KR100483369B1 - Pediococcus pentosaceus CBT-8 which produces antimicrobial materials for restraining Helicobacter pylori and Listeria monocytogenes, and Production method of antimicrobial materials from Pediococcus pentosaceus CBT-8, and Application process for functional foods and/or drugs using the strain and the antimicrobial materials - Google Patents
Pediococcus pentosaceus CBT-8 which produces antimicrobial materials for restraining Helicobacter pylori and Listeria monocytogenes, and Production method of antimicrobial materials from Pediococcus pentosaceus CBT-8, and Application process for functional foods and/or drugs using the strain and the antimicrobial materials Download PDFInfo
- Publication number
- KR100483369B1 KR100483369B1 KR10-2002-0062825A KR20020062825A KR100483369B1 KR 100483369 B1 KR100483369 B1 KR 100483369B1 KR 20020062825 A KR20020062825 A KR 20020062825A KR 100483369 B1 KR100483369 B1 KR 100483369B1
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- KR
- South Korea
- Prior art keywords
- antimicrobial
- helicobacter pylori
- pediococcus pentosaceus
- antimicrobial materials
- lactic acid
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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
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
본 발명은 페디오코커스 펜토사세우스(Pediococcus pentosaceus) CBT-8로 명명된 유산균주 및 이를 이용한 항균특성을 갖는 항균물질 제조 방법 및 그 제조된 항균물질을 기능성 식품 및 의약품에 이용하는 방법에 관한 것이다. 본 발명에 따르면, 위염증(궤양) 원인균인 헬리코박터 파일로리균 및 식중독 원인균인 리스테리아 모노사이토진균에 대한 감염예방 및 제균작용에 의한 치료효과를 동시에 수행할 수 있는 항균활성물질을 제조할 수 있으며, 이로부터 특정 원인균에 대한 감염예방 및 치료 기능을 갖는 기능성 식품이나 의약품을 생산 공급할 수 있는 장점을 가진다.The present invention relates to a lactic acid strain named Pediococcus pentosaceus (CBT-8), a method for producing an antimicrobial substance having antimicrobial properties using the same, and a method of using the prepared antimicrobial substance in functional foods and pharmaceuticals. . According to the present invention, it is possible to prepare an antimicrobial active material that can simultaneously carry out the treatment effect by the infection prevention and bactericidal effect against the bacterium Helicobacter pylori and the food poisoning cause Listeria monocytogenes, Since it has the advantage of producing and supplying functional foods or medicines having a function of preventing and treating infections against specific causative bacteria.
Description
본 발명은 페디오코커스 펜토사세우스(Pediococcus pentosaceus) CBT-8로 명명된 유산균주, 상기 유산균주를 이용하여 소정의 항균활성물질을 제조하는 방법과 그 제조된 항균물질, 상기 제조된 항균물질을 농축하는 방법과 그 방법에 따라 농축된 항균활성물질농축제, 상기 항균활성물질농축제를 건조시키는 방법과 그 방법에 따라 건조된 항균활성물질농축제건조물질 및 상기 항균활성물질농축액 또는 그 건조물질을 기능성 식품 및 의약품에 이용하는 방법에 관한 것이다.The present invention is a Lactobacillus strain named Pediococcus pentosaceus CBT-8, a method for producing a predetermined antimicrobial active material using the lactic acid strain and the antimicrobial material prepared, the antimicrobial material prepared And a method for drying the antimicrobial active material concentrate concentrated in accordance with the method, a method for drying the antimicrobial active material concentrate, and a dried antimicrobial active material concentrate in accordance with the method and the antimicrobial active material concentrate or drying thereof A method of using a substance in functional foods and pharmaceuticals.
헬리코박터 파일로리균(Helicobacter pylori)은 유아기 이후 경구를 통하여 감염되는 것이 일반적이며, 통계적으로 살펴보면 국내 성인의 70% 이상이 보균자로 추산되고 있다. 헬리코박터 파일로리균은 인체 내에서 위염증 및 위암 발생의 원인균으로 잘 알려져 있다. 그런데, 종래에는 위 또는 십이지장 염증이나 궤양증을 치료하기 위해서는 항생제, 위산억제제 및 위점막 보호제 등 3개 약제를 동시에 투여하는 3제 요법이 대중화되고 있으며, 일부에서는 그에 따른 치료 효과가 보고되고 있다. 그러나, 이러한 종래의 3제 요법은, 장내 유익균총 파괴에 따른 설사나 변비증의 동반, 투여된 약제에 대한 내성 균주가 등장하는 문제로 인한 약효능의 저하 및 반복적 치료를 위해서 과량 투여 등의 약제사용에 대한 부작용이 발생되고 있으며, 일단 치료되었더라도 다시 재발하는 등의 문제점도 지적되고 있다.Helicobacter pylori are generally bacteria (Helicobacter pylori) are infected via the oral since infancy, referring statistically is more than 70% of Korean adult estimated to be carriers. Helicobacter pylori is well known as a causative agent of gastric inflammation and gastric cancer in the human body. However, conventionally, a three-dose regimen for simultaneously administering three agents, such as antibiotics, gastric acid inhibitors, and gastric mucosa, has been popularized to treat gastric or duodenal inflammation or ulcer, and in some cases, a therapeutic effect thereof has been reported. However, such conventional three-drug therapy is used with drugs such as diarrhea or constipation due to intestinal beneficial flora destruction, drug efficacy such as excessive administration for repetitive treatment and a decrease in drug efficacy due to the emergence of resistant strains to the administered drugs. Side effects are occurring, and problems such as recurrence even after treatment have been pointed out.
유아의 경우에는 젖떼는 시기에서부터 감염이 시작되고, 유아기 이후라도 국물이나 음료 등을 상시적으로 음용하는 식습관을 갖는 성인 등의 경우에는 경구를 통한 감염 가능성이 높으며, 성인 보균자의 경우에는 약제에 의한 제균치료 후에도 일상생활에서 다시 감염될 위험에 빈번하게 노출되어 있어 있다. Infants begin to spread from the milking stage. Infants are more likely to be infected by oral in adults who have a dietary habit of drinking soup or beverages even after infancy. Even after disinfection treatment, they are frequently exposed to the risk of being infected again in their daily lives.
상기와 같이, 종래에 행해지고 있는 항생제 등 약제를 사용한 제균요법에 의한 위염증을 유발시키는 병원균을 제어하는 데에는 한계가 있기 때문에, 감염후 치료보다는 사전에 감염으로부터 예방하는 차원의 다른 시도가 필요하게 되었다.As described above, since there is a limit in controlling pathogens causing gastritis caused by bacteriostatic therapy using conventional medicines such as antibiotics, other attempts to prevent infection from infection are needed rather than post-infection treatment. .
이러한 감영 예방에 관한 시도는 종래의 항생제를 포함한 의약품에만 의존하는 감염 제어방식과는 달리, 균체, 균체파쇄액, 요소분해효소(Urease), p54 단백질 등을 면역원으로 이용하여 생성시킨 항체를 이용하거나 면역란을 이용하여 헬리코박터 파일로리균을 제어하기 위한 방향으로 연구되고 있다.In contrast, infection control methods, which rely solely on medicines containing antibiotics, utilize antibodies produced by using cells, cell lysate, urease, p54 protein, etc. as immunogens. It has been studied in the direction for controlling Helicobacter pylori bacteria using immune eggs.
또 다른 방법으로, 헬리코박터 파일로리균이 위장관 등에 부착되는 것을 방지시키기 위한 목적으로 제균성능이 있는 유산균이나 그로부터 생산된 항균 물질이 첨가된 각종 건강식품, 발효유 및 유제품이 소개되는 등 의약품이 아닌 기능성 식품을 사용하여 감염 예방이나 제균 또는 치료 목적이 달성될 수 있도록 하는 제품개발 연구가 진행되고 있다.Alternatively, functional foods other than pharmaceuticals, such as the introduction of various health foods, fermented milk and dairy products added with lactic acid bacteria or antibacterial substances produced therein for the purpose of preventing the adhesion of Helicobacter pylori to the gastrointestinal tract, etc. Research into product development is underway to ensure that the purpose of infection, prevention or disinfection or treatment can be achieved.
