KR20170109446A - Lactobacillus sp. strain having antimicrobial activity against microorganisms causing premature birth and vaginosis, antiviral activity againt HSV and improved vagina adhesion ability and uses thereof - Google Patents
Lactobacillus sp. strain having antimicrobial activity against microorganisms causing premature birth and vaginosis, antiviral activity againt HSV and improved vagina adhesion ability and uses thereof Download PDFInfo
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- KR20170109446A KR20170109446A KR1020160033594A KR20160033594A KR20170109446A KR 20170109446 A KR20170109446 A KR 20170109446A KR 1020160033594 A KR1020160033594 A KR 1020160033594A KR 20160033594 A KR20160033594 A KR 20160033594A KR 20170109446 A KR20170109446 A KR 20170109446A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A23Y2220/00—
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- A23Y2220/37—
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- A23Y2220/41—
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- C12R1/225—
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
Description
본 발명은 조산 및 질염 원인균에 대한 항균 활성, 성병 원인 바이러스에 대한 항바이러스 활성 및 질내 부착능이 우수한 락토바실러스 속 균주 및 이의 용도에 관한 것이다.The present invention relates to a strain of Lactobacillus sp. Having excellent antibacterial activity against prematurity and vaginitis causing bacteria, antiviral activity against viruses causing sexually transmitted diseases, and vaginal adherence, and uses thereof.
현재까지 조산의 치료를 위하여 조기진통의 산모에게 자궁수축 억제제 투여, 항생제 치료, 스테로이드제 및 프로게스테론 투여를 통해 진통을 억제하고 분만을 지연시켜왔다. 그러나, 조산의 증상이 나타난 이후에 치료를 수행하는 것보다는, 조산의 위험성을 미리 예측함으로써 조산위험 산모군을 선별하고, 이들에 대한 적절한 관리를 통하여 조산을 방지하거나 효과적인 치료방법을 개발하는 것이 필요하다. 또한, 조산의 시기가 빠를수록 신생아에게 후유증을 남길 가능성이 높고 후유증의 정도도 심하기 때문에 조산을 예측할 수 있다면 미숙아와 그로 인한 장애아의 발생률을 현저하게 줄일 수 있을 것으로 기대된다.Until now, for the treatment of premature labor, maternal use of ant contractions, antibiotics, steroids and progesterone have been used to suppress labor and delay delivery. However, it is necessary to select maternal risk pregnancies by anticipating the risk of premature birth rather than performing treatment after symptoms of preterm birth, and to prevent premature birth or to develop an effective treatment method through appropriate management Do. In addition, the earlier the preterm delivery period, the more likely it is to leave a sequel to the neonate and the degree of sequelae is so severe that it is expected that the incidence of preterm infants and children with disabilities can be significantly reduced if the preterm delivery can be predicted.
조산을 예측하기 위한 일반적인 접근 방법은, 산부인과학적으로, 인구통계학적으로 및 여러가지 증후군에 따라 특별한 주의를 기울여야 하는 여성군을 확인하는 것이었으나, 이러한 접근 방법은 그 방식이 민감하거나 특이적이지 않다는 문제가 있다. 이러한 문제점을 극복하기 위하여, 갑작스런 조산이나 양막파수를 예견하기 위한 여러가지 방법이 연구되고 있는 실정이다.A common approach to predicting preterm birth was to identify female populations that should be given special attention in terms of gynecologically, demographically, and various syndromes, but this approach is not as sensitive or specific as the approach . In order to overcome these problems, various methods for predicting sudden preterm birth and amniotic membrane wave have been studied.
특히, 본 발명에서는 가임기 여성이 임신을 할 경우 태아보호 및 감염예방을 위해 질내 미생물이 더욱 단순화되고 강력한 항균활성으로 외부 병원성균에 대한 질내 감염을 억제하는 사실을 토대로 조산 원인균인 가르드네렐라 바기날리스(Gardnerella vaginalis) 또는 우레아플라스마 우레아리티쿰(Ureaplasma urealyticum) 균주의 임산부 질내 분포도 조사를 통해 조산을 예측할 수 있을 것으로 판단하였고, 이러한 조산 원인균에 의한 조산을 예방하고자 조산 원인균에 대해 강력한 항균 활성을 가지는 질내 균주를 분리하고자 시도하였다.Particularly, in the present invention, the present invention is based on the fact that the vaginal microorganisms are further simplified and the strong antibacterial activity is inhibited in the vaginal infection against the external pathogenic bacteria for the protection of the fetus and the prevention of infection when the pregnant woman is pregnant, Gardnerella vaginalis ) or urea plasma urealyticum ( Ureaplasma urealyticum ) in the maternal vaginal cavity. In order to prevent preterm birth due to these preterm infants, vaginal strains with strong antimicrobial activity against prematurity bacteria .
한편, 한국등록특허 제0712286호에서는 '젖산균 복합제, 및 감염 및 염증 증상의 예방 및/또는 치료를 위한 그의 용도'이 개시되어 있고, 한국공개특허 제2001-0006273호에서는 '질의 락토바실러스 약제'가 개시되어 있으나, 본 발명에서와 같이, 조산 및 질염 원인균에 대한 항균 활성, 성병 원인 바이러스에 대한 항바이러스 활성 및 질내 부착능이 우수한 락토바실러스 속 균주 및 이의 용도에 대해서는 밝혀진 바가 없다.Korean Patent No. 0712286 discloses a combination of lactic acid bacteria and its use for prevention and / or treatment of infectious and inflammatory conditions, Korean Patent Laid-Open No. 2001-0006273 discloses a pharmaceutical composition comprising However, as in the present invention, the strain of Lactobacillus sp. Having excellent antimicrobial activity against prematurity and vaginitis causing bacteria, antiviral activity against viruses causing sexually transmitted diseases, and vaginal adhesion ability has not been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자는 가임기 여성이 임신을 할 경우 태아보호 및 감염예방을 위해 질내 미생물이 더욱 단순화되고 강력한 항균활성으로 외부 병원성균에 대한 질내 감염을 억제할 것이라는 사실에 착안하여, 우선 건강한 임산부들의 질내 상피세포로부터 채취한 점액을 절대혐기적 조건에서 배양한 후 1차적으로 병원성 균주에 생육억제 활성을 보이는 158주를 분리하였다. 상기 158주는 총 14종의 락토바실러스 속으로 분류되었고, 이들 중 활성이 우수한 12주를 2차적으로 선정하여 상기 12주의 균주 배양액의 에틸 아세테이트 추출물을 대상으로 총 15종의 각종 병원성 균주에 대한 항균활성을 측정한 결과, 락토바실러스 크리스파터스(Lactobacillus crispatus) AB70, 락토바실러스 젠세니(Lactobacillus jensenni) AD1-1 및 락토바실러스 가세리(Lactobacillus gasseri) AD2-3로 동정된 균주가 타 균주보다 우수한 항균활성을 나타내는 것을 확인하였다.SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method and a device for preventing vaginal infections in a vaginal epithelium, The mucus from vaginal epithelial cells of healthy pregnant women was cultivated in absolute anaerobic condition and then 158 strains showing primary growth inhibitory activity against pathogenic strains were isolated. The 158 strains were classified into 14 strains of Lactobacillus genus. Among them, 12 strains with excellent activity were selected secondarily. The ethyl acetate extracts of the culture strains of 12 strains were tested for antimicrobial activity against 15 kinds of pathogenic strains As a result, Lactobacillus spp. crispatus) AB70, was found to represent the Lactobacillus Zen Senigallia (Lactobacillus jensenni) AD1-1 and Lactobacillus biasing Li (excellent antibacterial activity that the strains identified as Lactobacillus gasseri) AD2-3 than other strains.
