KR100424790B1 - Lactic acid bacteria with inhibiting activities on helicobacter pylori - Google Patents
Lactic acid bacteria with inhibiting activities on helicobacter pylori Download PDFInfo
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- KR100424790B1 KR100424790B1 KR20010076750A KR20010076750A KR100424790B1 KR 100424790 B1 KR100424790 B1 KR 100424790B1 KR 20010076750 A KR20010076750 A KR 20010076750A KR 20010076750 A KR20010076750 A KR 20010076750A KR 100424790 B1 KR100424790 B1 KR 100424790B1
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- kccm
- lactobacillus
- strain
- bacteria
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Classifications
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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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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
- A23V2400/11—Lactobacillus
Abstract
본 발명은 헬리코박터 필로리의 활성을 억제하는 유산균에 관한 것으로, 헬리코박터 필로리의 위점막 부착을 억제시키고 헬리코박터 필로리의 성장을 저해시키는 균주, 락토바실러스 코프로필러스 PL 9001(KCCM-10245), 엔테로코커스 두란스 PL 9002(KCCM-10246), 스트렙토코커스 패칼리스 PL 9003(KCCM-10247), 락토바실러스 코프로필러스 PL 9004(KCCM-10248), 락토바실러스 퍼멘텀 PL 9005(KCCM-10250) 및 락토바실러스 퍼멘텀 PL 9006(KCCM-10251)를 제공한다. 본 발명의 균주들은 위궤양 억제제, 식품첨가제, 헬리코박터 감염 예방제 또는 치료제, 식중독 치료제, 세균감염 치료제 등으로 사용할 수 있다.The present invention relates to a lactic acid bacterium that inhibits the activity of Helicobacter pylori, a strain that inhibits the adhesion of gastric mucosa of Helicobacter pylori and inhibits the growth of Helicobacter pylori, Lactobacillus copropylus PL 9001 (KCCM-10245), Entero Caucas Durans PL 9002 (KCCM-10246), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus copropylus PL 9004 (KCCM-10248), Lactobacillus permanent PL 9005 (KCCM-10250) and Lactobacillus The company offers the company's new PL 9006 (KCCM-10251). Strains of the present invention can be used as a gastric ulcer inhibitor, food additives, Helicobacter infection prevention or treatment, food poisoning treatment, bacterial infection treatment and the like.
Description
본 발명은 헬리코박터 필로리의 활성을 억제하는 유산균에 관한 것으로서, 더욱 상세하게는 위궤양의 원인균인 헬리코박터 필로리의 위점막 부착을 억제할 수 있으며 성장도 억제할 수 있는 균주에 관한 것이다.The present invention relates to a lactic acid bacterium that inhibits the activity of Helicobacter pylori, and more particularly, to a strain capable of inhibiting gastric mucosa adhesion of Helicobacter pylori, a causative agent of gastric ulcer, and also inhibiting growth.
헬리코박터 필로리(Helicobacter pylori)는 그람음성 세균으로서 1983년 웨렌(Warren)과 마샬(Marshall)에 의하여 처음으로 사람의 위 점막조직에서 분리 및 동정되었다. 처음에는 캠필로박터 파이로리디스(Campylo bacter pyloridis)로 명명되었으나, 체내에서의 모양과 위내에서 주로 서식하는 유문부(pylorus)의 명칭을 인용하여 '헬리코박터 필로리'로 명명되었다.Helicobacter pylori is a Gram-negative bacterium that was first isolated and identified in human gastric mucosa by Warren and Marshall in 1983. It was originally named Campylo bacter pyloridis, but it was named Helicobacter pylori, citing the shape of the body and the name of the pylorus that inhabit the stomach.
헬리코박터 필로리는 위궤양의 원인균으로, 위암의 원인균이기도 하다.(Dubois, A. 1995. Spiral bacteria in the human stomach: the gastric Helicobacters. Emerg. Infect. Dis. 1: 790085 ; Slomiany, B. L. and A. Slomiany. 1992. Mechanism of Helicobacter pylori pathogenesis: focus on mucus. J. Clin. Gastroenterol. 14 Suppl 1 S114-21) 헬리코박터 필로리에 일단 감염되면 감염이 수십 년간 지속되고, 자연적으로 제거되는 경우는 거의 없어 헬리코박터 필로리는 만성 위염의 주원인이다.Helicobacter pylori is a causative agent of gastric ulcer, which is also a causative agent of gastric cancer (Dubois, A. 1995. Spiral bacteria in the human stomach: the gastric Helicobacters.Emerg. Infect.Dis. 1: 790085; Slomiany, BL and A. Slomiany Mechanism of Helicobacter pylori pathogenesis: focus on mucus.J. Clin.Gastroenterol. 14 Suppl 1 S114-21) Once infected with Helicobacter pylori, infection persists for decades and rarely eliminates naturally. Is the main cause of chronic gastritis.
헬리코박터 필로리의 감염은 식품 등과 함께 경구적으로 침입한 균이 위, 십이지장 점막에 부착함에 따라 시작된다. 헬리코박터 필로리의 병독인자로는 위내의 강산에서 생존하기 위해 분비하는 유레아제, 운동성을 유지하도록 하는 편모, 위 상피세포에 쉽게 부착하도록 도와주는 세포 표면의 외막단백질이 있다.Infection of Helicobacter pylori begins when orally invasive bacteria adhere to the stomach and duodenum mucosa along with foods. Helicobacter pylori virulence factors include urease secreting to survive in strong acids in the stomach, flagella to maintain motility, and outer membrane proteins on the cell surface to help adhere to gastric epithelial cells.
헬리코박터 필로리는 위점막에 부착시 사람의 적혈구에서 발현되는 것과 같은 항원에 부착된다.(Alkout, A. M., C. C. Blackwell, D. M. Weir, I. R. Poxton, R. A. Elton, W. Luman, and K. Palmer. 1997.Gastroenterol. 112: 1179001187 ; Boren, T., P. Flak, K. A. Roth, G. Larson, and S. Normark. 1993.Science262: 1892-1895 ; Clyne, M. and B. Drumm. 1997.Gastroenterol. 113: 72-80) 특히 O형 적혈구에 발현되는 레비스 항원(Lewis antigen)과 같은 항원이 위점막에도 발현되어 O형에서 위궤양 발생율이 높다.(Heneghan, M. A., A. P. Moran, K. M. Feeley, E. L. Egan, J. Goulding J, C. E. Connolly, and C. F. McCarthy. 1998.FEMS Immunol. Med. Microbiol. 20: 257-266 ; Kobayashi, K., J. Sakamoto, Y. Yamamura, T. Kito, H. Inagaki, T. Watanabe, and H. Nakazato. 1991.Nippon Geka Gakkai Zasshi92: 813-819 ; Kobayashi, K., J. Sakamoto, Y. Kito, Y. Yamamura, T. Koshikawa, M. Fugita, T. Watanabe, and H. Nakazato. 1993.Am. J. Gastroenterol. 88: 91900924)Helicobacter pylori attaches to antigens such as those expressed in human red blood cells when attached to the gastric mucosa (Alkout, AM, CC Blackwell, DM Weir, IR Poxton, RA Elton, W. Luman, and K. Palmer. 1997. Gastroenterol . 112: 1179001187; Boren, T., P. Flak, KA Roth, G. Larson, and S. Normark. 1993. Science 262: 1892-1895; Clyne, M. and B. Drumm. 1997. Gastroenterol . 113 : 72-80) In particular, antigens such as Levis antigen expressed in type O red blood cells are also expressed in gastric mucosa, resulting in a high incidence of gastric ulcer in type O. (Heneghan, MA, AP Moran, KM Feeley, EL Egan, J. Goulding J, CE Connolly, and CF McCarthy. 1998. FEMS Immunol.Med.Microbiol. 20: 257-266; Kobayashi, K., J. Sakamoto, Y. Yamamura, T. Kito, H. Inagaki, T. Watanabe, and H. Nakazato. 1991. Nippon Geka Gakkai Zasshi 92: 813-819; Kobayashi, K., J. Sakamoto, Y. Kito, Y. Yamamura, T. Koshikawa, M. Fugita, T. Watanabe, and H Nakazato. 1993. Am. J. Gastroenterol . 88: 91900924).
헬리코박터 필로리의 치료방법으로는 항생제, 프로톤 펌프 억제제, 위산 제거제를 이용한 방법을 사용하고 있으며, 헬리코박터 필로리의 대량 배양이 어려워 전체 세균을 이용한 백신 개발은 아직 이루어지지 않은 실정이다. 항생제를 이용한 방법은 약물 부작용이 있으며, 항생제에 대한 내성을 갖는 헬리코박터 필로리가 발생하는 문제점을 가지고 있다. 위산 제거제를 이용한 방법 역시 위산분비를 억제하는 방법으로 헬리코박터 필로리의 근본적인 치료가 되지 못한다. 또한 헬리코박터 필로리의 유레아제를 이용한 백신이 개발되었으나 효과가 탁월하지 않은 실정이다. 현재로써는 헬리코박터 필로리에 대한 백신은 헬리코박터 필로리의 까다로운 배양 조건과 작용 부위 때문에 앞으로도 개발이 쉽지 않을 것으로 생각된다.Helicobacter Philosophy is used as a method for the treatment of antibiotics, proton pump inhibitors, gastric acid eliminators, and the development of vaccines using whole bacteria has not been made yet due to the difficulty in mass culturing Helicobacter phyllori. Antibiotic methods have drug side effects and have a problem in that helicobacter pylori, which is resistant to antibiotics, occurs. Gastric acid remover is also a method of suppressing gastric acid secretion is not a fundamental treatment of Helicobacter Philosophy. In addition, a vaccine using urease of Helicobacter pylori was developed, but the effect is not excellent. At present, the vaccine against Helicobacter pylori may not be easy to develop due to the difficult culture conditions and site of action of Helicobacter pylori.
한편, 락토바실러스(Lactobacillus)는 락테이트와 아직 규명되지 않은 미지의 물질을 생산하여 헬리코박터 필로리를 억제하는 것으로 밝혀져 있으며, 현재까지는 락토바실러스 살리바리우스(Lactobacillus salivarius)의 경우 그 배양액이가장 헬리코박터 필로리 성장 억제 효과가 큰 것으로 알려져 있다.(Aiba, Y., N. Suzuki, A. M. Kabir, A. Takagi, and Y. Koga. 1998 Am. J. Gastroenterol. 93: 2097-2101) 또한 락토바실러스가 헬리코박터 필로리의 위점막 결합 부착되는 레비스 항원 (Lewis antigen) 결합을 억제하는 것으로 확인되었다.(Lee, Y., E. Shin, J. Lee, and J.H. Park. 1999. Lactobacillus acidophilus inhibits the Helicobacter pylori adherence.J. Microb. Biotech. 9: 794-797)On the other hand, Lactobacillus (Lactobacillus) has been shown to suppress the Helicobacter Philosophy by producing lactate and unknown substance has not yet been identified, until now Lactobacillus salivarius (Lactobacillus salivarius) its culture is the most Helicobacter Phillori It is known to have a large growth inhibitory effect (Aiba, Y., N. Suzuki, AM Kabir, A. Takagi, and Y. Koga. 1998 Am. J. Gastroenterol. 93: 2097-2101). Lori's gastric mucosal binding has been shown to inhibit the binding of the Levis antigen (Lee, Y., E. Shin, J. Lee, and JH Park. 1999. Lactobacillus acidophilus inhibits the Helicobacter pylori adherence. J. Microb.Biotech. 9: 794-797)
따라서, 본 발명은 위궤양을 억제할 수 있는 균주를 제공하는 것을 목적으로 한다.Therefore, an object of the present invention is to provide a strain capable of suppressing gastric ulcer.
또한 본 발명은 헬리코박터 필로리의 위점막 부착을 억제할 수 있는 균주를 제공하는 것을 목적으로 한다.It is another object of the present invention to provide a strain capable of inhibiting gastric mucosa adhesion of Helicobacter pylori.
또한 본 발명은 헬리코박터 필로리의 활성을 억제할 수 있는 균주를 제공하는 것을 목적으로 한다.It is another object of the present invention to provide a strain capable of inhibiting the activity of Helicobacter pylori.
또한 본 발명은 헬리코박터 필로리의 성장을 억제하는 물질을 생산하는 균주를 제공하는 것을 목적으로 한다.It is also an object of the present invention to provide a strain for producing a substance that inhibits the growth of Helicobacter pylori.
또한 본 발명은 식중독 균의 성장을 억제할 수 있는 균주를 제공하는 것을 목적으로 한다.It is another object of the present invention to provide a strain capable of inhibiting the growth of food poisoning bacteria.
또한 본 발명은 혐기성 세균 또는 여드름 유발균의 성장을 억제할 수 있는 균주를 제공하는 것을 목적으로 한다.It is also an object of the present invention to provide a strain capable of inhibiting the growth of anaerobic bacteria or acne-inducing bacteria.
또한 본 발명은 면역반응을 증강시키는 균주를 제공하는 것을 목적으로 한다.It is another object of the present invention to provide a strain for enhancing an immune response.