한편, 유산균은 면역증강기능, 장내 유익균총 유지, 정장기능 등을 갖고 있는 이외에도 헬리코박터 파일로리균이 위점막에 부착되는 것을 방지하는 역할은 물론 제균 작용까지도 수행하고 있으므로, 위염증 예방 및 병증 개선 작용을 동시에 발휘하는 것으로 알려져 있다.On the other hand, lactic acid bacteria have immune enhancing function, maintenance of intestinal beneficial flora, intestinal function, etc. In addition to preventing H. pylori from attaching to the gastric mucosa, they also act as a bactericidal, thus preventing gastritis and improving symptoms. It is known to exert simultaneously.
첫째, 헬리코박터 파일로리균은 요소분해효소(Urease) 활성을 갖고 있으므로, 위산 중화능력을 갖고 있으며, 유기산만의 작용으로는 목적하는 바와 같은 위염증 예방 및 제균과 관련된 활성도 높은 효과를 기대하기 어려우며, 둘째, 헬리코박터 파일로리균은 위점막 상피세포에 점착하여 생육하기 때문에 위점막 상피세포와 결합력이 뛰어난 세포구조를 지닌 특정 유산균이 먼저 부착부위를 선점함으로써 헬리코박터 파일로리균이 위점막에 부착되는 것을 방지하는 작용을 하므로 생균체 및 사균체에 대해서는 소정의 목적하는 바를 달성할 수 있지만, 이미 위점막에 헬리코박터 파일로리균이 부착되어 있는 감염자에게는 제균효과가 높지 않은 문제점이 있었으며, 마지막으로, 유산균이 생산하는 항균활성물질(antimicrobial peptide), 미확인(un-known) 항균활성물질 또는 면역강화 성분에 의한 제어기능으로서 헬리코박터 파일로리균이 위점막에 부착하는 것을 방지(예방)하거나 동시에 제균효과(치료)까지도 달성할 수 있으며 발효과정 및 물질 회수기술에 의해 농축 제조된 고농도의 항균활성물질을 이용하는 경우에는 헬리코박터 파일로리균이 야기시키는 위염증에 대해 양호한 예방효과는 물론 치료효과까지도 기대할 수 있게 되었다.First, because Helicobacter pylori has urease activity, it has neutralizing ability in acid, and it is difficult to expect high effect related to prevention and eradication of gastritis as desired by the action of organic acid only. , Helicobacter pylori adheres to the gastric mucosal epithelial cells, so that specific lactic acid bacteria having a cell structure superior in binding to gastric mucosal epithelial cells preoccupy the attachment site, thereby preventing the Helicobacter pylori bacteria from attaching to the gastric mucosa. Therefore, it is possible to achieve a predetermined purpose for live cells and dead cells, but there was a problem that the infection effect is not high for those infected with Helicobacter pylori attached to the gastric mucosa, and finally, the antibacterial active material produced by lactic acid bacteria (antimicrobial peptide), unknown antibacterial activity As a control function by substances or immunopotentiating ingredients, Helicobacter pylori bacteria can prevent (prevent) from attaching to gastric mucosa or at the same time achieve bactericidal effect (treatment) and high concentration antibacterial concentrated by fermentation process and material recovery technology In the case of using the active substance, it was possible to expect a good preventive effect as well as a therapeutic effect against the gastritis caused by Helicobacter pylori.
이상 살펴본 것처럼 유산균을 이용한 기능성식품으로서 헬리코박터 파일로리균을 제어하기 위해서는 위에 열거한 세가지 요인을 적절한 조건에서 적극 활용할 필요가 있다. 특히, 유산균이 생산하는 항균활성물질(Antimicrobial peptide, 박테리오신(Bacteriocin)이라 총칭하기도 함)은 고유의 분자량, 아미노산 염기서열 및 구조에 따라 침투할 수 있는 미생물 구조(세포막, 그람(Gram) 음성균의 경우 외막구조)에 따른 특이성이 있기 때문에 항균영역에 분명한 선택성이 있다. 따라서, 유산균이 생산하는 항균물질을 이용하는 경우에는 항생제에서 나타나는 유익균총 파괴와 같은 부작용이 발생하지 않으며, 그 작용기작이 타겟 미생물 세포막에 선택적으로 리크(leak)를 형성하는 것이므로 내성개발의 우려도 없으므로, 식품으로의 이용에 매우 적합한 안전한 생체 친화적인 특성을 지니고 있다. 또한, 항균활성물질 유도 및 고농도 발효기술과 물질분리 기술에 의하여 고활성을 지닌 제품생산이 가능하며, 유산균 생균제 및 다른 기능성 소재들과 병용이 가능한 물성을 지니고 있어 다양한 용도 적성이 개발될 수 있는 장점이 있다.As described above, in order to control Helicobacter pylori as a functional food using lactic acid bacteria, it is necessary to actively use the three factors listed above under appropriate conditions. In particular, the antimicrobial active substance (antimicrobial peptide, also known as bacteriocin) produced by lactic acid bacteria is a microorganism structure (cell membrane, Gram-negative bacteria that can penetrate according to its own molecular weight, amino acid sequence and structure). Because of the specificity according to the outer membrane structure), there is a clear selectivity in the antimicrobial region. Therefore, the use of the antimicrobial material produced by lactic acid bacteria does not cause side effects such as destruction of the beneficial flora caused by antibiotics, and since its action mechanism selectively forms a leak on the target microbial cell membrane, there is no fear of resistance development. It has a safe and bio-friendly property that is very suitable for use in foods. In addition, it is possible to produce products with high activity by induction of antimicrobial active substances, high concentration fermentation technology and material separation technology, and has the properties that can be used in combination with lactic acid bacteria probiotics and other functional materials. There is this.
한편, 리스테리아 모노사이토진은 주로 치즈 등 유가공품, 식육가공품 및 기타 냉장 유통 식품 등에 오염되어 저온유통 중에도 증식하여 식중독 사고를 일으키는 미생물로서 인체에 무해한 천연 식품보존제에 의한 제균 및 오염방지가 필요한 병원성 세균이다.On the other hand, Listeria monocytozin is a pathogenic bacterium that is contaminated with dairy products such as cheese, meat processed products and other refrigerated foods, and grows even during low temperature distribution, causing food poisoning accidents. .
전술한 종래의 문제점에 기초하여 본 발명이 이루고자 하는 기술적 과제는, 종래의 위염증 등을 치료하기 위한 3제 요법 등이 갖는 문제점을 해결하고 감염 후 치료 목적은 물론 감염 예방 목적을 달성할 수 있는 항균물질을 생성시키는 소정의 유산균주를 활용하고자 함에 있으며, 이러한 기술적 과제를 달성하기 위하여, 헬리코박터 파일로리균(Helicobacter pylori) 및 리스테리아 모노사이토진균(Listeria monocytogenes)의 생육을 억제하는 항균물질을 생산하는 페디오코커스 펜토사세우스(Pediococcus pentosaceus) CBT-8로 명명된 유산균주, 상기 유산균주를 이용하여 소정의 항균활성물질을 제조하는 방법과 그 제조된 항균활성물질, 상기 제조된 항균활성물질을 농축하는 방법과 그 방법에 따라 농축된 항균활성물질 농축제, 상기 항균활성물질 농축제를 건조시키는 방법과 그 방법에 따라 건조된 항균활성물질 농축제 건조물질 및 상기 항균활성물질농축액 또는 그 건조물질을 이용하여 제조된 기능성 식품 및 의약품을 제공하는 것을 목적으로 한다.The technical problem to be achieved by the present invention based on the above-mentioned conventional problems, to solve the problems with the three-therapy regimens for the treatment of conventional gastritis and the like, and to achieve the purpose of prevention of infection as well as treatment after infection. In order to utilize a certain lactic acid strain to produce an antimicrobial substance, in order to achieve this technical problem, pedi to produce an antimicrobial substance that inhibits the growth of Helicobacter pylori and Listeria monocytogenes ( Listeria monocytogenes ) Lactobacillus strain named Okoccus pentosaceus CBT-8, a method for producing a predetermined antimicrobial active substance using the lactic acid strain and the antimicrobial active substance prepared therein, the antimicrobial active substance is concentrated And the antimicrobial active material thickener concentrated according to the method, and when drying the antimicrobial active material thickener It is an object of the present invention to provide a functional food and medicines prepared using the antimicrobial active substance concentrate dry matter, and the antimicrobial active substance concentrate or dried material according to the dry method and a method thereof.
