KR101786626B1 - Composition for preventing Helicobacter pylori from attaching on the epithelial cells of stomach, the preparation therefor, and the functional beverage using them - Google Patents
Composition for preventing Helicobacter pylori from attaching on the epithelial cells of stomach, the preparation therefor, and the functional beverage using them Download PDFInfo
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- KR101786626B1 KR101786626B1 KR1020150168002A KR20150168002A KR101786626B1 KR 101786626 B1 KR101786626 B1 KR 101786626B1 KR 1020150168002 A KR1020150168002 A KR 1020150168002A KR 20150168002 A KR20150168002 A KR 20150168002A KR 101786626 B1 KR101786626 B1 KR 101786626B1
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- 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
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
- A23L2/52—Adding ingredients
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- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
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Abstract
본 발명은 헬리코박터 파이로리의 부착 억제능력을 가지는 청국장으로부터 분리되는 유산균 균주 조성물 및 이를 활용한 기능성 음료의 제조방법에 관한 것이다. 한편, 본 발명은 내산성 유산균 균주 획득을 위한 배양조건으로서 산도 조건 및 배양 배지 조건에 관한 것이다.
본 발명은 천연물질로부터 유래하는 유산균을 이용하여 헬리코박터균의 위점막 부착을 억제하는 조성물 및 그 용도를 제공하며, 자세하게는 청국장으로부터 유래하는 헬리코박터 파이로리의 부착 억제능력을 가지는 Enterococcus faecium J9 (KCCM 11735P)를 포함하는 유산균 균주의 배양방법 및 그 용도를 제공한다. The present invention relates to a lactic acid bacterial strain composition which is isolated from chungkukjang having an ability to inhibit adhesion of Helicobacter pylori, and a method for producing a functional beverage utilizing the same. On the other hand, the present invention relates to acidic conditions and culture medium conditions as culture conditions for obtaining an acid-resistant lactic acid bacteria strain.
The present invention provides a composition for inhibiting gastric mucosal adhesion of Helicobacter pylori by using a lactic acid bacterium derived from a natural substance, and its use. More specifically, the present invention provides Enterococcus faecium J9 (KCCM 11735P) having ability to inhibit adhesion of Helicobacter pylori derived from Chungkookjang, And a method for culturing a strain of a lactic acid bacterium comprising the same.
Description
본 발명은 헬리코박터 파이로리의 위점막 부착 억제능력을 가지는 청국장으로부터 분리되는 유산균 균주 조성물, 그 제조방법 및 이를 활용한 기능성 음료에 관한 것이다. The present invention relates to a lactic acid bacterial strain composition which is isolated from chungkukjang having the ability to inhibit gastric mucosal adhesion of Helicobacter pylori, a method for producing the same, and a functional beverage using the same.
더욱 자세하게는, 본 발명은 내산성 유산균 균주 획득을 위한 배양조건으로서 산도 조건 및 배양 배지 조건에 관한 것이다. More specifically, the present invention relates to acidic conditions and culture medium conditions as culture conditions for obtaining an acid-resistant lactic acid bacteria strain.
헬리코박터균(Helicobacter pylori, H. pylori)은 미호기성 나선형 그람음성간균으로 1983년 위점막에서 처음 배양된 이후 위궤양, 만성 전정부 위염, 위암 및 MALT (mucosa-as-sociated lymphoid tissue) 림프종의 원인균으로 알려졌으며, 각종 소화기계 질환뿐만 아니라 혈액, 심장 질환과도 관련이 있는 것으로 알려져 있다. Helicobacter pylori (H. pylori) is a microaerobic spiral gram-negative bacillus that was first cultured in the gastric mucosa in 1983 and has been the causative agent of gastric ulcer, chronic gastritis, gastric cancer, and mucosa-as-sociated lymphoid tissue (MALT) It is known that it is related not only to various digestive system diseases but also to blood and heart diseases.
헬리코박터 파이로리균은 몇 개의 편모를 가지고 있는 나선형의 세균으로 헬리코박터균은 위장점막에 주로 감염되어 위염, 위궤양, 십이지장 궤양, 위선암, 위림프종 등을 유발하는 원인으로 작용한다. 위장점막의 표면이나 위장의 점액에서 발견되며, 위장점막 세포 자체를 뚫고 감염되는 것은 매우 드물다. 강산성의 위장 환경에서 요소분해효소(urease)를 가지고 있는데, 이는 세균이 알칼리성 환경을 만들어 위장점막에서 살아가는 데 필수적인 역할을 한다. Helicobacter pylori is a helical bacterium with several flagella. Helicobacter pylori is mainly infected to the gastric mucosa and causes gastritis, gastric ulcer, duodenal ulcer, gastric cancer, gastric lymphoma and the like. It is found on the surface of the gastric mucosa and in the mucus of the stomach, and it is very rare to get through the gastrointestinal mucosa itself. It has urease in strongly acidic gastrointestinal environment, which plays an essential role for bacteria to live in the gastric mucosa by creating an alkaline environment.
헬리코박터균에 감염되어 있는 경우, 위암 발생 위험성이 3.8배 높은 것으로 보고되어 있으며, 1994년 세계보건기구에 의해 헬리코박터균은 분명한 발암 인자로 분류 되었다. It has been reported that the risk of stomach cancer is 3.8 times higher when infected with Helicobacter pylori. Helicobacter pylori was classified as a clear carcinogen by the World Health Organization in 1994.
따라서, 헬리코박터균 제균 치료가 위암예방에서 중요한 역할을 할 수 있을 것으로 기대하고 있다. 전세계적으로 헬리코박터균 감염률은 성인의 50% 정도인 것으로 알려져 있고, 특히 개발도상국에서는 헬리코박터균 감염률이 80∼90%까지 보고되고 있으며, 최근 우리나라 보고에 따르면 61.3%로 알려져 있다. Therefore, anticancer therapy of Helicobacter pylori is expected to play an important role in gastric cancer prevention. Globally, H. pylori infection rate is known to be about 50% of adults, and especially in developing countries, H. pylori infection rate is reported to be 80 ~ 90%, and according to our country, 61.3% is known.