특히, 상기 3종의 균주는 조산과 질염 원인균 및 성병 원인 바이러스에 강력한 항균/항바이러스 활성을 나타낼 뿐만 아니라 질내 상피세포 부착/정착성이 이미 알려진 특허 균주 및 표준 균주보다 우수한 것을 확인함으로써, 조산, 질염 및 성병 원인균에 대한 항균 및 항바이러스용 약학 조성물로 매우 유용한 점을 확인함으로써, 본 발명을 완성하였다.Particularly, the above three strains not only show strong antimicrobial / antiviral activity against prematurity, vaginitis and sexually transmitted disease viruses, but also confirm that the vaginal epithelial cell adhesion / fixation property is superior to the already known patent strains and standard strains, The present invention has been accomplished by ascertaining that it is very useful as an antimicrobial and antiviral pharmaceutical composition against vaginitis and sexually transmitted diseases.
상기 과제를 해결하기 위해, 본 발명은 조산 및 질염 원인균에 대한 항균 활성, 성병 원인 바이러스에 대한 항바이러스 활성 및 질내 부착능이 우수한 락토바실러스 크리스파터스(Lactobacillus crispatus), 락토바실러스 젠세니(Lactobacillus jensenni) 및 락토바실러스 가세리(Lactobacillus gasseri)로 이루어진 군으로부터 선택된 하나 이상의 락토바실러스 속 균주를 제공한다.In order to solve the above problems, the present invention is excellent Lactobacillus Cri Spa Tuscan (Lactobacillus crispatus), Lactobacillus Zen Senigallia (Lactobacillus jensenni) ability antiviral activity and vaginal attached to the antimicrobial activity, sexually transmitted disease causing viruses to premature birth and salt organisms And Lactobacillus gasseri. ≪ Desc /
또한, 본 발명은 상기 균주의 추출물을 제공한다.The present invention also provides an extract of said strain.
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 항균용 조성물을 제공한다.The present invention also relates to an antimicrobial agent containing at least one selected from the group consisting of the strain, the strain mixture, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture and the extract of the culture ≪ / RTI >
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 성병 원인 바이러스에 대한 항바이러스용 조성물을 제공한다.The present invention also relates to a method for treating a sexually transmitted disease comprising at least one member selected from the group consisting of the strain, the strain, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture, A composition for an antiviral against a causative virus is provided.
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 질염 및 성병의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also relates to a method for producing a vaginal yeast containing at least one member selected from the group consisting of the strain, the strain mixture, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture and the extract of the culture And a pharmaceutical composition for preventing or treating sexually transmitted diseases.
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 질염, 조산 및 성병의 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention also relates to a method for producing a vaginal yeast containing at least one member selected from the group consisting of the strain, the strain mixture, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture and the extract of the culture , A health functional food composition for preventing or ameliorating premature and sexually transmitted diseases.
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 조산의 예방용 조성물을 제공한다.Further, the present invention relates to a method for the production of a crude aspartame, which comprises at least one selected from the group consisting of the strain, the strain mixture, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture, Of the present invention.
또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는 조산 및 질염 원인균에 대한 항균용 미생물 제제를 제조하는 방법을 제공한다.In addition, the present invention provides a method for producing a microorganism preparation for antibiotics against prematurity and vaginitis causing bacteria, comprising the step of culturing the strain.
또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는 성병 원인 바이러스에 대한 항바이러스용 미생물 제제를 제조하는 방법을 제공한다.The present invention also provides a method for producing an antiviral microorganism preparation against a sexually transmitted disease virus comprising the step of culturing the strain.
본 발명은 건강한 한국인 임산부 질 내에서 절대혐기적으로 분리한 락토바실러스 크리스파터스(Lactobacillus crispatus) AB70, 락토바실러스 젠세니(Lactobacillus jensenni) AD1-1 및 락토바실러스 가세리(Lactobacillus gasseri) AD2-3 균주에 관한 것으로, 상기 3종의 균주는 조산과 질염 원인균 및 성병 원인 바이러스에 강력한 항균/항바이러스 활성을 나타낼 뿐만 아니라 질내 상피세포 부착/정착성이 이미 알려진 특허 균주 및 표준 균주보다 우수한 것을 확인하였다. 따라서, 이들 균주 및 균주 혼합물은 향후 세균 및 바이러스에 의한 성병을 비롯하여, 조산/질염 및 각종 여성생식기 질환 바이러스에 대한 치료제/치료보조제/예방제 등으로 보다 효과적으로 사용 가능하여 관련 산업에 매우 유용하다.The present invention relates to the use of Lactobacillus crispus ( Lactobacillus sp. crispatus AB70, Lactobacillus < RTI ID = 0.0 > The present invention relates to a strain of AD1-1 and Lactobacillus gasseri AD2-3, which exhibit strong antibacterial / antiviral activity against premature and vaginitis causing causative bacteria and sexually transmitted diseases, as well as vaginal epithelial cells Adhesion / fixability was superior to the patented and standard strains already known. Therefore, these strains and the mixture of strains are useful for related industries because they can be more effectively used as a therapeutic / therapeutic adjuvant / prophylactic agent against prematurity / vaginitis and various female genital disease viruses as well as sexually transmitted diseases caused by bacteria and viruses.
도 1은 본 발명에서 사용된 절대 혐기 임상시료 채취를 위한 모바일 시료 채취 장비를 나타낸다. (A, 가스 조절기; B, 가스발생 선; C, 알루미늄 가스 실린더 (혼합 가스, N2:H2:CO2=86:7:7); D, 가스 실린더 트롤리; E, 가압된 혐기적 자 (Oxoid); F, 팔라듐 (촉매제))
도 2는 본 발명을 통해 병원성 균주에 생육억제 활성을 보이는 1차 선정된 158주의 균종 분포도를 나타낸다.
도 3은 본 발명에서 분리한 AB70 균주를 포함한 분리 균주의 대장균(E. coli) 생육저해 활성환을 나타낸다.
도 4는 본 발명에서 2차 분리된 12주의 질내 균주의 15종의 병원성 세균에 대한 생육저해 활성을 나타낸다(숫자는 저해환의 지름(cm)을 나타냄).