본 발명이 이루고자 하는 기술적 과제의 달성을 위해 본 발명은 위염증 원인균인 헬리코박터 파일로리균과 식중독 원인균인 리스테리아 모노사이토진의 생육을 동시에 억제할 수 있는 소정의 항균활성물질을 생산하는 미생물 유산균인 페디오코커스 펜토사세우스(Pediococcus pentosaceus) CBT-8로 명명된 유산균을 제공하고, 그를 이용하여 소정의 항균특성을 갖는 항균활성물질을 제조하는 방법과 그 제조된 항균활성물질을 식품 또는 의약품에 이용하는 방법을 제공한다.In order to achieve the technical problem to be achieved by the present invention, the present invention is a microbial lactic acid bacteria pediococcus producing a predetermined antimicrobial active material that can simultaneously inhibit the growth of gastroenteritis cause bacteria Helicobacter pylori and food poisoning cause Listeria monocytogenes Pediococcus pentosaceus ( Pdiococcus pentosaceus ) provides a lactic acid bacterium named CBT-8, and using it to prepare an antimicrobial active material having a predetermined antimicrobial properties, and the method of using the prepared antimicrobial active material in food or medicine to provide.
구체적으로 살펴보면, 본 발명에서 제공되는 유산균주는 헬리코박터 파일로리균에 대한 생육 억제능과 리스테리아 모노사이토진균에 대한 생육 억제능을 갖는 항균활성물질을 생산하는 것을 특징으로 하는 페디오코커스 펜토사세우스 (Pediococcus pentosaceus) CBT-8로 명명된 유산균주이다.Specifically, the lactic acid bacteria strain provided in the present invention is characterized by producing an antimicrobial active substance having a growth inhibitory ability against Helicobacter pylori and a growth inhibitory activity against Listeria monocytofungus ( Pediococcus pentosaceus ) It is a lactic acid strain named CBT-8.
한편, 상기 페디오코커스 펜토사세우스(Pediococcus pentosaceus) CBT-8인 유산균주를 이용하여 헬리코박터 파일로리균과 리스테리아 모노사이토진균 모두에 항균활성도를 항균활성물질을 제조하는 방법은, (a) 페디오코커스 펜토사세우스 (Pediococcus pentosaceus) CBT-8인 유산균주를 준비하는 단계; (b) 상기 준비된 유산균주 중량대비 3 내지 5 중량%의 포도당, 0.5 내지 1 중량%의 효모추출물 (Yeast Extract), 1 내지 1,5 중량%의 육엑기스, 0.5 내지 1 중량%의 단백질가수분해물, 0.01 내지 0.1 중량%의 암모늄 시트레이트, 소듐 아세테이트, 디포타시움포스테이트, 마그네슘 셀페이트, 망간 셀페이트, 소듐 클로라이드 중 선택된 하나 이상의 이온결합성화화물을 첨가하여 물에 용해시키는 단계; (c) 상기 첨가물이 포함된 수용액을 살균하는 단계; 및 (d) 상기 살균된 수용액에 상기 (a) 단계에서 준비된 유산균주를 투입하고 혐기적 발효관에서 배양하는 단계를 포함하여 진행하는 것을 특징으로 한다.On the other hand, the pediococcus pentosaceus ( Pediococcus pentosaceus ) using a lactic acid strain of CBT-8 is a method for producing an antimicrobial active material antimicrobial activity to both Helicobacter pylori and Listeria monocytofungal, (a) Pedio Preparing a lactic acid strain which is Pediococcus pentosaceus CBT-8; (b) 3 to 5% by weight of glucose, 0.5 to 1% by weight of yeast extract (Yeast Extract), 1 to 1,5% by weight of meat extract, 0.5 to 1% by weight of protein hydrolyzate Dissolving in water by adding one or more ionic binders selected from 0.01 to 0.1% by weight of ammonium citrate, sodium acetate, dipotassium formate, magnesium sulphate, manganese sulphate, sodium chloride; (c) sterilizing the aqueous solution containing the additives; And (d) adding the lactic acid strain prepared in step (a) to the sterilized aqueous solution and culturing in an anaerobic fermentation tube.
한편, 상기 (d)단계 이후에, 상기 제조된 항균활성물질에 대해 고속원심분리 또는 여과장치를 이용하여 균체를 제거한 후 여액을 농축하는 단계를 더 포함하여 진행하면, 항균활성물질의 활성도를 향상시킬 수 있으므로 바람직하다. 더 구체적으로는, 상기 여과장치를 이용하여 농축하는 단계는, 제1여과단계에서는 균체와 최대배제분자량을 초과하는 물질을 제거하고, 제2여과단계로 최소배제분자량 미만의물질을 연속적으로 제거하여 소정 범위의 분자량을 갖는 항균물질만을 농축하는 2단 여과방법으로 수행하게 되면, 일정 범위 내의 분자량을 갖는 항균물질만을 선택적으로 농축할 수 있으므로 바람직하다.On the other hand, after the step (d), further comprising the step of concentrating the filtrate after removing the cells using a high-speed centrifugal separation or filtration device for the prepared antimicrobial active material, improve the activity of the antimicrobial active material It is preferable because it can be made. More specifically, the step of concentrating using the filtration device, in the first filtration step to remove the cells and the material exceeding the maximum exclusion molecular weight, and in the second filtration step to remove the material less than the minimum exclusion molecular weight continuously When performing the two-stage filtration method of concentrating only the antimicrobial substance having a predetermined range of molecular weight, it is preferable because only the antimicrobial substance having a molecular weight within a certain range can be selectively concentrated.
이와 더불어, 상기 농축단계진행 결과물인 항균활성물질 농축액에 대해 동결건조방식 또는 분사식 유동층 건조방법에 따라 건조시키는 단계를 더 포함하여 진행하면, 상용화되는 최종 제품의 성상에 따라 다양한 활용가능성을 확보할 수 있어 바람직하다.In addition, further comprising the step of drying according to the freeze-drying method or spray type fluidized bed drying method for the antimicrobial active material concentrate resulting from the concentration step progress, it is possible to secure a variety of applications depending on the properties of the commercialized final product It is preferable.
그리고, 본 발명이 제공하는 페디오코커스 펜토사세우스(Pediococcus pentosaceus) CBT-8인 유산균주로부터 생산되는 헬리코박터 파일로리균 및 리스테리아 모노사이토진에 대한 항균특성을 갖는 항균활성물질을 식품 또는 의약품에 첨가함으로써, 헬리코박터 파일로리균과 리스테리아 모노사이토진균에 대한 감염예방은 물론 치료효과를 달성할 수 있는 기능성 식품 또는 의약품을 개발할 수 있어 바람직하다.In addition, an antimicrobial active substance having antimicrobial properties against Helicobacter pylori and Listeria monocytogenes produced from a lactic acid strain of Pediococcus pentosaceus CBT-8 provided by the present invention is added to food or pharmaceuticals. By doing so, it is desirable to prevent the infection of Helicobacter pylori and Listeria monocytofungus, as well as to develop a functional food or medicine that can achieve a therapeutic effect.
이하, 본 발명을 구체적으로 설명하기 위해 실시예를 들어 설명하고, 발명에 대한 이해를 돕기 위해 첨부도면을 참조하여 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 아래에서 상술하는 실시예들에 한정되는 것으로 해석되어지지 않아야 한다. 본 발명의 실시예들은 당 업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해서 제공되어지는 것이다.Hereinafter, the present invention will be described in detail with reference to examples, and detailed description will be made with reference to the accompanying drawings in order to help understanding of the present invention. However, embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.