헬리코박터균 제균 치료는 양성자펌프억제제(proton pump inhibitor, PPI)를 기본으로 한 clarithromycin (500 mg bid)과 amoxicillin (1 g bid)의 병합요법을 일차 치료제로 사용하고 있다. 일차 치료제로 제균 치료를 시행한 경우 제균율이 55∼90%로 알려져 있고, 국내 한 연구에 따르면 표준일차 치료법의 제균율이 1991년부터 1998년까지 90% 이상을 보여 매우 효과적이었던 것에 반해 2000년 이후의 제균율이 80% 미만인 것으로 나타난다는 보고도 있어, 제균율을 높이기 위한 연구들이 진행 중이다. [The Korean Journal of Helicobacter and Upper Gastrointestinal Research, Vol. 11, No. 1, 26-36, June 2011] Helicobacter pylori eradication therapy uses a combination of clarithromycin (500 mg bid) and amoxicillin (1 g bid) based on a proton pump inhibitor (PPI) as a primary treatment. The incidence of eradication was known to be 55% to 90% in patients treated with primary treatment. According to a study conducted in Korea, the eradication rate of standard primary treatment was 90% or more from 1991 to 1998, There are reports that the eradication rate is less than 80%, and studies are ongoing to increase the eradication rate. [The Korean Journal of Helicobacter and Upper Gastrointestinal Research, Vol. 11, No. 1, 26-36, June 2011]
항생제 치료법에 있어서 제균율 저하의 원인은, 헬리코박터균이 위장점막 표면이나 점액에 존재하므로 항생제 유효성분이 헬리코박터균이 있는 곳까지 충분히 도달하지 못하는 경우가 많다는 점에 기인하는 것이며, 여러 차례 항생제에 노출이 된 적이 있는 경우에는 약물에 대한 내성이 발생하여 치료가 쉽지 않다는 원인 또한 존재한다. The reason for the lowering of the eradication rate in the antibiotic therapy is due to the presence of the antibiotic effective ingredient in the gastric mucosal surface or mucus in many cases where the Helicobacter bacterium does not sufficiently reach the place of the H. pylori bacteria, If there is an enemy, there is also a reason that the treatment is not easy due to the resistance to the drug.
한편, 항생제를 복용하다가 임의로 중단하는 경우에는 항생제 내성으로 인한 다음 치료가 어려워질 수 있고, 항생제를 병용했을 때 설사, 복통, 쓴맛, 오심 등의 부작용이 나타나는 문제점도 있다. On the other hand, if you stop taking antibiotics arbitrarily, the next treatment due to antibiotic resistance may become difficult, and side effects such as diarrhea, abdominal pain, bitter taste, and nausea may appear when antibiotics are added.
헬리코박터균의 위점막 부착 억제를 위한 항생제 치료의 대체로서, 생물학적 치료제에 대한 연구가 이루어지고 있으며, 이중 유산균에 대한 연구가 활발하게 진행되고 있다. As a substitute for antibiotic therapy for inhibiting gastric mucosal adhesion of Helicobacter pylori, research on biological therapeutic agents has been conducted, and studies on lactic acid bacteria have been actively conducted.
가장 많이 연구된 유산균에는 젖산을 생산하는 젖산간균속 세균(Lactobacillus spp.)과 비피도세균(Bifidobacterium spp.)이 있으며, 이중 Lactobacilli는 정상인의 위에 우세하게 존재하는 균으로 0∼103/mL가량 존재하는 것으로 알려져 있다. Lactobacillus spp. And Bifidobacterium spp. Are among the most studied lactic acid bacteria. Lactobacilli are predominantly present on normal individuals and present 0 ~ 10 3 / mL .
따라서, 본 발명은 상기 언급한 헬리코박터균의 위점막 부착 억제에 관한 항생제 약물치료의 문제점을 해결하기 위한 것으로서, 청국장으로부터 분리되는 유산균 균주 조성물, 그 제조방법 및 이를 활용한 기능성 음료를 제공함을 목적으로 한다. Accordingly, the object of the present invention is to solve the problem of antibiotic drug treatment for inhibiting adhesion of gastric mucosa to the gastric mucosa of the above-mentioned Helicobacter bacteria, and to provide a lactic acid bacteria strain composition isolated from Chungkukjang, a process for producing the same, and a functional beverage using the same do.
본 발명의 해결과제와 관련하여, 천연물질 활용의 측면에서 진행된 연구로서, 김치를 활용하는 방법, 발효홍삼을 이용하는 방법 등의 연구가 진행되었지만, 청국장으로부터 유래하는 유산균을 이용하는 연구는 아직까지 진행되지 않았다. In connection with the problem of the present invention, studies on the utilization of kimchi and the method of using fermented red ginseng have been carried out in order to utilize natural materials, but studies using lactic acid bacteria derived from Chungkookjang have not yet been conducted I did.
한편, 유산균을 활용하여 헬리코박터균을 제균하는 방법은, 강한 산성인 사람 위장 환경에서 유산균이 정상적으로 증식하며 활동하기에 매우 어렵다는 점과, 헬리코박터균의 구조적 특징상 생명력이 우수하여 균체의 완전한 사멸이 어렵다는 점에서, 문제점이 발생한다. On the other hand, the method of eradicating Helicobacter by using lactic acid bacteria is very difficult for the lactic acid bacteria to proliferate normally in a strong acidic gastrointestinal environment and it is difficult for the cells to die completely because of the excellent life of the Helicobacter In point, a problem arises.