도 5는 본 발명을 통해 분리된 3종 균주를 대상으로 아토포비움 바기내(Atopobium vaginae), 가르드네렐라 바기날리스(Gardnerella vaginalis), 칸디다 알비칸스(Candida albicans) 및 우레아플라스마 우레아리티쿰(Ureaplasma urealyticum)에 대한 항균활성을 나타낸다. AB70 : 본 발명의 락토바실러스 크리스파터스, AD1-1 : 본 발명의 락토바실러스 젠세니, AD2-3 : 본 발명의 락토바실러스 가세리, KLB46, GR-1, RC-14 : 이미 상품화된 특허균주
도 6은 본 발명을 통해 분리된 3종 균주를 대상으로 질 상피세포 부착/정착능을 조사한 결과이다. AB70 : 본 발명의 락토바실러스 크리스파터스, AD1-1 : 본 발명의 락토바실러스 젠세니, AD2-3 : 본 발명의 락토바실러스 가세리, KLB46, GR-1, RC-14 : 이미 상품화된 특허균주
도 7은 본 발명에서 분리한 AB70 균주의 HSV(herpes simplex virus)에 대한 항바이러스 활성 마우스 실험 결과이다.1 shows a mobile sampling device for collecting absolute anaerobic clinical samples used in the present invention. (A, gas regulator, B, gas generation line, C, aluminum gas cylinder (mixed gas, N 2 : H 2 : CO 2 = 86: 7: 7) D, gas cylinder trolley E, pressurized anaerobic (Oxoid), F, palladium (catalyst))
FIG. 2 shows a distribution chart of 158 strains of primary strains showing inhibitory activity against pathogenic strains through the present invention.
Fig. 3 shows the E. coli growth inhibitory activity of the isolated strain containing AB70 strain isolated in the present invention.
Fig. 4 shows growth inhibitory activity against 15 pathogenic bacteria of 12 vaginal strains secondaryly isolated in the present invention (the numbers indicate the diameter (cm) of inhibitory rings).
FIG. 5 is a graph showing the results of the three strains isolated from the present invention, showing that Atopobium vaginae , Gardnerella < RTI ID = 0.0 > vaginalis ), Candida albicans ( Candida albicans ) and Ureaplasma urealyticum . AD-1: Lactobacillus genicinus of the present invention, AD2-3: Lactobacillus taxa of the present invention, KLB46, GR-1, RC-14:
FIG. 6 shows the results of investigation of vaginal epithelial cell adhesion / fixation ability of the three strains isolated through the present invention. AD-1: Lactobacillus genicinus of the present invention, AD2-3: Lactobacillus taxa of the present invention, KLB46, GR-1, RC-14:
FIG. 7 shows the result of an antiviral activity mouse test for HSV (herpes simplex virus) of AB70 strain isolated in the present invention.
본 발명의 목적을 달성하기 위하여, 본 발명은 조산 및 질염 원인균에 대한 항균 활성, 성병 원인 바이러스에 대한 항바이러스 활성 및 질내 부착능이 우수한 락토바실러스 크리스파터스(Lactobacillus crispatus), 락토바실러스 젠세니(Lactobacillus jensenni) 및 락토바실러스 가세리(Lactobacillus gasseri)로 이루어진 군으로부터 선택된 하나 이상의 락토바실러스 속 균주를 제공한다.According to an aspect of the invention, the invention is excellent Lactobacillus Cri Spa Tuscan ability antiviral activity and attached to the vagina for the antibacterial activity, sexually transmitted viruses cause for premature labor and yeast infection causing bacteria (Lactobacillus crispatus , Lactobacillus < RTI ID = 0.0 > and at least one Lactobacillus sp. strain selected from the group consisting of Lactobacillus sp. jensenni and Lactobacillus gasseri .
본 발명에서는 가임기 여성이 임신을 할 경우 태아보호 및 감염예방을 위해 질내 미생물이 더욱 단순화되고 강력한 항균활성으로 외부 병원성균에 대한 질내 감염을 억제할 것이라는 사실로부터 건강한 임산부들의 질내 상피세포로부터 채취한 점액을 절대혐기적 조건에서 배양하여 분리하였고, 특히 조산 및 질염 원인균에 대한 항균 활성, 성병 원인 바이러스에 대한 항바이러스 활성 및 질내 부착능이 우수한 3종의 락토바실러스 속 균주를 분리하였다.In the present invention, from the fact that the vaginal microorganism is more simplified and strong antimicrobial activity is inhibited in the vaginal infection against the outer pathogenic bacteria when the pregnant woman is pregnant, the mucus collected from vaginal epithelial cells of healthy pregnant women Were isolated and cultured under an absolute anaerobic condition. In particular, three strains of Lactobacillus sp. Were isolated with excellent antimicrobial activity against prematurity and vaginitis, antiviral activity against viral disease virus and vaginal adherence.
상기 3종의 락토바실러스 속 균주는 락토바실러스 크리스파터스(Lactobacillus crispatus), 락토바실러스 젠세니(Lactobacillus jensenni) 및 락토바실러스 가세리(Lactobacillus gasseri) 균주로, 상기 락토바실러스 크리스파터스 균주와 락토바실러스 크리스파터스 균주, 락토바실러스 젠세니 균주 및 락토바실러스 가세리 균주의 3종 균주 혼합물을 각각 한국생명공학연구원에 2016년 2월 12일자로 기탁하였다.The three strains of the genus Lactobacillus are Lactobacillus < RTI ID = 0.0 > crispatus , Lactobacillus < RTI ID = 0.0 > jensenni) and Lactobacillus joined Lee (Lactobacillus gasseri ), a mixture of three strains of the Lactobacillus crassus strain, Lactobacillus crassus strain, Lactobacillus gensenii strain and Lactobacillus gasseri strain, respectively, were administered to Korean Institute of Biotechnology on Feb. 12, 2016 ≪ / RTI >
본 발명의 일 구현 예에 따른 균주에서, 상기 락토바실러스 크리스파터스(Lactobacillus crispatus) 균주는 기탁번호가 KCTC 12976BP일 수 있으나, 이에 제한되지 않는다.In the strain in accordance with an embodiment of the present invention, the Lactobacillus Cri Spa Tuscan (Lactobacillus crispatus ) strain may be, but is not limited to, KCTC 12976BP.
본 발명의 일 구현 예에 따른 균주에서, 상기 락토바실러스 크리스파터스(Lactobacillus crispatus) 균주, 락토바실러스 젠세니(Lactobacillus jensenni) 균주 및 락토바실러스 가세리(Lactobacillus gasseri) 균주가 혼합된 혼합균주는 기탁번호가 KCTC 12977BP인 것으로, 기탁증에는 "Vaginal Probiome"로 기재하였다.In the strain in accordance with an embodiment of the present invention, the Lactobacillus Cri Spa Tuscan (Lactobacillus crispatus ), Lactobacillus jensenni) strain and Lactobacillus biasing Li (Lactobacillus gasseri ), the deposit number is KCTC 12977BP, and the deposit is described as "Vaginal Probiome".