유산균 CBT-8의 균학적 성질의 동정Identification of Mycological Properties of Lactic Acid Bacteria CBT-8
본 발명에 따른 유산균주는 API 50 CHL을 사용한 당자화능을 시험해본 결과 하기 표 1과 같은 당자화능 결과를 얻을 수 있었다. Lactobacillus strain according to the present invention was tested for the glycosylation capacity using the API 50 CHL was obtained the glycosylation results as shown in Table 1.
또한, 상기 유산균주의 16S rDNA 염기서열을 분석하고, 분석결과는 별도의 염기서열목록을 도시한 도 1과 같다. 상기 분석된염기서열목록의 결과를 가지고, 발생학적 계보를 파악하기 위해 이미 알려져 있는 균주들과의 16S rDNA 염기서열을 비교해본 결과 하기 표 2와 같은 결과를 얻을 수 있었다. In addition, the 16S rDNA nucleotide sequence analysis of the lactic acid strain, and the analysis results are shown in Figure 1 showing a separate base sequence list. With the results of the analyzed base sequence list, comparing the 16S rDNA base sequence with the known strains in order to determine the developmental lineage was obtained as shown in Table 2 below.
한편, 본 발명에 따른 유산균주의 스트레인(strain)과 페디오코커스 유형의 스트레인(strain)인 페디오코커스 텍스트리니쿠스(KCTC3506), 페디오코커스 펜토사세우스(KCTC3507), 페디오코커스 파르부러스(KCTC 3615) 및 페디오코커스 이노피나투스(KCTC3653) 등과의 DNA-DNA 혼성(hybridization)을 시킨 경우, 본 발명에 따른 유산균주는 페디오코커스 펜토사세우스와 93%이상(100~50 ng/nu 범위에서의 평균)의 유전적 관련성을 보임을 하기 표 3과 도 1을 통해 확인할 수 있었다.On the other hand, strain of the lactic acid bacteria strain (strain) and pediococcus type strain (strain) of the Pediococcus textlinicus (KCTC3506), Pediococcus pentosaceus (KCTC3507), Pediococcus parburus (KCTC 3615) and Pediococcus Inopinatus (KCTC3653) and the DNA-DNA hybridization (hybridization), the lactic acid bacteria strain according to the present invention and Pediococcus pentosaceus 93% or more (100 ~ 50 ng / nu Genetic relevance of the mean) in the range was confirmed through Table 3 and FIG.
상기 표 1에 따른 당자화능 결과치와 상기 표 2에 따른 16S rDNA 염기서열의 유사성, 그리고 상기 표 3 및 도 2에 나타난 바와 같은 DNA-DNA 혼성결과를 종합적으로 동정해본 결과, 본 발명에 따른 유산균주는 페디오코커스 펜토사세우스의 변종으로 파악되며, 이러한 근거에 의해 그 발생학적 계보(Phylognetic tree)를 그려보면 도 3처럼 도시할 수 있다.As a result of comprehensive identification of the similarity between the results of glycosylation according to Table 1 and 16S rDNA nucleotide sequences according to Table 2, and DNA-DNA hybridization results as shown in Table 3 and FIG. 2, the lactic acid bacteria according to the present invention The strain is identified as a strain of Pediococcus pentosaceus, and based on this evidence, the phylogenetic tree can be depicted as shown in FIG. 3.
이상과 같이, 본 발명에 사용되는 미생물 유산균주는 당자화능 및 유전학적 분석에 의하여 페디오코커스 펜토사케우스(Pediococcus pentosaceus)로 동정되는 것으로서, 이를 구체적으로 페디오코커스 펜토사세우스(Pediococcus pentosaceus) CBT-8로 명명하고, 2002년 6월25 일부로 대한민국 미생물 기탁기관인 한국생명공학연구원 유전자은행에 기탁하여 균주기탁번호 KCTC 10297 BP를 부여받았다.As described above, the microbial lactic acid strains used in the present invention are identified as Pediococcus pentosaceus by the glycosylation ability and genetic analysis, and specifically, these are Pediococcus pentosaceus . It was named CBT-8, and as a part of June 25, 2002, it was deposited with the Korea Biotechnology Research Institute Gene Bank, a microorganism depositing institution of Korea, and was given strain accession number KCTC 10297 BP.
유산균 CBT-8의 항균영역 및 항균활성Antimicrobial Area and Antimicrobial Activity of Lactic Acid Bacteria CBT-8
본 발명에 따른 유산균인 페디오코커스 펜토사세우스 CBT-8이 갖고 있는 항균영역과 항균활성도를 페디오코커스 펜토사세우스 CBT-8의 배양여액을 사용하여 각 지시균의 배양평판에 대한 생육 저해 활성도를 측정하여 확인해 본 결과, 하기 표 4에 나타낸 바와 같다.The antimicrobial region and antimicrobial activity of the lactic acid bacterium Pediococcus pentosaceus CBT-8 according to the present invention was grown using the culture filtrate of Pediococcus pentosaceus CBT-8 on the culture plate of each indicator. As a result of measuring and confirming the inhibitory activity, it is as shown in Table 4 below.
상기 표 4에 나타난 결과로 확인해보면, 식중독 원인균인 리스테리아 모노사이토진균과 위염증 원인균인 헬리코박터 파일로리균의 생육을 강력하게 저해한다. As shown in the results shown in Table 4, it strongly inhibits the growth of Listeria monocytofungi as a food poisoning cause and Helicobacter pylori as a gastrointestinal cause.
본 발명에 따른 유산균주를 이용하여 최종 응용단계인 기능성 식품이나 의약품으로 활용하기 위한 농축된 건조분말을 형성하는 단계는, (a)유산균 CBT-8의 선발단계, (b)유산균 배양단계, (c)항균물질 발효단계, (d)발효액의 농축 및 젖산제거단계 및 (e)농축액의 건조분체화 단계로 순차적으로 진행(도 4 참조)되며, 이하에서는 이들 각각의 단계에 대해 보다 상세하게 설명하기로 한다.The step of forming a concentrated dry powder for use as a functional food or drug as a final application step using the lactic acid strain according to the present invention, (a) selection of lactic acid bacteria CBT-8, (b) lactic acid bacteria culture step, ( c) the antimicrobial fermentation step, (d) fermentation broth concentration and lactic acid removal step and (e) the dry powder of the concentrate step proceeds sequentially (see Figure 4), each of these steps will be described in more detail below Let's do it.
(a) 유산균 CBT-8의 선발단계(a) Selection stage of lactic acid bacteria CBT-8
지시균으로서 헬리코박터 파일로리균을 사용하여 10% 말 혈청액(Horse serum)이 첨가된 콜롬비아 블러드 아가(Colombia blood agar) 평판 배지에서 37℃, 10% CO2 조건으로 배양하고, 리스테리아 모노사이토진균에 대해서는 ATP 배지에서 37℃ 및 통성조건에서 배양한 여액을 사용한 생육 저해 활성도 측정을 통하여 각각 800Au/ml, 800Au/ml 이상의 항균활성을 발휘하는 유산종균을 선발한 후 발효생산 배지조건에서 배양하여 접종액으로 사용한다.Using Helicobacter pylori as an indicator, incubated at 37 ° C and 10% CO2 in Colombia blood agar plate medium supplemented with 10% horse serum, and ATP for Listeria monocytobacteria. Lactobacillus showing antimicrobial activity of 800Au / ml and 800Au / ml was selected by measuring the growth inhibitory activity using filtrates cultured at 37 ° C and permeable conditions, and then cultured under fermentation medium conditions and used as inoculum. do.
(b) 유산균 배양단계(b) lactic acid bacteria culture step
중량비 3 내지 5% 범위의 포도당, 0.5 내지 1% 범위의 효모추출물, 1 내지 1.5% 범위의 육엑기스, 0.5 내지 1% 범위의 단백가수분해물, 0.01% 내지 0.1% 범위의 암모니움 시트레이트, 소듐 아세테이트, 디포타슘 포스테이트, 마그네슘 설페이트, 망간 설페이트, 소듐 클로라이드 등을 포함하는 이온성분을 물에 용해시키고 혐기적 발효관에서 살균시킨 후, 상기 선발된 유산종균을 배양한다.Glucose in the range of 3 to 5% by weight, yeast extract in the range of 0.5 to 1%, hexaextract in the range of 1 to 1.5%, protein hydrolyzate in the range of 0.5 to 1%, ammonium citrate in the range of 0.01% to 0.1%, sodium Ionic components including acetate, dipotassium potassium state, magnesium sulfate, manganese sulfate, sodium chloride and the like are dissolved in water and sterilized in an anaerobic fermentation tube, followed by culturing the selected lactic acid seed.