따라서, 본 발명은 내산성을 가지는 유산균주의 획득을 위하여, 산도를 높인 유산균의 배양액이나 균체 분리 배양여액을 활용하는 방법을 제공하며, 나아가 이를 위한 유산균 배양배지의 조성비율을 제공한다. Accordingly, the present invention provides a method for utilizing lactic acid bacteria having increased acidity or a culture broth for the isolation of cultured bacteria to obtain a lactic acid bacteria having acid resistance, and further provides a composition ratio of the lactic acid bacteria culture medium for this.
본 발명이 이루고자 하는 기술적 과제들은 이상에서 언급한 기술적 과제들로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 본 발명의 기재로부터 당해 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다. The technical objects to be achieved by the present invention are not limited to the technical matters mentioned above, and other technical subjects which are not mentioned can be clearly understood by those skilled in the art from the description of the present invention .
상기와 같은 기술적 과제의 해결을 위하여, 본 발명은 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9 기탁번호 : KCCM 11735P)를 포함하는 헬리코박터 파이로리(Helicobacter pylori) 균주의 위점막 부착 억제용 조성물을 제공한다. In order to solve the above technical problems, the present invention provides a composition for inhibiting gastric mucosal adhesion of a Helicobacter pylori strain comprising Enterococcus pacem J9 strain (Enterococcus faecium J9 accession number: KCCM 11735P).
또한, 본 발명은 상기 조성물에 있어서, 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9 기탁번호 : KCCM 11735P)이외에도, 엔테로코커스 페칼리스(Enterococcus faecalis), 락토바실러스 플랜타룸(Lactobacillus plantarum), 락토바실러스 아시도필러스(Lactobacillus acidophilus), 락토바실러스 파라카세이(Lactobacillus paracasei), 락토바실러스 루테우리(Lactobacillus ruteuri), 비피도박테리움 비피디움(Bifidobacterium bifidium) 및 이들이 하나 이상 포함된 군에서 선택되는 어느 하나를 더 포함하는 것을 특징으로 하는 헬리코박터 파이로리(Helicobacter pylori) 균주의 위점막 부착 억제용 조성물을 제공한다. In addition to the Enterococcus faecium J9 strain (Enterococcus faecium J9 accession number: KCCM 11735P), the composition of the present invention may further comprise one or more selected from the group consisting of Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus acidophilus Wherein the microorganism further comprises one selected from the group consisting of Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus ruteuri, Bifidobacterium bifidium, and one or more of them Wherein the Helicobacter pylori strains are used for the prevention of gastric mucosal adhesion of Helicobacter pylori strains.
또한 본 발명은 상기 조성물이 유산균 배양액 또는 배양여액인 것을 특징으로 하며, 아울러, 배양액 또는 배양여액의 pH 가 3.0 내지 4.5 인 것을 특징으로 한다. Further, the present invention is characterized in that the composition is a culture solution of lactic acid bacteria or culture filtrate, and the pH of the culture solution or culture filtrate is 3.0 to 4.5.
한편, 본 발명은 헬리코박터 파이로리(Helicobacter pylori) 균주의 위점막 부착 억제용 조성물의 제조방법으로서, 배양배지원액, 탄소원 및 질소원을 포함하는 배양배지의 제조단계; 상기 배양배지에 엔테로코커스 패슘 J9 균주를 접종하는 단계; 및 상기 접종된 배지를 배양하는 단계를 제공한다. The present invention also provides a method of producing a composition for inhibiting stomach adherence of Helicobacter pylori strain, comprising the steps of: preparing a culture medium containing culture supporter, carbon source and nitrogen source; Inoculating said culture medium with Enterococcus paclitus J9 strain; And culturing the inoculated medium.
또한, 본 발명은 상기 제조방법에 있어서, 상기 탄소원 및 질소원을 포함하는 배양배지는 C(탄소) : N(질소) 비율이 0.5 내지 5.0 범위인 것을 특징으로 하며, 상기 탄소원 및 질소원을 포함하는 배양배지는 배지 100 중량부에 대하여 탄소원이 2 내지 5 중량부와 질소원이 1 내지 4 중량부인 것을 특징으로 한다. Further, the present invention is characterized in that the culture medium containing the carbon source and the nitrogen source has a C (carbon): N (nitrogen) ratio in the range of 0.5 to 5.0, and the culture comprising the carbon source and the nitrogen source The medium is characterized in that the carbon source is 2 to 5 parts by weight and the nitrogen source is 1 to 4 parts by weight based on 100 parts by weight of the medium.
또한, 본 발명은 상기 제조방법에 있어서, 상기 배양배지의 탄소원이 글루코스인 것을 특징으로 하며, 상기 배양배지의 질소원이 이스트 추출물 또는 펩톤인 것을 특징으로 한다. Further, the present invention is characterized in that in the above production method, the carbon source of the culture medium is glucose, and the nitrogen source of the culture medium is yeast extract or peptone.
한편, 본 발명의 다른 일측면으로서, 헬리코박터 파이로리(Helicobacter pylori) 균주의 위점막 부착 억제효과를 가지는 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9 기탁번호 : KCCM 11735P) 자체를 제공하며, 유산균 배양액을 활용한 헬리코박터 파이로리(Helicobacter pylori) 균주의 위점막 부착 억제 기능을 가지는 기능성 음료를 제공한다. As another aspect of the present invention, there is provided an enterococcus paclitaxel J9 strain (Enterococcus faecium J9 accession number: KCCM 11735P) itself having an effect of inhibiting gastric mucosal adhesion of Helicobacter pylori strain, A functional beverage having a gastric mucosal adhesion-inhibiting function of a Helicobacter pylori strain is provided.
