KR102224072B1 - Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application - Google Patents
Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application Download PDFInfo
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- KR102224072B1 KR102224072B1 KR1020190101226A KR20190101226A KR102224072B1 KR 102224072 B1 KR102224072 B1 KR 102224072B1 KR 1020190101226 A KR1020190101226 A KR 1020190101226A KR 20190101226 A KR20190101226 A KR 20190101226A KR 102224072 B1 KR102224072 B1 KR 102224072B1
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Abstract
본 발명은 건강한 성인에서 분리된 비피도박테리엄 롱검 아종 롱검(Bifidobacterium longum subsp. longum) BCBL-583에 관한 것으로서, 본 발명의 분리된 균주는 인체에 무해한 유전인자가 없으며, 내산소성, 내열성, 장내정착능이 우수하며, 혈중콜레스테롤 농도를 감소시키고, 면역조절능력을 가져, 프로바이오틱 활성을 갖는 식품 및 의약품에 이용될 수 있다. The present invention relates to Bifidobacterium longum subsp. longum BCBL-583 isolated from healthy adults, and the isolated strain of the present invention does not have genetic factors harmless to the human body, and is resistant to oxygen, heat, and intestines. It has excellent fixation ability, reduces blood cholesterol concentration, has immunomodulatory ability, and can be used in foods and pharmaceuticals having probiotic activity.
Description
본 발명은 면역 조절, 혈중콜레스테롤 감소 등 건강 증진에 도움을 주며, 한국 건강한 성인으로부터 분리한 신규한 비피도 박테리엄 롱검 아종(Bifidobacterium longum subsp. longum) 균주의 분리 및 이의 용도에 관한 것이다. The present invention relates to the isolation and use of a novel Bifidobacterium longum subsp. longum strain isolated from healthy adults in Korea, which aids in health promotion such as immune regulation and reduction of blood cholesterol.
프로바이오틱스(Probiotics)란 인간이나 동물 등 숙주의 건강에 유익한 효과를 나타내는 미생물 또는 그 성분으로, 바람직하게는 유산균이다. 프로바이오틱스는 균주 특이적(strain specific)이며 같은 종과 속에 해당하는 균주라도 효능은 매우 상이하다. 프로바이오틱스는 섭취하게 되면 장에 정착하여, 정장작용 등의 일차적인 기능성 외에도 면역증강, 대사증후군, 뇌건강 등 신체 전반에 도움을 주는 기능이 보고되어 있다. 프로바이오틱스 제품으로는 생균제 제품과 사균제 제품, 박테리오신 함유 제품, 유전자 재조합 제품 등이 있다. 생균제의 경우 균체 자체가 인체의 소장 및 대장에 정착하여 생리활성에 도움을 주는 제품이며, 사균제는 균체가 분비한 유용 물질들이 인체에서 유익한 효과를 나타낸다. 프로바이오틱스가 장에 도달하여 유익한 유산균 증식, 유해균 억제, 원활한 배변활동의 기능을 나타내려면 하루에 108~1010 colony forming unit (CFU)를 섭취하도록 권고하고 있다. Probiotics are microorganisms or components thereof that exhibit beneficial effects on the health of a host such as humans or animals, and are preferably lactic acid bacteria. Probiotics are strain specific, and even strains corresponding to the same species and genus have very different efficacy. When ingested, probiotics settle in the intestine, and in addition to primary functions such as intestinal function, functions that help the whole body such as immunity enhancement, metabolic syndrome, and brain health have been reported. Probiotics products include probiotics and dead fungicides, bacteriocin-containing products, and genetically modified products. In the case of probiotics, the cells themselves settle in the small and large intestine of the human body to help physiological activity, and the dead fungicides show beneficial effects in the human body by the useful substances secreted by the cells. In order for probiotics to reach the intestine and show the function of beneficial lactic acid bacteria proliferation, harmful bacteria suppression, and smooth bowel activity, it is recommended to consume 10 8 ~ 10 10 colony forming units (CFU) per day.
서구화된 식습관으로 인해 고칼로리, 고지방 식품의 섭취가 늘어나면서 비만 및 각종 성인질환 발병률이 높아지고 있으며, 장내의 유익한 유산균의 수가 줄어들고 있다. 유익한 유산균의 수가 줄어들게 되면 유해균의 수가 증가되고, 세균 다양성 불균형이 일어난다. 세균 다양성 불균형으로 인해 최근 국내에서 발병률이 증가하는 질병으로는, 염증성 장질환(Inflammatory bowel disease, IBD)중 하나인 크론병(Crohn’s disease, CD)이 있다. CD는 미국 및 유럽과 같은 고지방, 저식이섬유 식단위주의 국가에서 흔히 발병하던 질환으로, CD와 같은 궤양성 대장염은 재발과 호전을 반복하는 원인 불명의 만성적 질환이다. 상기와 같은 염증성 장질환은 기존의 치료로는 완치가 어려운 난치성 질환이다. IBD의 발병원인 중 하나는 마우스에서 관찰 되었으며, IBD가 유발된 마우스는 장내 공생세균 중 헬리코박터 헵파티쿠스(Helicobacter hepaticus)와 박테로아이데스(Bacteroides)가 증가돼 있음이 확인되었다. 이들 세균은 선천 및 획득 면역계를 자극하여 염증 반응을 유도하여, CD와 같은 궤양성 대장염을 유발하는 것으로 확인되었다. 장내 유해균들이 과다 증식하면서, 제한된 에너지원을 모두 소비하고, 유익균이 정상적으로 자리 잡지 못하게 되면 세균 다양성 불균형이 초래된다. CD와 같은 질병을 치료하기 위한 기존의 치료방법은 유해균을 사멸시켜 장내 세균 불균형을 개선하기 위해 항생제를 이용하였다. 그러나, 항생제를 이용한 치료방법은 유익균 또한 사멸시키기 때문에, 장내 세균 불균형이 회복되기 어려운 실정이다. IBD와 같은 장내 세균 불균형 질환을 치료하기 위해, 무너진 세균 불균형을 회복하는 방법들이 도입되고 있으며, 그 중 프로바이오틱스 의약품을 경구 및 직접 투여하는 방법이 연구되고 있다. As the intake of high-calorie and high-fat foods increases due to westernized eating habits, the incidence of obesity and various adult diseases is increasing, and the number of beneficial lactic acid bacteria in the intestine is decreasing. When the number of beneficial lactic acid bacteria decreases, the number of harmful bacteria increases, resulting in an imbalance in bacterial diversity. Crohn's disease (CD), one of inflammatory bowel disease (IBD), is a disease that has recently increased incidence in Korea due to an imbalance in bacterial diversity. CD is a common disease in countries with high fat and low fiber diets such as the United States and Europe, and ulcerative colitis such as CD is a chronic disease of unknown cause that repeats recurrence and improvement. Inflammatory bowel disease as described above is a refractory disease that is difficult to cure with conventional treatment. One of the causes of IBD was observed in mice, and it was confirmed that IBD-induced mice had an increase in Helicobacter hepaticus and Bacteroides among the intestinal symbiotic bacteria. It has been found that these bacteria stimulate the innate and acquired immune systems to induce an inflammatory response, causing ulcerative colitis such as CD. When harmful bacteria in the intestine over-proliferate, consume all of the limited energy sources, and when beneficial bacteria are not properly positioned, an imbalance in bacterial diversity is caused. Existing treatment methods for treating diseases such as CD have used antibiotics to improve the intestinal bacterial imbalance by killing harmful bacteria. However, since the treatment method using antibiotics also kills beneficial bacteria, it is difficult to recover the intestinal bacterial imbalance. In order to treat an intestinal bacterial imbalance disease such as IBD, methods for restoring a collapsed bacterial imbalance are being introduced, and among them, a method of oral or direct administration of probiotic drugs is being studied.