(c) 항균물질 발효단계(c) antimicrobial fermentation stage
항균물질 발효공정의 경시변화는 도 5에 도시하였으며, 상기 도 5에서 "L.m"은 리스테리아 모노사이토진의 항균활성도를 나타내며, "H.p"는 헬리코박터 파일로리균의 항균활성도를 구별하기 위한 식별자로 기재되었다. 상기 도 5에 도시된 바에 따르면, 발효 개시 후, 16시간 경과할 때, 위염증 원인균인 헬리코박터 파일로리균에 대하여는 최대 1600 Au/ml의 저해활성을 나타내고, 리스테리아 모노사이토진에 대해서는 1600 Au/ml의 저해활성을 나타내는 항균활성 물질이 생산됨을 알 수 있다. Time-dependent change of the antimicrobial fermentation process is shown in Figure 5, "L.m" in Figure 5 represents the antimicrobial activity of Listeria monocytogene, "H.p" was described as an identifier for distinguishing the antimicrobial activity of Helicobacter pylori. As shown in FIG. 5, 16 hours after the start of fermentation, the inhibitory activity of H. pylori, which is the causative agent of gastritis, showed up to 1600 Au / ml of inhibitory activity, and 1600 Au / ml of Listeria monocytogene. It can be seen that an antimicrobial active material that exhibits inhibitory activity is produced.
(d) 발효액의 농축 및 젖산제거단계(d) concentration of fermentation broth and removal of lactic acid
상기 발효단계를 종료한 후, 발효액은 15,000 RPM 이상의 고속원심분리기나 균체분리용 한외여과기에 의해 균체로부터 분리시킨다. 균체가 분리된 발효여액은 배제분자량 10,000 정도인 한외여과막(Ultra filtration membrane)에 1차 여과하고 투과액에 대하여 배제분자량 1000 정도인 나노미터여과막(Nanometer membrane)에 의하여 분리 추출함으로써 분자량 1,000에서 10,000사이의 활성분획만을 포집하여 농축시킨다.After the fermentation step is completed, the fermentation broth is separated from the cells by a high-speed centrifuge or cell ultrafiltration for separating cells. The fermented filtrate from which the cells were separated was first filtered through an ultra filtration membrane having an exclusion molecular weight of 10,000, and separated and extracted by a nanometer membrane having an exclusion molecular weight of 1000 with respect to the permeate. Only the active fractions of are collected and concentrated.
상기 농축액에 대해서는 5배량의 무균수를 연속적으로 가하며 pH가 6.0이상이 될 때까지 다이아 여과(Dia-filtration)하여 유기산 성분을 완전 제거하고 유기산이 제거된 최종 농축액의 부피는 발효원액에 대비하여 1/10의 부피로 조절한다. 발효액으로부터 항균물질을 분리 포집하고 젖산을 제거하는 공정에서의 항균활성물질의 경시변화는 하기 표 5에 나타내었으며, 최종 농축액의 항균활성 자료는 도 6에 도시되어 있으며, 상기 도 6은 본 발명에 따른 유산균인 페디오코커스 펜토사세우스 CBT-8의 배양농축액에 대한 생육저지환(Clear zone) 시험 결과를 나타낸 사진이다.5 times of sterile water is added to the concentrate continuously. Diafiltration is performed until the pH is 6.0 or higher to completely remove the organic acid component, and the volume of the final concentrate from which the organic acid is removed is 1 compared to the fermentation stock solution. Adjust to volume of / 10. The time-dependent change of the antimicrobial active material in the process of separating and collecting the antimicrobial material from the fermentation broth and removing lactic acid is shown in Table 5 below. The antimicrobial activity data of the final concentrate are shown in FIG. 6, and FIG. This is a photograph showing the growth zone test (Clear zone) test results for the culture concentrate of the P. coccus pentosaceus CBT-8 according to the lactic acid bacteria.
(e) 농축액의 건조분체화 단계(e) Dry Powderation of Concentrate
상기 농축액의 중량 대비 1 내지 10%의 유당, 말토덱스트린, 만니톨, 탈지분유를 혼합하여 용해시키고 80℃의 온도 조건에서 교반시키면서, 2회 30분간 열처리 함으로써 기타 잡균 및 잔존 유산균 등을 살균한 후, 동결건조기에 투입하여 건조시키거나 또는 분사식 유동층건조기를 이용하여 건조시킨 후, 건조물을 분쇄하여 분말을 제조한다. 이후, 응용제품의 용도에 맞추어 추가로 첨가되는 각종 부형제 및 희석제의 종류와 그 첨가량 등을 다양한 형태로 변형시켜 사용한다.After dissolving 1 to 10% of lactose, maltodextrin, mannitol, and skim milk powder by weight of the concentrate, dissolving and stirring at 80 ° C., the mixture was heat treated twice for 30 minutes to sterilize other various bacteria and residual lactic acid bacteria, and the like. After drying by adding to a lyophilizer or drying using a spray type fluidized bed dryer, the dried product is ground to prepare a powder. Thereafter, the types and amounts of various excipients and diluents added in addition to the use of the application product are used in various forms.
본 발명에 따른 유산균을 활용하여 위염증 원인균인 헬리코박터 파일로리균과 식중독 원인균인 리스테리아 모노사이토진 균의 생육을 저해하는 항균활성물질을 제조하는 방법을 실시예를 들어 보다 구체적으로 설명하기로 한다.Using the lactic acid bacteria according to the present invention will be described in more detail with reference to the method for producing an antimicrobial active material that inhibits the growth of the bacterium causing bacteria Helicobacter pylori and food poisoning causing Listeria monocytogene bacteria.
실시예 1Example 1
먼저, 포도당 25㎏, 육엑기스 5㎏, 효모추출물 2.5㎏, 카제이펩톤 2.5㎏, 소이펩톤 2.5㎏, 디포타슘 포스페이트 0.25㎏, 암모니움 시트레이트 0.25㎏, 소듐 아세테이트 0.25㎏, 마그네슘 설페이트 0.05㎏, 망간 설페이트 0.05㎏을 물 500L에 첨가하여 용해시키고 800L 용량의 혐기적 발효관에 이송하여 121℃에서 15분간 살균하여 상온으로 냉각시킨 후 미리 배양한 종균 10L를 접종하여 20시간 발효시킨다. First, glucose 25 kg, meat extract 5 kg, yeast extract 2.5 kg, casei peptone 2.5 kg, soy peptone 2.5 kg, dipotassium phosphate 0.25 kg, ammonium citrate 0.25 kg, sodium acetate 0.25 kg, magnesium sulfate 0.05 kg, 0.05 kg of manganese sulfate was added to 500 L of water, dissolved, and transferred to an anaerobic fermentation tube of 800 L, sterilized at 121 ° C. for 15 minutes, cooled to room temperature, and inoculated with 10 L of pre-cultivated spawn for 20 hours.
이어서, 상기 발효액을 60L/h의 유속으로 연속식 원심분리기에 이송시키면서 균체를 제거하고, 배양여액을 배제분자량 10,000의 한외여과기 필터에 통과시키고 투과액을 회수하여 다시 배제분자량 1,000의 나노미터 필터에 통과시켜 분자량 1000 내지 10,000사이의 항균물질 농축액을 50L를 획득한다. Subsequently, the cells were removed while transferring the fermentation broth to a continuous centrifuge at a flow rate of 60 L / h, the culture filtrate was passed through an ultrafilter filter with a molecular weight of 10,000 excluded, and the permeate was recovered. Pass through to obtain 50 L of antimicrobial concentrate with a molecular weight of 1000 to 10,000.