본 발명의 일 구현 예에 따른 균주에서, 상기 조산 원인균은 바람직하게는 가르드네렐라 바기날리스(Gardnerella vaginalis) 또는 우레아플라스마 우레아리티쿰(Ureaplasma urealyticum)일 수 있고, 상기 질염 원인균은 바람직하게는 칸디다 알비칸스(Candida albicans), 아토포비움 바기내(Atopobium vaginae), 가르드네렐라 바기날리스(Gardnerella vaginalis) 또는 우레아플라스마 우레아리티쿰(Ureaplasma urealyticum)일 수 있고, 상기 성병 원인 바이러스는 바람직하게는 HSV(herpes simplex virus)일 수 있으나, 이에 제한되지 않는다.In a strain according to an embodiment of the present invention, said prenatal bacteria are preferably selected from the group consisting of Gardnerella < RTI ID = 0.0 > vaginalis ) or Ureaplasma urealyticum , and the vaginitis causing bacteria are preferably Candida albicans ( Candida albicans ), Atopobium vaginae , Gardnerella < RTI ID = 0.0 > vaginalis ) or Ureaplasma urealyticum , and the sexually transmitted disease virus is preferably, but not limited to, herpes simplex virus (HSV).
본 발명의 일 구현 예에 따른 균주에서, 상기 균주는 추가로 슈도모나스 애루기노사(Pseudomonas aeruginosa), 스타필로코커스 아우레우스(Staphylococus aureus), 비브리오 파라해모리티쿠스(Vibrio parahaemolyticus), 바실러스 세레우스(Bacillus cereus), 예르시니아 엔테로콜리티카(Yersinia enterocolitica), 세라티아 마르세센스(Serratia marcescens), 리스테리아 모노시토게네스(Listeria monocytogenes), 바실러스 서틸리스(Bacillus subtilis), 대장균(Escherichia coli), 프로피오니박테리움 에크니(Propionibacterium acnes) 및 스트렙토코커스 뮤탄스(Streptococcus mutans) 균주에 대한 항균 활성을 가질 수 있으나, 이에 제한되지 않는다.In a strain according to an embodiment of the present invention, the strain further comprises Pseudomonas aeruginosa aeruginosa), Staphylococcus aureus (Staphylococus aureus , Vibrio parahaemolyticus), Bacillus cereus (Bacillus cereus , Yersinia enterocolitica , Serratia marcensis, marcescens), L. monocytogenes Cistercian's Ness (Listeria monocytogenes), Bacillus subtilis books (Bacillus subtilis , Escherichia coli , Propionibacterium < RTI ID = 0.0 > acnes ) and Streptococcus mutans but are not limited to, an antimicrobial activity against a mutans strain.
또한, 본 발명은 상기 균주의 추출물을 제공한다. 상기 균주의 추출물은 물, 유기용매 등을 이용하여 추출할 수 있으며, 예를 들면, 물, 탄소수 1 내지 4의 저급 알코올, 헥산, 클로로포름, 에틸아세테이트 또는 이들의 혼합용매 등을 이용하여 추출할 수 있으며, 바람직하게는 에틸 아세테이트(ethyl acetate)로 추출할 수 있으나, 이에 제한되지 않는다.The present invention also provides an extract of said strain. The extract of the strain may be extracted with water, an organic solvent or the like, and may be extracted using, for example, water, a lower alcohol having 1 to 4 carbon atoms, hexane, chloroform, ethyl acetate, And may be extracted with ethyl acetate, but is not limited thereto.
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 항균용 조성물을 제공한다.The present invention also relates to an antimicrobial agent containing at least one selected from the group consisting of the strain, the strain mixture, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture and the extract of the culture ≪ / RTI >
본 발명의 일 구현 예에 따른 항균용 조성물에서, 상기 균주 배양물의 추출물은 바람직하게는 에틸 아세테이트(ethyl acetate) 추출물일 수 있으나, 이에 제한되지 않는다.In the antimicrobial composition according to one embodiment of the present invention, the extract of the strain culture may be, but is not limited to, ethyl acetate extract.
본 발명에서 배양물은 본 발명의 균주를 배양 배지 또는 배양액에서 배양한 것을 의미하며, 상기 배양물은 본 발명의 균주를 포함하거나 포함하지 않을 수 있다. 상기 배양물은 그 제형이 액체 또는 고체일 수 있으나, 이에 제한되는 것은 아니다.The culture in the present invention means that the strain of the present invention is cultured in a culture medium or a culture solution, and the culture may or may not include the strain of the present invention. The culture may be, but is not limited to, a liquid or solid formulation.
본 발명에서 "항균 (antimicrobial)"의 의미는 특정 농도에서 미생물의 성장 또는 생존을 감소, 방지, 억제, 또는 제거하는 능력을 의미한다. 항균용 조성물이란 항미생물제를 총칭하는 의미인 항생제와 같은 의미일 수 있고, 항균제, 살균제, 방부제, 보존제 또는 제균제와 같은 의미일 수 있으며, 바람직하게는 그람양성균, 그람음성균, 진균 (효모 및 곰팡이)으로 이루어진 군에서 선택된 1 종 이상의 미생물의 발육과 생활 기능을 저지 또는 억제할 수 있는 물질 즉, 항세균 및 항진균 효력이 있는 물질을 의미한다.The term "antimicrobial" in the present invention means the ability to reduce, prevent, inhibit, or eliminate the growth or survival of a microorganism at a specific concentration. The antimicrobial composition may have the same meaning as antibiotics, which is generically referred to as an antimicrobial agent, and may have the same meaning as an antimicrobial agent, a bactericide, an antiseptic, a preservative or a bactericidal agent and is preferably a gram-positive bacteria, a gram-negative bacterium, ), That is, a substance capable of inhibiting or inhibiting the growth and the function of living organisms of one or more microorganisms selected from the group consisting of microorganisms and antifungal agents.
본 발명의 항균용 조성물은 질염 또는 조산 원인균 또는 기타 상기 유해 미생물에 감염된 부분에 직접 투여하거나 적절한 형태의 조성물로 제조하여 투여할 수 있다. 본 발명의 항균용 조성물의 투여량은 투여 대상에서 질염 또는 조산 원인균 또는 기타 상기 유해 미생물을 사멸시키는 효과를 발휘하기 위해 요구되는 정도의 양을 의미하는데, 제형의 종류 및 투여 대상의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 경로, 투여 시간 및 기간을 비롯한 다양한 인자에 따라 조절될 수 있다. The antimicrobial composition of the present invention may be directly administered to vaginal infections or causative bacteria or other parts infected with the harmful microorganisms, or may be prepared into a composition in a suitable form and administered. The dosage of the antimicrobial composition of the present invention means an amount required to exert the effect of killing the infectious agent or the causative bacteria or other harmful microorganisms in the subject to be administered. The kind of the formulation and the age, General health status, sex and diet, route of administration, time and duration of administration, and the like.
본 발명의 일 구현 예에 따른 항균용 조성물에서, 상기 조성물은 바람직하게는 식품, 식품 첨가제, 사료 첨가제 또는 의약품 형태일 수 있다.In the antimicrobial composition according to one embodiment of the present invention, the composition may preferably be in the form of a food, a food additive, a feed additive or a medicament.
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 성병 원인 바이러스에 대한 항바이러스용 조성물을 제공한다.The present invention also relates to a method for treating a sexually transmitted disease comprising at least one member selected from the group consisting of the strain, the strain, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture, A composition for an antiviral against a causative virus is provided.