성인성 질환을 유발시키는 요인 중에서도 가장 주된 요인은 동물성 지방의 과다한 섭취로 인한 혈중 고콜레스테롤이다. 고콜레스테롤 혈증은 고지혈증, 동맥경화증, 지질 관련 대사증후군과 같은 질환의 원인이 되고 있다. 유산균은 혈중 콜레스테롤을 감소시키는 것으로 알려져 있으며, 유산균에 의한 콜레스테롤 흡수의 감소 기작을 세 가지로 설명할 수 있다(대한민국 등록특허 제10-0930251호 참조). 첫 번째는 유산균이 장내에 정착 증식하면서 콜레스테롤을 흡착하여 배설시키는 것이고, 두 번째는 유산균이 담즙산을 흡착하여 배설시키는 것이다. 콜레스테롤은 담즙산의 전구체이므로 유산균에 의해 담즙산이 배출되면, 부족한 담즙산을 보충하기 위해 콜레스테롤을 이용하게 되어, 결과적으로 장내 콜레스테롤 농도를 감소시킬 수 있다. 그리고 세 번째 기작은 유산균이 포합담즙산(conjugated bile acid)을 탈포합시키는 작용이다. 간에서 생성된 프리담즙산(free bile acid)은 소장에서 타우린 또는 글라이신과 결합하여 포합담즙산이 된다. 포합담즙산은 지질 용해도가 높아, 소장에서 콜레스테롤 등의 지질 흡수를 용이하게 한다. 그러므로, 포합담즙산이 많은 경우 흡수되는 콜레스테롤 양이 많아져 혈중 콜레스테롤 농도가 높아진다. 그러나, 탈포합된 담즙산은 지질 용해도가 낮아 콜레스테롤 등 지질의 혈중 이행을 저하시키는 작용을 한다. 따라서, 포합담즙산을 탈포합시키는 담즙산 분해효소(bile salt hydrolase)를 갖는 균주는 지질 용해도를 낮추어 소장에서 콜레스테롤의 흡수를 감소시킬 수 있다. Among the factors that cause adult diseases, the main factor is high cholesterol in blood due to excessive consumption of animal fat. Hypercholesterolemia is a cause of diseases such as hyperlipidemia, arteriosclerosis, and lipid-related metabolic syndrome. Lactic acid bacteria are known to reduce blood cholesterol, and three mechanisms for reducing cholesterol absorption by lactic acid bacteria can be explained (refer to Korean Patent Registration No. 10-0930251). The first is for lactic acid bacteria to settle and proliferate in the intestine and adsorb and excrete cholesterol, and the second is for lactic acid bacteria to adsorb and excrete bile acids. Cholesterol is a precursor of bile acids, so when bile acids are discharged by lactic acid bacteria, cholesterol is used to compensate for insufficient bile acids, and as a result, cholesterol concentration in the intestine can be reduced. And the third mechanism is the action of lactic acid bacteria to deconjugate conjugated bile acid. The free bile acid produced in the liver is combined with taurine or glycine in the small intestine to form a conjugated bile acid. Conjugated bile acids have high lipid solubility and facilitate the absorption of lipids such as cholesterol in the small intestine. Therefore, when there is a large amount of conjugated bile acids, the amount of absorbed cholesterol increases, resulting in a high blood cholesterol concentration. However, deconjugated bile acids have low lipid solubility and act to lower the transition of lipids such as cholesterol into the blood. Therefore, a strain having a bile salt hydrolase that deconjugates the conjugated bile acid can reduce the absorption of cholesterol in the small intestine by lowering the lipid solubility.
인체의 소화관에는 많은 세균들이 서식하고 있으며, 이러한 세균의 대부분은 장내세균총을 이루고 있으며 장에 존재하는 다양한 면역세포와의 상호작용을 통해 인체의 면역조절에 영향을 미치고 있다. 그리고 이 세균들은 장 건강에 도움을 주는 유익균과 건강에 해로운 유해균으로 나눌 수 있다. 유익균으로 알려진 균속은 락토바실러스(Lactobacillus), 비피도박테리엄(Bifidobacterium), 스트렙토코커스(Streptococcus), 스포로락토바실러스(Sporolactobacillus) 등이 알려져 있으며, 상기 유익균들이 대장균(E. coli), 헬리코박터(Helicobacter), 블라우티아(Blautia)와 같은 유해균보다 우세균으로 우점하고 있다. 장내 미생물이 균형을 유지할 때 건강을 유지할 수 있으며, 인체의 면역세포들은 외부에 침입하는 항원(미생물 포함)과 내적으로 생기는 이상 세포를 제어하여 건강을 유지하도록 해준다. 이 면역세포의 70%이상이 소화관에 위치하고 있고, 소화관에 이미 서식하고 있는 미생물 또는 외부에서 침입하는 미생물들을 제어한다. 인체의 면역조절 작용에 문제가 생기는 경우 면역력 저하나 면역 과민반응이 초래되고, 그로인해 암, IBD, CD, 알러지 질환, 아토피, 비만, 당뇨병 등과 같은 질환이 유발될 수 있다. 따라서 상기 질환들을 예방하고, 치료하기 위해서 소화관의 면역계를 정상적으로 잘 작동하도록 하는 것이 중요하다. 장내 균총과 숙주인 인간의 건강과 밀접한 관련성이 제시되고 있는 만큼, 장내 미생물의 종류와 특성을 확인하고, 군집으로서 장내 미생물을 이해하는 일은 매우 중요하다.Many bacteria live in the digestive tract of the human body, and most of these bacteria make up the intestinal flora and influence the immune regulation of the human body through interaction with various immune cells present in the intestine. And these bacteria can be divided into beneficial bacteria that help intestinal health and harmful bacteria that are harmful to health. Species known yuikgyun are Lactobacillus bacteria (Lactobacillus), William Bifidobacterium (Bifidobacterium), Streptococcus (Streptococcus), and in sports Lactobacillus (Sporolactobacillus), etc. are known, have the yuikgyun coli (E. coli), Helicobacter (Helicobacter ), it has been dominated by Blau thiazole (woosegyun more harmful bacteria like Blautia). Health can be maintained when the intestinal microbes are in balance, and the body's immune cells control antigens (including microbes) that invade outside and abnormal cells that occur inside to maintain health. More than 70% of these immune cells are located in the digestive tract and control microorganisms already inhabiting the digestive tract or invading microorganisms from the outside. When a problem occurs in the immune regulation function of the human body, immunity decreases or immune hypersensitivity reactions are caused, and diseases such as cancer, IBD, CD, allergic diseases, atopy, obesity, and diabetes may be caused. Therefore, it is important to make the immune system of the digestive tract function normally in order to prevent and treat the above diseases. As closely related to the intestinal flora and the human health as the host, it is very important to identify the types and characteristics of intestinal microbes, and to understand the intestinal microbes as a community.
프로바이오틱스로서 효과가 있기 위해서는 경구로 섭취될 때, 내산성 및 내담즙성이 우수해야 하며, 장내 정착능이 우수하여야 한다. 유산균은 인체의 소화계에 공생하면서 섬유질 및 복합단백질들을 분해하여 중요한 영양성분으로 전환시키는 역할을 하게 된다. 유산균은 장내에서 정착하면서 유해균의 생장을 억제하고, 유익균 및 장내의 정상균무리(Nomal flora)의 수를 유지시켜주며, 항당뇨, 항고지혈증 효과, 발암억제, 대장염 억제 및 숙주 면역체계의 비특이적 활성 등의 효과를 나타낸다. 이러한 유산균의 다양한 생리활성이 알려지면서, 인체에 안전하고 생리활성 조절이 우수한 유산균 균주를 개발하여, 기능성 식품, 화장품 첨가제 및 의약품으로 적용하려는 연구가 활발하게 진행되고 있다.In order to be effective as a probiotic, when taken orally, acid resistance and bile resistance should be excellent, and intestinal fixation ability should be excellent. Lactic acid bacteria coexist in the digestive system of the human body, decomposing fiber and complex proteins and converting them into important nutrients. Lactobacillus settles in the intestine, inhibits the growth of harmful bacteria, maintains the number of beneficial bacteria and normal flora in the intestine, has antidiabetic, antihyperlipidemic effect, inhibits carcinogenesis, inhibits colitis, and non-specific activity of the host immune system, etc. Shows the effect of. As various physiological activities of such lactic acid bacteria are known, studies to develop lactic acid bacteria strains that are safe for the human body and have excellent physiological activity control and are applied as functional foods, cosmetic additives, and pharmaceuticals are being actively conducted.
예를 들어, 대한민국 등록특허공보 제10-0472865호에는 면역증가능, 콜레스테롤 감소능, 항돌연변이능이 우수한 비피도박테리엄 인판티스 PL9506 (B. infantis PL9506) KCCM-10406이 개시되어 있다. 또한, 대한민국 등록 특허공보 제10-1239806호에는 면역증강 및 항산화 활성을 가지는 신규한 락토바실러스 플란타룸 HY7712(L. plantarum HY7712)이 개시되어 있다. 또한, 대한민국 특허공보 제10-1868517호에는 리파제 생산능력이 뛰어나고, 항생제 내성 전이의 위험이 없으며, 식중독균 및 혐기성균 성장 억제능이 뛰어나, 배변활동 및 장 건강 상태를 증진시키는 생균제 또는 유산균 제제등으로 이용할 수 있는 락토바실러스 퍼멘텀 PL9119(L. fermentum PL9119)이 기재되어 있다. For example, Korean Patent Publication No. 10-0472865 discloses Bifidobacterium Infantis PL9506 ( B. infantis PL9506) KCCM-10406, which is excellent in immunogenicity, cholesterol reducing ability, and antimutagenicity. In addition, Korean Patent Publication No. 10-1239806 discloses a novel Lactobacillus plantarum HY7712 (L. plantarum HY7712) having immunity enhancing and antioxidant activity. In addition, Korean Patent Publication No. 10-1868517 has excellent lipase production capacity, no risk of antibiotic resistance transfer, and excellent ability to inhibit the growth of food poisoning and anaerobic bacteria, and can be used as probiotics or lactic acid bacteria preparations to improve bowel activity and intestinal health. A possible Lactobacillus fermentum PL9119 (L. fermentum PL9119) is described.