청국장에서 유래하는 본 발명의 균주는 헬리코박터 파이로리의 위점막 부착을 억제하는 활성이 있고 항균력이 우수한 특징이 있다. 따라서, 본 발명의 유산균은 식품 첨가제로서 우수한 효과를 가지는바, 기능성 음료 제조 등의 용도로 활용 가능하다. The strain of the present invention derived from Chungkookjang has an activity of inhibiting gastric mucosal adhesion of Helicobacter pylori and is excellent in antibacterial activity. Therefore, the lactic acid bacterium of the present invention has an excellent effect as a food additive, and thus can be utilized for the production of functional beverages and the like.
도 1은 본 발명의 실시예 1에 따른 유산균의 16s rRNA 서열분석 결과를 나타내는 것이다.
도 2는 본 발명의 실시예 1에 따른 유산균의 16s rRNA 서열분석 결과에 기초로 한 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9 기탁번호 : KCCM 11735P) 의 계통발생 관계를 나타내는 계통도이다.
도 3은 본 발명의 실시예 2에서 제조한 유산균 배양여액 처리에 따른 헬리코박터 파이로리균의 부착능력을 비교한 실시예 3의 FACS 분석결과이다.
도 4는 본 발명의 실시예 3에 따른 부착된 헬리코박터균의 1차, 2차, 3차 실험 결과에서 유산균 및 대조군의 수를 비교하는 결과이다. 1 shows the result of 16s rRNA sequencing of a lactic acid bacterium according to Example 1 of the present invention.
FIG. 2 is a flow diagram showing the phylogeny relationship of the Enterococcus pacem J9 strain (Enterococcus faecium J9 accession number: KCCM 11735P) based on the 16s rRNA sequencing analysis of the lactic acid bacteria according to Example 1 of the present invention.
3 is a FACS analysis result of Example 3 in which the adhesion ability of Helicobacter pylori according to the lactic acid bacteria culture filtrate treatment prepared in Example 2 of the present invention was compared.
FIG. 4 shows the results of comparing the number of lactic acid bacteria and the control group in the results of primary, secondary, and tertiary tests of the attached Helicobacter pylori according to Example 3 of the present invention.
본 발명은 헬리코박터균의 위점막 부착 억제에 관한 생물학적 경감으로서, 청국장으로부터 분리되는 유산균 균주 조성물, 그 제조방법 및 이를 활용한 기능성 음료를 제공함을 목적으로 한다. The present invention is a biological alleviation of inhibition of adhesion of gastric mucosa to Helicobacter germs. It is intended to provide a lactic acid bacterial strain composition separated from chungkukjang, a method for producing the same, and a functional beverage utilizing the same.
더욱 자세하게는, 본 발명은 내산성 유산균 균주 획득을 위한 배양조건으로서 산도 조건 및 배양 배지 조건에 관한 것이다. More specifically, the present invention relates to acidic conditions and culture medium conditions as culture conditions for obtaining an acid-resistant lactic acid bacteria strain.
이하에서는, 본 발명에 의한 헬리코박터 파이로리의 위점막 부착 억제능력을 가지는 조성물 및 그 제조방법의 바람직한 실시예를 상세하게 설명한다. 이들 실시예는 본 발명에 대한 이해를 돕기 위한 것일 뿐이며, 어떤 의미로든 본 발명의 범위가 이들에 의해 제한되는 것은 아니다. Hereinafter, preferred embodiments of a composition having the gastric mucosal adhesion inhibitory ability of Helicobacter pylori according to the present invention and a method for producing the same will be described in detail. These examples are only for the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.
본 발명의 분야에서 통상의 지식을 가진 자가 청구범위에 기재된 권리범위 내에서 다양한 변형과 개작을 할 수 있다는 것은 자명하다.
It will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the appended claims.
실시예 (1) : 유산균의 분리 및 동정 Example (1): Isolation and Identification of Lactic Acid Bacteria
1) 청국장으로부터 유산균의 분리 1) Isolation of lactic acid bacteria from Chungkukjang
수집된 청국장으로부터 MRS 아가에 도말하는 작업을 수차례 반복 실시하여 5가지 종류의 유산균을 선발하였다. 이중 배양이 잘되고 산도가 높은 배양 균주를 선택하여 한국 미생물 보존센터에 16s rDNA Sequencing 의뢰하였고, NCBI BLAST 프로그램으로 비교하여 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9 기탁번호 : KCCM 11735P)인 것으로 나타났다. Five kinds of Lactobacillus were selected from the collected Chungkookjang by repeating the process of spraying on MRS agar several times. The 16S rDNA sequencing was selected by the Korean Society for Microbiology and Biotechnology, and the enterococcus pacema J9 strain (Enterococcus faecium J9 accession number: KCCM 11735P) was compared with the NCBI BLAST program.
2) 16s rDNA 서열분석을 통한 유산균 동정 2) Identification of lactic acid bacteria by 16s rDNA sequencing
분리된 유산균의 최종적인 동정을 위하여 16s rRNA 서열 분석을 수행하였다. 서열 분석을 위해 분리된 유산균을 Lactobacilli MRS broth에 배양하고, 배양액 1.5 mL를 취해 원심 분리한 후 0.8% 멸균 생리식염수로 수세하였다. 16s rRNA sequence analysis was performed for the final identification of isolated lactic acid bacteria. For sequencing, isolated lactic acid bacteria were cultured in Lactobacilli MRS broth, and 1.5 mL of the culture broth was centrifuged and then rinsed with 0.8% sterile physiological saline.
