07% based on the median compared to the control group (p<0.01) (Figure 7f).
11.11. 항염 효과 실험Anti-inflammatory effect experiment
11-1.11-1. 아질산염 측정Nitrite measurement
산화질소(NO) 합성의 지표로 사용되는 아질산염(NO2-)의 농도는 이전에 보고된 대로 Griess 시약(Promega, USA)을 사용하여 측정되었다(Zerin et al., 2015). 요약하면, RAW264.7 세포를 96-웰 세포 배양 플레이트에 웰당 1 x 105 cells/ml의 밀도로 분주하고 24시간 동안 배양한 후 M. tuberculosis H37Rv로 감염시켰다. 1x PBS로 3회 세척한 후, 세포를 24, 48, 72시간 동안 프로바이오틱 추출물로 처리하였다. L-NG-모노-메틸아르기닌(L-NMMA)은 산화질소 합성 효소 억제제로 사용되었다. 인큐베이션 후 세포 배양 상층액 50 ㎕를 새로운 96-웰 플레이트에 옮기고, 동량의 Griess 시약 용액과 혼합하고, 실온에서 10분 동안 인큐베이션하고, 마이크로플레이트 리더로 540 nm에서 측정하였다.The concentration of nitrite (NO 2 -), used as an indicator of nitric oxide (NO) synthesis, was measured using Griess reagent (Promega, USA) as previously reported (Zerin et al., 2015). In summary, RAW264.7 cells were seeded in a 96-well cell culture plate at a density of 1 x 10 5 cells/ml per well, cultured for 24 hours, and then infected with M. tuberculosis H37Rv. After washing three times with 1x PBS, cells were treated with probiotic extract for 24, 48, and 72 hours. LN G -Mono-methylarginine (L-NMMA) was used as a nitric oxide synthase inhibitor. After incubation, 50 μl of the cell culture supernatant was transferred to a new 96-well plate, mixed with an equal amount of Griess reagent solution, incubated at room temperature for 10 minutes, and measured at 540 nm with a microplate reader.
11-2.11-2. PMC201의 항염 효과Anti-inflammatory effect of PMC201
아질산염 생성은 결핵균에 감염된 대식세포를 PMC201로 처리하여 측정하였다(도 8). 결핵균 감염 3일 후 Raw264.7 세포에서 아질산염 생성이 유도되었다. 3일 동안 M. tuberculosis에 감염된 대식세포에서 3.9×107 CFU/ml, 1.9×107 CFU/ml, 0.9×107 CFU/ml에 해당하는 PMC201 추출물을 처리한 시료에서 PMC201 추출물로 처리하지 않은 샘플과 비교하여 아질산염의 양이 유의하게 낮았다. (각각 34.8%, 31.1% 및 23%). 산화질소 합성 억제제인 L-NMMA 5mM을 처리한 시료에서 결핵균에 감염된 대식세포에서 아질산염의 감소가 나타났다. Nitrite production was measured by treating macrophages infected with Mycobacterium tuberculosis with PMC201 (Figure 8). Nitrite production was induced in Raw264.7 cells 3 days after Mycobacterium tuberculosis infection. In macrophages infected with M. tuberculosis for 3 days, in samples treated with PMC201 extract corresponding to 3.9×10 7 CFU/ml, 1.9×10 7 CFU/ml, and 0.9×10 7 CFU/ml, The amount of nitrite was significantly lower compared to the sample. (34.8%, 31.1% and 23% respectively). In samples treated with 5mM L-NMMA, a nitric oxide synthesis inhibitor, a decrease in nitrite was observed in macrophages infected with Mycobacterium tuberculosis.
Claims (11)
마이코박테리움 투베르쿨로시스(M. tuberculosis) 균에 대한 항균 활성이 있는 항균용 조성물.
Containing a Lactobacillus crispatus strain or a culture medium thereof with the accession number of KCTC 14673BP,
An antibacterial composition having antibacterial activity against Mycobacterium tuberculosis ( M. tuberculosis ).
상기 항균용 조성물은 생체에 이용되는, 항균용 조성물.
According to paragraph 1,
The antibacterial composition is an antibacterial composition used in living organisms.
상기 항균용 조성물은 생체 외 환경에 이용되는, 항균용 조성물.
According to paragraph 1,
The antibacterial composition is an antibacterial composition used in an in vitro environment.
결핵 치료 또는 예방용 약학적 조성물.
Containing a Lactobacillus crispatus strain or a culture medium thereof with the accession number of KCTC 14673BP,
Pharmaceutical composition for treating or preventing tuberculosis.
결핵 예방 또는 개선용 식품 조성물.
Containing a Lactobacillus crispatus strain or a culture medium thereof with the accession number of KCTC 14673BP,
Food composition for preventing or improving tuberculosis.
상기 식품 조성물은 건강 기능 식품 조성물인, 결핵 예방 또는 개선용 식품 조성물.
According to clause 9,
The food composition is a health functional food composition, a food composition for preventing or improving tuberculosis.
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