유산균을 이용한 기술은 가장 오래된 바이오관련 기술로서 국내에서도 그동안 많은 연구가 이루어져 왔고, 이를 이용한 산업으로서, 발효유, 기능성식품, 의약용 정장제, 동물용 의약품 및 화장품 등 다양하게 활용되고 있다. 유산균은 중요한 산업용 균주로 알려져 있으며, 그중에서도 비피도박테리엄 속 균주는 인체의 장내에 서식하는 정상 미생물 군집의 주요한 균주로서, 건강한 소화기관과 장내 환경을 유지하는 데 있어서 중요한 것으로 알려져 왔다. 미국의 공중건강 가이드라인(U.S. Public Health Service guidelines)에 따르면, 현재 미국 균주 기탁기관(ATCC)에 기탁된 비피도박테리엄 균주 모두 인체나 동물에 질병을 유발할 잠재적 위험에 대해서는 알려진 것이 없다고 인정되는 ‘생물 안전 수준(Bio-safty Level) 1’로 분류되어 있다. 상기 설명한 유산균의 기능으로 인해 인체용 의약품 및 동물용 의약품과 사료첨가제로서 그 이용도 늘고 있어, 우수한 특성을 가지는 유산균의 개발이 요구되고 있다. The technology using lactic acid bacteria is the oldest bio-related technology, and a lot of research has been conducted in Korea so far, and as an industry using it, fermented milk, functional foods, medical intestinal products, veterinary medicines and cosmetics are widely used. Lactobacillus is known as an important industrial strain, and among them, Bifidobacterium strain is a major strain of the normal microbial community living in the intestine of the human body, and has been known to be important in maintaining a healthy digestive system and intestinal environment. According to the US Public Health Service guidelines, it is recognized that none of the Bifidobacterium strains currently deposited with the US Strains Depository (ATCC) are known about the potential risk of causing disease in humans or animals. It is classified as'Bio-safty Level 1'. Due to the functions of the lactic acid bacteria described above, the use of them as pharmaceuticals for humans, pharmaceuticals for animals, and feed additives is increasing, and the development of lactic acid bacteria having excellent properties is required.
기존의 상업적 유산균 균주는 외국 유래 혹은 동물 유래 유산균제로서 한국인이 섭취할 경우 장내 생존력이나 그 기능성이 떨어질 위험이 있다. 또한 한국에서 분리된 유산균들의 경우 시험관 내(In vitro)에서만 기능성을 검증하여, 그 효능을 확인하였기 때문에 사람이 섭취할 경우 어떠한 기능적인 특성이나 안전성에 대한 문제를 증명할 수 없다. 또한 이러한 균주들은 유전체 수준에서 안전성을 확인하지 않았기 때문에 사람에게서 분리가 되었음에도 불구하고 플라스미드 등을 통한 유해인자를 보유 혹은 전달할 가능성이 있기 때문에, 유전체 수준에서의 안전성 검사가 필수적 이다. 또한 생물체 내(In vivo)에서 기능성 및 장내 균총의 변화를 확인한 경우도 없기 때문에 장내에 존재하는 균주들에 대한 상호 작용 뿐만 아니라 숙주에게 미치는 영향에 대하여 확인할 필요가 있다. 이러한 조건들은 산업적 균주로 사용되기 위해 모두 충족되어야 하며 한 가지만 충족되어 산업적으로 사용되는 일은 매우 위험한 일이다. Existing commercial lactic acid bacteria strains are foreign-derived or animal-derived lactic acid bacteria, and when consumed by Koreans, there is a risk that intestinal viability or its functionality may decrease. In addition, in the case of lactic acid bacteria isolated in Korea, the functionality was verified only in vitro, and its efficacy was verified, so any functional characteristics or safety problems cannot be proved when ingested by humans. In addition, since these strains have not been confirmed for safety at the genome level, even though they are isolated from humans, they may possess or transmit harmful factors through plasmids, so safety testing at the genome level is essential. In addition, since there is no case of confirming changes in functionality and intestinal flora in vivo, it is necessary to confirm not only the interactions with the strains present in the intestine but also the effects on the host. All of these conditions must be met in order to be used as an industrial strain, and it is very dangerous to be used industrially because only one is met.
따라서, 새로운 프로바이오틱스 균주를 한국인으로부터 분리하고 in vitro 상에서의 기능성 확인 및 in vivo 상에서의 기능성 검증을 통해 기능성이 확인되어야 한다. 기능성이 확인된 균주로부터 유전체 수준에서의 안전성검증까지 수행하여, 산업용 균주로 사용하기 적합한 균주를 발굴하여야 한다. 이에, 본 발명은 건강한 한국 성인들로부터 비피도박테리엄 롱검 아종을 분리하였고, in vitro 및 in vivo 수준에서의 프로바이오틱스로서의 기능성 평가와 유전체 수준에서의 안전성을 확인하였다. 따라서 본 발명은 상기 분리된 비피도박테리엄 롱검 아종 균주를 다양한 용도로서 사용할 수 있음을 확인함으로써 본 발명을 완성하였다.Therefore, a new probiotic strain should be isolated from Koreans, and the functionality should be confirmed through functional verification in vitro and functional verification in vivo. It is necessary to find a strain suitable for use as an industrial strain by carrying out safety verification at the genome level from the strain with which the functionality has been confirmed. Accordingly, the present invention isolated Bifidobacterium longum subspecies from healthy Korean adults, and confirmed the functional evaluation as a probiotic at the in vitro and in vivo levels and safety at the genomic level. Therefore, the present invention completed the present invention by confirming that the isolated Bifidobacterium longum subspecies strain can be used for various purposes.
(특허문헌 1) KR10-0930251 B (Patent Document 1) KR10-0930251 B
(특허문헌 2) KR10-0472865 B (Patent Document 2) KR10-0472865 B
(특허문헌 3) KR10-1239806 B (Patent Document 3) KR10-1239806 B
(특허문헌 4) KR10-1868517 B (Patent Document 4) KR10-1868517 B
본 발명의 목적은 한국인에게 잘 정착할 수 있도록 건강한 한국인 성인으로부터 분리한 신규한 비피도박테리엄 롱검 아종 롱검(Bifodibacterium longum subsp. longum) 균주를 제공하는 것이다.It is an object of the present invention to provide a novel Bifidobacterium longum subsp. longum strain isolated from healthy Korean adults so that they can settle well in Koreans.
본 발명의 또 다른 목적은 신규한 비피도박테리엄 롱검 아종 롱검 균주 또는, 이의 배양액을 유효성분으로 하는 약학적 조성물, 건강기능식품 및 일반 식품을 제공하는 것이다.Another object of the present invention is to provide a novel Bifidobacterium longum subspecies Longum strain or a pharmaceutical composition, a health functional food, and a general food using a culture medium thereof as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 신규한 비피도박테리엄 롱검 아종 롱검(Bifidobacterium longum subsp. longum)균주를 제공한다.In order to achieve the above object, the present invention provides a novel Bifidobacterium longum subsp. longum strain.
또한, 본 발명은 신규한 비피도박테리엄 롱검 아종 롱검, 또는 이들의 배양액을 유효성분으로 함유하는 생균제 조성물을 제공한다.In addition, the present invention provides a probiotic composition containing a novel Bifidobacterium longum subspecies longum, or a culture solution thereof, as an active ingredient.
따라서, 본 발명은 신규한 비피도박테리엄 롱검 아종 롱검, 또는 이들의 배양액을 유효성분으로 함유하며 면역 조절과, 혈중콜레스테롤 감소 등을 위한 약학적 조성물, 건강기능식품 또는 일반 식품 조성물을 제공한다.Accordingly, the present invention provides a pharmaceutical composition, a health functional food or a general food composition containing a novel Bifidobacterium longum subspecies longum, or a culture medium thereof, as an active ingredient, and for immune regulation and reduction of blood cholesterol.
본 발명의 신규한 비피도박테리엄 롱검 아종 롱검(Bifidobacterium longum subsp. longum)BCBL-583 균주 또는 이의 배양액은 산소에 대한 내성과, 열안정성, 장내 정착능과 장내 효소에 대한 내성이 우수하여, 섭취 시 장에 효과적으로 정착이 가능하다. 또한, 본 발명의 신규한 비피도박테리엄 롱검 아종 롱검 균주는 혈중 콜레스테롤 감소능력과 면역 조절능력을 보유하고 있어, 식품을 통해 섭취할 경우 건강기능 개선을 기대할 수 있다. The novel Bifidobacterium longum subsp. longum BCBL-583 strain of the present invention or its culture medium has excellent resistance to oxygen, heat stability, intestinal fixation ability and resistance to intestinal enzymes, and ingestion It can be settled effectively in the market. In addition, the novel Bifidobacterium longum subspecies Longum strain of the present invention has the ability to reduce blood cholesterol and immune regulation, and thus, when ingested through food, it can be expected to improve health functions.
도 1은 본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주와 표준균주와의 산소내성을 비교한 것이다.
도 2는 본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주와 표준균주의 45℃에서의 내열성을 확인한 것이다.
도 3은 본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주와 표준균주의 내산성 및 내 담즙성을 확인한 것이다.
도 4는 본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주와 표준균주의 장내정착능을 평가한 것이다.
도 5는 본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주와 표준균주의 배지 내 콜레스테롤 감소능을 확인한 것이다.
도 6은 본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주와 표준균주에 의한 Raw 264.7세포의 사이토카인 반응성을 확인한 것이다.
도 7은 본 발명의 In vivo 실험에서 5개의 마우스 실험군에 따른, 급여 사료 및 투여한 균주를 나타낸 것이다.
도 8은 본 발명에서의 식이 및 섭취 균주에 따른 면역조절능력의 in vivo 결과이다.
도 9는 본 발명에서의 식이 및 섭취 균주에 따른 콜레스테롤 감소의 in vivo 결과이다.
도 10은 본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583의 유전자 지도를 나타낸 것이다.
도 11은 본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583과 비피도박테리엄 19종의 상동성을 분석한 것이다.
도 12은 본 발명의 섭취 균주에 따른, 주차별 균총 조성의 변화를 나타낸 것이다.
도 13는 본 발명의 섭취 균주에 따른, 주차별 균총의 변화를 나타낸 것이다.1 is a comparison of the oxygen resistance between the Bifidobacterium longum subspecies Longum BCBL-583 strain and the standard strain of the present invention.
Figure 2 shows the heat resistance of the Bifidobacterium longum subspecies Longum BCBL-583 strain and standard strain at 45°C of the present invention.