이후, 상기 농축액에 대하여 다시 5배량의 무균수를 연속적으로 가하며 배제분자량 1,000의 나노미터 필터에 통과시켜 투과액과 농축액을 재분리한다. 이 공정에서 잔존유기산, 색소, 불용성 성분 등이 완전히 제거되었고, 헬리코박터 파일로리균 및 리스테리아 모노사이토진균에 대하여 항균활성을 지닌 분자량 1000 내지 10,000사이의 항균물질분획이 선택적으로 분리농축되어진다. Thereafter, 5 times aseptic water is continuously added to the concentrate, and the permeate and the concentrate are separated again by passing through a nanometer filter having a molecular weight of 1,000. In this process, residual organic acids, pigments, insoluble components, and the like are completely removed, and an antimicrobial fraction having a molecular weight of 1000 to 10,000 having an antimicrobial activity against Helicobacter pylori and Listeria monocytobacteria is selectively concentrated.
상기 분리 농축된 항균물질 포집액은 최종 부피를 발효원액의 1/10수준으로 농축하면, 위염증 및 식중독 원인균에 대한 항균활성 또한 10배로 농축된다. 항균물질 분리 농축액의 중량에 대비하여 5 내지 50% 범위의 만니톨, 유당 등의 수용성 부형제를 사용하여 동결건조 또는 분사식 유동층 건조하여 수용성 분말화한다. 이때 항균활성은 최종 분말의 경우 1,600 내지 16,000 Au/g이 되도록 희석비를 조정할 수 있다. When the separated concentrated concentrated antimicrobial substance is concentrated to the 1/10 level of the fermentation stock solution, the antimicrobial activity against gastroenteritis and food poisoning causative bacteria is also concentrated 10 times. The water-soluble powder is lyophilized or spray-liquid dried using a water-soluble excipient such as mannitol and lactose in the range of 5 to 50% relative to the weight of the antimicrobial substance separation concentrate. In this case, the antimicrobial activity may be adjusted to a dilution ratio of 1,600 to 16,000 Au / g for the final powder.
이상의 본 발명에 따른 유산균주로부터 제조된 항균물질과 그 농축과정 등을 진행하여 제조된 최종 산물인 항균활성물질은, 식중독 원인균인 리스테리아 모노사이토진과 위염증 원인균인 헬리코박터 파일로리균의 생육을 강력하게 저해하고, 장질환 유발균인 살모넬라 갈리나룸과 여드름증 원인균인 프로피오니박테리움 아크네스 등에도 동시에 항균활성을 나타내고 있음을 알아본 바와 같으므로, 이를 유해균의 감염예방이나 치료목적으로 제조된 소정의 식품 또는 의약품에 활용할 수 있으며, 경우에 따라서는 제조에서부터 섭취에 따른 소비과정에 상당한 기간이 소요되는 식품 등에서 유해균을 차단시키기 위한 목적으로 식품보존제로도 활용될 수 있음을 다음 실험예 1 내지 3을 통해 구체적으로 알아보기로 한다.The antimicrobial material prepared from the lactic acid strain according to the present invention and the final product prepared by proceeding its concentration process, antimicrobial active material, strongly inhibit the growth of the food poisoning causing bacteria Listeria monocytogene and gastritis causative bacteria Helicobacter pylori In addition, as shown to show the antimicrobial activity to Salmonella gallinarum (intestinal disease causing bacteria) and Propionibacterium acnes (causes acne causing bacteria) at the same time, it is a predetermined food prepared for the prevention or treatment of harmful bacteria Or it can be used in medicine, and in some cases it can also be used as a food preservative for the purpose of blocking harmful bacteria in foods, etc., which takes a considerable period of time from the manufacture to consumption through the following Experimental Examples 1 to 3 Let's find out in detail.
실험예 1Experimental Example 1
본 실험예 1에서는 감염균으로 헬리코박터 파일로리균을 이용하고, 이에 대한 감염방지 및 제균효과를 확인하기 위해, 본 발명에 따른 페디오코커스 펜토사세우스 CBT-8 생균과 항균활성물질(상품명 "SAFELAC") 분리건조물 등 총 2개의 시료를 이용하였으며, 대상 임상물로는 6주령의 사막모래쥐(Gerbil)를 이용하였으며, 병소감염테스트(ELISA Test)에 의하여 항체가의 변화를 관찰하였다. In Experimental Example 1, the bacterium Helicobacter pylori was used as an infectious bacterium, and in order to confirm the infection prevention and bactericidal effect thereof, the bacteriophage and the antimicrobial activity of Pediococcus pentosaceus CBT-8 according to the present invention (brand name "SAFELAC" A total of two samples, such as isolated dry matter, were used. Six-week-old desert sand rats (Gerbil) were used, and the change in antibody titer was observed by ELISA test.
이때, 각 실험구는 비투여 대조구(A군), 감염균에 의한 감염구(B군), 항균활성물질 경구투여후 감염균으로 감염시도구(C군), 감염균에 감염된 후 항균활성물질에 의한 치료시도구(D군), 감염균과 항균활성물질 동시 투여구(E군) 등 총 5개의 실험구로 구분하여 부착방지 및 제균효과를 동시에 관찰하였다. 실험동물은 각 실험구별로 5마리씩 구성하였다. 이러한 실험을 위한 실험군의 구성은 하기 표 6에 따라 구성하였다.At this time, each experimental group was treated with a non-administrative control group (Group A), an infected group (Infected Group B), orally administered with an antimicrobial active material, and then treated with an antimicrobial active material after being infected with an infectious agent (Group C) and an infectious agent. Anti-adhesion and bactericidal effects were observed at the same time by dividing into 5 experimental groups including tool (D group), infectious bacteria and antimicrobial active material (E group). Experimental animals consisted of five animals for each experiment. The configuration of the experimental group for this experiment was configured according to Table 6.
각 실험군에 따른 기간별 항체가의 변화에 대해서는 하기 표 7에 나타내었다.The change in antibody titers according to time periods according to each experimental group is shown in Table 7 below.
상기 표 5 및 표 6에 나타낸 바와 같이, 실험대조구와 각 실험구에 대해 개별적이고 구체적으로 진행된 실험내용과 그 결과에 대해 설명하면 다음과 같다.As shown in Table 5 and Table 6, the experimental control and the details of the experiment and the results of the experiment proceeded individually and specifically for each experimental section as follows.
상기 실험예 1에서, 상기 대조구(A군)를 제외한 나머지 실험군(B, C, D 및 E군)의 경우 생균의 경구 투여를 통해 감염시도를 행하는 헬리코박터 파일로리균의 농도는 108 cells/0.3㎖를 사용하였고, 항균물질 분리건조물의 경우는 항균 활성도가 50 Au/0.2㎖인 것을 사용하였다. In Experimental Example 1, except for the control group (Group A), the rest of the experimental group (B, C, D and E group) concentration of Helicobacter pylori bacteria to try infection through oral administration of live bacteria is 108 cells / 0.3ml In the case of the dried antimicrobial substance, antimicrobial activity of 50 Au / 0.2ml was used.
한편, 헬리코박터 파일로리균의 감염은 7일간 경구투여 후 항체가를 측정하여 감염을 확인하였고(B군), 부착방지능을 검토하기 위한 실험구(C군)의 경우에는 항균물질 분리건조물 시료를 30일간 경구투여 후, 그 투여를 중지하고 헬리코박터 파일로리균의 감염을 시도하였다. On the other hand, the infection of Helicobacter pylori was confirmed by the antibody value after oral administration for 7 days (B group), and in the case of the experimental group (Group C) to examine the anti-adhesion ability, 30 samples of the isolates were isolated. After daily oral administration, the administration was discontinued and infection with Helicobacter pylori was attempted.