도 1은 적혈구에서 추출한 당지질에 헬리코박터 필로리를 결합시킨 것으로, 당지질의 양이 증가할수록(0 ug, 0.14 ug, 1.4 ug, 14 ug, 70 ug, 140 ug, 280 ug) 헬리코박터 필로리 부착량이 증가함을 확인한 것이고,Figure 1 is a Helicobacter Philoly coupled to the glycolipids extracted from red blood cells, the amount of Helicobacter Philosophy increases as the amount of glycolipid increases (0 ug, 0.14 ug, 1.4 ug, 14 ug, 70 ug, 140 ug, 280 ug) Confirming that
도 2는 본 발명의 PL 균주들이 헬리코박터 필로리의 당지질 부착을 억제하는지에 대하여 측정한 것이고,2 is a measure of whether the PL strains of the present invention inhibits glycolipid adhesion of Helicobacter pylori,
도 3은 PL 균주들의 헬리코박터 위점막 부착 억제능을 확인한 사진이고,Figure 3 is a photograph confirming the inhibition of Helicobacter gastric mucosa adhesion of PL strains,
도 4는 위세포인 MKN-45에 부착된 PL 균주를 나타낸 사진이고,Figure 4 is a photograph showing a PL strain attached to the gastric cells MKN-45,
도 5는 본 발명의 PL 균주들이 생산하는 물질에 의해 헬리코박터의 증식이 억제됨을 나타낸 것이고,Figure 5 shows that the proliferation of Helicobacter is suppressed by the material produced by the PL strains of the present invention,
도 6은 각 PL 균주별 헬리코박터 성장 억제환의 지름을 나타낸 것이고,Figure 6 shows the diameter of the Helicobacter growth inhibitory ring for each PL strain,
도 7은 PL 균주 배양여액에 의한 헬리코박터 필로리 성장억제정도를 나타낸 그래프이고,7 is a graph showing the degree of Helicobacter pylori growth inhibition by PL strain culture filtrate,
도 8은 본 발명의 락토바실러스 코프로필러스 PL 9001의 식중독균 성장억제능을 그래프로 나타낸 것이고,8 is a graph showing the growth inhibitory activity of food poisoning bacteria of Lactobacillus copropylus PL 9001 of the present invention,
도 9는 본 발명의 엔테로코커스 두란스 PL 9002의 식중독균 성장억제능을 그래프로 나타낸 것이고,9 is a graph showing the growth inhibitory activity of food poisoning bacteria of Enterococcus Durans PL 9002 of the present invention,
도 10은 본 발명의 스트렙토코커스 패칼리스 PL 9003의 식중독균 성장억제능을 그래프로 나타낸 것이고,10 is a graph showing the growth inhibitory activity of food poisoning bacteria of Streptococcus faecalis PL 9003 of the present invention,
도 11은 본 발명의 락토바실러스 코프로필러스 PL 9004의 식중독균 성장억제능을 그래프로 나타낸 것이고,11 is a graph showing the growth inhibitory ability of food poisoning bacteria of Lactobacillus copropylus PL 9004 of the present invention,
도 12는 본 발명의 락토바실러스 퍼멘텀 PL 9005의 식중독균 성장억제능을 그래프로 나타낸 것이고,12 is a graph showing the growth inhibitory activity of food poisoning bacteria of Lactobacillus permanentment PL 9005 of the present invention,
도 13은 본 발명의 락토바실러스 퍼멘텀 PL 9006의 식중독균 성장억제능을 그래프로 나타낸 것이고,13 is a graph showing the growth inhibitory activity of food poisoning bacteria of Lactobacillus permanent PL 9006 of the present invention,
도 14는 본 발명의 PL 균주들에 의한 여드름균 성장억제능을 그래프로 도시한 것이고,14 is a graph showing the growth inhibitory ability of acne by PL strains of the present invention,
도 15는 본 발명의 PL 균주들의 면역증강효과를 나타낸 그래프이고,15 is a graph showing the immune enhancing effect of the PL strains of the present invention,
도 16은 장세포인 Caco-2에 부착된 PL 균주를 나타낸 사진이다.Figure 16 is a photograph showing a PL strain attached to Caco-2 which is enterocytes.
상기의 목적을 달성하기 위하여, 본 발명은 헬리코박터 필로리 위점막부착억제능을 나타내는 락토바실러스 코프로필러스 PL 9004(KCCM-10248)를 제공한다.In order to achieve the above object, the present invention provides Lactobacillus copropylus PL 9004 (KCCM-10248) exhibiting a Helicobacter pylori gastric mucosa adhesion inhibitory ability.
또한 본 발명은 헬리코박터 필로리의 성장을 억제하는 락토바실러스 코프로필러스 PL 9004(KCCM-10248)를 제공한다.The present invention also provides Lactobacillus copropylus PL 9004 (KCCM-10248), which inhibits the growth of Helicobacter pylori.
또한 본 발명은 락토바실러스 코프로필러스 PL 9004(KCCM-10248)를 포함하는 세균성장 저해용 조성물을 제공한다.The present invention also provides a composition for inhibiting bacterial growth comprising Lactobacillus copropylus PL 9004 (KCCM-10248).
또한 본 발명은 락토바실러스 코프로필러스 PL 9004(KCCM-10248) 또는 이의 배양여액을 포함하는 화장품 조성물을 제공한다.The present invention also provides a cosmetic composition comprising Lactobacillus copropylus PL 9004 (KCCM-10248) or a culture filtrate thereof.
또한 본 발명은 락토바실러스 코프로필러스 PL 9004(KCCM-10248) 또는 이의 배양여액을 포함하는 식품첨가제를 제공한다.The present invention also provides a food additive comprising Lactobacillus copropylus PL 9004 (KCCM-10248) or a culture filtrate thereof.
또한 본 발명은 락토바실러스 코프로필러스 PL 9004(KCCM-10248)를 종균으로 사용하여 제조된 유제품을 제공한다.The present invention also provides a dairy product prepared using Lactobacillus copropylus PL 9004 (KCCM-10248) as a seed.
또한 본 발명은 락토바실러스 코프로필러스 PL 9004(KCCM-10248) 또는 이의 배양여액을 포함하는 면역증강용 조성물을 제공한다.In another aspect, the present invention provides a composition for immuno-enhancing comprising Lactobacillus copropylus PL 9004 (KCCM-10248) or a culture filtrate thereof.
또한 본 발명은 락토바실러스 코프로필러스 PL 9004(KCCM-10248) 또는 이의 배양여액을 포함하는 정장용 조성물을 제공한다.The present invention also provides a formal composition comprising Lactobacillus copropylus PL 9004 (KCCM-10248) or a culture filtrate thereof.
이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 헬리코박터 필로리의 위점막 부착을 억제시키는 균주를 제공한다.상기 균주는 유산균으로 락토바실러스 속 균주 및 엔테로코커스 속 균주가 바람직하다. 더욱 바람직하게는 상기 균주가 락토바실러스 코프로필러스(Lactobacillus coprophilus), 엔테로코커스 두란스(Enterococcus durans), 스트렙토코커스 패칼리스(Streptococcus faecalis) 또는 락토바실러스 퍼멘텀(Lactobacillus fermentum)이고, 가장 바람직하게는 락토바실러스 코프로필러스 PL 9001, 엔테로코커스 두란스 PL 9002, 스트렙토코커스 패칼리스 PL 9003, 락토바실러스 코프로필러스 PL 9004, 락토바실러스 퍼멘텀 PL 9005 또는 락토바실러스 퍼멘텀 PL 9006이다.The present invention provides a strain for inhibiting gastric mucosa adhesion of Helicobacter Philo. The strain is preferably Lactobacillus sp. And Enterococcus sp. More preferably, the strain is Lactobacillus coprophilus , Enterococcus durans , Streptococcus faecalis or Lactobacillus fermentum , and most preferably Lactobacillus fermentum . Bacillus copropylus PL 9001, Enterococcus Durans PL 9002, Streptococcus faecalis PL 9003, Lactobacillus copropylus PL 9004, Lactobacillus permanent PL 9005 or Lactobacillus permanent PL 9006.
상기 PL 균주들(락토바실러스 코프로필러스 PL 9001, 엔테로코커스 두란스 PL 9002, 스트렙토코커스 패칼리스 PL 9003, 락토바실러스 코프로필러스 PL 9004, 락토바실러스 퍼멘텀 PL 9005 및 락토바실러스 퍼멘텀 PL 9006)은 한국미생물보존센터에 국제기탁하여 기탁번호를 부여받았다. 국제기탁번호로는 락토바실러스 코프로필러스 PL 9001는 KCCM-10245이고, 엔테로코커스 두란스 PL 9002는 KCCM-10246이고, 스트렙토코커스 패칼리스 PL 9003은 KCCM-10247이고, 락토바실러스 코프로필러스 PL 9004는 KCCM-10248이고, 락토바실러스 퍼멘텀 PL 9005는 KCCM-10250이고, 락토바실러스 퍼멘텀 PL 9006은 KCCM-10251이다.The PL strains (Lactobacillus copropylus PL 9001, Enterococcus Durans PL 9002, Streptococcus faecalis PL 9003, Lactobacillus copropylus PL 9004, Lactobacillus fermentum PL 9005 and Lactobacillus fermentum PL 9006) It has been deposited with the Korea Center for Microbiological Conservation and has been given a deposit number. The international accession number is Lactobacillus copropylus PL 9001 is KCCM-10245, Enterococcus Durans PL 9002 is KCCM-10246, Streptococcus faecalis PL 9003 is KCCM-10247, and Lactobacillus copropylus PL 9004 is KCCM-10248, Lactobacillus permanent PL 9005 is KCCM-10250, and Lactobacillus permanent PL 9006 is KCCM-10251.
또한 본 발명은 위궤양 억제제를 제공한다. 위궤양 억제제는 락토바실러스 속 균주 또는 엔테로코커스 속 균주들을 단독 또는 혼합하여 포함하는 것을 특징으로 하고, 더욱 바람직하게로는 상기 PL 균주들을 포함한다. 락토바실러스 속 균주 또는 엔테로코커스속 균주들은 헬리코박터 필로리 성장을 억제할 뿐만 아니라 헬리코박터 필로리 위막 부착능을 억제시킨다. 상기한 효과는 락토바실러스 코프로필러스 PL 9001(KCCM-10245), 엔테로코커스 두란스 PL 9002(KCCM-10246), 스트렙토코커스 패칼리스 PL 9003 (KCCM-10247), 락토바실러스 코프로필러스 PL 9004(KCCM-10248), 락토바실러스 퍼멘텀 PL 9005 (KCCM-10250) 및 락토바실러스 퍼멘텀 PL 9006 (KCCM-10251)에 대하여 실험하여 확인하였다. 또한 PL 균주들은 헬리코박터 필로리 성장을 억제 또는 헬리코박터 필로리 위막 부착능을 억제시키는 물질을 생산하므로, PL 균주 배양여액 역시 위궤양 치료제로 사용할 수 있다. PL 균주들은 내항생제성, 내산성 및 내담즙성을 가지므로 생체내에서 안정한 상태를 유지할 수 있으며, 생균, 건조균 또는 사균 형태로 사용될 수 있다.The present invention also provides a gastric ulcer inhibitor. The gastric ulcer inhibitor is characterized in that it comprises the Lactobacillus strain or Enterococcus strains alone or in combination, more preferably comprises the PL strains. Lactobacillus strains or Enterococcus strains not only inhibit Helicobacter pylori growth but also inhibit Helicobacter pilori epithelial adhesion. The above effects are Lactobacillus copropylus PL 9001 (KCCM-10245), Enterococcus Durans PL 9002 (KCCM-10246), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus copropylus PL 9004 (KCCM -10248), Lactobacillus permanent PL 9005 (KCCM-10250) and Lactobacillus permanent PL 9006 (KCCM-10251). In addition, the PL strains produce a substance that inhibits the growth of Helicobacter pylori or inhibits the adhesion of Helicobacter pili, so that the PL strain culture filtrate can also be used as a gastric ulcer treatment. Since PL strains have antibiotic resistance, acid resistance, and bile resistance, they can maintain a stable state in vivo and can be used in the form of live, dry or dead bacteria.
본 발명의 위궤양 억제제는 어떠한 제형으로도 적용가능하며, 제조한 제형은 경구용, 비경구용, 도포용으로 사용 할 수 있다. 상기 제형은 주사용 또는 경구용으로 조제하는 것이 바람직하고, 경구용으로 조제하는 것이 가장 바람직하다. 상기 위궤양 억제제는 단일제 또는 복합제제로 제조할 수 있으며 복합제일 경우 약리학적으로 허용가능한 담체를 더욱 포함시켜 제조한다. 또한 제제에 포함되는 균주는 제조하는 제형에 따라 함량비를 조절하는 것이 바람직하다.Gastric ulcer inhibitors of the present invention can be applied to any formulation, the formulation can be used for oral, parenteral, application. The formulation is preferably prepared by injection or oral, most preferably by oral use. The gastric ulcer inhibitor may be prepared by a single agent or a combination, and in the case of a combination, it may be prepared by further including a pharmacologically acceptable carrier. In addition, the strain contained in the formulation is preferably adjusted to the content ratio according to the formulation to be prepared.
상기 PL 균주들의 배양여액은 통상적인 배양배지상에서 배양한 배양물을 여과 또는 원심분리하여 균주를 제거한 여액이 바람직하고, 더욱 바람직하게는 MRS 액체배지에 PL 균주를 접종하고 37 ℃에서 16시간 내지 48시간 배양한 다음 원심분리로 분리한 상등액이다.The filtrate of the PL strain is preferably the filtrate from which the strain is removed by filtration or centrifugation of the culture cultured on a conventional culture medium, more preferably inoculated PL strain in MRS liquid medium and 16 to 48 hours at 37 ℃ Supernatant separated by centrifugation after time incubation.
또한 본 발명의 락토바실러스 속 균주 및 엔테로코커스 속 균주는 다른 세균의 활성을 억제할 용도로 더욱 사용할 수 있다. 바람직하게는 PL 균주(PL9001,PL9002, PL9003, PL9004, PL9005 또는 PL9006), 세포벽 파쇄 분획, 및 PL 균주 배양여액을 혐기성 세균, 식중독 유발균, 여드름 유발균 등의 증식을 방지하는 용도로 사용하는 것이다.In addition, the strains of the genus Lactobacillus and the genus Enterococcus can be further used for the purpose of inhibiting the activity of other bacteria. Preferably, PL strains (PL9001, PL9002, PL9003, PL9004, PL9005 or PL9006), cell wall disruption fractions, and PL strain culture filtrates are used to prevent the growth of anaerobic bacteria, food poisoning bacteria, and acne causing bacteria. .
상기 혐기성 세균은 통상적인 병원성 세균으로 파상풍균, 가스괴저균, 클로로스트리듐균이 바람직하고, 더욱 바람직하게는 클로로스트리듐균이다.The anaerobic bacterium is a common pathogenic bacterium, preferably tetanus bacteria, gastroenteritis bacteria, or chlorotridium bacteria, and more preferably chlorotridium bacteria.
상기 식중독 유발균은 리스테리아증, 이질, 설사, 0157, 식중독을 유발하는 균이다.The food poisoning bacteria are Listeriosis, dysentery, diarrhea, 0157, bacteria causing food poisoning.
또한 본 발명의 PL 균주들은 면역성을 증가시킬 수 있는 활성을 가져, 건강증진을 위한 용도로 활용될 수 있으며, 위세포 부착 및 장세포 부착활성을 가진다. 본 발명의 PL 균주들은 위세포 부착 및 장세포 부착으로 위 및 장에 서식하여 세균의 성장을 저해하는 정장 작용을 수행한다.In addition, the PL strains of the present invention have activity that can increase immunity, can be used for health promotion, and have gastric cell adhesion and enteric cell adhesion activity. PL strains of the present invention inhabit the stomach and intestine by gastric cell adhesion and enterocyte adhesion to perform a formal function of inhibiting bacterial growth.