3 is a view confirming the acid resistance and bile resistance of the Bifidobacterium Longum subspecies Longum BCBL-583 strain and the standard strain of the present invention.
Figure 4 is an evaluation of the intestinal fixation ability of the Bifidobacterium longum subspecies Longum BCBL-583 strain and the standard strain of the present invention.
Figure 5 shows the cholesterol-reducing ability in the medium of the Bifidobacterium longum subspecies Longum BCBL-583 strain and the standard strain of the present invention.
6 is a view confirming the cytokine reactivity of Raw 264.7 cells by the Bifidobacterium Longum subspecies Longum BCBL-583 strain and the standard strain of the present invention.
Figure 7 shows the feed feed and administered strains according to the five mouse experimental groups in the in vivo experiment of the present invention.
8 is an in vivo result of immunomodulatory ability according to diet and ingestion strains in the present invention.
9 is an in vivo result of cholesterol reduction according to diet and ingestion strains in the present invention.
Figure 10 shows a genetic map of the Bifidobacterium Longum subspecies Longum BCBL-583 of the present invention.
11 is an analysis of the homology of the Bifidobacterium longum subspecies Longum BCBL-583 and the Bifidobacterium 19 species of the present invention.
Figure 12 shows the change in the composition of the colony for each week according to the intake strain of the present invention.
13 shows the change of the colony for each week according to the ingested strain of the present invention.
이하, 본 발명에서 사용한 용어를 설명한다.Hereinafter, terms used in the present invention will be described.
본 발명에서 사용되는 용어 “배양물”이란 미생물을 공지의 액체 배지 또는 고체 배지에서 배양시켜 수득한 산물을 의미하며, 미생물이 포함되는 개념이다.The term “culture” used in the present invention refers to a product obtained by culturing a microorganism in a known liquid medium or solid medium, and is a concept including microorganisms.
본 발명에서 “약제학적으로 허용가능한”이란 생물체를 상당히 자극하지 않고 투여 활성 물질의 생물학적 활성 및 특성을 저해하지 않는 것을 의미한다.In the present invention, “pharmaceutically acceptable” means not significantly irritating an organism and not inhibiting the biological activity and properties of the administered active substance.
본 발명에서 사용되는 용어 “예방”은 본 발명의 조성물의 투여로 특정 질환의 증상을 억제하거나 진행을 지연시키는 모든 행위를 의미한다.The term “prevention” as used in the present invention refers to any action that suppresses or delays the progression of a symptom of a specific disease by administration of the composition of the present invention.
본 발명에서 사용되는 용어 “치료”는 본 발명의 조성물의 투여로 특정 질환의 증상을 호전 또는 이롭게 변경시키는 모든 행위를 의미한다.The term "treatment" used in the present invention refers to any action that improves or beneficially alters the symptoms of a specific disease by administering the composition of the present invention.
본 발명에서 사용되는 용어 “개선”은 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소 시키는 모든 행위를 의미한다.As used herein, the term “improvement” refers to any action that at least reduces the severity of a parameter related to the condition being treated, for example, symptoms.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 신규한 비피도박테리엄 롱검 아종 롱검을 제공한다.The present invention provides a novel Bifidobacterium longum subspecies long gum.
상기 본 발명의 균주는 비피도박테리엄 롱검 아종 롱검 BCBL-583으로서, 한국미생물자원센터에 수탁번호 KCTC 13859BP로 기탁된 균주이다.The strain of the present invention is Bifidobacterium longum subspecies Longum BCBL-583, which is a strain deposited with the Korea Microbial Resource Center under the accession number KCTC 13859BP.
더욱 구체적으로, 본 발명의 발명자들은 20대의 건강한 14명의 성인으로부터 분변을 수집하였다. 분변 수집에 앞서 3개월간 항생제를 복용하지 않았고, 3주간 프로바이오틱스 제품을 섭취하지 않은 대상으로부터 분변을 수집하였다. 수집된 분변은 희석배수 106이 되도록 펩톤수(Peptone water)로 연속 희석하여, 비피도박테리엄 선택배지인 비피도박테리엄 아이오딘아세테이트 배지(BIM-25)에 평판 도말하여 형성된 콜로니를 확보하였다. 확보된 콜로니는 MRS 액체배지에 접종하여 배양시킨 후 비피도박테리엄의 특이적 효소인 과당-6-인산 포스포케톨라제(Fructose-6-phosphate phosphoketolase)에 대한 활성을 측정하여, 활성이 나타나는 균주를 분리하였다. More specifically, the inventors of the present invention collected feces from 14 healthy adults in their twenties. Before fecal collection, feces were collected from subjects who did not take antibiotics for 3 months and did not take probiotic products for 3 weeks. Collected feces were serially diluted with peptone water to a dilution factor of 10 6 , and plated on Bifidobacterium iodine acetate medium (BIM-25), which is a Bifidobacterium selective medium, to obtain colonies formed. . The obtained colonies were inoculated in MRS liquid medium and cultured, and then the activity against fructose-6-phosphate phosphoketolase, a specific enzyme of Bifidobacterium, was measured. Was separated.
상기 분리된 균주는, MRS 액체배지에 접종하여 배양시킨 후 전장 DNA를 분리하여 16S rRNA universal primer를 이용하여 중합효소연쇄반응(PCR)을 사용하여 16S rRNA의 염기서열을 증폭시켰다증폭된 PCR산물은 카필러리 시퀀싱(Capillary sequencing)을 이용하여 염기서열을 획득하였다. 상기 16S rRNA는 서열번호 1의 염기서열을 포함한다. 획득된 염기서열은 NCBI의 BLASTN 프로그램을 이용하여 분자생물학적 동정을 실시하였다. 동정 결과 상기 분리균주는 비피도박테리엄 롱검의 아종 균주였고, 본 발명에서는 비피도박테리엄 롱검 아종 롱검 BCBL-583 으로 명명하였으며, 한국생명공학연구원 미생물자원센터(Korea Collection of Type Culture, KCTC)에 수탁번호 KCTC13859BP로 기탁하였다.The isolated strain was inoculated in MRS liquid medium and cultured, and then the full-length DNA was separated and the nucleotide sequence of 16S rRNA was amplified using a 16S rRNA universal primer. The base sequence was obtained using capillary sequencing. The 16S rRNA includes the nucleotide sequence of SEQ ID NO: 1. The obtained nucleotide sequence was identified by molecular biology using NCBI's BLASTN program. As a result of the identification, the isolated strain was a subspecies strain of Bifidobacterium longum, and in the present invention, it was named as Bifidobacterium longum subspecies Longum BCBL-583, and was referred to the Korea Collection of Type Culture (KCTC). It was deposited under the accession number KCTC13859BP.
또한, 본 발명은 수탁번호 KCTC13859BP로 기탁된 신규한 비피도박테리엄 롱검 아종 롱검 BCBL-583, 또는 이들의 배양액을 유효성분으로 함유하는 생균제 조성물을 제공한다.In addition, the present invention provides a novel Bifidobacterium longum subspecies Longum BCBL-583 deposited under accession number KCTC13859BP, or a probiotic composition containing a culture solution thereof as an active ingredient.
본 발명의 조성물은 통상적인 생균제 조성물의 제조방법에 따라 제조될 수 있으며, 일반적으로, 배양현탁액이나 건조분말 형태일 수 있다. 또한, 주성분인 상기 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주, 또는 이의 배양액의 유효량에 1종 또는 2종 이상의 약제학적으로 허용 가능한 통상적인 담체 또는 1종 또는 2종 이상의 첨가제를 선택하여 통상적인 제형의 조성물로 제조할 수 있다.The composition of the present invention may be prepared according to a conventional method for preparing a probiotic composition, and generally, may be in the form of a culture suspension or a dry powder. In addition, one or two or more pharmaceutically acceptable conventional carriers or one or two or more additives are selected in the effective amount of the Bifidobacterium longum subspecies Longum BCBL-583 strain, or a culture solution thereof, as the main component. It can be prepared as a formulation composition.
담체는 희석제, 활택제, 결합제, 붕해제, 감미제, 안정제, 방부제 중에서 1종 또는 2종 이상을 선택하여 사용할 수 있으며, 첨가제로는 향료, 비타민류, 항산화제 중에서 1종 또는 2종 이상을 선택하여 사용할 수 있다.The carrier can be used by selecting one or two or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and one or two or more of flavors, vitamins, and antioxidants can be selected as additives. Can be used.