그리고 genomic DNA kit를 사용하여 chromosomal DNA를 추출하여 PCR을 위한 주형 DNA로 사용하였으며, 세균의 16s rRNA에 대한 universal primer로서 F-341(CCTACGGGAGGCAGCAG) 및 786-R(GACTACCAGGGTATCTAATC)를 사용하였다. PCR 수행이후 DNA 증폭산물을 확인하고, 다시 정제하였다. The genomic DNA kit was used to extract chromosomal DNA and used as template DNA for PCR. F-341 (CCTACGGGAGGCAGCAG) and 786-R (GACTACCAGGGTATCTAATC) were used as universal primers for bacterial 16s rRNA. After PCR, DNA amplification products were identified and further purified.
염기서열은 DNA 염기서열 분석 자동화기기를 활용하였으며, 프라이머는 상기 언급한 것과 동일하게 사용하였다. 16S rRNA 서열의 상동성 분석은 National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/)의 BLAST 서치 프로그램을 이용하여 DNA 데이타베이스와 비교하였다. The nucleotide sequence was determined using a DNA sequencing automated apparatus, and primers were used in the same manner as described above. Homology analysis of the 16S rRNA sequence was compared with the DNA database using the BLAST search program of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
또한, 동정된 유산균의 계통학적 분석도구로서 Clustal X, BioEdit, MEGA 4(www.megasoftware.net/)를 이용하였으며, 이를 통하여 염기서열 간의 유전적 거리와 계통도를 확인하였다. In addition, Clustal X, BioEdit, and MEGA 4 (www.megasoftware.net/) were used as phylogenetic analysis tools for the identified lactic acid bacteria.
도 1은 본 발명의 실시예 (1)에 따른 유산균의 16s rRNA 서열분석 결과를 나타내는 것이며, 도 2는 본 발명의 실시예 (1)에 따른 유산균의 16s rRNA 서열분석 결과에 기초로 한 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9 기탁번호 : KCCM 11735P)의 계통발생 관계를 나타내는 계통도이다. Fig. 1 shows the result of 16s rRNA sequencing of a lactic acid bacterium according to Example (1) of the present invention. Fig. 2 shows the results of 16s rRNA sequence analysis of lactic acid bacteria according to Example (1) (Enterococcus faecium J9 accession number: KCCM 11735P).
3) 유산균의 분리 및 동정 결과 3) Isolation and Identification of Lactic Acid Bacteria
유산균의 분리 및 동정 결과로서, 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9)를 포함하는 총 7개의 유산균 균주를 분리 선발할 수 있었다. As a result of the isolation and identification of the lactic acid bacteria, a total of seven lactic acid bacterial strains including the Enterococcus pacem J9 strain (Enterococcus faecium J9) could be isolated and selected.
Lactobacilli MRS 아가에서 배양된 집락을 형태학적인 특성에 따라 분리한 후, 그램 시험, 카탈라아제 시험, 현미경 관찰 등을 통하여 젖산균 의심 대상 균주를 선발하였다. 그리고 API 50 CHL kit 를 이용하여 당 발효특성 등의 생화학적 특성을 확인하고, 그 결과를 동정 프로그램을 통하여 재확인하였다. The colonies cultured on Lactobacilli MRS agar were separated according to their morphological characteristics and strains suspected to be lactic acid bacteria were selected through a gram test, a catalase test, and a microscopic observation. Biochemical characteristics such as sugar fermentation characteristics were confirmed using API 50 CHL kit, and the results were confirmed through identification program.
그 결과, 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9 기탁번호 : KCCM 11735P) 이외에도 엔테로코커스 페칼리스(Enterococcus faecalis), 락토바실러스 플랜타룸(Lactobacillus plantarum), 락토바실러스 아시도필러스(Lactobacillus acidophilus), 락토바실러스 파라카세이(Lactobacillus paracasei), 락토바실러스 루테우리(Lactobacillus ruteuri), 비피도박테리움 비피디움(Bifidobacterium bifidium)의 유산균이 존재하는 것으로 확인되었다. As a result, in addition to Enterococcus pacem J9 strain (Enterococcus faecium J9 accession number: KCCM 11735P), Enterococcus faecalis, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus ruteuri, and Bifidobacterium bifidium lactic acid bacteria were found to be present.
상기한 유산균은 헬리코박터 파이로리의 생육을 억제하고 내산성, 내염성 및 항균 활성을 가지는 특징이 있다. 특히, 청국장에서 분리한 본 발명의 유산균주는 pH 3.0 내지 4.5 범위에서 생리활성을 가지는 특징을 갖는다. The above-mentioned lactic acid bacteria are characterized by inhibiting the growth of Helicobacter pylori and having acid resistance, salt resistance and antibacterial activity. In particular, the lactic acid bacteria of the present invention isolated from chungkukjang have a physiological activity at pH 3.0 to 4.5.
한편, 엔테로코커스 패슘 J9 균주는 2015년 7월 23일자로 미생물 기탁기관인 한국 미생물 보존센터에 기탁하였고, 기탁번호 KCCM 11735P를 부여받았다.
On the other hand, the enterococcus paclitaxel strain J9 was deposited at the Korean Microorganism Conservation Center, which is a microorganism depository institution, on July 23, 2015, and received the deposit number KCCM 11735P.
실시예 (2) : 헬리코박터 파리로리의 위점막 부착 저해 성능 실험 Example (2): Inhibitory effect on gastric mucosa adhesion of Helicobacter pylori
1) 유산균 배양여액의 준비 및 산도 측정 1) Preparation of lactic acid bacteria culture filtrate and acidity measurement
먼저, 배양배지로서 배지 100 중량부에 대하여, 탄소원 (Glucose) 2 내지 5 중량부와 질소원 (Y.ext, Peptone류를 총합) 1 내지 4 중량부를 포함시켜 배지를 조성하고, 산도 pH 6.2 로 하여 온도 121℃ 조건에서 15분간 멸균하였다. First, as a culture medium, a culture medium containing 2 to 5 parts by weight of a carbon source (Glucose) and 1 to 4 parts by weight of a nitrogen source (total of Y.ext, peptones) was prepared with respect to 100 parts by weight of the medium, And sterilized at a temperature of 121 캜 for 15 minutes.