따라서, 본 발명의 PL 균주들은 헬리코박터 필로리 활성 저해, 세균의 활성저해, 면역증강 또는 정장작용을 위한 조성물로 사용할 수 있으며, 상기 조성물은 사료, 사료 첨가제, 식품, 식품첨가제, 약제 또는 화장품제 등으로 사용할 수 있다. 또한 상기 조성물은 PL 균주들의 파쇄된 세포벽 분획, 생균, 사균, 건조균 또는 배양여액을 유효성분으로 포함시킬 수 있으며, 부형제 또는 담체를 더욱 포함할 수 있다. 조성물내의 PL 균주 함량은 조성물의 용도, 제형에 따라 다르다. 상기 조성물이 부형제 또는 담체를 포함하는 경우, 조성물내 PL 균주함량은 0.001 중량% 내지 90 중량%가 바람직하며, 이에 한정되는 것은 아니다. 본 발명의 락토바실러스 속 균주 및 엔테로코커스 속 균주는 독성검사 결과, 안전한 시료로 확인되었다.Therefore, the PL strains of the present invention can be used as a composition for inhibiting Helicobacter phyllori activity, inhibiting the activity of bacteria, enhancing or suiting action, the composition is a feed, feed additives, food, food additives, drugs or cosmetics, etc. Can be used as In addition, the composition may include crushed cell wall fractions of PL strains, live bacteria, dead bacteria, dry bacteria or culture filtrate as an active ingredient, and may further include an excipient or a carrier. The PL strain content in the composition depends on the use and formulation of the composition. When the composition includes an excipient or a carrier, the PL strain content in the composition is preferably 0.001% to 90% by weight, but is not limited thereto. Lactobacillus strain and Enterococcus strain of the present invention was confirmed as a safe sample, as a result of the toxicity test.
상기 사료 또는 사료첨가제는 통상의 사료에 PL 균주를 0.001 내지 50 중량%로 포함하는 것이나, 이에 한정되진 않는다. 사료 또는 사료첨가제는 음료 또는 고형분으로 제조할 수 있다.The feed or feed additive includes a PL strain of 0.001 to 50% by weight in a conventional feed, but is not limited thereto. Feed or feed additives may be prepared as beverages or solids.
상기 식품 또는 식품첨가제는 요구르트, 이유식, 치즈, 유제품, 김치, 각종 음료 등 모든 식품이 바람직하고, 또한 본 발명의 PL 균주를 종균으로 사용하여 제조된 것도 바람직하다.The food or food additive is preferably all foods such as yogurt, baby food, cheese, dairy products, kimchi, various beverages, and also preferably produced using the PL strain of the present invention as a seed.
상기 약제는 단일제로 제조할 수 있으며, 1종 이상의 허용가능한 약리학적 조성물을 더욱 포함하여 복합제로 제조할 수 있다. 또한 약제는 약리학적으로 유용한 것으로 알려진 적합한 약학용 희석제를 더욱 포함할 수 있으며, 바람직한 희석제는 식염수, 완충 식염수, 덱스트로스, 물, 글리세롤, 및 에탄올로 이루어진 군으로부터 1종이상 선택된 것이다. 하지만 상기 희석제는 이에 국한되지 않는다. 당해 기술 분야에 알려진 적합한 제제는 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 기재되어 있다. 약제의 사용량은 사용용도 및 질병의 상태에 따라 달리 적용하는 것이 바람직하다. 치료의 종류와 정도, 다른 약물의 복용여부(예, 화학요법제), 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율 등의 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 좋다. 더욱 바람직하게, 약제의 단위 투여량은 1 내지 800 mg이 좋고, 가장 바람직하게는 40 내지 400 mg이다. 또한 환자, 질환의 종류 및 질환의 정도에 따라 용량 및 투여방법이 달라지기는 하나, 일반적으로 일일 0.1 내지 20 mg/kg으로 투여할 수 있으며, 선택적으로는 0.2 내지 10 mg/kg을 투여하고 1일 1-3회 투여하는 것이 바람직하다.The medicament may be prepared in a single agent and may be prepared in combination, further comprising one or more acceptable pharmacological compositions. The medicament may further comprise a suitable pharmaceutical diluent known to be pharmacologically useful, with the preferred diluent being at least one selected from the group consisting of saline, buffered saline, dextrose, water, glycerol, and ethanol. However, the diluent is not limited thereto. Suitable formulations known in the art are described in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. The amount of the drug is preferably applied differently depending on the purpose of use and the condition of the disease. Various factors and medicinal fields, including the type and severity of treatment, whether other medications are used (e.g. chemotherapy), the patient's age, weight, general health, sex and diet, time of administration, route of administration, and rate of composition Depending on the well-known analogy factor, it is best to apply differently. More preferably, the unit dose of the medicament is preferably 1 to 800 mg, most preferably 40 to 400 mg. In addition, depending on the patient, the type of disease and the degree of the disease, the dosage and administration method, but can be generally administered at 0.1 to 20 mg / kg daily, optionally 0.2 to 10 mg / kg and 1 It is preferable to administer 1-3 times a day.
상기 약제는 경구, 비경구 또는 도포법으로 투여할 수 있다. 비경구 투여는 직장, 국소, 정맥내, 복강내, 근육내, 동맥내, 경피, 비측내, 흡입, 안구내 또는 피내 주사 및 주입을 포함하는 투여 방식을 의미한다. 약제 제형은 어떠한 제형으로도 적용가능하며, 경구용 및 주사용 형태(용액, 현탁액 또는 유탁액)로 조제하는 것이 바람직하고, 정제, 캅셀제, 연질캅셀제, 수액제, 과립제, 환제 등 경구용으로 조제하는 것이 가장 바람직하다. PL 균주를 포함하는 약제는 연질캅셀에 부형제없이 충전하거나 미립된 고체 담체, 액체 담체 또는 그 양자와 균일하게 충분히 접촉시키고, 바람직한 제제로 성형할 수 있다. 상기 담체의 예로는 전분, 물, 식염수, 링거액 및 덱스트로스 용액이 있으며, 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 것을 사용할 수 있다.The agent can be administered orally, parenterally or by application. Parenteral administration means administration modes including rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal injection and infusion. Pharmaceutical formulations may be applied in any formulation, preferably in oral and injectable form (solutions, suspensions or emulsions), orally or in tablets, capsules, soft capsules, infusions, granules, pills, and the like. Most preferred. A medicament comprising the PL strain may be uniformly sufficiently brought into contact with the solid capsule, the liquid carrier or both, without the excipient or filled with the soft capsule, and molded into the desired formulation. Examples of such carriers include starch, water, saline, Ringer's solution and dextrose solution, and those disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.
상기 PL 균주들을 포함하는 화장품제는 PL 균주들을 0.001 내지 20 중량%로 포함하는 것이 바람직하나, 이에 한정되는 것은 아니다. 상기 화장품제는 통상적인 피부화장료에 배합되는 보통의 성분, 예를 들면 유분, 물, 계면활성제, 보습제, 저급알콜, 증점제, 킬레이트제, 색소, 방부제, 향료 등을 필요한 만큼 적용 배합하는 것이 가능하다. 상기 화장료 조성물은 피부에 사용하는 것으로서, 스킨, 로션, 영양크림, 맛사지크림, 에센스, 팩, 파우더, 연고, 서스펜션, 에멀젼 또는 스프레이 등의 통상의 화장료 형태로 제조될 수 있으며, 이들 각 제형에 적합하고 당업계에 주지된 각종의 통상적인 담체와 첨가제를 포함할 수 있다.Cosmetics containing the PL strains preferably include PL strains in 0.001 to 20% by weight, but is not limited thereto. The cosmetics may be applied and blended with the usual ingredients, such as oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., as necessary. . The cosmetic composition is used for the skin, and may be prepared in a common cosmetic form such as skins, lotions, nourishing creams, massage creams, essences, packs, powders, ointments, suspensions, emulsions, or sprays, and is suitable for each of these formulations. And various conventional carriers and additives well known in the art.
또한 본 발명은 락토바실러스 속 균주 또는 엔테로코커스 속 균주에서 생성되는 유해세균 성장억제물질을 제공한다. 상기 유해세균 성장억제물질은 헬리코박터 필로리, 혐기성 세균, 여드름 유발균, 또는 식중독 유발균의 성장을 저해하는 물질로, 상기 균주를 배양하였을 때 배양여액에서 수득되어질 수 있다. 따라서 락토바실러스 속 균주 또는 엔테로코커스 속 균주에서 생성되는 유해세균 성장억제물질 역시 세균저해용 및 면역증강용으로 사용할 수 있다.In another aspect, the present invention provides a harmful bacterium growth inhibitory substance produced from the strain Lactobacillus or Enterococcus strain. The harmful bacterium growth inhibitory substance is a substance that inhibits the growth of Helicobacter Philoly, anaerobic bacteria, acne-causing bacteria, or food poisoning-causing bacteria, can be obtained in the culture filtrate when the strain is cultured. Therefore, harmful bacterium growth inhibitory substances generated from Lactobacillus sp. Or Enterococcus spp. Strains may also be used for bacteriostatic and immune enhancing.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 하기의 실시예에 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.
실시예 1: 균주의 분리Example 1 Isolation of Strains
MRS broth(Difco, bacto proteose peptone No.3 10 g, bacto beef extract 10 g, bacto yeast extract 5 g, bacto dextrose 20 g, polysorbate 80 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate 0.1 g, manganese sulfate 0.05 g, dipotassium phosphate 2 g /L)에 브롬페놀 블루(bromphenol blue)를 0.002 %로 첨가한 MRS+BPB 배지를 준비하였다. 김치 시료는 펩톤수로 십배수 희석하여 0.1 ㎖씩 MRS+BPB 배지에 접종한 후 유리막대로 도말하였고, 영유아의 분변은 면봉으로 채취한 뒤 MRS+BPB 배지에 접종하였다. 배양은 25 ℃의 항온배양기에서 3-4일간 배양한 후 생성된 콜로니를 관찰하여 유산균을 분리하였다.MRS broth (Difco, bacto proteose peptone No. 3 10 g, bacto beef extract 10 g, bacto yeast extract 5 g, bacto dextrose 20 g, polysorbate 80 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate 0.1 g, 0.05 g of manganese sulfate and 2 g / L of dipotassium phosphate were prepared with MRS + BPB medium in which bromphenol blue was added at 0.002%. Kimchi samples were diluted 10-fold with peptone water and inoculated in 0.1 ml of MRS + BPB medium and plated with glass rods. The feces of infants and toddlers were collected with a cotton swab and inoculated in MRS + BPB medium. The culture was incubated for 3-4 days in an incubator at 25 ℃ after observing the resulting colonies were isolated lactic acid bacteria.
유산균의 집락 형태를 구별하기 위해 pH 3.0일 때 황색, pH 4.6일 때 자색을나타내는 BPB(bromphenol blue)를 MRS 고체배지에 첨가하여 집락의 BPB 착색정도로 유산균을 감별하였다. BPB에 의한 착색 정도는 유산 생성정도와 pH 내성, 그리고 수명에 따라 형성된다.P. acidolactic와S. faecalis는 정상유산발효균주이며,L. mesenteroides와L. brevis는 이상유산발효를 하며,L. plantarum는 임의정상유산발효를 하는 것으로 알려져 있다.S. faecalis는 정상유산발효를 하므로 유산을 많이 생성하기 때문에 백색의 집락이 형성되고, 배지는 담황색으로 변화된다. 이상유산발효를 하는L. mesenteroides는 유산생성이 적으므로 집락의 색상은 전체적으로 암청색으로 환이 없고 집락의 크기가 작게 나타난다.In order to distinguish the colony form of lactic acid bacteria, BPB (bromphenol blue), which is yellow at pH 3.0 and purple at pH 4.6, was added to MRS solid medium, and the lactic acid bacteria were discriminated by the degree of BPB coloring of colonies. The degree of pigmentation by BPB depends on the degree of lactic acid formation, pH tolerance, and lifespan. P. acidolactic and S. faecalis are normal lactic acid fermentation strains, L. mesenteroides and L. brevis are known as biphasic fermentation, and L. plantarum is known to ferment randomly. S. faecalis produces a lactic acid because fermentation of normal lactic acid produces white colonies and the medium turns pale yellow. L. mesenteroides, which are fermented by abnormal abortions, have low lactic acid production, so the color of colonies is dark blue, and there are no rings and the size of colonies is small.
락토바실러스는 전체적으로 담청색을 띄거나 중앙에 암청색의 환이 있고, 또는 전체적으로 흰색이며 집락의 크기가 큰 것으로 구분하였다. 그리고P. acidolactic와L. mesenteroides가 암청색으로 되는 이유는 짧은 수명(life time)과 pH에 기인한다. 그 예로L. mesenteroides는 pH 4.8 이하에서는 생장 할 수 없다.Lactobacillus was divided into either light blue or dark blue rings in the center, or white and large colonies. The reason why P. acidolactic and L. mesenteroides become dark blue is due to short life time and pH. For example, L. mesenteroides cannot grow below pH 4.8.
본 균주들은 모두 0.3 mm이상의 흰색의 집락을 형성하여 락토바실러스로 구분하였다.All of these strains formed a colony of white more than 0.3 mm and classified as Lactobacillus.
단일 콜로니로 분리한 후 버기스 매뉴얼(Bergy's manual of systematic bacteriology)에 준하여 형태학적, 생화학적 특성을 조사하여, 그람염색과 카탈라제반응을 확인한 다음, API system (La Balme-les-Grottes, France)으로 동정하였다. 면봉을 이용하여 콜로니를 취한 후 멸균 증류수 2 ㎖에 부유시켰다. 부유액은 API 50 CHL 배지에 첨가하여 균질화시켰다. 균질화된 API 50 CHL 배지는 API50 CH의 50개의 튜브에 200 ㎕씩 접종하고 미네랄 오일로 튜브 위를 덮어 준 후 37 ℃에서 48시간 동안 배양시킨 후 결과를 관찰하였다. API 50 CH 및 API 50 CHL로 49종의 탄수화물 발효패턴을 확인한 후 이 결과를 ATB 동정 컴퓨터 시스템(identification computer system, bio Merieux France)에 입력하여 동정하였다. 분리한 균 중 제 1 균의 동정 결과는 표 1과 같다.After separation into single colonies, the morphological and biochemical properties were examined according to the Bergy's manual of systematic bacteriology, and the gram staining and catalase reactions were confirmed.Then, the API system (La Balme-les-Grottes, France) was used. I identified it. Colonies were taken using a cotton swab and then suspended in 2 ml of sterile distilled water. Suspension was added to API 50 CHL medium and homogenized. Homogenized API 50 CHL medium was inoculated with 200 μl in 50 tubes of API50 CH, the tube was topped with mineral oil and incubated at 37 ° C. for 48 hours, and the results were observed. After confirming 49 carbohydrate fermentation patterns with API 50 CH and API 50 CHL, the results were identified by entering the ATB identification computer system (bio Merieux France). The results of the identification of the first strain among the isolates are shown in Table 1.