본 발명의 추출물이 상기와 같은 항바이러스용으로 이용되기 위해서는, 식품학 또는 약제학적 분야에서 공지된 다양한 방법에 의해 제조될 수 있으며 그 자체 또는 식품학적으로 허용되는 담체, 부형제, 희석제 등과 혼합하여 경구로 섭취할 수 있는 어떤 식품 형태로도 제조될 수 있다. 바람직하게는 음료, 환, 과립, 정제 또는 캅셀 형태이다.In order to use the extract of the present invention for such an antiviral, it may be prepared by various methods known in the field of food science or pharmacy and may be prepared by mixing with itself or a pharmaceutically acceptable carrier, excipient, diluent or the like, It can be produced in any food form that can be ingested. Preferably in the form of beverage, ring, granule, tablet or capsule.
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 질염 및 성병의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also relates to a method for producing a vaginal yeast containing at least one member selected from the group consisting of the strain, the strain mixture, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture and the extract of the culture And a pharmaceutical composition for preventing or treating sexually transmitted diseases.
본 발명의 상기 약학조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.
본 발명에 따른 상기 약학조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 본 발명에 따른 상기 약학조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함한 다양한 화합물 혹은 혼합물을 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 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, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜(propylene glycol), 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, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition according to the present invention may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to conventional methods Can be used. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, Various compounds or mixtures including calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil . In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 질염, 조산 및 성병의 예방 또는 개선용 건강기능식품 조성물을 제공한다.The present invention also relates to a method for producing a vaginal yeast containing at least one member selected from the group consisting of the strain, the strain mixture, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture and the extract of the culture , A health functional food composition for preventing or ameliorating premature and sexually transmitted diseases.
상기에서 언급한 본 발명의 상기 균주 또는 이의 배양액 등을 건강기능식품 조성물로 사용할 경우, 상기 균주 또는 이의 배양액을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있으며, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그 사용 목적 (예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다.When the above-mentioned strain of the present invention or a culture solution thereof or the like is used as a health functional food composition, the strain or a culture thereof may be directly added or used together with other foods or food ingredients, . The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).
본 발명의 식품(또는 건강기능식품)은, 식품 제조 시에 통상적으로 첨가되고 식품학적으로 허용되는 성분을 더 포함할 수 있다. 예컨대, 음료수로 제조되는 경우에는 본 발명의 혼합 추출물 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙 등에서 하나 이상의 성분을 추가로 포함시킬 수 있다.The food (or the health functional food) of the present invention may further comprise ingredients that are conventionally added at the time of food production and which are pharmaceutically acceptable. For example, in the case of beverage preparation, one or more components may be further contained in citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, etc., in addition to the mixed extract of the present invention.
본 발명에 따른 식품(또는 건강기능식품)의 유효성분으로 포함될 수 있는 양은 항균 또는 항바이러스용 식품을 원하는 사람의 연령, 성별, 체중, 상태, 질병의 증상에 따라 적절히 선택될 수 있으며, 바람직하게는 성인기준 1일 0.01 g 내지 10.0 g 정도로 포함되는 것이 좋으며, 이러한 함량을 갖는 식품을 섭취함으로써 항바이러스 효과를 얻을 수 있다.The amount of the food (or health functional food) according to the present invention that can be included as an active ingredient can be appropriately selected according to the age, sex, weight, condition, and symptom of a person who desires antimicrobial or antiviral food, Is preferably contained in an amount of 0.01 to 10.0 g per day on an adult basis, and an antiviral effect can be obtained by ingesting foods having such a content.
또한, 본 발명은 상기 균주, 상기 균주 혼합물, 상기 균주 또는 균주 혼합물의 배양물, 상기 배양물의 농축액, 상기 배양물의 건조물 및 상기 배양물의 추출물로 이루어지는 군에서 선택된 1종 이상을 유효성분으로 함유하는 조산의 예방용 조성물을 제공한다.Further, the present invention relates to a method for the production of a crude aspartame, which comprises at least one selected from the group consisting of the strain, the strain mixture, the culture of the strain or the mixture of strains, the concentrate of the culture, the dried product of the culture, Of the present invention.
상기에서 언급한 본 발명의 상기 균주 또는 이의 배양액 등으로 조산을 예방하는 방법은 본 발명의 락토바실러스 크리스파터스(Lactobacillus crispatus), 락토바실러스 젠세니(Lactobacillus jensenni) 및 락토바실러스 가세리(Lactobacillus gasseri) 균주가 조산 원인균인 가르드네렐라 바기날리스(Gardnerella vaginalis) 또는 우레아플라스마 우레아리티쿰(Ureaplasma urealyticum) 균주에 대해 강력한 항균 활성을 나타내므로, 본 발명의 락토바실러스 크리스파터스, 락토바실러스 젠세니 또는 락토바실러스 가세리 균주 또는 이들의 혼합 균주를 임산부의 질 내부에 처리함으로 인해 가능한 것이다.The method for preventing premature birth by the above-mentioned strain of the present invention or a culture solution thereof or the like is a method of preventing Lactobacillus crispus crispatus , Lactobacillus < RTI ID = 0.0 > jensenni) and Lactobacillus joined Lee (Lactobacillus gasseri ) strain is a causative agent of Gardnerella ( Gardnerella < RTI ID = 0.0 > vaginalis ) or Ureaplasma urealyticum strains. Therefore, the Lactobacillus crispus, Lactobacillus gensenii, or Lactobacillus gasseri strains of the present invention, or a mixed strain thereof, It is possible to process it internally.
또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는 조산 및 질염 원인균에 대한 항균용 미생물 제제를 제조하는 방법을 제공한다.In addition, the present invention provides a method for producing a microorganism preparation for antibiotics against prematurity and vaginitis causing bacteria, comprising the step of culturing the strain.
또한, 본 발명은 상기 균주를 배양하는 단계를 포함하는 성병 원인 바이러스에 대한 항바이러스용 미생물 제제를 제조하는 방법을 제공한다.The present invention also provides a method for producing an antiviral microorganism preparation against a sexually transmitted disease virus comprising the step of culturing the strain.
본 발명의 균주를 배양하는 방법은 당업계에 통상적으로 이용되는 방법에 따라 배양할 수 있다.The method for culturing the strain of the present invention can be carried out according to a method commonly used in the art.
본 발명의 조산 및 질염 원인균에 대한 항균용 미생물 제제 또는 성병 원인 바이러스에 대한 항바이러스용 미생물 제제는 유효성분으로서 락토바실러스 크리스파터스, 락토바실러스 젠세니 및 락토바실러스 가세리 균주를 이용하여 제조될 수 있다. 본 발명에 따른 항균용 또는 항바이러스 미생물 제제는 용액, 분말, 현탁액, 분산액, 에멀젼, 유성 분산액, 페이스트, 분진, 분사제 또는 과립제로 제조할 수 있으나, 이에 제한되는 것은 아니다.The microorganism preparation for antibiotics or the antiviral antiviral agent against the sexually transmitted disease virus according to the present invention can be prepared by using Lactobacillus crassus, Lactobacillus gensensi and Lactobacillus gasseri strains as active ingredients have. The antimicrobial or antiviral microorganism preparation according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oil dispersion, paste, dust, spray or granule, but is not limited thereto.