이후 Enterococcus faecium J9을 접종하고, 17시간 배양한 다음 15,000 rpm으로 원심분리하여 유산균체를 제거한 배양여액을 준비하였다. Then, Enterococcus faecium J9 was inoculated, cultured for 17 hours, and centrifuged at 15,000 rpm to prepare a culture filtrate in which the lactic acid bacteria were removed.
상기 언급한 바와 같이, 배양배지는 탄소원 및 질소원을 포함하는 것으로서, 이를 C(탄소) : N(질소) 비율로 나타내면 0.5 내지 5.0 범위인 것을 특징으로 한다. As mentioned above, the culture medium contains a carbon source and a nitrogen source, and is expressed by a ratio of C (carbon): N (nitrogen), which is in the range of 0.5 to 5.0.
한편, 본 발명은 상기 배양배지의 탄소원이 글루코스인 것을 특징으로 하며, 질소원은 이스트 추출물 또는 펩톤인 것을 특징으로 한다. Meanwhile, the present invention is characterized in that the carbon source of the culture medium is glucose, and the nitrogen source is yeast extract or peptone.
배양배지에서 유산균을 획득하는 조건은 산성 조건으로서 이는 유산균 배양에 의하여 발생하는 유기산 함량에 의하여 결정되었다. The conditions for obtaining lactic acid bacteria in the culture medium were acidic conditions, which were determined by the content of organic acids generated by the cultivation of lactic acid bacteria.
유기산은 산성의 특성을 나타내는 유기물질로서, 일반적으로 초산(Acetic acid), 인산(Phosphoric acid), 푸마르산(Fumaric acid), 젖산(Lactic acid), 구연산(Citric acid), 사과산(Malic acid), 뷰티르산(Butyric acid), 개미산(Formic acid), 프로피온산(Propionic acid) 등을 포함한다. 본 발명의 실시예에서는, 배양배지를 조성하고 pH 6.2 조건에서 121℃ 조건 15분간 멸균한 후, 유산균을 접종하였다. 이후, 17시간 배양한 후, 젖산의 함량을 정량하여 산도 조건을 결정하였다. Organic acid is an organic substance that shows the characteristic of acidity. It is generally used as an organic substance, and it is generally used as an organic substance such as acetic acid, phosphoric acid, fumaric acid, lactic acid, citric acid, malic acid, Butyric acid, formic acid, propionic acid, and the like. In the examples of the present invention, a culture medium was prepared and sterilized at 121 ° C for 15 minutes under pH 6.2 conditions, and lactic acid bacteria were inoculated. After incubation for 17 hours, the content of lactic acid was determined to determine acidity conditions.
배양배지에 유산균 접종 이후 3시간이 경과하면, 산도가 pH 4.5로 나타나며, 접종 이후 17시간이 경과하면, 산도는 pH 4.0로 나타났다. 이때의 유기산 함량은 1.84 % v/v 인 것으로 나타났다. After 3 hours of inoculation with lactic acid bacteria in the culture medium, the acidity showed a pH of 4.5, and after 17 hours of inoculation, the acidity showed a pH of 4.0. At this time, the organic acid content was found to be 1.84% v / v.
유기산 함량에 의한 산도 측정과 관련하여, 배양액 30ml를 0.1M NaOH 용액으로 pH가 8.3이 될 때까지 적정하여 소비된 0.1M NaOH 소비량을 구하여 젖산의 함량을 결정하였다. With respect to the acidity measurement by the organic acid content, 30 ml of the culture liquid was titrated with 0.1 M NaOH solution until the pH reached 8.3, and the consumed 0.1 M NaOH consumption was determined to determine the content of lactic acid.
이 경우, 배양액의 유산균 개체수가 2.5 X 1010 CFU/g 에 이르러 균체가 최적으로 배양되었음을 확인하였다. 헬리코박터 파이로리의 위점막 부착 억제 효과의 확인을 위하여, 배양액 자체 또는 배양여액을 사용하는 것이 가능하다. 본 실시예에서는 배양여액을 활용하였는데, 분리절차로서 배양액을 대상으로 하여 15,000g 조건 원심분리기를 이용하여 균체를 분리하고 배양여액을 회수하여 사용하였다. In this case, the number of lactic acid bacteria in the culture reached 2.5 X 10 10 CFU / g, confirming that the cells were cultured optimally. In order to confirm the gastric mucosal adhesion inhibitory effect of Helicobacter pylori, it is possible to use the culture medium itself or the culture filtrate. In this embodiment, the culture filtrate was used. As a separation procedure, the culture broth was separated from the cells using a centrifugal separator under a condition of 15,000 g, and the culture filtrate was recovered.
2) 위벽 상피세포 배양 2) Gastric epithelial cell culture
위벽 상피세포 배양을 위하여, RPMI-1640 배지를 준비하는데, RPMI-1640 배지에 송아지 혈청 10%, 항생제-항진균제 1%가 함유되도록 첨가하였다. For gastric epithelial cell culture, RPMI-1640 medium was prepared, which was supplemented with 10% calf serum and 1% antibiotic-antifungal agent in RPMI-1640 medium.
준비된 위벽 상피세포 배양용 RPMI-1640 배지를 2ml를 넣고, 여기에 위벽 상피세포(MKN-28 또는 MKN-45)를 접종하였다. 접종하는 세포 배양접시당 세포수는 1 X 105 이 되는 것이 바람직하다. 2 ml of RPMI-1640 medium for culture of prepared gastric epithelial cells was inoculated with gastric epithelial cells (MKN-28 or MKN-45). It is preferable that the number of cells per inoculated cell culture dish is 1 × 10 5 .