동정한 결과 제 1균주는 락토바실러스 코프로필러스(Lacto. coprophilus)에 99.9 %, 락토바실러스 브레비스(Lacto. brevis)에 0.1 % 동일성을 가짐을 확인하였다. 또한 제 1균주의 16S rRNA를 Microseq 16S rRNA gene kit(Perkin Elmer Applied Biosystem)로 염기서열을 분석하여 서열번호 1의 염기서열을 확인하였다. 서열번호 1의 염기서열은 BLAST 검색결과(http://www.ncbi.nlm.nih.gov/blast) 락토바실러스 코프로필러스의 16S rRNA의 염기서열과 동일하였다. 락토바실러스 코프러필러스는 바이젤라 콘퓨자(Weissella confusa) 또는 락토바실러스 콘퓨서스(L. confusus)라고도 한다. 상기 균주는 락토바실러스 PL 9001(KFCC-11240)로 한국종균협회에 국내기탁하였고, 동일 균주를 락토바실러스 코프로필러스 PL 9001(KCCM-10245)로 한국미생물보존센터에 국제기탁하였다.As a result, it was confirmed that the first strain had 99.9% identity to Lacto. Coprophilus and 0.1% identity to Lacto. Brevis. In addition, the nucleotide sequence of SEQ ID NO: 1 was confirmed by analyzing the nucleotide sequence of the 16S rRNA of the first strain with a Microseq 16S rRNA gene kit (Perkin Elmer Applied Biosystem). The base sequence of SEQ ID NO: 1 was identical to the base sequence of 16S rRNA of Lactobacillus copropylus (http://www.ncbi.nlm.nih.gov/blast). Lactobacillus coprophilus is also known as Weissella confusa or L. confusus . The strain was domestically deposited with the Korean spawn association as Lactobacillus PL 9001 (KFCC-11240), and the same strain was internationally deposited with the Korea Microbial Conservation Center as Lactobacillus copropylus PL 9001 (KCCM-10245).
제 2균은 API STREP Kit을 사용하여 동정하였고, 동정결과는 표 2와 같다.The second strain was identified using the API STREP Kit, and the identification results are shown in Table 2.
상기 제 2 균주는 API STREP KIT으로는 동정이 되지 않아 16S rRNA 염기서열을 분석하였다. 그 결과 서열번호 2의 염기서열을 가지고 있었으며, 엔테로코커스 두란스(Enterococcus durans)의 16S RNA 염기서열과 동일하였다. 따라서 가장 정확한 염기서열분석결과를 토대로, 제 2균주를 엔테로코커스 두란스 PL 9002로 명명하였다. 상기 균주는 엔테로코커스 두란스로 PL 9002(KFCC-11241)로 한국종균협회에 국내기탁하였고, 동일 균주를 엔테로코커스 듀란스로 PL 9002(KCCM-10246)로 한국미생물보존센터에 국제기탁하였다.The second strain was not identified by API STREP KIT, and 16S rRNA sequences were analyzed. As a result, it had the nucleotide sequence of SEQ ID NO: 2, and was identical to the 16S RNA nucleotide sequence of Enterococcus durans . Therefore, based on the most accurate sequencing results, the second strain was named Enterococcus Durans PL 9002. The strain was domestically deposited with the Enterococcus Durans PL 9002 (KFCC-11241) to the Korean spawn association, and the same strain was internationally deposited with the Enterococcus Durans PL 9002 (KCCM-10246).
제 3균의 동정결과는 표 3과 같다.The identification results of the third bacteria are shown in Table 3.
동정한 결과 제 3균주는 스트렙토코커스 페칼리스(Streptococcus faecalis, Enterococcus faecalis)와 99.1% 동일한 것으로 동정되었다. 상기 균주는 스트렙토코커스 페칼리스 PL 9003(KFCC-11242)로 한국종균협회에 국내기탁하였고, 동일 균주를 스트렙토코커스 페칼리스 PL 9003 (KCCM-10247)로 한국미생물보존센터에 국제기탁하였다.As a result, the third strain was identified as 99.1% identical to Streptococcus faecalis (Enterococcus faecalis ). The strain was domestically deposited to the Korean spawn association with Streptococcus pecalis PL 9003 (KFCC-11242), and the same strain was internationally deposited to the Korea Microbial Conservation Center with Streptococcus pecalis PL 9003 (KCCM-10247).
상기 제 3균주의 16S rRNA 분석결과 서열번호 3의 염기서열을 가지고 있었으며, 스트렙토코커스 패칼리스의 16S RNA와 염기서열이 동일하였다. 제 4균의 동정결과는 표 4와 같다.16S rRNA analysis of the third strain had a nucleotide sequence of SEQ ID NO: 3, the base sequence and 16S RNA of Streptococcus faecalis was identical. Identification results of the fourth strain is shown in Table 4.
동정한 결과 제 4균주는 락토바실러스 코프로필러스(Lacto. coprophilus)에 98.2 %, 락토바실러스 브레비스(Lacto. brevis)에 1.3 % 동일하였다. 또한 제 4균주의 16S RNA는 서열번호 4로 확인되었고, 서열번호 4의 염기서열은 BLAST 검색결과(http://www.ncbi.nlm.nih.gov/blast) 락토바실러스 코프로필러스의 16S rRNA의 염기서열과 동일하였다. 상기 균주는 락토바실러스 PL 9004(KFCC-11243)로 한국종균협회에 국내기탁하였고, 동일 균주를 락토바실러스 코프로필러스 PL 9004(KCCM-10248)로 한국미생물보존센터에 국제기탁하였다.As a result, the fourth strain was the same as 98.2% in Lacto. Coprophilus and 1.3% in Lacto. Brevis. In addition, 16S RNA of the fourth strain was identified by SEQ ID NO: 4, and the nucleotide sequence of SEQ ID NO: 4 was found in BLAST search results (http://www.ncbi.nlm.nih.gov/blast) Lactobacillus copropylus 16S rRNA It was identical to the nucleotide sequence of. The strain was domestically deposited with the Korean spawn association as Lactobacillus PL 9004 (KFCC-11243), and the same strain was internationally deposited with the Korea Microorganism Conservation Center as the Lactobacillus copropylus PL 9004 (KCCM-10248).
제 5균의 동정결과는 표 5와 같다.Identification results of the fifth strain is shown in Table 5.
동정한 결과 제 5균주는 락토바실러스 퍼멘텀(Lacto. fermentum)에 93.2 %Leuoconostoc lactis와 6.7 % 동일하였다. 또한 제 5균주의 16S rRNA는 서열번호 5로 나타났으며, 서열번호 5의 염기서열을 BLAST 검색한 결과(http://www.ncbi.nlm.nih.gov/blast) 락토바실러스 퍼멘텀의 16S rRNA의 염기서열과 일치하였다. 상기 균주는 락토바실러스 퍼멘텀 PL 9005 (KCCM-10250)로 한국미생물보존센터에 기탁하였다.As a result, the fifth strain was 6.7% identical to 93.2% Leuoconostoc lactis in Lacto. Fermentum . In addition, 16S rRNA of the fifth strain was shown in SEQ ID NO: 5, BLAST search of the nucleotide sequence of SEQ ID NO: 5 (http://www.ncbi.nlm.nih.gov/blast) Lactobacillus fermentum 16S It matched the nucleotide sequence of rRNA. The strain was deposited with the Korea Microorganism Conservation Center as Lactobacillus permanent PL 9005 (KCCM-10250).
제 6균의 동정결과는 표 6과 같다.The identification results of the sixth strain are shown in Table 6.
동정한 결과 제 6균주는 락토바실러스 퍼멘텀(Lacto. fermentum)에 94.4 %, 락토바실러스 락티스에 5.4 % 동일하였다. 또한 제 6균주의 16S rRNA는 서열번호 5로 나타났으며, 서열번호 6의 염기서열을 BLAST 검색한 결과(http://www.ncbi.nlm.nih.gov/blast) 락토바실러스 퍼멘텀의 16S rRNA의 염기서열과 동일하였다. 상기 균주는 락토바실러스 퍼멘텀 PL 9006 (KCCM-10251)로 한국미생물보존센터에 기탁하였다.As a result, the sixth strain was equal to 94.4% in Lacto. Fermentum and 5.4% in Lactobacillus lactis. In addition, 16S rRNA of strain 6 was found to be SEQ ID NO: 5, BLAST search of the nucleotide sequence of SEQ ID NO: 6 (http://www.ncbi.nlm.nih.gov/blast) Lactobacillus fermentum 16S It was identical to the nucleotide sequence of rRNA. The strain was deposited with the Korea Microorganism Conservation Center as Lactobacillus permanent PL 9006 (KCCM-10251).
실시예 2: 헬리코박터 필로리 위점막 부착 억제능 실험Example 2 Helicobacter Philosophy Gastric Mucoadhesion Inhibition Test
(1) 균주 준비(1) Strain Preparation
헬리코박터 필로리(ATCC 43504)는 Brucella 고형배지(Brucella broth, fungizone(2.5 mg/ml amphotericin B), Skirrow's supplement(0.16 mg/ml polymyxin B, 5 mg/ml vancomycin, 2.5 mg/ml trimethoprim), 10 % horse serum, 1.5 % agar)에 접종하여 5 내지 10 % 이산화탄소 배양기에서 37 ℃, 48시간 배양하였다. 배양된 균체는 배지로부터 긁어모아 인산완충액(PBS, pH7.4)으로 2-3회 세척하고, 소량의 10 mM Tris-Cl 완충용액에 현탁시켜 사용하기 전까지 -20 ℃에 보관하였다.Helicobacter pylori (ATCC 43504) is a Brucella solid medium (Brucella broth, fungizone (2.5 mg / ml amphotericin B), Skirrow's supplement (0.16 mg / ml polymyxin B, 5 mg / ml vancomycin, 2.5 mg / ml trimethoprim), 10% horse serum, 1.5% agar) was incubated for 48 hours at 37 ℃ in 5-10% carbon dioxide incubator. The cultured cells were scraped from the medium, washed 2-3 times with phosphate buffer (PBS, pH7.4), suspended in a small amount of 10 mM Tris-Cl buffer and stored at -20 ° C until use.
실시예 1의 균주들은 통칭하여 이하 PL 균주로 나타낸다. PL 균주들(락토바실러스 코프로필러스 PL 9001(KCCM-10245), 엔테로코커스 두란스 PL 9002(KCCM-10246), 스트렙토코커스 패칼리스 PL 9003 (KCCM-10247), 락토바실러스 코프로필러스 PL 9004(KCCM-10248), 락토바실러스 퍼멘텀 PL 9005(KCCM-10250), 락토바실러스 퍼멘텀 9006(KCCM-10251))을 MRS 액체배지에 접종하여 37 ℃에서 24시간 배양한 후 원심분리로 가라앉힌 균체를 PBS(pH 7.4)로 2-3회 세척하고, 소량의 10 mM Tris-Cl 완충용액에 균체를 현탁시켜 사용하기 전까지 -20 ℃에 보관하였다.The strains of Example 1 are collectively referred to as PL strains below. PL strains (Lactobacillus copropylus PL 9001 (KCCM-10245), Enterococcus Durans PL 9002 (KCCM-10246), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus copropylus PL 9004 (KCCM -10248), Lactobacillus Permantum PL 9005 (KCCM-10250), Lactobacillus Permantum 9006 (KCCM-10251)) were inoculated in MRS liquid medium and incubated at 37 ° C for 24 hours, followed by centrifugation. After washing 2-3 times with (pH 7.4), the cells were suspended in a small amount of 10 mM Tris-Cl buffer and stored at -20 ° C until use.
(2) 당지질(Glycolipid) 추출 및 분리(2) Glycolipid Extraction and Separation
혈액형이 O형인 사람의 적혈구 농축액으로부터 당지질을 발표된 논문에 기술된 방법으로 추출, 분리하였다.(Lee, Y., E. Shin, J. Lee, and J.H. Park. 1999. J. Microb. Biotech. 9: 794-797) 적혈구 농축액을 최소량의 물에 풀어서 -70 ℃에서 얼렸다가 녹이는 과정을 반복하여 적혈구를 파괴한 다음, 정치시켜 상등액 표면의 부유물은 제거하고 나머지 부분을 취하는 추출과정을 실시하였다. 추출은 클로로포름과 메탄올이 2:1(v/v) 비율로 혼합된 용액으로 수행하였으며, 상기 용액을 혼합하여 분리된 층에서 하층(lipid 함유)을 취하여 회전식 증발기(Rotary Evaporator)에서 건조시켰다. 건조시킨 고형물을 2 % 메탄올이 함유된 클로로포름 용액에 용해시켜 규산(silicic acid; H2SiO3) 컬럼으로 분리하였다. 시료는 클로로포름으로 충진된 규산 컬럼에 넣고, 클로로포름, 아세톤/메탄올(3:1, v/v), 메탄올용액을 순차적으로 컬럼에 흘려 분획으로 분리시켰다. 그 중 메탄올로 용출된 분획을 취하여 증류기로 건조시킨 후 소량의 메탄올에 다시 녹여 적당량 미세원심분리 튜브(microcentrifuge tube)에 나누어 분주하여 -70 ℃에서 보관하였다.Glycolipids were extracted and isolated from red blood cell concentrates of humans with type O blood (Lee, Y., E. Shin, J. Lee, and JH Park. 1999. J. Microb. 9: 794-797) The red blood cell concentrate was dissolved in a minimum amount of water, frozen at -70 ° C, and dissolved. The red blood cells were destroyed by standing, and then allowed to stand to remove the suspended matter on the surface of the supernatant, followed by an extraction process. Extraction was carried out with a solution in which chloroform and methanol were mixed at a ratio of 2: 1 (v / v), and the solution was mixed and dried in a rotary evaporator taking a lower layer (containing a lipid) in a separated layer. The dried solid was dissolved in a chloroform solution containing 2% methanol and separated by a silicic acid (H 2 SiO 3 ) column. The sample was placed in a silicic acid column filled with chloroform, and chloroform, acetone / methanol (3: 1, v / v) and methanol solution were sequentially flowed through the column to separate the fractions. Among them, the fractions eluted with methanol were taken, dried in a distillation, dissolved in a small amount of methanol, divided into appropriate amounts of microcentrifuge tubes, and stored at -70 ° C.