이하 본 발명을 하기 실시예에 의하여 더욱 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
실시예Example 1. 건강한 한국인 임산부 1. Healthy Korean pregnant women 질내Vagina 시료 채취 Sampling
건양대병원 산부인과에 정기검진을 받기 위해 내원한 건강한 한국인 임산부들의 동의를 받아 이들 임산부들의 질내 시료를 채취하였다. 시료를 채취하는 방법은 다음과 같다. 병원에서 질내 시료 채취에 사용하는 자궁내 경부도말 브러쉬(endocervical brush)를 사용하여 임산부 질내 상피세포에 있는 점액을 채취한 후 브러쉬를 살균된 50 mL 튜브에 넣고 뚜껑을 닫지 않은 채로 바로 가압형 혐기 자(jar) (3.5 L HP0011A, Oxoid, UK) 안에 넣고 혐기 자(jar)의 뚜껑을 닫는다. 그리고 질소:수소:이산화탄소 86:7:7의 비율로 섞인 혼합가스를 15분간 주입하여 혐기 자(jar) 내의 공기를 산소가 포함되지 않은 혼합가스로 대체하였다. 혐기 자(jar) 내에 남아 있는 미량의 산소는 혐기 자(jar) 내의 팔라디움 촉매와 수소가 반응하여 물로 환원되어 제거하였다. 이와 같은 장비의 구성은 도 1에 나타내었으며, 이는 본 발명자의 연구실에서 자체 제작하였고, 이를 채취한 임상시료의 절대혐기적 보관 및 운반을 위한 모바일 시료 채취장비라 명명하였다(도 1).The vaginal specimens of these pregnant women were collected with the consent of healthy Korean pregnant women who came to visit the obstetrics clinic at Konyang University Hospital. The method of sampling is as follows. The mucus from epithelial cells in the vaginal epithelium was collected using an endocervical brush, which was used for vaginal sampling in the hospital. The brush was placed in a sterilized 50 mL tube and the pressure-type anaerobic Place it in a jar (3.5 L HP0011A, Oxoid, UK) and close the lid of the anaerobic jar. Then, a mixed gas of nitrogen: hydrogen: carbon dioxide 86: 7: 7 was injected for 15 minutes to replace the air in the anaerobic jar with an oxygen-free mixed gas. Trace amounts of oxygen remaining in the anaerobic jar were reduced by the reaction of hydrogen with the palladium catalyst in the anaerobic jar. The structure of such a device is shown in FIG. 1, which was prepared in-house by the inventor's laboratory and named as a mobile sample collection bin for absolute anaerobic storage and transportation of the collected clinical samples (FIG. 1).
실시예Example 2. 건강한 한국인 임산부 2. Healthy Korean pregnant women 질내Vagina 시료에서 혐기성 미생물 분리 Anaerobic microbial separation from sample
상기 실시예 1에서 설명한 방법으로 채취한 시료는 시료 채취 직후 바로 한국생명공학연구원 실험실로 전달되어 시료가 들어있는 혐기 자(jar)를 혼합가스 (질소:수소:이산화탄소 = 86:7:7)로 채워져 있는 절대혐기챔버 (Coy Laboratory Product, USA) 안에 넣고 미리 준비한 절대혐기적으로 제조된 DSM 104 아가 플레이트 (1.5% w/V) 상에 질내 시료를 도말하여 37℃에서 48시간 배양 후 형성된 콜로니를 선발였다. DSM 104 배지의 조성은 아래 그림과 같다.The sample collected by the method described in Example 1 was immediately transferred to a laboratory of the Korea Research Institute of Bioscience and Biotechnology as soon as the sample was collected and the anaerobic jar containing the sample was mixed with the mixed gas (nitrogen: hydrogen: carbon dioxide = 86: 7: 7) A colony formed after culturing the vaginal specimen on a DSM 104 agar plate (1.5% w / V) prepared in advance in an absolute anaerobic chamber (Coy Laboratory Product, USA) It was a starter. The composition of the DSM 104 medium is shown in the figure below.
Haemin solution : 1ml 1N NaOH에 50㎎ Haemin을 녹임Haemin solution: 1ml Na NaOH to dissolve 50mg Haemin
Vitamin K1 solution : 20ml 95% 에탄올에 0.1ml Vitamin K1을 녹임Vitamin K 1 solution: 20 ml Vitamin K 1 dissolved in 0.1 ml of 95% ethanol
총 300여 개의 콜로니를 한국인 임산부로부터 단일 콜로니 분리 방법을 통해 순수 분리하였으며, 콜로니 PCR을 통해 이들 분리된 균주의 분자생물학적 동정을 실시하였다. 동정된 균주는 총 29종으로 이 중 유산균 15종, 비유산균 14종으로 확인되었다. 이들 분리된 300주의 한국인 임산부 질내 미생물에 대하여 1차적으로 바실러스 서틸리스 및 병원성 대장균 균주를 시험균주로 사용하여 이들 시험균주의 생육을 억제하는 질내 미생물 균주를 고체배지에서의 페이퍼 디스크 분석을 이용한 생육억제환의 생성여부를 측정하여 총 158주를 선정하였다.A total of 300 colonies were isolated from Korean pregnant women by single colony isolation method and molecular biology identification of these isolated strains was carried out by colony PCR. A total of 29 isolates were identified, of which 15 were lactic acid bacteria and 14 were non lactic acid bacteria. Using the bacillus subtilis and pathogenic Escherichia coli strains as test strains, the vaginal microbial strains inhibiting the growth of these test strains were isolated from the 300-week-old Korean mothers of pregnant women by using paper disc analysis in solid medium A total of 158 weeks were selected by measuring the generation of inhibitory rings.
선정된 158주(선정된 158주는 총 14종의 락토바실러스 속으로 분류됨, 도 2) 중 활성이 우수한 12주를 2차 선정하고 이들 선정된 균주에 대해서는 총 15종의 병원성 세균의 생육억제 정도를 정량적으로 세밀히 측정하게 되었다. 선정된 질내 미생물 12주는 100 mL씩 5일간 액체배양하여 배양액의 동량의 에틸 아세테이트를 처리하여 1시간 반응시킨 후 유기용매층을 분리하여 건조하였다. 유기용매를 모두 제거한 후 메탄올 125㎕에 용해시키고, 용해된 추출액 중 25㎕를 15종의 각종 병원성 균주에 대한 생육억제환의 지름을 측정하여(도 3) 생육억제 정도가 가장 우수한 AB70 균주를 3차로 선정하게 되었다. The selected 158 strains (the 158 selected strains were classified into 14 strains of Lactobacillus, Fig. 2), and the 12 strains with excellent activity were selected second, and the growth inhibition of 15 pathogenic bacteria In a quantitative manner. The selected vaginal microorganisms 12 were cultured for 5 days in 100 mL increments. The culture was treated with the same amount of ethyl acetate and reacted for 1 hour. The organic solvent layer was separated and dried. After removal of all the organic solvent, 125 μl of methanol was dissolved, and 25 μl of the dissolved extract was used to measure the diameter of the growth inhibition rings of 15 kinds of pathogenic strains (FIG. 3) Respectively.