본 발명에 있어서, 담체 및 첨가제는 그 종류에 제한되는 것은 아니며, 구체적으로는 희석제로 유당(lactose monohydrate), 트레할로스(Trehalose), 옥수수 전분(corn starch), 콩기름(soybean oil), 미결정 셀룰로오스(microcrystalline cellulose) 또는 만니톨(D-mannitorl), 활택제로는 스테아린산 마 그네슘(magnesiumstearate) 또는 탈크(talc), 결합제로는 폴리비닐피롤리돈(PVP:polyvinyipyrolidone) 또는 하이드록시프로필셀룰로오스(HPC: hydroxypropylcellulose) 중에서 선택할 수 있다. 또한, 붕해제로는 카르복시메틸셀룰로오스칼슘(Ca-CMC: carboxymethylcellulose calcium), 전분글리콜산나트륨(sodium starchglycolate), 폴라크릴린칼륨(polacrylin potassium) 또는 크로스포비돈(cross-linkedpolyvinylpyrrolidone), 감미제로는 백당, 과당, 소르비톨(sorbitol) 또는 아스파탐(aspartame), 안정제로는 카르복시메틸셀룰로오스나트륨(Na-CMC: carboxymethylcellulosesodium), 베타-싸이크로덱스트린(β-cyclodextrin), 백납(white bee's wax) 또는 잔탄검(xanthan gum) 중에서 선택할 수 있으며, 방부제로는 파라옥시안식향산메칠(methyl p-hydroxy benzoate, methlparaben), 파라옥시안식향산프로필(propyl p-hydroxybenzoate, propylparaben), 또는 소르빈산칼륨(potassium sorbate) 중에서 선택할 수 있다.In the present invention, the carrier and the additive are not limited to the type, specifically, as a diluent, lactose monohydrate, trehalose, corn starch, soybean oil, microcrystalline cellulose. cellulose) or mannitol (D-mannitorl), magnesium stearate or talc as a lubricant, polyvinyipyrolidone (PVP) or hydroxypropylcellulose (HPC) as a binder You can choose. In addition, as a disintegrant, carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, pollacrylin potassium, or cross-linked polyvinylpyrrolidone, as a sweetener, sugar, Fructose, sorbitol or aspartame, as a stabilizer, carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin, white bee's wax, or xanthan gum ), and the preservative may be selected from methyl p-hydroxy benzoate (methlparaben), propyl p-hydroxybenzoate (propylparaben), or potassium sorbate.
또한, 본 발명은 수탁번호 KCTC13859BP로 기탁된 비피도박테리엄 롱검 아종 롱검 BCBL-583, 또는 이들의 배양액을 유효성분으로 함유하는 약학적 조성물, 건강기능식품 조성물 또는 일반 식품 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition, a health functional food composition or a general food composition containing Bifidobacterium longum subspecies Longum BCBL-583, or a culture solution thereof, deposited as accession number KCTC13859BP as an active ingredient.
상기 미생물의 배양액에는 미생물이 생산하는 콜레스테롤 감소 물질이 포함되어 있다.The culture medium of the microorganism contains a cholesterol reducing substance produced by the microorganism.
또한, 상기 본 발명에 따른 조성물은 본 발명의 균주와 함께 섭취하기 적합하고, 섭취 시 유해 미생물의 생육을 억제하며, 장내 균총의 균형을 개선시키는 다른 종류의 공지된 미생물을 추가로 포함할 수 있다.In addition, the composition according to the present invention is suitable for ingestion with the strain of the present invention, inhibits the growth of harmful microorganisms when ingested, and may further include other types of known microorganisms that improve the balance of the intestinal flora. .
상기 분리균주는 내산소성, 내열성, 내담즙성, 내산성, 장내 정착능이 우수하며, 혈중콜레스테롤 감소효과 및 면역세포에서 항염증성 사이토카인 IL-4, IL-10의 분비의 증가 또는 전염증성 사이토카인 TNF-α, 및 IL-6의 분비를 감소시켜 면역조절능력을 가지는 균주이다. The isolated strain has excellent oxygen resistance, heat resistance, bile resistance, acid resistance, and intestinal fixation ability, and has the effect of reducing blood cholesterol and increasing the secretion of anti-inflammatory cytokines IL-4 and IL-10 in immune cells, or TNF, a pro-inflammatory cytokine. It is a strain having immunomodulatory ability by reducing the secretion of -α and IL-6.
상기 균주 또는 이의 배양액은 장내 균총의 균형을 통한 장 건강 활성을 증진시키는 효과를 가진다.The strain or a culture medium thereof has an effect of enhancing intestinal health activity through the balance of intestinal flora.
본 발명의 상기 약학적 조성물은 대사성 질환에 대한 예방 또는 치료 효과를 포함한다. 본 발명의 수탁번호 KCTC13859BP로 기탁된 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주는, 앞서 언급한 바, 혈중 콜레스테롤 감소 효과를 가지므로, 이러한 효과를 통하여, 본 발명의 균주는 대사성 질환에 대한 예방 또는 치료 효과를 가질 수 있다. 상기 대사성 질환은 이에 제한되는 것은 아니나, 당뇨, 고혈압, 고지혈증, 동맥경화증, 고콜레스테롤혈증 또는 비만 중에서 선택되는 1 이상의 질환을 의미한다. 본 발명의 일 실시예에서는 세포 및 동물실험을 통하여 혈중 콜레스테롤 감소 효과를 확인하였다.The pharmaceutical composition of the present invention includes a preventive or therapeutic effect on metabolic diseases. The Bifidobacterium longum subspecies Longum BCBL-583 strain deposited with the accession number KCTC13859BP of the present invention, as mentioned above, has an effect of reducing blood cholesterol, through this effect, the strain of the present invention is prevented against metabolic diseases. Or it may have a therapeutic effect. The metabolic disease is not limited thereto, but refers to one or more diseases selected from diabetes, hypertension, hyperlipidemia, arteriosclerosis, hypercholesterolemia, or obesity. In an embodiment of the present invention, the effect of reducing blood cholesterol was confirmed through cell and animal experiments.
또한, 본 발명의 약학적 조성물은, 면역 조절기능을 포함한다. 본 발명의 균주가 가지는 “면역 조절기능” 이란, 면역 증강 뿐 아니라, 면역 과민반응 감소 활성을 가지는 것으로 해석될 수 있다. 면역 과민반응은 특정 항원에 대해 과도한 면역반응이 일어나 조직 손상 등의 부적절한 피해를 주는 면역반응을 말하는 것으로서 면역 과민반응과 관련한 질환으로는 알레르기, 아토피성 피부염, 천식, 건초열, 전신성 아나필락틱 쇼크, 적아세포증, 류마티스성 관절염, 사구체신염, 루프스, 아르투스반응, 접촉성 피부염, 조직이식 거부반응 등이 포함될 수 있다. In addition, the pharmaceutical composition of the present invention contains an immune modulating function. The "immune modulating function" of the strain of the present invention can be interpreted as having an immune enhancing activity as well as reducing an immune hypersensitivity reaction. Immune hypersensitivity reaction refers to an immune response that causes inappropriate damage such as tissue damage due to excessive immune reaction against a specific antigen. Diseases related to immune hypersensitivity reaction include allergy, atopic dermatitis, asthma, hay fever, systemic anaphylactic shock, It may include erythroblastosis, rheumatoid arthritis, glomerulonephritis, lupus, Artus reaction, contact dermatitis, tissue transplant rejection, and the like.
본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주는 상기 열거된 질환과 관련한 면역 과민반응을 조절할 수 있도록, 전염증성 사이토카인의 분비 감소 및 항염증성 사이토카인의 분비를 증가시키는 기능을 가지는 것을 특징으로 한다. 즉, 본 발명의 일 실시예를 통하여, 염증이 유발된 세포에서 상기 BCBL-583 균주가 공지된 다른 균주에 비하여 항염증성 사이토카인인 IL-4 및 IL-10 의 분비를 증가시키고, 이에 비하여 전염증성 사이토카인인 TNF-α 및 IL-6의 분비를 크게 감소시킨 것을 바탕으로, 본 발명의 BCBL-583 균주는 면역 조절 기능을 가질 수 있음을 확인하였다. The Bifidobacterium longum subspecies Longum BCBL-583 strain of the present invention has the function of reducing the secretion of pro-inflammatory cytokines and increasing the secretion of anti-inflammatory cytokines so as to control immune hypersensitivity reactions associated with the diseases listed above. It is characterized. That is, through an embodiment of the present invention, the BCBL-583 strain increases the secretion of anti-inflammatory cytokines IL-4 and IL-10 compared to other known strains in cells in which inflammation is induced. Based on the significant reduction in the secretion of inflammatory cytokines TNF-α and IL-6, it was confirmed that the BCBL-583 strain of the present invention can have an immunomodulatory function.
본 발명의 상기 건강기능식품은 인체에 유용한 기능을 가진 원료의 복합물이나 특정한 기능을 가진 성분을 사용하여 제조한 식품을 말하는 것으로서, 본 발명에서는 상기 검토한 약학적 조성물과 같이, 혈중 콜레스테롤 감소 또는 면역기능 조절 효과를 가지는 것을 특징으로 한다. 상기 혈중 콜레스테롤 감소 효과를 통하여 본 발명의 건강기능식품은 대사성 질환에 대한 예방 또는 개선용도로서 활용될 수 있다. The health functional food of the present invention refers to a food manufactured using a complex of raw materials having a useful function or a component having a specific function in the human body.In the present invention, like the pharmaceutical composition examined above, blood cholesterol reduction or immunity It is characterized by having a function control effect. Through the effect of reducing cholesterol in the blood, the health functional food of the present invention can be used for prevention or improvement of metabolic diseases.
본 발명의 건강기능식품은 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주 또는 이의 배양액을 유효성분으로 하는 차, 젤리, 즙, 엑기스, 음료 등을 포함할 수 있으며, 상기 검토한 기능을 포함하고 있는 것이라면 가공 형태에 제한이 있는 것은 아니다.The health functional food of the present invention may include tea, jelly, juice, extract, beverage, etc. using the Bifidobacterium longum subspecies Longum BCBL-583 strain or a culture solution thereof as an active ingredient, and includes the functions examined above. If it is, there are no restrictions on the form of processing.