(3) 경쟁적 지질 결합실험(Competitive Lipid Binding Assay)(3) Competitive Lipid Binding Assay
부착억제능 측정은 기발표된 논문에 기재된 방법에 따라 수행하였다.(Lee, Y., E. Shin, J. Lee, and J.H. Park. 1999. J. Microb. Biotech. 9: 794-797) 추출한 당지질(14 ug/5 ul)을 박막크로마토그래피(TLC) 플레이트(Merck, Kreselgel60, EM Separations, Gibbstown, NJ, U.S.A)에 점적시킨 후 비특이적인 부착을 방지하기 위해 3 % 젤라틴이 함유된 10 mM Tris-Cl(pH7.6) 완충용액에 잠기게 하여 37 ℃에서 2시간 동안 교반시켰다. 2시간 후 10 mM Tris-Cl(pH 7.6) 완충용액을 사용하여 TLC 플레이트를 2회 씻어낸 다음 PL균주(OD600=1, CFU 2.4 X 108) 현탁액을 주입하고, 5-30분 경과한 뒤에 헬리코박터 필로리를 최종 CFU가 2.0 X 108되도록 주입하여 2시간 동안 교반시켰다. 또한 락토바실러스를 제외하고 헬리코박터 필로리만 상기와 동일하게 TLC 플레이트에 주입하여 2시간 동안 교반하였다. 2시간 후 TLC 플레이트를 완충용액으로 10분씩 3회 씻어내고, 1차 항체인 헬리코박터 필로리 토끼항혈청(rabbit-antiserum, 1:600)을 첨가하여 2시간 동안 상온에서 교반하여 반응시키고, 완충용액으로 씻어낸 후 2차 항체인 항-토끼 IgG(1:1,000)를 첨가하여 상온에서 1시간동안 교반하여 반응시켰다. 1시간 후 완충용액으로 씻어낸 TLC 플레이트에 BCIP/NBT 용액을 첨가하여 발색을 관찰하였다. 적혈구에서 추출한 당지질에 헬리코박터 필로리의 부착정도의 발색정도로 확인할 수 있었다.Adhesion inhibition was measured according to the method described in published papers (Lee, Y., E. Shin, J. Lee, and JH Park. 1999. J. Microb. Biotech. 9: 794-797) Extracted glycolipids (14 ug / 5 ul) was inoculated onto thin layer chromatography (TLC) plates (Merck, Kreselgel60, EM Separations, Gibbstown, NJ, USA) and then 10 mM Tris- containing 3% gelatin to prevent nonspecific adhesion. It was immersed in Cl (pH 7.6) buffer and stirred for 2 hours at 37 ℃. After 2 hours, wash the TLC plate twice with 10 mM Tris-Cl (pH 7.6) buffer and inject the PL strain (OD600 = 1, CFU 2.4 X 10 8 ) suspension, and after 5-30 minutes, Helicobacter Philoly was injected with a final CFU of 2.0 × 10 8 and stirred for 2 hours. Also, except for Lactobacillus, only Helicobacter pylori was injected into the TLC plate in the same manner as above and stirred for 2 hours. After 2 hours, the TLC plate was washed three times with a buffer solution for 10 minutes, and the primary antibody Helicobacter Philoly rabbit antiserum (rabbit-antiserum, 1: 600) was added and stirred at room temperature for 2 hours, followed by reaction with a buffer solution. After washing, a secondary antibody, anti-rabbit IgG (1: 1,000) was added thereto, and reacted by stirring at room temperature for 1 hour. After 1 hour, color development was observed by adding BCIP / NBT solution to TLC plates washed with buffer solution. The degree of adhesion of Helicobacter pylori to the glycolipids extracted from red blood cells was confirmed.
도 1은 적혈구에서 추출한 당지질에 헬리코박터 필로리를 결합시킨 것으로, 당지질의 양이 증가할수록(0 ug, 0.14 ug, 1.4 ug, 14 ug, 70 ug, 140 ug, 280 ug) 헬리코박터 필로리 부착량이 증가함을 확인할 수 있었다.Figure 1 is a Helicobacter Philoly coupled to the glycolipids extracted from red blood cells, the amount of Helicobacter Philosophy increases as the amount of glycolipid increases (0 ug, 0.14 ug, 1.4 ug, 14 ug, 70 ug, 140 ug, 280 ug) Could confirm.
도 2는 본 발명의 6종의 균주가 헬리코박터 필로리의 당지질 부착을 억제하는지에 대하여 측정한 것으로, PL균주(락토바실러스 코프로필러스 PL 9001(KCCM-10245), 엔테로코커스 두란스 PL 9002(KCCM-10246), 스트렙토코커스 패칼리스 PL 9003(KCCM-10247), 락토바실러스 코프로필러스 PL 9004(KCCM-10248), 락토바실러스 퍼멘텀 PL 9005(KCCM-10250), 락토바실러스 퍼멘텀 PL 9006(KCCM-10251))와 헬리코박터 필로리를 각각 TLC 플레이트에 주입한 것이다. 도 2에서 나타난 바와 같이, 본 실험에서 분리한 균주는 헬리코박터 필로리의 당지질 부착을 억제시켰다.Figure 2 is a measure of whether the six strains of the present invention inhibit the glycolipid adhesion of Helicobacter Philo, PL strain (Lactobacillus copropylus PL 9001 (KCCM-10245), Enterococcus Durans PL 9002 (KCCM) -10246), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus copropylus PL 9004 (KCCM-10248), Lactobacillus permanent PL 9005 (KCCM-10250), Lactobacillus permanent PL 9006 (KCCM- 10251) and Helicobacter pylori were injected into the TLC plate, respectively. As shown in Figure 2, the strain isolated in this experiment inhibited the glycolipid adhesion of Helicobacter Philo.
(4) 헬리코박터 필로리의 위점막 부착능 억제실험(4) Inhibition test of gastric mucosa adhesion of Helicobacter pylori
살아있는 유산균과 75 ℃에서 15분간 가열한 사균을 준비하였다. MKN-45(Human Gastric adenocarcinoma) 세포주에 생균과 사균을 처리하고, 헬리코박터 필로리의 위점막 부착에 대한 억제능을 확인하였다.Live lactic acid bacteria and dead bacteria heated at 75 ° C. for 15 minutes were prepared. MKN-45 (Human Gastric adenocarcinoma) cell lines were treated with live bacteria and dead bacteria, and Helicobacter pylori was confirmed to inhibit the gastric mucosa adhesion.
MKN-45 세포주를 2 g/L의 소듐 바이카보네이트, 10 % 열-비활성화된 FBS 및 항생제 안티마이코틱을 포함하는 RPMI-1640 배지(pH 7.2)에서 배양하였다. 3 X 105세포를 2 ml의 배양배지에 접종하고, 30 mm 배양접시에 세포 단일층으로 배양하였다. 배지는 매일 교환해 주었다. 6일간 배양 후 2 ml PBS로 단일세포군을 2회 세척하였다.MKN-45 cell lines were cultured in RPMI-1640 medium, pH 7.2, containing 2 g / L sodium bicarbonate, 10% heat-inactivated FBS and antibiotic antimycotic. 3 × 10 5 cells were seeded in 2 ml of culture medium and incubated with a cell monolayer in a 30 mm culture dish. The medium was changed every day. After culturing for 6 days, single cell groups were washed twice with 2 ml PBS.
1 x 107CFU/mL의 유산균 및 사균을 각각 완전배지 2 mL에 혼합하고, 혼합액은 배양접시에 둔 후 37 ℃, 5 % CO2-95 % 공기 조건상에서 배양하였다. 60분간 배양 후 세포 단일층은 멸균된 PBS로 2회 세척하고 메탄올로 고정시켰다. FITC가 표지된 항-헬리코박터 이차항체를 세포에 처리하고 형광현미경으로 관찰하였다.1 x 10 7 CFU / mL of lactic acid bacteria and dead bacteria were mixed in 2 mL of complete medium, and the mixed solution was placed in a culture dish and incubated at 37 ° C and 5% CO 2 -95% air condition. After incubation for 60 minutes, the cell monolayers were washed twice with sterile PBS and fixed with methanol. FITC-labeled anti-helicobacter secondary antibodies were treated with cells and observed by fluorescence microscopy.
도 3은 PL 균주들의 헬리코박터 위점막 부착 억제능을 확인한 사진으로, 형광물질로 표지된 노란색의 헬리코박터를 관찰한 것이다. a는 MKN -45 세포이고, b는 MKN-45에 헬리코박터 필로리를 처리한 것이고, c는 MKN-45에 헬리코박터 필로리 및 PL9001 균주를 처리한 것이고, d는 MKN-45에 헬리코박터 필로리 및 PL9001 사균을 처리한 것이고, e는 MKN-45에 헬리코박터 필로리 및 PL9003 균주를 처리한 것이고, f는 MKN-45에 헬리코박터 필로리 및 PL9003 사균을 처리한 것이고, g는 MKN-45에 헬리코박터 필로리 및 PL9004 균주를 처리한 것이고, h는 MKN-45에 헬리코박터 필로리 및 PL9004 사균을 처리한 것이고, i는 MKN-45에 헬리코박터 필로리 및 PL9005 균주를 처리한 것이고, j는 MKN-45에 헬리코박터 필로리 및 PL9005 사균을 처리한 것이고, k는 MKN-45에 헬리코박터 필로리 및 PL9006 균주를 처리한 것이고, l는 MKN-45에 헬리코박터 필로리 및 PL9006 사균을 처리한 것이다. MKN-45는 푸른색을 띄며, 헬리코박터 필로리는 노란색으로 관찰된다. 흑백인 경우 헬리코박터 필로리는 MKN-45 세포에 비해 밝게 나타난다.Figure 3 is a photograph confirming the inhibitory ability of Helicobacter gastric mucosa adhesion of PL strains, observed a yellow Helicobacter labeled with a fluorescent material. a is MKN-45 cells, b is MKN-45 treated with Helicobacter pili, c is MKN-45 treated with Helicobacter pili and PL9001 strain, d is MKN-45 with Helicobacter pili and PL9001 E. coli was treated with MKN-45, Helicobacter Philophyll and PL9003 strain, f is MKN-45 treated with Helicobacter Philophyll and PL9003 bacterium, g is MKN-45 Helicobacter Philo. PL9004 strain, h is MKN-45 treated with Helicobacter Philophyll and PL9004 kill, i is MKN-45 treated with Helicobacter Philosophy and PL9005 strain, j is MKN-45 Helicobacter Philosophy And PL9005 bacteria, k is MKN-45 treated with Helicobacter Philosophy and PL9006 strain, l is MKN-45 treated with Helicobacter Philosophy and PL9006 bacteria. MKN-45 is blue and Helicobacter pylori is yellow. In black and white, Helicobacter pylori appears brighter than MKN-45 cells.
도 3에서 MKN-45 세포에 부착되는 헬리코박터 필로리가 PL9001 생균 또는 사균 처리시 위점막에 부착되지 못함을 확인할 수 있었다. 따라서, PL9001는 헬리코박터 필로리의 위 서식을 저해하여 헬리코박터 필로리에 의한 질병을 치료 및 예방한다.In FIG. 3, the Helicobacter pylori attached to the MKN-45 cells could not be attached to the gastric mucosa upon treatment with PL9001 live or dead bacteria. Thus, PL9001 inhibits the gastrointestinal uptake of Helicobacter pylori, thereby treating and preventing diseases caused by Helicobacter pylori.
실시예 3: 유산균의 생존에 따른 위세포 부착력 실험Example 3: Gastric cell adhesion test according to the survival of lactic acid bacteria
살아있는 유산균과 75 ℃에서 15분간 가열한 사균을 준비하였다. MKN-45(Human Gastric adenocarcinoma) 세포주에 생균과 사균의 부착력 실험을 실시하였다.Live lactic acid bacteria and dead bacteria heated at 75 ° C. for 15 minutes were prepared. Adhesion test of live and dead bacteria was carried out on MKN-45 (Human Gastric adenocarcinoma) cell line.
1 x 107CFU/mL의 PL 균주를 완전배지 2 mL에 혼합하고, 혼합액은 배양접시에 둔 후 37 ℃, 5 % CO2-95 % 공기 조건상에서 배양하였다. 60분간 배양 후 세포 단일층은 멸균된 PBS로 2회 세척하고 메탄올로 고정시켰다. 그람염색하고 세포는 광현미경에서 관찰하였다.PL strain of 1 × 10 7 CFU / mL was mixed in 2 mL of complete medium, and the mixed solution was placed in a culture dish and incubated at 37 ° C., 5% CO 2 -95% air conditions. After incubation for 60 minutes, the cell monolayers were washed twice with sterile PBS and fixed with methanol. Gram staining and cells were observed under light microscopy.
도 4는 본 발명의 PL 균주들의 생균과 사균의 위벽세포 부착력을 확인한 사진이다. (a)는 PL9001 생균, (a')는 PL9001 사균, (b)는 PL9003 생균, (b')는 PL9003 사균, (c)는 PL9004 생균, (c')는 PL9004 사균, (d)는 PL9005 생균, (d')는 PL9005 사균, (e)는 PL9006 생균, (e')는 PL9006 사균을 나타낸 것이다. 본 발명의 PL 균주들은 생존에 상관없이 위세포 부착력을 나타내었다.Figure 4 is a photograph confirming the adhesion of gastric wall cells of viable and dead bacteria of PL strains of the present invention. (a) live PL9001, (a ') live PL9001, (b) live PL9003, (b') live PL9003, (c) live PL9004, (c ') live PL9004, (d) PL9005 Live bacteria, (d ') shows PL9005 dead bacteria, (e) PL9006 live bacteria, and (e') shows PL9006 dead bacteria. PL strains of the present invention showed gastric cell adhesion regardless of survival.