그 결과, 헬리코박터 파일로리균의 사막모래쥐 모델에서의 헬리코박터 파일로리균을 경구 투여 종료 후 약 3주 경과시에 항체가가 3.4 이상으로 상승하여 감염이 확인되었으며(B군), 항균물질 분리건조물 시료를 먼저 경구투여한 후 감염을 시도한 실험구(C군)의 경우 항체가 상승이 나타나지 않아 부착방지 및 감염방어 효과가 있음을 확인하였다. 또한, 감염 후의 제균효과를 알아보기 위한 실험구(D군)에서는 30일 간의 시료투여 종료시점부터 항체의 감소가 발생하기 시작하여 3주 후 무처리구인 대조구(A군) 수준으로 감소하였다. 그리고, 헬리코박터 파일로리균과 항균물질 분리건조물 시료를 동시에 투여한 실험구(E군)의 경우에도 항체 상승이 발생되지 않아 감염방어 효과를 확인할 수 있었다. As a result, about 3 weeks after orally administering Helicobacter pylori in the desert sand rat model of Helicobacter pylori, the antibody titer rose to 3.4 or higher (Group B). In the experimental group (group C) that attempted infection after first oral administration, the antibody did not show an increase, and it was confirmed that there was an anti-adhesion and infection protection effect. In addition, in the experimental group (group D) to determine the bactericidal effect after infection, the antibody began to decrease from the end of 30 days of sample administration and decreased to the control (group A) level after 3 weeks. In addition, even in the experimental group (E group) to which the Helicobacter pylori bacteria and the antimicrobial substance isolated and dried samples were administered at the same time, there was no increase in the antibody so that the infection protection effect could be confirmed.
이상과 같은 실험을 통해서 살펴본, 헬리코박터 파일로리균에 대한 감염방어 (부착방지) 및 제균효과는 유산균 생균 시료에서도 같은 경향을 나타내고 있음을 확인하였으므로, 유산균 및 그 항균물질이 동시에 위염증의 예방과 치료효과를 발휘할 수 있는 것을 보여 주고 있다. 유산균의 경우에는 위점막 상피세포에 정착하기 좋은 세포구조를 지니어 헬리코박터 파일로리균의 부착지점을 경쟁적으로 선점하거나 위내에 정착 및 서식하여 항균물질 등을 분비하여 헬리코박터 파일로리균에 대한 부착억제 및 제균효과가 나타나는 것이며, 항균물질의 경우에도 위내 잔존하여 같은 효과를 나타내는 것으로서 이상의 결과에서 유산균 및 항균물질의 단독 사용 또는 동시사용에 의하여 헬리코박터 파일로리균에 대한 감염방어 및 위염증 제어효과를 발휘하고 있는 것을 확인하였다.Through the above experiments, it was confirmed that the infection defense (anti-adhesion) and bactericidal effect against Helicobacter pylori showed the same tendency in the probiotic samples of lactic acid bacteria, so the lactic acid bacteria and its antimicrobial substances simultaneously prevent and treat gastritis. Showing what can be exercised. In the case of lactic acid bacteria, the cell structure that is good to settle in gastric mucosal epithelial cells is competitively preoccupied with the point of attachment of the genotype Helicobacter pylori, or it is fixed and inhabited in the stomach to secrete antimicrobial substances to inhibit adhesion and bactericidal effect against Helicobacter pylori. In the above results, it was confirmed that the antimicrobial substance had the same effect as the remaining effect in the stomach and the control of infection and gastric inflammation against Helicobacter pylori by the use or simultaneous use of lactic acid bacteria and antimicrobial substances. It was.
실험예 2Experimental Example 2
본 실험예 2에서는 헬리코박터 파일로리균의 보균자에 대한 요소호기시험에 관한 것으로서, 위내시경검사, 요소분해효소(rapid urease) 검사, 13C-요소호기검사에 의하여 헬리코박터 파일로리균의 감염이 확인되고, 최소 1개월 전 약물치료 경력이 없으며 간, 담도, 췌장계의 만성질환자가 아닌 성인남녀 40명을 대상으로 하여 인체에서의 헬리코박터 파일로리균에 대한 제균효과를 검토하였다.In Experimental Example 2, the urea breath test for the carriers of Helicobacter pylori bacteria, the infection of Helicobacter pylori was confirmed by gastroscopy, rapid urease test, 13 C-urea breath test, We reviewed the bactericidal effect of Helicobacter pylori in humans on 40 adult men and women who had no history of drug treatment a month ago and who were not chronic diseases of the liver, biliary tract, and pancreas.
상기 실험예 2에서 이용되는 경구 투여 시료는 페디오코커스 펜토사세우스 CBT-8 생균 1010개와 항균 활성이 10,000Au/ml인 항균물질이 포함된 팩(pack)을 1일 2회씩 4주간 복용시켰으며, 시료 복용 전과 후의 13C-요소호기검사에 의한 DOB30의 수치를 관찰하여 도 7에 나타내었다. 모든 측정치는 데이터베이스 통계 프로그램 (Dbstat program)을 이용하여 통계처리하였고, 페어드 티-테스트(paired t-test)를 사용하여 p값이 0.05미만인 경우 통계적으로 유의한 것으로 판정하였다. 그 결과, 시료 복용 전과 후의 13C-요소호기검사에 의한 DOB30의 수치는 평균 26에서 15.3으로 감소하여 약 42%의 감소가 관찰되어(도 7 참조) 제균작용이 진행되고 있음을 알 수 있으며, 특별한 부작용은 나타나지 않았음을 확인하였다. 또한, 동물실험 및 인비보(in-vivo) 타입의 임상실험결과에서도 부착방지(감염방어에 따른 위염증 예방) 및 치료효과(제균작용에 따른 병증개선)가 유의성 있게 인정되었음을 확인하였다.The oral administration sample used in Experimental Example 2 is a pack containing 10 10 live bacteria of Pediococcus pentosaceus CBT-8 and an antimicrobial activity of 10,000 Au / ml, twice a day for 4 weeks. The DOB30 level was observed by 13C-urea breath test before and after taking the sample and is shown in FIG. 7. All measurements were statistically processed using the Dbstat program and determined to be statistically significant when the p-value was less than 0.05 using a paired t-test. As a result, the DOB30 level by 13 C-urea breath test before and after taking the sample decreased from an average of 26 to 15.3, a decrease of about 42% was observed (see FIG. 7). It was confirmed that no special side effects appeared. In addition, the results of animal experiments and in-vivo-type clinical trials confirmed that adhesion prevention (prevention of gastritis due to infection defense) and treatment effect (improved pathology by bactericidal action) were significantly recognized.
실험예 3Experimental Example 3
본 실험예 3에서는 상용되고 있는 유제품중의 하나인 치즈에서의 리스테리아 모노사이토진에 대한 제균효과 검토하기 위하여, 유럽산(프랑스산과 이탈리아산) 치즈의 표면에 리스테리아 모노사이토진이 104/g 정도로 인위적 오염을 시키고, 본 발명에 따른 항균물질 농축분체(상품명: 1% SAFELAC, (주)쎌바이오텍사 생산) 용액을 치즈표면에 고루 분무한 뒤 상온(25℃)과 냉장온도(4℃이하에서 각각 방치하면서 생균수의 변화를 관찰하였다.In this Experimental Example 3, in order to examine the bactericidal effect on Listeria monocytogene in cheese, one of the dairy products that are commercially available, Listeria monocytogene was contaminated about 10 4 / g on the surface of European (French and Italian) cheeses. After spraying the solution of the concentrated antimicrobial substance according to the present invention (brand name: 1% SAFELAC, produced by Bio Biotech Co., Ltd.) evenly on the surface of cheese, it is allowed to stand at room temperature (25 ℃) and refrigeration temperature (4 ℃ or less). The change in viable cell count was observed.
이때, 대조구로서는 리스테리아 모노사이토진을 오염시킨 후에 상기 항균물질 농축분체 용액으로 처리하지 않은 것을 사용하였다. 상기 실험예 3에서 사용된 치즈는 두가지 종류로서, 하는 프랑스산 치즈이고, 다른 하나는 이탈리아산 치즈를 이용하였으며, 그 결과를 구분하여 하기 표 8에 도시하였다.At this time, as a control, the one that was not treated with the antimicrobial concentrated powder solution after contaminating Listeria monocytogene was used. The cheese used in Experimental Example 3 is two kinds, French cheese made, and the other was used Italian cheese, and the results are shown in Table 8 below.