위벽 상피세포가 접종된 세포 배양접시를 5% 이산화탄소 존재하의 배양기에 넣어 37℃ 에서 2일 내지 3일 동안 배양하였다. Cell culture dishes inoculated with gastric epithelial cells were cultured in an incubator in the presence of 5% carbon dioxide at 37 ° C for 2 to 3 days.
위벽 상피세포가 완전 단층(confluent monolayer)을 이루는 면적이 세포배양접시의 80% 정도에 이르면 세포 배양접시로부터 배지를 제거하였다. When the area of the gastric epithelial cells forming a confluent monolayer reached about 80% of the cell culture dish, the culture medium was removed from the cell culture dish.
위벽 상피세포가 부착되어 있는 배양접시를 인산염 완충 식염수로 3회 세척하였다. The culture dish with gastric epithelial cells attached was washed three times with phosphate buffered saline.
3) 헬리코박터균 배양 3) Culture of Helicobacter
헬리코박터균 배양을 위하여, 브루셀라 혈액 배지를 준비하는데, 시판하는 브루셀라 배지(Brucella broth/agar)에 말 혈청 10%, 스키로 보충제 0.2%, 암포테리신 B 0.2%가 함유되도록 첨가하였다. For Brucella broth culture, 10% horse serum, 0.2% skolor supplement and 0.2% amphotericin B were added to commercially available brucella broth / agar.
이후, 준비된 브루셀라 혈액 배지에 헬리코박터 파이로리(ATCC 43504)를 접종하고, 10% 이산화탄소 배양기에 넣어 37℃ 에서 3일 동안 배양한 다음 시험에 사용하였다.
Then, the prepared Brucella blood culture medium was inoculated with Helicobacter pylori (ATCC 43504), cultured in a 10% carbon dioxide incubator at 37 ° C for 3 days, and then used for the test.
실시예 (3) : 헬리코박터 저해 in vitro 효과 확인 Example (3): Confirmation of in vitro effect of helicobacter inhibition
측정의 대상이 되는 유산균 한 종류당 최소한 4개의 세포 배양접시를 준비하였다. At least four cell culture dishes per kind of lactic acid bacteria to be measured were prepared.
1. 음성 대조군용 1 : 위벽 상피세포만 배양한 것 1. For negative control 1: cultured only gastric epithelial cells
2. 음성 대조군용 2 : 위벽 상피세포와 유산균 배양여액을 배양한 것2. For negative control 2: cultured gastric epithelial cells and culture solution of lactic acid bacteria
3. 양성 대조군용 : 위벽 상피세포와 헬리코박터 파이로리를 배양한 것3. For positive control: cultured gastric epithelial cells and Helicobacter pylori
4. 부착 억제 기능 측정용 : 위벽 상피세포, 헬리코박터 파이로리 및 유산균 배양여액을 모두 배양한 것 ( H.pylori 1 ml + 유산균 100 ug / ml처리) 4. Adhesion Suppression Function: For cultured gastric epithelial cells, Helicobacter pylori and lactic acid bacteria culture filtrate (treated with 1 ml of H.pylori + 100 μg / ml of lactic acid bacteria)
상기 준비한 4개의 배양접시를 최소 3회 이상 반복 실험하였다. The prepared four culture dishes were repeatedly tested at least three times.
헬리코박터의 위벽 상피세포 부착저해 효과의 확인을 위하여, FACS 분석을 진행하였다. FACS analysis was performed to confirm the inhibitory effect of Helicobacter on gastric epithelial cell adhesion.
도 3은 본 발명의 실시예 (1)에서 제조한 유산균 배양여액 처리에 따른 헬리코박터 파이로리균의 부착능력을 비교한 FACS 분석결과이며, 도 4는 본 발명의 실시예 (3)에 따른 1차, 2차, 3차 실험 결과에서 위벽에 부착된 헬리코박터균의 수를 대조군과 비교하는 결과이다. FIG. 3 is a FACS analysis result comparing the adhesion ability of Helicobacter pylori according to the lactic acid bacteria culture filtrate treatment prepared in Example (1) of the present invention. The results of the second and third experiments show that the number of Helicobacter bacteria attached to the stomach wall is compared with the control group.
헬리코박터의 위벽 상피세포 부착저해 효과의 확인을 위하여, 유산균 배양여액으로 처리한 군과 대조군의 세포를 떼어내어 FACS 분석을 통하여 헬리코박터 수를 측정하였고, 도 3 및 도 4 에서와 같이 유산균 처리군에서 헬리코박터의 수가 현저하게 감소함을 확인하였다. In order to confirm the inhibition effect of gastric epithelial cell adhesion on Helicobacter pylori, the number of Helicobacter pylori was measured by FACS analysis after removing the cells from the group treated with the lactic acid bacteria culture solution and the control group. As shown in FIGS. 3 and 4, Was significantly decreased.
도 4는 배양접시중 위벽 상피세포와 헬리코박터 파이로리를 배양한 양성 대조군용 접시와, 위벽 상피세포, 헬리코박터 파이로리 및 유산균 배양여액을 모두 배양한 부착 억제 기능 측정용 접시에 대하여, 3회 반복 실험을 통하여 얻은 헬리코박터 파이로리의 수를 비교한 결과이다. FIG. 4 is a graph showing the results of three repeated experiments on a plate for measurement of adherence-suppression function in which both gastric epithelial cells and Helicobacter pylori were cultured in a culture plate, a plate for a positive control group in which culture of gastric epithelial cells, H. pylori and lactic acid bacteria was cultured This is the result of comparing the number of obtained Helicobacter pylori.