실시예 4Example 4
PL 균주들을 각각 5 M 리튬클로라이드로 처리하여 세포벽에 존재하는 단백질을 제거한 다음 위벽 세포 부착력을 실험하였다.(Mark S. TURNER, Peter TIMMS,Louise M. HAFNER, and Philip M. GIFFARD (1997)Journal of Bacteriololgy, vol. 179, No. 10, p. 3310-3316) 현미경상에서 MKN-45 세포주에 부착된 세포 수를 측정하여 하기 표 7에 나타내었다. 리튬클로라이드로 처리한 경우 부착된 유산균의 수가 줄어든 것으로 나타나, PL 균주들의 세포벽에 존재하는 단백질이 위벽세포의 부착에 관여함을 확인할 수 있었다.Each process in 5 M lithium chloride, the PL strains removing the proteins present in the cell wall was the next experimental gastric wall cell adhesion. (Mark S. TURNER, Peter TIMMS , Louise M. HAFNER, and Philip M. GIFFARD (1997) Journal of Bacteriololgy , vol. 179, No. 10, p. 3310-3316) The number of cells attached to the MKN-45 cell line under a microscope was measured and shown in Table 7. When treated with lithium chloride, the number of lactic acid bacteria attached was reduced, indicating that proteins present in the cell walls of PL strains were involved in gastric wall adhesion.
실시예 5: 헬리코박터 필로리 성장억제능 실험Example 5: Helicobacter pylori growth inhibitory experiment
(1) PL 균주 배양여액에 의한 헬리코박터 성장 억제실험(1) Helicobacter growth inhibition test by PL strain culture filtrate
PL 균주들(락토바실러스 코프로필러스 PL 9001 (KCCM-10245), 엔테로코커스 두란스 PL 9002(KCCM-10246), 스트렙토코커스 패칼리스 PL 9003(KCCM-10247), 락토바실러스 코프로필러스 PL 9004(KCCM-10248), 락토바실러스 퍼멘텀 PL 9005(KCCM-10250), 락토바실러스 퍼멘텀 PL 9006 (KCCM-10251)) 을 MRS 액체배지에 접종하여 37 ℃에서 24시간 배양한 후 원심분리하여 상등액을 분리한 다음 상기 상등액을 배양여액으로 준비하였다.PL strains (Lactobacillus copropylus PL 9001 (KCCM-10245), Enterococcus Durans PL 9002 (KCCM-10246), Streptococcus faecalis PL 9003 (KCCM-10247), Lactobacillus copropylus PL 9004 (KCCM -10248), Lactobacillus pertumtum PL 9005 (KCCM-10250), Lactobacillus pertumtum PL 9006 (KCCM-10251)) were inoculated in MRS liquid medium and incubated at 37 ° C for 24 hours, followed by centrifugation to separate the supernatant. Next, the supernatant was prepared as a culture filtrate.
PL 균주 배양여액에 의한 헬리코박터 성장 억제실험을 실시하였다. Brucella 고형배지(Brucella broth에 멸균된 파스퇴르 파이펫을 이용하여 구멍(well)을 만들었다. 고형배지에 헬리코박터 필로리를 도말하고, 구멍에 PL 균주 배양여액(spent culture: 배양한 후 균을 제거한 배양여액)을 첨가하였다. 이산화탄소 배양기(5 % 내지 10 % CO2)에서 배양하고 이틀 뒤 배양액여내의 억제 물질로 인하여 헬리코박터 필로리의 성장이 억제된 환의 지름을 측정하였다.Helicobacter growth inhibition experiment by PL strain culture filtrate was performed. The wells were made using a pasteur pipette sterilized in Brucella broth. Helicobacter Philophyte was smeared in a solid medium, and PL strain culture filtrate (spent culture) was removed from the culture medium. Incubated in a carbon dioxide incubator (5% to 10% CO 2 ), and two days later, the diameter of the ring in which the growth of Helicobacter pylori was inhibited by the inhibitor in the culture medium was measured.
도 5는 PL균주 배양여액의 헬리코박터 필로리 성장 억제능을 측정한 사진이고, 도 6은 PL 균주 배양여액에 의한 헬리코박터 필로리 성장 억제환의 지름을 나타낸 것이다. PL 9001은 락토바실러스 코프로필러스 PL 9001(KCCM-10245), PL 9002는 엔테로코커스 두란스 PL 9002 (KCCM-10246), PL 9003은 스트렙토코커스 패칼리스 PL 9003 (KCCM-10247), PL 9004는 락토바실러스 코프로필러스 PL 9004 (KCCM-10248)이다. 도 6의 결과에서 알 수 있듯이, PL 균주들은 헬리코박터 필로리의 성장을 억제하는 물질을 분비함을 할 수 있다.Figure 5 is a photograph of the Helicobacter Philophyllium growth inhibitory ability of the strain cultured PL strain, Figure 6 shows the diameter of the Helicobacter phyllori growth inhibitory ring by the PL strain culture filtrate. PL 9001 is Lactobacillus copropylus PL 9001 (KCCM-10245), PL 9002 is Enterococcus Durans PL 9002 (KCCM-10246), PL 9003 is Streptococcus faecalis PL 9003 (KCCM-10247), and PL 9004 is Lactobacillus Bacillus copropylus PL 9004 (KCCM-10248). As can be seen from the results of Figure 6, PL strains can secrete a substance that inhibits the growth of Helicobacter Philophyll.
(2) 동결건조된 PL 균주 배양여액의 헬리코박터 성장억제(2) Helicobacter growth inhibition of lyophilized PL strain culture filtrate
PL균주 MRS 배양여액 4 ml에 멸균된 MQ 2 ml을 첨가하여 총 6 ml로 맞추어 동결건조시켰다. 동결건조된 배양여액 시료는 6 ml(동량)의 skim milk(10 mg/ml)에 다시 풀어서 1 ml 씩 취하고, 헬리코박터 필로피(OD625=1.0)를 10 ul씩 첨가하여 1시간동안 37 ℃ 이산화탄소 배양기에서 배양하였다. 원심분리(15000 rpm, 10 min)하여 상등액을 멸균MQ로 1/2 희석한 후 인돌페놀(indophenol)방법에 의한 유레아제(urease)성분을 측정하였다. 측정된 수치는 헬리코박터 필로리로부터 유래한 유레아제 활성에 비례하므로 시료상에 존재하는 헬리코박터 필로리 균의 양을 알 수 있다. 이러한 원리를 통하여 헬리코박터 필로리에 대한 PL균주 배양여액의처리에 따른 헬리코박터 성장억제 효과를 측정하였다.2 ml of sterile MQ was added to 4 ml of PL strain MRS culture filtrate and lyophilized to a total of 6 ml. The lyophilized culture filtrate samples were taken again in 6 ml (same amount) skim milk (10 mg / ml), taken 1 ml each, and 10 ul of Helicobacter Philophy (OD625 = 1.0) was added at 37 ° C for 2 hours. Incubated at. After centrifugation (15000 rpm, 10 min), the supernatant was diluted 1/2 with sterile MQ and the urease component was measured by the indophenol method. Since the measured value is proportional to the urease activity derived from Helicobacter Philophyllium, the amount of Helicobacter Philophyllium present in the sample can be known. Through this principle, the effect of Helicobacter growth inhibition according to the treatment of PL strain culture filtrate on Helicobacter Philophyllium was measured.
도 7은 본 발명의 PL 균주 배양여액에 의한 헬리코박터 필로리 성장억제정도를 나타낸 그래프로, 동결건조된 PL 균주 배양여액이 헬리코박터 필러리 성장억제능을 유지함을 알 수 있었다.Figure 7 is a graph showing the degree of Helicobacter Philophyll growth inhibition by the PL strain culture filtrate of the present invention, it can be seen that the lyophilized PL strain culture filtrate maintains the Helicobacter pillar growth inhibitory ability.
실시예 6: 독성검사Example 6: Toxicity Test
실시예의 PL 균주들에 대한 경구독성시험은 식품의약품안전청고시 제19990061호 '의약품 등의 독성시험기준(1999. 12. 22)'에 의거하여 실시하였다.Oral toxicity test of the PL strains of the Example was carried out in accordance with the Food and Drug Safety Notice No. 19990061 'Toxicity Test Standards for Drugs (Dec. 22, 1999)'.
5 주령(암컷 : 100 ∼ 120g / 수컷 : 110 ∼ 130g)의 랫드(Sprague- Dawley)를 (260 W x 20 L x 80H mm) 케이지에 온도 23+2 ℃, 상대습도 50+10 %의 조건으로 사육하였다.Sprague-Dawley rats of 5 weeks of age (female: 100-120 g / male: 110-130 g) (260 W x 20 L x 80 H mm) were placed in a cage at a temperature of 23 + 2 ° C and a relative humidity of 50 + 10%. Breeding.
국립보건안전연구원 예규 10호 "의약품 등의 독성시험기준(1988. 10. 29.)"2)에 따르면 경구투여시 LD50치가 5,000 mg/kg 이상이면 저독성으로 해석하고 있다. 따라서 본 시험에서는 생균제제의 LD50치를 밝히기 위하여 랫드에 투여할 수 있는 최대용량인 체중 kg 당 5,000 mg을 투여용량(투여액량 ; 20 ml/kg B.W.)으로 설정하였다.National Health and Safety Institute Established Rule No. 10 is oral administration, according to "Toxicity Criteria (1988. 10. 29), such as drugs," 2) LD 50 value of 5,000 mg / kg or more is interpreted as a low toxicity. Therefore, in this study, to determine the LD 50 value of probiotic, 5,000 mg / kg body weight, which is the maximum dose that can be administered to rats, was set as the dosage (dose amount; 20 ml / kg BW).
상기 표 8의 방법으로 14일간 실시하였으나, 사망예는 전혀 관찰되지 않아 LD50치의 산정은 불가능하였다.Although 14 days were carried out by the method of Table 8, no death was observed, and therefore, it was impossible to calculate the LD 50 value.
표 9는 실험군의 체중변화를 나타낸 것으로, 유의성 있는 체중차이는 전혀 관찰되지 않았다.Table 9 shows the weight change of the experimental group, no significant weight difference was observed.
또한 모든 생존동물의 부검을 실시하였으나 유의할 만한 육안적인 병변은 관찰되지 않았다.(표 10)Autopsy of all surviving animals was also performed, but no significant gross lesions were observed (Table 10).
따라서, 실시예의 PL 균주는 경구독성 및 부작용을 유발하지 않는 안전한 시료로 확인되었다.Thus, the PL strains of the examples were identified as safe samples that did not cause oral toxicity and side effects.
실시예 7: 식중독 성장억제실험Example 7: Food Poisoning Growth Inhibition Test
각 PL 균주의 MRS 배양여액과 2배 농도의 BHI 배양액을 동량 혼합하였다. 여기에 BHI(Brain Heart Infusion : Brain Hear, Infusion form, 6.0 g, Peptic digest of animal tissue 6.0 g, sodium chloride 5.0 g, dextrose 3.0 g, pancreatic digest of gelatin 14.5 g, disodium phosphate 2.5 g)에서 성장한 살모넬라 티피무리움(Salmonella typhimurium), 살모넬라 엔테리티디스(Salmonella. enteriditis),E. coliO157:H7, 에로모나스 히드로필라(Aeromonas hydrophila), 리스테리아 모노시토제네스(Listeria monocytogenes)을 최종농도가 1 % 되도록 접종하여 37 ℃에서 배양하였다. 접종 후 6시간과 24시간에 배양액 내의 각각의 생균수를 측정하였다. 이때 리스테리아 모노시토제네스(L.M 이라 기재함)는 혈액한천배지에, 살모넬라 티피무리움(S.T라 기재함), 살모넬라 엔테리티디스(S.E라 기재함), E. coli O157:H7(O157이라 기재함), 및 에로모나스 히드로필라(A.H라 기재함)는 MacConkey 배지에 접종하여 O.D600값으로 생균수를 측정하였다.MRS culture filtrate of each PL strain and BHI culture solution of 2 times concentration were mixed in equal amounts. Salmonella typhie grown in BHI (Brain Heart Infusion: Brain Hear, Infusion form, 6.0 g, Peptic digest of animal tissue 6.0 g, sodium chloride 5.0 g, dextrose 3.0 g, pancreatic digest of gelatin 14.5 g, disodium phosphate 2.5 g) Salmonella typhimurium , Salmonella enteriditis , E. coli O157: H7, Aeromonas hydrophila , Listeria monocytogenes were inoculated to a final concentration of 1%. Incubated at 37 ° C. Each viable cell count was measured at 6 and 24 hours after inoculation. Listeria monocytogenes (described as LM) is described in blood agar medium, Salmonella typhimurium (described as ST), Salmonella enteritidis (described as SE), E. coli O157: H7 (described as O157). ), And eromonas hydrophila (described as AH) were inoculated in MacConkey medium to determine viable counts with an OD 600 value.
표 11은 PL 9001 배양여액에 의한 식중독 균주 성장 O.D600값을 나타낸 것이다.Table 11 shows the food poisoning strain growth OD 600 value by PL 9001 culture filtrate.
도 8은 본 발명의 락토바실러스 코프로필러스 PL9001의 식중독균 성장억제능을 그래프로 나타낸 것으로, 표 11의 결과를 나타낸(0시간일 때 O.D600값을 100으로 하여 환산) 것이다. 표 11 및 도 8에서 알수 있듯이, 락토바실러스 코프로필러스 PL 9001은 식중독균을 억제하는 물질을 생산하여 식중독균의 성장을 억제하였다.8 is a graph showing the growth inhibitory ability of food poisoning bacteria of Lactobacillus copropylus PL9001 of the present invention, which shows the results of Table 11 (converted to OD 600 value 100 at 0 hours). As can be seen in Table 11 and Figure 8, Lactobacillus copropylus PL 9001 produced a substance that inhibits food poisoning bacteria to inhibit the growth of food poisoning bacteria.
또한 엔테로코커스 두란스 PL 9002는 식중독균의 성장을 억제하였다. 측정결과는 하기 표 12와 도 9에 나타내었다.Enterococcus Durans PL 9002 also inhibited the growth of food poisoning bacteria. The measurement results are shown in Table 12 and FIG. 9.