AB70 균주는 특히 조산/질염과 관계가 있는 질내 병원성 균주인 아토포비움 바기내(Atopobium vaginae, 질염균), 가르드네렐라 바기날리스(Gardnerella vaginalis, 조산/질염)에 대해 가장 우수한 생육저해능을 보였으며(도 4), 질염균 이외에도 슈도모나스 애루기노사(Pseudomonas aeruginosa, 녹농균), 리스테리아 모노시토게네스(Listeria monocytogenes, 수막염/뇌막염균), 프로피오니박테리움 에크니(Propionibacterium acnes, 여드름균), 병원성 대장균 및 스트렙토코커스 뮤탄스(Streptococcus mutans, 충치균)에 대하여 가장 우수한 생육억제 활성을 나타냈다(도 4).The AB70 strain is particularly susceptible to viral pathogens such as Atopobium vaginae , and Gardnerella vaginalis (FIG. 4). In addition to the vaginitis infections, Pseudomonas aeruginosa ( Pseudomonas aeruginosa) aeruginosa , P. aeruginosa), Listeria monocytogenes ( Listeria monocytogenes , meningitis / meningitis), Propionibacterium < RTI ID = 0.0 > acnes , acne), pathogenic Escherichia coli, and Streptococcus mutans (tooth decay) (FIG. 4).
여성은 질내 미생물의 분포에 따라 크게 5가지 그룹으로 나누어지는데, 이들 그룹은 다음과 같다; 제 1 그룹 락토바실러스 크리스파터스(Lactobacillus crispatus) 우점 그룹, 제 2 그룹 락토바실러스 가세리(Lactobacillus gasseri), 제 3 그룹 락토바실러스 이너스(Lactobacillus iners) 우점 그룹, 제 4 그룹 다양성 그룹 (특정 유산균이 지배하지 않고 각종 혐기성 세균이 지배하는 그룹), 제 5 그룹 락토바실러스 젠세니(Lactobacillus jensenni) 우점 그룹. 한국인 여성 150명의 질내 미생물 분포를 NGS로 조사한 본 발명자의 결과에서도 유사한 결과를 보였는데, 특히 락토바실러스 크리스파터스가 지배하는 그룹이 가장 많았으며, 그 다음으로 락토바실러스 이너스 그룹, 락토바실러스 가세리 그룹, 그리고 락토바실러스 젠세니 그룹 순으로 많았으며, 건강한 여성의 경우에는 다양성 그룹이 관찰되지 않았으나, 질염 증상이 있거나 고위험 임신 경험이 있는 여성의 경우 다양성 그룹이 관찰되었다.Women are divided into five groups according to the distribution of vaginal microorganisms, which are as follows; A first group Lactobacillus Cri Spa Tuscan (Lactobacillus crispatus) dominated group, the second group Lactobacillus biasing Li (Lactobacillus gasseri ), the third group Lactobacillus iners ) dominant group, a fourth group of diversity group (a group dominated by various anaerobic bacteria without being dominated by specific lactobacilli), a fifth group of Lactobacillus dominance group. The results of the present inventors who examined the distribution of vaginal microorganisms in 150 Korean women by NGS showed similar results. In particular, the group dominated by Lactobacillus crassus was the most dominant group, followed by Lactobacillus annulus group, Lactobacillus gasseri group , And Lactobacillus gensenii. Diversity groups were not observed in healthy women, but diversity groups were observed in women with symptoms of vaginitis or high - risk pregnancy.
락토바실러스 이너스 그룹의 경우, 락토바실러스 이너스 균주의 항균활성이 다른 그룹의 균주에 비해 현저히 떨어지며, 이들 그룹의 경우에도 고위험임신 가능성이 높은 것으로 알려져 있다. In the case of Lactobacillus reinflexes, the antimicrobial activity of Lactobacillus strain is significantly lower than that of other strains, and these groups are also known to be highly likely to have high-risk pregnancies.
본 연구그룹의 실험결과에서도 최종 선정된 12주에 락토바실러스 이너스 균주는 선정되지 않았으며, 락토바실러스 크리스파터스 AB70 균주가 각종 병원성 세균에 대해 가장 우수한 항균활성을 나타냈다. 그러나 조산의 주원인 균주로 추정되는 우레아플라스마 우레아리티쿰(Ureaplasma urealyticum, 조산/질염균) 균주와 진균인 칸디다 알비칸스(Candida albicans, 질염균)에 대해서는 락토바실러스 젠세니 그룹인 AD1-1 균주와 락토바실러스 가세리 그룹인 AD2-3 균주가 우수한 항균활성을 보였으며, 따라서 이들 세 균주 AB70, AD1-1 및 AD2-3 균주 각각 또는 이들 균주배양물/추출물의 혼합체를 여성의 조산/질염 예방/치료제로 사용가능할 것으로 예상된다.Lactobacillus sp. Strain AB70 was found to be the most effective antibiotic against various pathogenic bacteria in the experimental group of this study group. However, Ureaplasma, which is presumed to be the main cause of prematurity, urealyticum , and Candida albicans , which is a fungus, is classified as Lactobacillus genoseni AD1-1 and Lactobacillus gasseri group AD2-3 showed excellent antimicrobial activity. Therefore, each of these three strains AB70, AD1-1 and AD2-3, or a mixture of cultures / extracts of these strains, It is expected to be used as a prenatal / vaginitis prevention / remedy for women.
선정된 3주와 기존에 질염치료로 보고된 특허 및 상품화된 균주인 락토바실러스 크리스파터스(Lactobacillus crispatus) KLB46, 락토바실러스 람노서스(Lactobacillus rhamnosus) GR-1, 락토바실러스 루테리(Lactobacillus reuteri) RC-14 균주에 대해 특히 조산/질염 원인균주에 대한 항균활성을 비교실험하였다. 실험결과 AB70 균주가 조산/질염 원인균에 대해 가장 우수한 항균활성능을 보였다 (도 5). 이는 건강한 임산부의 경우 임신 전보다 질내 미생물분포가 더욱 단순해지면서 강력한 항균활성으로 외부 병원성균에 대한 질내 감염을 억제할 것으로 추정한 기존 보고와 일치하는 내용이다.The selected three weeks and the patented and commercialized strains previously reported as vaginitis therapy, Lactobacillus crispatus KLB46, Lactobacillus rhamnosus GR-1, Lactobacillus ( Lactobacillus < RTI ID = 0.0 > The antimicrobial activity of RC-14 strains was investigated. As a result, the AB70 strain exhibited the best antimicrobial activity against the causative agent of prematurity / vaginism (FIG. 5). This is consistent with previous reports that healthy pregnant women are more likely to inhibit vaginal infections of external pathogens with strong antimicrobial activity as the distribution of vaginal microorganisms becomes simpler than before the pregnancy.