또한, 본 발명의 상기 식품은 건강기능식품과 구별되는 일반 식품으로서, 아이스크림류, 우유, 두유, 요구르트, 치즈를 포함하는 유제품, 및 두유제품, 음료, 육류, 소세지, 빵, 초콜릿 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 각종 수프, 음료수, 차, 드링크제, 알코올 음료, 캡슐 또는 스틱 포장의 유산균 제제로 구성된 군으로부터 선택할 수 있다.In addition, the food of the present invention is a general food that is distinguished from health functional foods, such as ice cream, milk, soy milk, yogurt, dairy products including cheese, and soy milk products, beverages, meat, sausages, bread, chocolate candies, snacks, It can be selected from the group consisting of confectionery, pizza, ramen, other noodles, chewing gum, various soups, beverages, tea, drinks, alcoholic beverages, capsules or lactic acid bacteria formulations in stick packaging.
본 발명의 신규한 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주, 또는 이의 배양액은 내산성 및 내담즙성이 우수하고, 유전체 수준에서의 안전성이 있으며, 장내 정착능, 콜레스테롤 감소능 및 면역조절 활성이 뛰어나므로 상기 균주 및 이의 배양액은 대사성 질환 또는 면역기능 조절을 위한 약학적 조성물, 건강기능 식품 조성물 또는 일반 식품 조성물 등 다양한 형태로 활용될 수 있다. 또한, 상기 기능을 활용할 수 있는 생균제, 유산균 제제, 발효유제품, 사료 첨가제 및 화장품 등의 첨가제로서 제한 없이 활용될 수 있다. The novel Bifidobacterium longum subspecies Longum BCBL-583 strain of the present invention, or its culture medium, has excellent acid resistance and bile resistance, safety at the genomic level, and has intestinal fixation ability, cholesterol reduction ability, and immunomodulatory activity. Since it is excellent, the strain and its culture medium can be used in various forms, such as a pharmaceutical composition for metabolic disease or immune function control, a health functional food composition, or a general food composition. In addition, it may be used without limitation as additives such as probiotics, lactic acid bacteria preparations, fermented milk products, feed additives and cosmetics that can utilize the above functions.
상기 식품에 대한 일 례로서, 본 발명의 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주, 이의 배양물 또는 추출물을 포함하는 음료 조성물의 경우, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토오스 슈크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다.As an example of the food, in the case of a beverage composition comprising the Bifidobacterium longum subspecies Longum BCBL-583 strain of the present invention, a culture or extract thereof, various flavoring agents or natural carbohydrates, etc. It may contain as an additional component. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used.
상기 외의 본 발명의 상기 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주, 이의 배양물 또는 추출물을 포함하는 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. The composition comprising the Bifidobacterium longum subspecies Longum BCBL-583 strain of the present invention other than the above, a culture or extract thereof, includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained. These components may be used independently or in combination.
아울러 상기 본 발명의 균주, 이의 배양물 또는 추출물을 포함하는 조성물은 성분의 함량에 있어 크게 제한되는 것은 아니며, 본 발명에서 목적하는 기능을 나타낼 수 있는 양으로 포함할 수 있다. 즉, 유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. In addition, the composition comprising the strain of the present invention, a culture product or extract thereof is not significantly limited in terms of the content of the component, and may be included in an amount capable of exhibiting the desired function in the present invention. That is, the mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).
이하 본 발명을, 실시예를 통하여 보다 구체적으로 설명한다. Hereinafter, the present invention will be described in more detail through examples.
다만, 하기 실시예는 본 발명의 기술적 특징을 명확하게 예시하기 위한 것 일뿐, 본 발명의 보호범위를 한정하는 것은 아니다.However, the following examples are only intended to clearly illustrate the technical features of the present invention, and do not limit the protection scope of the present invention.
<실시예 1> 비피도박테리엄 롱검 아종 롱검 BCBL-583 in vitro 기능성 평가<Example 1> Bifidobacterium longum subspecies Longum BCBL-583 in vitro functional evaluation
<1-1> 산소내성 평가<1-1> Oxygen resistance evaluation
상기 분리된 균주는 산소내성 평가를 위해, MRS 고체배지에 획선 도말하였고, 대조군으로 표준 균주 비피도박테리엄 롱검 ATCC15707 및 비피도박테리엄 롱검 ATCC15708을 산소가 존재하는 조건에서 24시간 배양 하였다. 그 결과를 도 1에 나타내었으며, 고체배지에서는 세 균주 모두 성장하지 못하였다. 상기 분리된 균주 및 표준 균주를 5 ml의 MRS액체배지에 접종하여 진탕 배양하여 성장에 따른 흡광도를 측정한 결과, 24시간 배양하였을 경우, 본 발명의 분리된 균주가 표준 균주보다 높은 성장률을 보였다. The isolated strain was streaked on MRS solid medium for oxygen tolerance evaluation, and standard strains Bifidobacterium longum ATCC15707 and Bifidobacterium longum ATCC15708 were cultured for 24 hours in the presence of oxygen as controls. The results are shown in Fig. 1, and all three strains could not be grown in the solid medium. The isolated strain and the standard strain were inoculated in 5 ml of MRS liquid medium and cultured with shaking to measure the absorbance according to growth. When cultured for 24 hours, the isolated strain of the present invention showed a higher growth rate than the standard strain.
<1-2> 내열성 평가<1-2> Heat resistance evaluation
본 발명의 분리균주의 상업적 활용에 있어 공정상 열처리가 수행될 경우 균주의 사멸 및 균수의 저하가 일어 날 수 있다. 따라서 본 발명의 선발된 균주에 대한 내열성을 평가하고자 37℃, 42℃, 45℃ 및 60℃에서 열 충격을 3시간 수행했으며, 그 결과를 도 2에 나타내었다. 열 충격 후 균수 회복을 위해 37℃에서 12시간 배양하였을 때, 본 발명의 분리균주는 45℃까지 99%의 균수 회복을 보여 주었으나 60℃에서의 균수 회복률은 10% 미만이었다.In the commercial use of the isolated strains of the present invention, when heat treatment is performed in the process, the killing of the strain and the decrease in the number of bacteria may occur. Therefore, in order to evaluate the heat resistance of the selected strains of the present invention, thermal shock was performed at 37°C, 42°C, 45°C and 60°C for 3 hours, and the results are shown in FIG. 2. When cultured at 37°C for 12 hours to recover the number of bacteria after heat shock, the isolates of the present invention showed 99% recovery of the number of bacteria up to 45°C, but the recovery rate of the number of bacteria at 60°C was less than 10%.
<1-3> 위산 및 내담즙성 평가<1-3> Evaluation of gastric acid and bile resistance
유산균은 섭취하는 도중 다양한 pH에 노출 되며, 특히 장까지 도달하기 위해서는 위산 및 담즙산에 대한 내성능이 가장 중요하다. 본 발명에서는 분리균주의 위산 및 담즙산에 대한 내성을 평가하였고 그 결과를 도 3에 나타내었다. 분리균주 및 표준균주를 각각 37℃에서 3시간동안 위산에서 처리한 후, 세척하여 담즙산염에서 동일 온도, 시간동안 처리하였다. 위산 및 담즙산염을 처리하지 않은 분리균주와 표준균주를 37℃에서 6시간 동안 배양하여 상기 내성평가의 대조군으로 사용하였다. 상기 분리 균주는 위산 및 담즙산에 대한 내성이 표준 균주와 비슷한 수준으로 나타났다. Lactobacillus is exposed to various pHs during ingestion, and in particular, in order to reach the intestine, resistance to stomach and bile acids is the most important. In the present invention, the isolated strains were evaluated for resistance to gastric acid and bile acids, and the results are shown in FIG. 3. The isolated and standard strains were each treated with gastric acid at 37° C. for 3 hours, washed, and treated with bile salt for the same temperature and time. The isolated strains and the standard strains not treated with gastric acid and bile salts were cultured at 37°C for 6 hours and used as a control group for the tolerance evaluation. The isolated strains showed similar levels of resistance to gastric acid and bile acids as in the standard strain.
<1-4> 장내 정착능 평가<1-4> Evaluation of intestinal settlement ability
장에 도달한 분리균주의 장내 정착능은, 장내 환경의 변화 및 프로바이오틱스로서의 기능에 중요하다. 장내 표피세포의 표면에 존재하는 뮤신(mucin)은 비피도박테리엄이 장내 정착을 할 때 결합하는 장소를 제공하므로, 뮤신 부착(mucin adhesion test)을 이용하여, 상기 분리 균주의 장내 정착능을 평가 하였다. 96-well plate에 뮤신을 주입하고 40℃에서 12시간 이상 배양한 후, 배양된 뮤신에 분리균주 및 표준균주를 각각 넣고, 37℃에서 3시간동안 배양하였다. 배양 후 부착되지 않은 여분의 균주를 세척하여 제거한 뒤 부착된 균주는 트리톤 X-100으로 회수하여 연속 희석 한 후 MRS고체 배지에 평판도말 하여 생균수를 측정하였다. 장내 정착능의 결과는 도 4에 나타내었다.The ability to settle in the intestine of the isolated strain that has reached the intestine is important for changes in the intestinal environment and for its function as a probiotic. Since mucin present on the surface of intestinal epidermal cells provides a place to bind when Bifidobacterium settles in the intestine, a mucin adhesion test is used to evaluate the intestinal fixation ability of the isolated strain. I did. Mucin was injected into a 96-well plate and cultured at 40° C. for 12 hours or more, and then the isolated and standard strains were respectively added to the cultured mucin, followed by incubation at 37° C. for 3 hours. After cultivation, the unattached extra strain was washed and removed, and the attached strain was recovered with Triton X-100, serially diluted, and plated on MRS solid medium to measure the number of viable cells. The results of the intestinal fixation ability are shown in FIG. 4.