본 실험예 3에서의 대조구 1 및 2의 경우, 상온조건에서는 리스테리아 모노사이토진이 실험기간 내내 계속 증식하여 100배 가량 증식하였고, 냉장조건에서는 증식하지는 않았으나 생존상태를 유지하였다. 한편, 항균물질 농축분체 용액을 처리한 실험구 1 및 2의 경우에는 온도조건에 관계없이 리스테리아 모노사이토진이 점점 감소하여 종국에는 사멸하는 경향을 나타내고 있음을 확인하였다. 따라서, 제조 및 유통기간 중 리스테리아 모노사이토진에 의한 오염발생 가능성이 높은 식품에 대하여 본 발명에 따른 항균물질 농축분체용액을 식품보존제로 사용할 수 있음을 확인할 수 있다.In the control samples 1 and 2 in Experimental Example 3, Listeria monocytogenes continued to proliferate throughout the experimental period at room temperature, and proliferated about 100-fold, but did not proliferate under refrigerated conditions, but survived. On the other hand, in the experimental groups 1 and 2 treated with the concentrated powder solution of the antimicrobial agent, it was confirmed that the listeria monocytozin gradually decreased and eventually disappeared regardless of the temperature conditions. Therefore, it can be confirmed that the antimicrobial concentrated powder solution according to the present invention can be used as a food preservative for foods that are highly likely to be contaminated by Listeria monocytogenes during manufacturing and distribution.
실시예 2 페디오코커스 펜토사세우스(Example 2 Pediococcus pentosaceus ( Pediococcus pentosacePediococcus pentosace us) CBT-8균 항균물질 농축건조물을 이용한 기능성 식품의 제조us) Preparation of functional food using CBT-8 antimicrobial concentrate
혼합유산균 13.35%, 무수결정포도당 34.5%, 오렌지맛 8%, 페디오코커스 펜토사세우스(Pediococcus pentosaceus) CBT-8 균 항균물질 농축건조물 6%, 비타민C 2%, 분산제 (스테아린산 마그네슘) 0.1%, FOS (fructo-oligo saccharide) 12%, 퍼미자임(일종의 소화효소로서 알파 아밀라아제) 0.15%, 알로에 1.4%, 식물성분말크림 2% 및 순수 22.5%를 혼합하여 통상의 방법으로 기능성 식품을 제조하였다.Mixed Lactobacillus 13.35%, Crystalline Glucose 34.5%, Orange Flavor 8%, Pediococcus pentosaceus CBT-8 Bacteria Antimicrobial Concentrate 6%, Vitamin C 2%, Dispersant (Magnesium Stearate) 0.1% Functional foods were prepared by mixing FOS (fructo-oligo saccharide) 12%, Permizyme (alpha amylase as a digestive enzyme) 0.15%, Aloe 1.4%, plant ingredient cream 2% and pure 22.5%.
이상에서 설명된 본 발명의 최적 실시예들이 개시되었다. 여기서 특정한 용어들이 사용되었으나, 이는 단지 당업자에게 본 발명을 상세히 설명하기 위한 목적에서 사용된 것이지 의미한정이나 특허청구범위에 기재된 본 발명의 범위를 제한하기 위해 사용된 것이 아니다. Optimal embodiments of the present invention described above have been disclosed. Although specific terms have been used herein, they are used only for the purpose of describing the present invention in detail to those skilled in the art and are not intended to limit the scope of the present invention as defined in the claims or the claims.
본 발명에 따른 유산균주로부터 제조된 향균물질을 위염증 원인균인 헬리코박터 파일로리균에 대하여 특이적인 생육 저해활성을 지니며 동시에 식중독의 원인균인 리스테리아 모노사이토진균에 대해서도 특이적인 제균활성을 지니고 있다. 또한, 상기 항균물질을 발효과정을 거쳐 농축 건조시켜 제조된 건조분말은 수용성 항균성분의 분리농축물로서 다양한 식품, 의약품 및 식품보존제 제조에 적합한 물성을 지니고 있으며, 이를 응용하여 제조된 기능성 식품이나 의약품을 이용하는 경우에는 헬리코박터 파일로리균 및 리스테리아 모노사이토진균에 대한 생육억제 및 제균작용을 발휘하므로, 감염에 대한 치료 및 예방 효과를 달성할 수 있는 효과를 발휘할 수 있다.The antimicrobial material prepared from the lactic acid strain according to the present invention has specific growth inhibitory activity against Helicobacter pylori, which is a causative agent of gastritis, and at the same time, has a specific bactericidal activity against Listeria monocytogene, which is a causative agent of food poisoning. In addition, the dry powder prepared by concentrating and drying the antimicrobial material through the fermentation process is a separate concentrate of the water-soluble antimicrobial component has a physical property suitable for the production of various foods, medicines and food preservatives, functional foods or drugs prepared by applying this In the case of using, since it exhibits growth inhibition and bactericidal action against Helicobacter pylori and Listeria monocytofungus, it can exert an effect that can achieve a therapeutic and preventive effect against infection.
도 1은 본 발명에 따른 유산균주의 16S rDNA 염기서열1 is a 16S rDNA nucleotide sequence of the lactic acid strain according to the present invention
도 2는 본 발명에 따른 유전학적 동정을 살피기 위한 DNA-DNA 혼성결과도Figure 2 is a DNA-DNA hybrid results for examining the genetic identification according to the present invention
도 3은 본 발명에 따른 유산균주의 발생학적 계보도3 is a developmental genealogy diagram of the lactic acid bacteria strain according to the present invention
도 4는 본 발명에 따른 항균활성물질 제조 흐름도Figure 4 is a flow chart for producing an antimicrobial active material according to the present invention
도 5는 본 발명에 따른 항균활성물질의 발효공정 경시변화 그래프5 is a graph of changes over time of the fermentation process of the antimicrobial actives according to the present invention
도 6은 본 발명에 따른 항균활성물질의 생육저지환 시험결과의 사진Figure 6 is a photograph of the growth inhibition test results of the antimicrobial active material according to the present invention
도 7은 본 발명에 따른 항균활성물질의 DOB수치 저하효과를 나타내는 그래프Figure 7 is a graph showing the effect of reducing the DOB value of the antimicrobial active material according to the present invention
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| JP2003354432A JP3810403B2 (en) | 2002-10-15 | 2003-10-14 | A lactic acid strain named Pediococcus pentosaceus CBT-8 that produces an antibacterial active substance that suppresses the growth of Helicobacter pylori and Listeria monocytogenes, an antibacterial property using the same Method for producing antibacterial substance and method for using antibacterial substance for live bacteria in functional foods and pharmaceuticals |
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| CN103704741A (en) * | 2014-01-03 | 2014-04-09 | 扬州大学 | Culture method of leavening agent for high-density goose meat sausages |
| KR101915949B1 (en) * | 2018-01-09 | 2018-11-07 | 주식회사 쎌바이오텍 | Gene expression cassette and expression vector comprising the same |
| CN113332316A (en) * | 2021-06-29 | 2021-09-03 | 山东沐曦生物科技有限公司 | Helicobacter pylori inhibiting composition |
| CN115851551B (en) * | 2022-12-29 | 2023-08-01 | 广西爱生生命科技有限公司 | Pediococcus pentosaceus A21358 with helicobacter pylori resisting effect and application thereof |
-
2002
- 2002-10-15 KR KR10-2002-0062825A patent/KR100483369B1/en active IP Right Grant
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2003
- 2003-10-14 JP JP2003354432A patent/JP3810403B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101611829B1 (en) | 2015-06-08 | 2016-04-14 | 주식회사 쎌바이오텍 | Use of pediococcus pentosaceus cbt sl4 in the prevention or treatment of obesity and obesity-related metabolic syndrome and composition comprising the same |
| KR101611830B1 (en) * | 2015-06-08 | 2016-04-27 | 주식회사 쎌바이오텍 | Use of bifidobacterium breve cbt br3 in the prevention or treatment of obesity and obesity-related metabolic syndrome and composition comprising the same |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2004135669A (en) | 2004-05-13 |
| KR20040033616A (en) | 2004-04-28 |
| JP3810403B2 (en) | 2006-08-16 |
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