선정된 질내 미생물이 아무리 항균활성이 우수하다고 하더라도 실제 질내에 부착하고 정착하지 못한다면, 그 조산/질염 치료/예방효과는 기대하기 어렵다. 선정된 질내 미생물의 질내 정착성을 실험하기 위하여 사람의 질 상피세포인 세포주 (vagina epithelial cell, VK2/E6E7)를 미국 ATCC로부터 구입하여 세포 배양을 하였으며, 상기 설명한 다른 특허 및 상품 균주에 대해서도 질내 부착/정착성 실험을 하고 이를 비교하였다. 부착실험은 37℃에서 24시간 배양된 시험균주를 400 rpm에서 10분간 원심분리하고 균체를 RPM1640 배지로 세척한 후 사람의 질 상피세포가 배양된 6웰 플레이트에 각각 100㎕씩 접종하였다. 1시간 동안 CO2 배양기에서 배양 후 RPM1640 배지로 3회 세척하고 남은 균주수를 10X trypsin-EDTA 150㎕로 부착된 균주를 탈착하고 2 ml의 RPM1640 배지로 균주를 회수하여 회수된 균주의 cfu/ml 값을 측정하였다. 실험결과 본 발명을 통해 확보된 균주들은 모두 비교균주에 비해 월등한 질내 부착/정착성을 나타내었으며(도 6), 이는 건강한 임산부의 질내에서 분리한 질내 미생물이 일반 미생물에 비해 탁월한 질내 부착/정착성을 지니게 됨을 나타낸다.Even if the selected vaginal microorganisms have excellent antimicrobial activity, it is difficult to expect the antimicrobial activity / preventive effect of the vaginosis / vaginitis if it can not adhere and settle in the actual vagina. In order to test vaginal fixation properties of selected vaginal microorganisms, vagina epithelial cells (VK2 / E6E7) were purchased from ATCC, USA and cell cultures were carried out. For the other patents and product strains described above, / Fixability test and compared them. In the adherence test, the test strains cultured at 37 ° C for 24 hours were centrifuged at 400 rpm for 10 minutes, and the cells were washed with RPM1640 medium and 100 μl each was inoculated into 6 well plates cultured with human vaginal epithelial cells. After incubation for 1 hour in a CO 2 incubator, the cells were washed three times with RPM 1640 medium, and the remaining strains were desalted with 150 μl of 10 × trypsin-EDTA, and recovered with 2 ml of RPM1640 medium to recover cfu / ml Respectively. As a result, the strains obtained from the present invention exhibited superior vaginal adhesion / fixation (Fig. 6) compared to the comparative strains, indicating that vaginal microorganisms isolated from the vaginal vagina of healthy pregnant women had superior vaginal adhesion / fixation It shows that you have sex.
최종 선정된 3주의 HSV에 대한 항바이러스 활성을 확인하기 위한 마우스 질내에서의 인 비보(in vivo) 실험을 행하였다. 실험에 사용된 쥐는 암컷 C57BL/6 J 마우스이며, 이들 쥐를 항생제 처리를 통해 질내 미생물을 제거한 후 HSV에 감염시킬 경우 항생제 처리를 하지 않은 쥐에 비해 바이러스 감염에 취약하여 일찍 죽는 것을 관찰하였다(참고문헌: Dysbiosis-induced IL-33 contributes to impaired antiviral immunity in the genital mucosa. 2016. PNAS on line published). 항생제 처리를 통해 HSV 감염에 취약해진 마우스에 본 발명을 통해 개발된 AB70 균주 100㎕ (1010 cfu/ml)를 처리할 경우 HSV 감염에 대해 저항성을 나타냄을 확인하였다 (도 7).In vivo experiments were performed in the mouse vagina to confirm the antiviral activity against HSV of the final three selected weeks. The mice used in the experiment were female C57BL / 6J mice, and HSV infected mice were found to be susceptible to viral infections earlier than those not treated with antibiotics. Dysbiosis-induced IL-33 contributes to impaired antiviral immunity in the genital mucosa. 2016. PNAS on line published). It was confirmed that treating 100 μl (10 10 cfu / ml) of AB70 strain developed through the present invention against mice susceptible to HSV infection through antibiotic treatment showed resistance to HSV infection (FIG. 7).
실시예Example 3. 본 발명에서 최종 선발한 3. In the present invention, AB70AB70 , , AD1AD1 -1 및 -1 and AD2AD2 -3의 3종 균주의 동정 방법 및 결과Identification and identification of three strains
최종 선정된 3주의 분자생물학적 동정을 위하여 16S rRNA 유전자를 타겟으로 하는 유니버셜 프라이머인 27F (5'-AGA GTT TGA TCM TGG CTC A-3': 서열번호 1)와 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3': 서열번호 2)을 사용하였고, 직접 콜로니 PCR 법으로 PCR 산물을 얻은 후 BIOFACT 사(BIOFACT, Korea)에 의뢰하여 16S rRNA 유전자의 염기서열 분석을 수행하였다. 분석하여 얻은 염기 서열들은 EZbiocloud (http://www.ezbiocloud.net/)에서 동정검색을 통해 염기서열을 확인하였다. 그 결과 AB70 균주는 락토바실러스 크리스파터스(Lactobacillus crispatus) DSM 20584T와 100%, AD1-1 균주는 락토바실러스 젠세니(Lactobacillus jensenni) ATCC 25258T와 99.85% 그리고 AD2-3 균주는 락토바실러스 가세리(Lactobacillus gasseri) ATCC 33323T와 99.72%의 상동성을 나타내었다.(5'-TAC GGY TAC CTT (SEQ ID NO: 1)) and 1492R (5'-TAC GGY TAC CTT) were used as the universal primers targeting the 16S rRNA gene GTT ACG ACT T-3 ': SEQ ID NO: 2) was used, and the PCR product was obtained by direct colony PCR method, and the sequence analysis of the 16S rRNA gene was performed by BIOFACT (BIOFACT, Korea). The nucleotide sequences were analyzed and identified by EZbiocloud (http://www.ezbiocloud.net/). As a result, the AB70 strain was identified as Lactobacillus crispatus )
<110> Korea Research Institute of Bioscience and Biotechnology <120> Lactobacillus sp. strain having antimicrobial activity against microorganisms causing premature birth and vaginosis, antiviral activity againt HSV and improved vagina adhesion ability and uses thereof <130> PN16049 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 agagtttgat cmtggctca 19 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tacggytacc ttgttacgac tt 22 <110> Korea Research Institute of Bioscience and Biotechnology <120> Lactobacillus sp. strain having antimicrobial activity against microorganisms causing premature birth and vaginosis, antiviral activity againt HSV and improved vaginal adhesion ability and uses the <130> PN16049 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 agagtttgat cmtggctca 19 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tacggytacc ttgttacgac tt 22
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| KR102139687B1 (en) * | 2019-05-03 | 2020-07-30 | 주식회사 메디오젠 | Lactobacillus MG4288 for the treatment or improvement of vaginitis with antimicrobial effect on Gardnerella vaginalis and Candida albicans |
| CN111893057A (en) * | 2020-06-29 | 2020-11-06 | 哈尔滨美华生物技术股份有限公司 | Lactobacillus crispatus for preventing and treating female urogenital infection and application thereof |
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| CN111893057A (en) * | 2020-06-29 | 2020-11-06 | 哈尔滨美华生物技术股份有限公司 | Lactobacillus crispatus for preventing and treating female urogenital infection and application thereof |
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