<1-5> 콜레스테롤 감소능 평가<1-5> Cholesterol reduction ability evaluation
비피도박테리엄의 콜레스테롤 감소능을 평가하기 위해 콜레스테롤이 포함된 MRS배지에 분리균주 및 표준균주를 배양하여, 콜레스테롤 감소능을 평가하였다. 콜레스테롤이 포함된 MRS액체 배지에 분리균주 및 표준균주를 접종하고 37℃에서 24시간 배양하였다. 배양액은 균체를 원심 분리 및 여과지를 이용하여 제거하고, 배지 내의 잔존 콜레스테롤을 직접 측정하였다. 상기 본 발명의 분리균주의 콜레스테롤 감소능이 표준균주 보다 우수하다는 것을 확인하였다. 콜레스테롤 감소능 평가에 대한 결과는 도 5에 나타내었다. In order to evaluate the cholesterol-reducing ability of Bifidobacterium, the isolated and standard strains were cultured in MRS medium containing cholesterol, and the cholesterol-reducing ability was evaluated. The isolated and standard strains were inoculated in MRS liquid medium containing cholesterol, and cultured at 37°C for 24 hours. The culture medium was centrifuged and removed using a filter paper, and the remaining cholesterol in the medium was directly measured. It was confirmed that the cholesterol-reducing ability of the isolated strain of the present invention was superior to that of the standard strain. The results for the evaluation of cholesterol reduction ability are shown in FIG. 5.
<1-6> 면역조절능력 평가<1-6> Evaluation of immunomodulatory ability
대식세포주인 Raw 264.7세포는 비피도박테리엄 균주와 접촉 시 이를 항원으로 인식하여 그에 따른 사이토카인을 분비하게 되며, 면역 증진 또는 안정화와 관련된 사이토카인의 반응성을 확인할 수 있다. 따라서, Raw 264.7세포를 15번 계대배양하여 활성화를 시켜준 후, 108 CFU/ml의 분리균주 및 표준균주를 접종하고 5%의 이산화탄소를 공급하여 37℃에서 24시간 배양하였다. 면역조절능력을 평가하기 위해서 양성 대조군으로 대장균의 외부다당류(LPS)를 사용하였다. 배양 후 배양액의 상층액을 분리하여, 면역 물질인 3종의 사이토카인을 효소면역침강법 키트(ELISA kit)를 활용하여 측정 하였다. 그 결과는 도 6에 나타내었다. TNF-α는 수지상세포를 성숙시켜 T세포의 활성과 성장 등을 조절하는 염증성 사이토카인으로 양성 대조군인 대장균의 LPS와 유의한 수준으로 반응이 높게 나타나는 것을 확인 하였다. IL-6은 B세포의 분화 촉진, 항체 분비 자극, 면역글로블린g(Ig) 생성 유도 및 T세포의 분화 증식에 관여하는 사이토카인으로 분리균주에 대한 반응이 존재함을 확인 할 수 있었다. IL-10의 경우 전형적인 항 염증성 사이토카인으로 알려져 있으며, 항원 제시를 낮추고, 대식세포 활성화를 억제하여 친염증성 분자 및 케모카인 생성 억제를 통해 면역 활동을 조절하는 것으로 알려져 있으며, 분리균주에 대한 반응이 존재하는 것으로 나타났다.When raw 264.7 cells, a macrophage cell line, are in contact with the Bifidobacterium strain, they recognize it as an antigen and secrete cytokines accordingly, and the reactivity of cytokines related to immunity enhancement or stabilization can be confirmed. Therefore, after subculturing Raw 264.7 cells 15 times for activation, 10 8 CFU/ml of isolated and standard strains were inoculated, and 5% of carbon dioxide was supplied and cultured at 37°C for 24 hours. To evaluate the immunomodulatory ability, an external polysaccharide (LPS) of E. coli was used as a positive control. After cultivation, the supernatant of the culture medium was separated, and three kinds of cytokines, which are immune substances, were measured using an enzyme immunoprecipitation kit (ELISA kit). The results are shown in FIG. 6. TNF-α is an inflammatory cytokine that regulates the activity and growth of T cells by maturing dendritic cells, and it was confirmed that the reaction was high with a significant level with LPS of E. coli, a positive control. IL-6 is a cytokine involved in promoting differentiation of B cells, stimulating antibody secretion, inducing immunoglobulin g (Ig) production, and proliferating differentiation of T cells, and it was confirmed that there is a response to the isolated strain. In the case of IL-10, it is known as a typical anti-inflammatory cytokine, and it is known to regulate immune activity by lowering antigen presentation and inhibiting macrophage activation, thereby suppressing pro-inflammatory molecule and chemokine production. Appeared to be.
<실시예 2> 비피도박테리엄 롱검 아종 롱검 BCBL-583 in vivo 기능성 평가<Example 2> Bifidobacterium longum subspecies Longum BCBL-583 in vivo functional evaluation
<2-1> 동물실험 계획<2-1> Animal experiment plan
생후 5주된 마우스(C57BL/6, male) 25마리를 5개의 군으로 나누어 실험을 진행하였다. 5주된 마우스를 1주일간 지방함량 10%인 일반사료를 투여하여 적응기간을 가진 후, 2주차부터 1번 그룹은 일반사료를 급여하고 나머지 그룹은 지방함량 60%의 고지방 사료를 8주간 급여하며, 모든 케이지 별로 매일 MRS배지 및 균주를 투여하였다. 상기 실험에 사용한 균주는 본 발명의 분리균주 및 표준균주를 투여 하였다. 매일 마우스 체중 1Kg당 108CFU/ml씩 균주를 투여하였다. 각 개체별 몸무게를 매주 2번씩 측정하여 몸무게 대비 균주투여량을 계산하였고, 분변은 1주차 및 5주차에 수집하여 -80℃에서 보관하였다. 18시간 공복을 유지한 후에 혈액을 채취하였다. 9주차에 모든 개체를 희생하여 비장을 적출하고 대장으로부터 분변을 수집하였다. 또한, 희생 후 심장으로부터 전혈을 확보하고, 수집된 분변으로부터 총 DNA를 분리하여 메타지놈(metagenome)분석을 수행하였다. 적출된 비장으로부터 세포를 분화시켜 이로부터 분비된 사이토카인을 측정하였다.After birth The experiment was conducted by dividing 25 mice (C57BL/6, male) of 5 weeks old into 5 groups. Five-week-old mice were given a period of adaptation by administering a normal diet with a fat content of 10% for 1 week, and then the first group from the 2nd week was fed regular feed, and the rest of the group fed a high-fat diet with a fat content of 60% for 8 weeks. MRS medium and strain were administered daily for each cage. The strain used in the experiment was administered with the isolated strain and the standard strain of the present invention. The strain was administered at 10 8 CFU/ml per 1Kg of mouse body weight every day. The body weight of each individual was measured twice a week to calculate the strain dose relative to the body weight, and feces were collected at
<2-2> 9주차 희생 후 실험<2-2> Experiment after sacrifice in the 9th week
적출된 마우스의 비장을 3 ml의 HBSS buffer에 침지시킨 후 조직을 메스를 이용하여 파쇄하였다. 파쇄된 비장 조직은 70 mash cell strainer에 통과시켜 세포들을 분리 하였고, 분리된 세포들은 10 ml의 염화암모늄용액과 석어 분쇄된 얼음에 10분간 반응시켜 혈액을 제거하였다. 300rpm, 4℃에서 5분간 원심분리하여 침전된 펠렛을 1 ml의 글루타민이 첨가된 10% FBS배지에 재부유 시켰다. 분리된 비장세포는 토마 혈구계를 활용하여 계수한 후 배수에 맞춰 배지로 희석한 후 각각 well에 분주하여, 37℃에서 5%의 이산화탄소를 유지한 채 72시간 배양하였다. 배양된 세포가 생산하는 사이토카인을 측정하기 위하여 상층액을 모두 회수하였고, 상층액을 마우스 ELISA kit을 활용하여 TNF-α, IL-6, IL-4 및 IL-10을 측정하였다. 사이토카인을 측정한 결과 도 8에서 나타낸 바와 같이, TNF-α 및 IL-6는 음성 대조군인 일반사료를 섭취한 그룹과 유사한 수준으로 수치가 떨어졌으며, IL-4의 경우 음성 대조군에 비하여 높은 양이 검출되었다. IL-10의 경우 음성 대조군에 비하여 높은 양이 검출 되었으나 통계학적으로 유의적인 차이를 보이지 않았다. 따라서, 고지방 식이로 인해 증가된 염증성 사이토카인인 TNF-α와 IL-6에 대하여 본 발명의 분리균주는 그 수치를 낮춰주는 역할을 하며, 이에 따라 항염증성 사이토카인인 IL-4와 IL-10을 증가시키는 역할을 하는 것을 확인 하였다.The spleen of the extracted mouse was immersed in 3 ml of HBSS buffer, and the tissue was crushed using a scalpel. The crushed spleen tissue was passed through a 70 mash cell strainer to separate the cells, and the separated cells were reacted with 10 ml of ammonium chloride solution and crushed ice for 10 minutes to remove blood. The pellet precipitated by centrifugation at 300 rpm for 5 minutes at 4° C. was resuspended in 10% FBS medium to which 1 ml of glutamine was added. The separated splenocytes were counted using a Toma hemocytometer, diluted with a medium according to the number of folds, and dispensed into each well, and cultured for 72 hours while maintaining 5% carbon dioxide at 37°C. In order to measure the cytokines produced by the cultured cells, all the supernatant was recovered, and the supernatant was measured for TNF-α, IL-6, IL-4, and IL-10 using a mouse ELISA kit. As a result of measuring cytokines, as shown in FIG. 8, the levels of TNF-α and IL-6 fell to a level similar to that of the group in which the general diet, which was the negative control, was consumed, and in the case of IL-4, higher amounts compared to the negative control. Was detected. In the case of IL-10, a higher amount was detected than that of the negative control group, but there was no statistically significant difference. Therefore, with respect to the inflammatory cytokines TNF-α and IL-6, which are increased due to a high fat diet, the isolates of the present invention serve to lower the levels, and accordingly, the anti-inflammatory cytokines IL-4 and IL-10 It was confirmed that it plays a role in increasing the value.