또한 스트렙토코커스 패칼리스 PL 9003에 의한 식중독 균 성장억제정도를 측정하였다. 하기 표 13은 스트렙토코커스 패칼리스 PL 9003 배양여액상에서 식중독균의 O.D600값을 나타낸것이고, 도 10은 표 13의 결과를 그래프로 나타낸 것이다. 스트렙토코커스 패칼리스 PL 9003은 식중독균의 성장을 크게 억제하는 물질을 생성함을 알 수 있었다.In addition, the growth inhibition of food poisoning bacteria by Streptococcus faecalis PL 9003 was measured. Table 13 below shows the OD 600 values of food poisoning bacteria in Streptococcus faecalis PL 9003 culture filtrate, and FIG. 10 shows the results of Table 13 graphically. Streptococcus faecalis PL 9003 was found to produce a substance that greatly inhibits the growth of food poisoning bacteria.
또한 락토바실러스 코프로필러스 PL 9004에 의한 식중독 균 성장억제정도를 측정하였다. 하기 표 14는 락토바실러스 코프로필러스 PL 9004 배양여액상에서 식중독균의 O.D600값을 나타낸 것이고, 도 11은 표 14의 결과를 그래프로 나타낸 것이다. 락토바실러스 코프로필러스 PL 9004은 식중독균의 성장을 크게 억제시키나, 시간이 경과됨에 따라 식중독균 성장 억제능이 약화되었다.In addition, the growth inhibition of food poisoning bacteria by Lactobacillus copropylus PL 9004 was measured. Table 14 below shows the OD 600 values of food poisoning bacteria in Lactobacillus copropylus PL 9004 culture filtrate, and FIG. 11 graphically shows the results of Table 14. Lactobacillus copropylus PL 9004 significantly inhibited the growth of food poisoning bacteria, but over time, the ability to suppress food poisoning growth was weakened.
또한 락토바실러스 퍼멘텀 PL 9005에 의한 식중독 균 성장억제정도를 측정하였다. 하기 표 15는 락토바실러스 퍼멘텀 PL 9005 배양여액상에서 식중독균의 O.D600값을 나타낸 것이고, 도 12는 리스테리아증에 대한 락토바실러스 퍼멘텀 PL 9005의 성장억제능을 그래프로 나타낸 것이다. 락토바실러스 퍼멘텀 PL 9005은 식중독균의 성장을 크게 억제시켰다.In addition, the degree of growth inhibition of food poisoning bacteria by Lactobacillus permanent PL 9005 was measured. Table 15 shows the OD 600 value of the food poisoning bacteria in the Lactobacillus Permantum PL 9005 culture filtrate, Figure 12 shows the growth inhibitory ability of Lactobacillus Permantum PL 9005 against Listeria. Lactobacillus permanent PL 9005 significantly inhibited the growth of food poisoning bacteria.
또한 락토바실러스 퍼멘텀 PL 9006에 의한 식중독 균 성장억제정도를 측정하였다. 하기 표 16은 락토바실러스 퍼멘텀 PL 9006 배양여액상에서 식중독균의 O.D600값을 나타낸것이고, 도 13은 리스테리아증에 대한 락토바실러스 퍼멘텀 PL 9006의 성장억제능을 그래프로 나타낸 것이다. 락토바실러스 퍼멘텀 PL 9006은 식중독균의 성장을 크게 억제시켰다.In addition, the degree of growth inhibition of food poisoning bacteria by Lactobacillus permanent PL 9006 was measured. Table 16 shows the OD 600 value of the food poisoning bacteria in the Lactobacillus Permantum PL 9006 culture filtrate, Figure 13 shows the growth inhibitory ability of Lactobacillus Permantum PL 9006 against Listeriosis. Lactobacillus permanent PL 9006 significantly inhibited the growth of food poisoning bacteria.
상기에서 나타난 바와 같이, 본 발명의 PL균주들이 식중독을 유발하는 균주의 성장을 억제하는 물질을 생산함을 확인할 수 있었다. 또한 시간의 경과에 따라 감소되는 효과는 식중독균 배지내에 투여한 한정된 억제물질이 모두 소모된 것에 의한 것으로, 계속적으로 PL 균주를 사용할 경우 계속적인 식중독균 성장억제효과를 나타낼 수 있을 것이다.As shown above, the PL strains of the present invention was confirmed to produce a substance that inhibits the growth of strains that cause food poisoning. In addition, the effect that decreases over time is due to the exhaustion of all the limited inhibitors administered in the food poisoning medium, if the continuous use of the PL strain may exhibit a continuous food poisoning growth inhibitory effect.
실시예 8: 여드름균 성장 억제능Example 8 Acne Bacteria Growth Inhibition
피부에 정상으로 존재하는 균의 하나로, 여드름생성에 관여하는 프로피오니박테륨 아크네(Propionibacterium acne, KCTC3314)를 액티노마이세스 브로스(actinomyces broth, 5 ml)에 접종하고, 위에 멸균된 파라핀오일을 덮어서 2일간 혐기적으로 정치배양하였다.(BR Vowels, S Yang, JJ Leyden. 1995. Inductionof proinflammatory cytokines by a soluble factor of Profionibacterium acnes: Implications for chronic inflammatory acne. Infection and Immunity 63: 3158-3165) MRS 액체배지에 배양한 PL 배양여액 45 ml에 프로피오니박테륨 아크네 배양물 5 ml을 혼합하였다. 혼합액 50 ul를 새로운 액티노마이세스 액체배지 5 ml에 접종하여 2일간 혐기적으로 정치배양하였다. 배양액내의 생균수를 측정하기 위하여 배양액을 0.05 % L-시스테인(Cysteine)이 함유된 혐기성 세균용 희석 용액으로 희석하고(OD600=2.4 일 때 약 9.6 X 108CFU/ml), 이중 100 ul를 취하여 액티노마이세스 고형 평판배지에 접종한 후 혐기성 배양기(anaerobic jar)에 넣어 37 ℃에서 6-7일간 배양하여 형성된 콜로니를 측정하여 생균수를 확인하였다. 도 14는 본 발명의 PL 균주에 의한 여드름균 성장억제능을 그래프로 도시한 것으로, 락토바실러스 코프로필러스 PL 9001는 프로피오니박테륨 아크네 성장을 거의 억제하지 못하였고, 엔테로코커스 두란스 PL 9002 및 스트렙토코커스 패칼리스 PL 9003는 프로피오니박테륨 아크네 성장을 완전히 억제하였으며, 락토바실러스 코프로필러스 PL 9004, 락토바실러스 퍼멘텀 PL 9005 및 락토바실러스 퍼멘텀 PL 9006은 프로피오니박테륨 아크네 성장을 억제하였다.One of the normal bacteria on the skin, Propionibacterium acne (KCTC3314), which is involved in acne production, is inoculated into actinomyces broth (5 ml) and covered with paraffin oil sterilized above. (BR Vowels, S Yang, JJ Leyden. 1995. Induction of proinflammatory cytokines by a soluble factor of Profionibacterium acnes: Implications for chronic inflammatory acne.Infection and Immunity 63: 3158-3165) MRS liquid medium 5 ml of propionibacterium acne culture was mixed with 45 ml of PL culture filtrate. 50 ul of the mixed solution was inoculated in 5 ml of fresh Actinomyces liquid medium and stationary anaerobicly for 2 days. To determine the number of viable cells in the culture, the culture was diluted with a dilute solution for anaerobic bacteria containing 0.05% L-Cysteine (approximately 9.6 X 10 8 CFU / ml at OD 600 = 2.4), of which 100 ul was added. After inoculation into the actinomyces solid plate medium and put into an anaerobic jar (anaerobic jar) incubated for 6-7 days at 37 ℃ by measuring the colonies formed to determine the number of viable cells. Figure 14 is a graph showing the growth inhibitory ability of acne bacteria by PL strain of the present invention, Lactobacillus copropylus PL 9001 hardly inhibited the growth of propionibacterium acne, Enterococcus Durans PL 9002 and Strepto Caucas Pacalis PL 9003 completely inhibited propionibacterium acne growth, while Lactobacillus copropylus PL 9004, Lactobacillus permanent PL 9005 and Lactobacillus permanent PL 9006 inhibited propionibacterium acne growth.
실시예 9: 혐기성 균의 성장억제Example 9 Inhibition of Growth of Anaerobic Bacteria
클로스트로디움 페르프린젠스(Clostrodium perfringens)를 헤민(Hemin, 0.01 g/L)과 L-시스테인(0.5 g/L)이 함유된 BHI 액체배지에 접종하고 37 ℃에서 24시간 혐기적으로 배양하였다.(Balows A Hausler WJ Herrmann KL Isenberg HD ShadomyHJ. Chapter 50. Clostridium. p505-521. Manual of Clinical Microbiology, 5th ed. ASM Washington D.C. U.S.A.) PL균주 배양여액과 2배 농도 BHI 배양액의 동량 혼합액하여 클로스트로디움 페르프린젠스의 최종농도가 1 % 되도록 접종하여 배양액 위에 파라핀 오일을 덮어 혐기적으로 37 ℃에서 배양하였다. 24시간후 배양액을 희석한 후 혈액한천배지에 접종하여 혐기적 배양기에서 37 ℃, 48시간 배양한 후 생균수를 측정하여 log 값으로 나타내었다. Clostrodium perfringens was inoculated into BHI liquid medium containing hemin (Hemin, 0.01 g / L) and L-cysteine (0.5 g / L) and incubated anaerobicly at 37 ° C. for 24 hours. (Balows A Hausler WJ Herrmann KL Isenberg HD ShadomyHJ. Chapter 50. Clostridium. P505-521. Manual of Clinical Microbiology, 5th ed. ASM Washington DCUSA) Clostridium perl by the same mixture of PL culture medium and double concentration BHI culture The final concentration of the prince was inoculated to 1% and incubated anaerobically at 37 ℃ covered with paraffin oil on the culture medium. After 24 hours, the culture solution was diluted, and then inoculated into blood agar medium, and cultured at 37 ° C. for 48 hours in an anaerobic incubator.
본 발명의 PL 균주 모두 혐기성 균주의 성장의 억제할 수 있는 물질을 생산하는 것으로 확인되었다.All of the PL strains of the present invention were found to produce substances capable of inhibiting the growth of anaerobic strains.
실시예 10: 면역증강효과Example 10: Immune Boosting Effect
본 발명의 PL 균주들에 의한 면역증강효과를 측정하였다. 마크로파지에 유산균을 첨가하였을 때 마크로파지가 생산하는 사이토카인(tumor necrosis factor-α, IL-6)의 증가율을 측정하였다.The immunopotentiating effect of the PL strains of the present invention was measured. When the lactic acid bacteria were added to macrophages, the increase rate of cytokine (tumor necrosis factor-α, IL-6) produced by macrophages was measured.
마이크로웰에 정제한 토끼 항-TNF-α또는 IL-6를 포함하는 코팅 완충액 50 ul로 피복하고 냉장 온도에서 하룻밤 방치하였다. PBST(tween80+phosphate buffer)용액으로 피복된 마이크로웰을 3회 세척한 다음 3 % BSA-PBST를 넣은 후 30분동안 반응시켰다. 마이크로웰을 PBST로 4회 세척하고 TNF-α standard/IL-6 standard(recombinant mouse TNF-α/IL-6, SEROTEC, UK) 또는 하룻밤 PL 균주를 첨가하여 배양한 마크로파지(macrophage) 상등액을 50 ul씩 첨가한 후 37 ℃에서 1시간 반응시켰다. 마이크로웰은 PBST로 4회, 멸균 증류수로 1회 세척하고, 3 % BSA-PBST에 바이오티닐레이티드 렛 항-마우스 TNF-α 또는 IL-6(biotinylated rat anti-mouse TNF-α/IL-6, 1 ug/ml)를 첨가하여 50 ul 씩 분주한 후 상온에서 1시간 반응시켰다. 마이크로웰을 PBST로 6회, 멸균 증류수로 1회 세척하고, 스트렙타비딘 퍼록시데이즈(streptavidin peroxidase)을 분주하여 1시간 반응시켰다. 마이크로웰은 PBST로 8회, 멸균 증류수로 2회 세척하고, TMB substrate(25 ml citric-phosphate buffer+400 ul TMB stock+100 ul 1 % H2O2)를 첨가한 후 발색할 때 까지 상온에 놓아두었다. 마이크로웰에 6 N H2SO4를 넣어 반응을 종료시킨 후 OD450에서 면역반응성을 측정하였다.Microwells were coated with 50 ul of coating buffer containing purified rabbit anti-TNF-α or IL-6 and left overnight at refrigeration temperature. Microwells coated with PBST (tween80 + phosphate buffer) solution were washed three times, and then reacted for 30 minutes after adding 3% BSA-PBST. Microwells were washed four times with PBST and 50 ul of macrophage supernatant incubated with TNF-α standard / IL-6 standard (recombinant mouse TNF-α / IL-6, SEROTEC, UK) or overnight PL strain. After each addition, the mixture was reacted at 37 ° C for 1 hour. Microwells were washed four times with PBST and once with sterile distilled water and biotinylated rat anti-mouse TNF-α / IL-6 in 3% BSA-PBST. , 1 ug / ml) was added, and 50 ul was dispensed for 1 hour at room temperature. The microwells were washed 6 times with PBST and once with sterile distilled water, and streptavidin peroxidase was dispensed for 1 hour. Microwells were washed 8 times with PBST and 2 times with sterile distilled water, and after adding TMB substrate (25 ml citric-phosphate buffer + 400 ul TMB stock + 100 ul 1% H 2 O 2 ) at room temperature until color development I let it go. 6 NH 2 SO 4 was added to the microwell to terminate the reaction, and immunoreactivity was measured at OD 450 .
도 15 및 표 18은 PL 균주들에 의한 면역증가정도를 나타낸 것으로, 본 발명의 PL 균주들은 TNF-α 및 IL-6의 생성을 증가시킴을 확인할 수 있었다.15 and Table 18 show the degree of immunity increase by PL strains, PL strains of the present invention was confirmed to increase the production of TNF-α and IL-6.
따라서, 본 발명의 유산균은 면역 증강제로 사용될 수 있으며 특히 어린이와 환자들과 같이 면역이 약화된 사람들의 면역을 증가시키는 건강보조식품 및 치료제로 사용될 수 있다.Therefore, the lactic acid bacteria of the present invention can be used as an immune enhancer, and in particular, as a dietary supplement and a therapeutic agent for increasing immunity of immunized people, such as children and patients.