유산균 처리에 의한 부착억제 효과를 확인한 결과, 저농도의 유산균 배양여액을 처리한 접시에서, 위벽 상피세포로부터 제균된 헬리코박터균이 59%~76% 정도 범위에 있는 것으로 확인되어, 높은 저해활성을 가지는 것으로 나타났다. As a result of confirming the adhesion inhibition effect by the lactic acid bacteria treatment, it was confirmed that the Helicobacter bacteria isolated from gastric epithelial cells were in the range of about 59% to 76% in the plate treated with the low concentration of the lactic acid bacteria culture filtrate, appear.
<110> j9 BIO Agricultural Association Corporation
<120> Composition for preventing Helicobacter pylori from attaching on
the epithelial cells of stomach, the preparation therefor, and
the functional beverage using them
<130> DP20150429
<140> 10-2015-0168002
<141> 2015-11-27
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 1384
<212> RNA
<213> Enterococcus faecium
<400> 1
agcttgctcc accggaaaaa gaggagtggc gaacgggtga gtaacacgtg ggtaacctgc 60
ccatcagaag gggataacac ttggaaacag gtgctaatac cgtataacaa tcgaaaccgc 120
atggttttga tttgaaaggc gctttcgggt gtcgctgatg gatggacccg cggtgcatta 180
gctagttggt gaggtaacgg ctcaccaagg ccacgatgca tagccgacct gagagggtga 240
tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca gtagggaatc 300
ttcggcaatg gacgaaagtc tgaccgagca acgccgcgtg agtgaagaag gttttcggat 360
cgtaaaactc tgttgttaga gaagaacaag gatgagagta actgttcatc ccttgacggt 420
atctaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca 480
agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggtttcttaa gtctgatgtg 540
aaagcccccg gctcaaccgg ggagggtcat tggaaactgg gagacttgag tgcagaagag 600
gagagtggaa ttccatgtgt agcggtgaaa tgcgtagata tatggaggaa caccagtggc 660
gaaggcggct ctctggtctg taactgacgc tgaggctcga aagcgtgggg agcaaacagg 720
attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttgg agggtttccg 780
cccttcagtg ctgcagctaa cgcattaagc actccgcctg gggagtacga ccgcaaggtt 840
gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900
gcaacgcgaa gaaccttacc aggtcttgac atcctttgac cactctagag atagagcttc 960
cccttcgggg gcaaagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020
tgggttaagt cccgcaacga gcgcaaccct tattgttagt tgccatcatt cagttgggca 1080
ctctagcaag actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg 1140
ccccttatga cctgggctac acacgtgcta caatgggaag tacaacgagt tgcgaagtcg 1200
cgaggctaag ctaatctctt aaagcttctc tcagttcgga ttgcaggctg caactcgcct 1260
gcatgaagcc ggaatcgcta gtaatcgcgg atcagcacgc cgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgaggtg 1380
aggt 1384
<110> j9 BIO Agricultural Association Corporation
<120> Composition for preventing Helicobacter pylori from attaching on
the epithelial cells of stomach, the preparation therefor, and
the functional beverage using them
<130> DP20150429
<140> 10-2015-0168002
<141> 2015-11-27
<160> 1
<170> KoPatentin 3.0
<210> 1
<211> 1384
<212> RNA
<213> Enterococcus faecium
<400> 1
Claims (13)
상기 배양배지에 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9 기탁번호 : KCCM 11735P)를 접종하는 단계; 및
상기 접종된 배지를 배양하는 단계;를 포함하고,
상기 엔테로코커스 패슘 J9 균주(Enterococcus faecium J9 기탁번호 : KCCM 11735P)는 청국장으로부터 유래하는 pH가 3.0 내지 4.5 범위의 배양액 또는 배양여액을 포함하며,
상기 탄소원 및 질소원을 포함하는 배양배지는 C(탄소):N(질소) 비율이 0.5 내지 5.0 범위인 것인 헬리코박터 파이로리(Helicobacter pylori)의 위점막 부착 억제용 조성물의 제조방법. A step of preparing a culture medium containing a culture medium supplement, a carbon source and a nitrogen source;
Inoculating the culture medium with Enterococcus pacmus J9 strain (Enterococcus faecium J9 accession number: KCCM 11735P); And
Culturing the inoculated medium,
The Enterococcus pacem J9 strain (Enterococcus faecium J9 accession number: KCCM 11735P) includes a culture medium or culture filtrate having a pH of 3.0 to 4.5 derived from chungkukjang,
Wherein the culture medium containing the carbon source and the nitrogen source has a C (carbon): N (nitrogen) ratio in the range of 0.5 to 5.0.
상기 탄소원 및 질소원을 포함하는 배양배지는 배지 100 중량부에 대하여 탄소원이 2 내지 5 중량부와 질소원이 1 내지 4 중량부인 것을 특징으로 하는 헬리코박터 파이로리(Helicobacter pylori)의 위점막 부착 억제용 조성물의 제조방법. 8. The method of claim 7,
Wherein the culture medium containing the carbon source and the nitrogen source comprises 2 to 5 parts by weight of carbon source and 1 to 4 parts by weight of nitrogen source per 100 parts by weight of the culture medium. Way.
상기 배양배지의 탄소원이 글루코스인 것을 특징으로 하는 헬리코박터 파이로리(Helicobacter pylori)의 위점막 부착 억제용 조성물의 제조방법. 8. The method of claim 7,
Wherein the carbon source of the culture medium is glucose. ≪ RTI ID = 0.0 > 11. < / RTI >
상기 배양배지의 질소원이 이스트 추출물 또는 펩톤인 것을 특징으로 하는 헬리코박터 파이로리(Helicobacter pylori)의 위점막 부착 억제용 조성물의 제조방법. 8. The method of claim 7,
Wherein the nitrogen source of the culture medium is yeast extract or peptone. ≪ RTI ID = 0.0 > 8. < / RTI >
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