실시예 11: 내산성 및 내담즙성 검증Example 11: Validation of Acid and Bile Resistance
시스테인이 첨가된 MRS 배지는 4 N HCl과 0.1 N NaOH로 pH를 7, 4.5, 4.0로 적정한 다음 멸균하여 사용하였으며, 담즙의 영향은 옥스갈(oxgall powder)를 0 %, 0.25 %, 0.50 % 농도로 배지에 첨가하여 멸균한 후 사용하였다. 배지에 활성화된 균액(OD=1)을 1 %로 접종시키고, 24시간 간격으로 620 nm에서 흡광도를 측정하였다. 하기 표 19는 내산성, 내담즙성을 나타낸 것으로, PL 9003이 내산실험에서 산에 약한 것으로 나타났으나 나머지 PL 9001, PL 9002, PL 9004, PL 9005, PL 9006은 내산성이 강했으며 PL 균주 모두 강한 내담즙성을 나타내었다. 즉, 6종의 PL 균주들은 위 및 장에서 안전함을 알 수 있었다.(Conway PL Gorback SL goldin BR. 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J. Dairy Sci. 70: 1-12. : Ibrahim SA Bezkorovainy A. 1993. Survival of bifidobacteria in the presence of bile salt. J. Sci. Food Agric. 62: 351-354)Cysteine-added MRS medium was titrated with 4 N HCl and 0.1 N NaOH to pH 7, 4.5, 4.0 and sterilized. The effect of bile was 0%, 0.25%, and 0.50% of oxgall powder. After sterilization was added to the medium used. Activated bacterial solution (OD = 1) was inoculated into the medium at 1%, and absorbance was measured at 620 nm at 24 hour intervals. Table 19 shows acid resistance and bile resistance, but PL 9003 was found to be weak in acid in acid tests, but the remaining PL 9001, PL 9002, PL 9004, PL 9005, and PL 9006 were strong in acid resistance, and all of PL strains were strong. It showed bile resistance. That is, six PL strains were found to be safe in the stomach and intestine (Conway PL Gorback SL goldin BR. 1987. Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells.J. Dairy Sci. 70: 1-12 .: Ibrahim SA Bezkorovainy A. 1993. Survival of bifidobacteria in the presence of bile salt.J. Sci. Food Agric. 62: 351-354)
실시예 12: 항생제 내성 측정Example 12 Antibiotic Resistance Measurement
PL 균주들에 대한 항생제 내성을 측정하였다. PL 균주를 MRS 고체배지에 접종하고, 여기에 표 18의 항생제가 포함된 필터(직경 6 mm)를 올려놓고 24-48시간 배양하여 항생제에 의한 형성된 억제환의 크기를 측정하였다. 억제환의 크기가 작을수록 항생제에 대한 내성이 크다는 것을 의미한다. 하기 표 20은 PL 균주에 대한 항생제 내성정도를 나타낸 것이다.Antibiotic resistance to PL strains was measured. PL strains were inoculated in MRS solid medium, and the filter (6 mm in diameter) containing the antibiotics of Table 18 was placed thereon and cultured for 24-48 hours to determine the size of the inhibitory ring formed by the antibiotics. The smaller the size of the inhibitory ring, the higher the resistance to antibiotics. Table 20 below shows antibiotic resistance to PL strains.
상기의 결과와 같이, 본 발명의 PL 균주들은 높은 항생제내성을 가진다.As a result of the above, the PL strains of the present invention have high antibiotic resistance.
실시예 13: 유산균의 장세포 부착력 확인 실험Example 13: Enteric cell adhesion confirmation experiment of lactic acid bacteria
Caco-2 세포주를 2 g/L의 소듐 바이카보네이트, 10 % 열-비활성화된 FBS 및 항생제 안티마이코틱을 포함하는 RPMI-1640 배지(pH 7.2)에서 배양하였다. 3 X 105세포를 2 ml의 배양배지에 접종하고, 30 mm 배양접시에 세포 단일층으로 배양하였다. 배지는 매일 교환해 주었다. 6일간 배양 후 2 ml PBS로 단일세포군을 2회 세척하였다.Caco-2 cell lines were cultured in RPMI-1640 medium (pH 7.2) containing 2 g / L sodium bicarbonate, 10% heat-inactivated FBS and antibiotic antimycotic. 3 × 10 5 cells were seeded in 2 ml of culture medium and incubated with a cell monolayer in a 30 mm culture dish. The medium was changed every day. After culturing for 6 days, single cell groups were washed twice with 2 ml PBS.
1 x 107CFU/mL의 유산균을 완전배지 2 mL에 혼합하고, 혼합액은 배양접시에 둔 후 37 ℃, 5 % CO2-95 % 공기 조건상에서 배양하였다. 60분간 배양 후 세포 단일층은 멸균된 PBS로 2회 세척하고 메탄올로 고정시켰다. 그람염색하고 세포는 광현미경에서 관찰하였다. 현미경상에서 20개의 필드에서 관찰되는 균수를 측정하였고, 총수가 400개 이상일 경우 부착으로 판정하였다.(C.N.JACOBSEN, V.ROSENFELDT NIELSEN, A.E.HAYFORD, P.L.MØLLER, K.F.MICHAELSEN, A.PAERREGAARD, B.SANDSTROM, M.TVEDE, and M.JAKOBSEN(1999)Applied and Environmental Microbiology,p.4949-4956 ; R.Bibiloni, P.F.Perez, and G.L.DeAntoni (1999)Anaerobe5, 483-485) 하기 표 21에 관찰된 유산균 수를 나타낸 것으로, 본 발명의 PL 유산균들은 뛰어난 장세포 부착력을 나타내었다.1 x 10 7 CFU / mL of lactic acid bacteria were mixed in 2 mL of complete medium, and the mixed solution was placed in a culture dish and incubated at 37 ° C and 5% CO 2 -95% air condition. After incubation for 60 minutes, the cell monolayers were washed twice with sterile PBS and fixed with methanol. Gram staining and cells were observed under light microscopy. The number of bacteria observed in 20 fields on the microscope was measured, and when the total number was more than 400, it was judged as attachment. (CNJACOBSEN, V.ROSENFELDT NIELSEN, AEHAYFORD, PLMØLLER, KFMICHAELSEN, A.PAERREGAARD, B.SANDSTROM, M.TVEDE, and M. JAKOBSEN (1999) Applied and Environmental Microbiology, p. 4949-4956; R. Bibiloni, PFPerez, and GLDeAntoni (1999) Anaerobe 5, 483-485). PL lactic acid bacteria showed excellent enterocyte adhesion.
도 16은 유산균의 장세포 부착정도를 확인한 사진으로, 대조군(a)에 비하여 PL9001(b), PL9003(c), PL9004(d), PL9005(e) 및 PL9006(f)을 장세포에 첨가하였을 때 장세포에 유산균이 부착됨을 관찰되었다.Figure 16 is a photograph confirming the degree of adhesion of enterocytes of lactic acid bacteria, PL900 (b), PL9003 (c), PL9004 (d), PL9005 (e) and PL9006 (f) was added to the enterocytes compared to the control (a) When lactic acid bacteria adhered to the intestinal cells.
비피도박테리움 인펀티스 및 비피도박테리움 롱검은 장세포 부착력이 우수한 것으로 입증된 것으로, 본 발명의 PL 균주들 역시 장세포 부착력이 우수하여 정장작용을 함을 확인할 수 있었다.Bifidobacterium inpontis and Bifidobacterium long gum was proved to be excellent in the adhesion of enterocytes, PL strains of the present invention was also confirmed that it has a good intestinal cell adhesion to work in the form of.
실시예 16: 화장품 제조Example 16: Cosmetic Preparation
스킨skin
PL9001 건조분말 0.02 중량%, 글리세린 5.0 중량%, 1,3-부틸렌글리콜 3.0 중량%, PEG1500 1.0 중량%, 알란토인 0.1 중량%, DL-판테놀 0.3 중량%, EDTA-2NA 0.02 중량%, 벤조페논-9 0.04 중량%, 소듐히아루로네이트 5.0 중량%, 에탄올 10.0 중량%, 옥틸도데세스-16 중량%, 폴리솔베이트 20 중량%, 및 미량의 방부제, 향, 색소와 잔량의 정제수를 혼합하여 스킨을 제조하였다.PL9001 dry powder 0.02% by weight, glycerin 5.0% by weight, 1,3-butylene glycol 3.0% by weight, PEG1500 1.0% by weight, allantoin 0.1% by weight, DL-panthenol 0.3% by weight, EDTA-2NA 0.02% by weight, benzophenone- 9 0.04% by weight, sodium hyaluronate 5.0% by weight, ethanol 10.0% by weight, octyldodeces-16% by weight, polysorbate by 20% by weight, and a small amount of preservatives, fragrances, pigments and residual amount of purified water Prepared.
로션 제조Lotion manufacturers
PL9001 건조분말 0.01 중량%, 글리세린 5.0 중량%, 1,3-부칠렌글리콜 3.0 중량%, 소듐히아루로네이트 5.0 중량%, 에탄올 10.0 중량%, 폴리솔베이트 60 중량%, 글리세릴스테아레이트 에이스 1.5 중량%, 세테아릴알콜 1.5 중량%, 라놀린 1.5 중량%, 솔비탄스테아레이트 0.5 중량%, 경하식물유 1.0 중량%, 광물유 5.0 중량%, 스쿠알란 3.0 중량%, 트리옥타노인 2.0 중량%, 디메치콘 0.8 중량%, 초산토코페롤 0.5 중량%, 카르복시비닐폴리머 0.12 중량%, 트리에탄올아민 0.12 중량%, 및 미량의 방부제, 향, 색소와 잔량의 정제수를 혼합하여 로션을 제조하였다.PL9001 0.01 wt% dry powder, glycerin 5.0 wt%, 1,3-butylene glycol 3.0 wt%, sodium hyaluronate 5.0 wt%, ethanol 10.0 wt%, polysorbate 60 wt%, glyceryl stearate ace 1.5 wt% %, Cetearyl alcohol 1.5 wt%, lanolin 1.5 wt%, sorbitan stearate 0.5 wt%, light vegetable oil 1.0 wt%, mineral oil 5.0 wt%, squalane 3.0 wt%, trioctanoin 2.0 wt%, dimethicone 0.8 wt% %, Tocopherol acetate 0.5% by weight, carboxyvinyl polymer 0.12% by weight, triethanolamine 0.12% by weight, and a small amount of preservatives, flavors, pigments and the residual amount of purified water were mixed.
상기에 언급한 바와 같이, 본 발명의 락토바실러스 코프로필러스 PL 9001,엔테로코커스 두란스 PL 9002, 스트렙토코커스 패칼리스 PL 9003, 락토바실러스 코프로필러스 PL 9004, 락토바실러스 퍼멘텀 PL 9005, 락토바실러스 퍼멘텀 PL 9006은 헬리코박터 필로리의 성장 및 위막 부착능을 억제한다. 또한 본 발명의 PL 균주, PL 균주 파쇄물, 또는 PL 균주 배양여액은 헬리코박터의 감염을 예방 및 치료하는 용도로 사용할 수 있으며, PL 균주 및 PL 균주 배양여액은 식중독 성장억제, 여드름균 성장억제, 혐기성균 성장억제 및 면역증강효과를 나타내어 감염 예방 및 치료의 목적으로 사용할 수 있다.As mentioned above, the Lactobacillus copropylus PL 9001 of the present invention, Enterococcus Durans PL 9002, Streptococcus faecalis PL 9003, Lactobacillus copropylus PL 9004, Lactobacillus fermentum PL 9005, Lactobacillus fur Menthium PL 9006 inhibits the growth and adhesion of the membrane to Helicobacter pylori. In addition, the PL strain, PL strain lysate, or PL strain culture filtrate of the present invention can be used for the purpose of preventing and treating infection of Helicobacter, PL strain and PL strain culture filtrate, food poisoning growth inhibition, acne bacteria growth inhibition, anaerobic bacteria It can be used for the purpose of preventing and treating infections by showing growth inhibition and immune enhancing effects.
Claims (16)
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PCT/KR2001/002126 WO2002045727A1 (en) | 2000-12-08 | 2001-12-07 | Lactic acid bacteria inhibiting adhesion of helicobacter pylori to gastric mucosa |
AU2002217571A AU2002217571A1 (en) | 2000-12-08 | 2001-12-07 | Lactic acid bacteria inhibiting adhesion of helicobacter pylori to gastric mucosa |
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KR101349452B1 (en) * | 2005-12-22 | 2014-01-08 | 오르가노발란스 게엠베하 | Novel lactobacillus strains and their use against helicobacter pylori |
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JPH09241173A (en) * | 1996-03-01 | 1997-09-16 | Wakamoto Pharmaceut Co Ltd | Anti-gastritis agent, antiulcer agent and fermented food containing lactobacillus as active ingredient |
WO1998055131A1 (en) * | 1997-06-02 | 1998-12-10 | Essum Ab | Pharmaceutical preparation comprising lactobacillus casei rhamnosus |
KR20000056981A (en) * | 1999-02-08 | 2000-09-15 | 이은선 | Food for preventing and treating gastritis, gastric and duodenal ulcers |
KR20010081822A (en) * | 2000-02-19 | 2001-08-29 | 이은선 | Lactobacillus acidophilus HY 2177, Lactobacillus casei HY 2743 having antibiotic activation for Helicobacter pylori and Lactobacillus product thereof |
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JPH09241173A (en) * | 1996-03-01 | 1997-09-16 | Wakamoto Pharmaceut Co Ltd | Anti-gastritis agent, antiulcer agent and fermented food containing lactobacillus as active ingredient |
WO1998055131A1 (en) * | 1997-06-02 | 1998-12-10 | Essum Ab | Pharmaceutical preparation comprising lactobacillus casei rhamnosus |
KR20000056981A (en) * | 1999-02-08 | 2000-09-15 | 이은선 | Food for preventing and treating gastritis, gastric and duodenal ulcers |
KR20010081822A (en) * | 2000-02-19 | 2001-08-29 | 이은선 | Lactobacillus acidophilus HY 2177, Lactobacillus casei HY 2743 having antibiotic activation for Helicobacter pylori and Lactobacillus product thereof |
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Journal of microbiology and biotechnology, 9(6), 1999.12, 794~797, 동일발명자 * |
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KR101349452B1 (en) * | 2005-12-22 | 2014-01-08 | 오르가노발란스 게엠베하 | Novel lactobacillus strains and their use against helicobacter pylori |
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