또한, 희생 후 확보된 혈액으로부터 혈중 콜레스테롤 농도를 키트를 사용하여 측정하였다. In vivo실험에서의 콜레스테롤 감소의 결과는 도 9에 나타내었다. 일반사료를 먹인 군은 고지방사료를 먹인 군에 비하여 콜레스테롤 수치가 낮은 것을 확인하였다. 특히 본 발명의 분리균주를 투여한 군은 다른 균주를 투여한 그룹보다 유의적으로 낮은 콜레스테롤 수치가 검출되었다. 고밀도(HDL) 콜레스테롤을 측정한 결과 고지방 사료만 투여한 군보다, 균주와 같이 투여한 군에서 높은 HDL 콜레스테롤이 확인되었다. HDL 콜레스테롤 측정에 따른 저밀도(LDL)콜레스테롤 계산값은 본 발명의 분리균주를 투여한 군에서 유의적으로 낮은 것을 확인하였다.In addition, blood cholesterol concentration from the blood obtained after sacrifice was measured using a kit. The results of cholesterol reduction in the in vivo experiment are shown in FIG. 9. It was confirmed that the group fed the general diet had lower cholesterol levels than the group fed the high fat diet. In particular, significantly lower cholesterol levels were detected in the group administered with the isolates of the present invention than in the group administered with other strains. As a result of measuring high-density (HDL) cholesterol, higher HDL cholesterol was found in the group administered with the strain than in the group administered with only the high-fat diet. It was confirmed that the calculated value of low-density (LDL) cholesterol according to HDL cholesterol measurement was significantly lower in the group to which the isolate of the present invention was administered.
<실시예 3> 안전성 평가<Example 3> Safety evaluation
<3-1> 유전체 수준의 안전성 검증<3-1> Genome-level safety verification
본 발명의 분리균주의 전장유전체 분석 결과 2,393,285 bp의 크로모좀 DNA(chromosomal DNA)를 가지며 플라스미드는 갖지 않는 것으로 확인되었다. 1,999개의 단백질 암호와 유전자를 발견하였으며 68개의 RNA가 확인되었다. 분리균주의 유전체 수준에서 유해인자를 확인한 결과, 인체에 유해한 유전자를 보유하고 있지 않았다. 상기 분리균주의 유전자 지도를 도 10에 나타내었다.As a result of full-length genome analysis of the isolate of the present invention, it was confirmed that it had 2,393,285 bp of chromosomal DNA and no plasmid. 1,999 protein codes and genes were discovered, and 68 RNAs were identified. As a result of confirming the harmful factor at the genome level of the isolated strain, it did not contain a gene that is harmful to the human body. The genetic map of the isolated strain is shown in FIG. 10.
전장유전체 분석이 완료된 비피도박테리엄 롱검의 유전체를 확보하여 이들의 전장유전체상의 상동성을 확인하였다. 19종의 비피도박테리엄의 전장 유전체와 분리 균주의 전장유전체의 상동성의 비교한 결과 다른 균주들과 1% (ANI value) 차이가 나는 것으로 확인 되었다. 19종의 비피도박테리엄과 분리균주의 상동성 비교는 도 11에 나타내었다.The genomes of Bifidobacterium longum, which had been analyzed for full-length genomes, were secured, and homology of their full-length genomes was confirmed. As a result of comparing the homology of the full-length genome of 19 species of Bifidobacterium and the full-length genome of the isolated strains, it was confirmed that there was a difference of 1% (ANI value) from other strains. A comparison of homology of 19 types of Bifidobacterium and isolates is shown in FIG. 11.
<3-2> 장내 환경 변화 효과 검증<3-2> Verification of the effect of changes in the environment in the venue
마우스의 주차에 따른 분변 샘플의 균총 조성을 확인하여 그 결과를 도 12 및 도 13에 나타내었다. 0주차의 샘플들에서는 50% 내외가 아커만시아(Akkermansia) 균주로 확인 되었으며, 그 비율은 5주와 9주로 넘어 갈수록 점차 줄어드는 것으로 확인되었다. 블라우티아(Blautia)의 경우 9주차가 될수록 증가하는 것을 볼 수 있었으나, 이 결과가 균주에 의한 영향인지 확인 할 수 없었다. 식이섬유 분해에 도움을 주는 균주들로 알려진 락초노스피라세아(Lachnoospiraceae)는 9주차에서 현저히 줄어드는 것을 확인 할 수 있었다. 루미노클로스트리디움(Ruminoclostridium)의 경우 마우스 장내에서 많이 발견되는 박테리아로써 본 발명에서의 경우 주차가 지날수록 발견되는 양이 증가하였다. 실제 본 발명에서 투여하였던 비피도박테리엄의 경우 5주차에서 상대적으로 많은 양이 발견되었다. 9주차에서는 본 발명의 분리 균주의 비율이 낮아지고, 표준균주인 ATCC15708의 비율이 높게 나타났다. 헬리코박터(Helicobacter) 비율은 MRS만을 투여한 군에서 가장 높게 나타났으며 비피도박테리엄을 섭취한 군에서는 그 비율이 낮아진 것을 확인할 수 있었다.The colony composition of the fecal sample according to the parking of the mouse was confirmed, and the results are shown in FIGS. 12 and 13. Around 50% of the samples at
<110> Binggrae Co., Ltd. University-Industry Cooperation Group of Kyung Hee University <120> Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application <130> PN1905-279 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1515 <212> DNA <213> Bifidobacterium longum <400> 1 ttcgattctg gctcaggatg aacgctggcg gcgtgcttaa cacatgcaag tcgaacggga 60 tccatcaggc tttgcttggt ggtgagagtg gcgaacgggt gagtaatgcg tgaccgacct 120 gccccataca ccggaatagc tcctggaaac gggtggtaat gccggatgct ccagttgatc 180 gcatggtctt ctgggaaagc tttcgcggta tgggatgggg tcgcgtccta tcagcttgac 240 ggcggggtaa cggcccaccg tggcttcgac gggtagccgg cctgagaggg cgaccggcca 300 cattgggact gagatacggc ccagactcct acgggaggca gcagtgggga atattgcaca 360 atgggcgcaa gcctgatgca gcgacgccgc gtgagggatg gaggccttcg ggttgtaaac 420 ctcttttatc ggggagcaag cgtgagtgag tttacccgtt gaataagcac cggctaacta 480 cgtgccagca gccgcggtaa tacgtagggt gcaagcgtta tccggaatta ttgggcgtaa 540 agggctcgta ggcggttcgt cgcgtccggt gtgaaagtcc atcgcttaac ggtggatccg 600 cgccgggtac gggcgggctt gagtgcggta ggggagactg gaattcccgg tgtaacggtg 660 gaatgtgtag atatcgggaa gaacaccaat ggcgaaggca ggtctctggg ccgttactga 720 cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780 aaacggtgga tgctggatgt ggggcccgtt ccacgggttc cgtgtcggag ctaacgcgtt 840 aagcatcccg cctggggagt acggccgcaa ggctaaaact caaagaaatt gacgggggcc 900 cgcacaagcg gcggagcatg cggattaatt cgatgcaacg cgaagaacct tacctgggct 960 tgacatgttc ccgacggtcg tagagatacg gcttcccttc ggggcgggtt cacaggtggt 1020 gcatggtcgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 cctcgccccg tgttgccagc ggattatgcc gggaactcac gggggaccgc cggggttaac 1140 tcggaggaag gtggggatga cgtcagatca tcatgcccct tacgtccagg gcttcacgca 1200 tgctacaatg gccggtacaa cgggatgcga cgcggcgacg cggagcggat ccctgaaaac 1260 cggtctcagt tcggatcgca gtctgcaact cgactgcgtg aaggcggagt cgctagtaat 1320 cgcgaatcag caacgtcgcg gtgaatgcgt tcccgggcct tgtacacacc gcccgtcaag 1380 tcatgaaagt gggcagcacc cgaagccggt ggcctaaccc cttgtgggat ggagccgtct 1440 aaggtgaggc tcgtgattgg gactaagtcg taacaaggta gccgtaccgg aaggtgcggc 1500 tggatcacct ccttt 1515 <110> Binggrae Co., Ltd. University-Industry Cooperation Group of Kyung Hee University <120> Bifidobacterium longum subsp. longum having both abilities of reducing total cholesterol in serum and immune regulation and its application <130> PN1905-279 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1515 <212> DNA <213> Bifidobacterium longum <400> 1 ttcgattctg gctcaggatg aacgctggcg gcgtgcttaa cacatgcaag tcgaacggga 60 tccatcaggc tttgcttggt ggtgagagtg gcgaacgggt gagtaatgcg tgaccgacct 120 gccccataca ccggaatagc tcctggaaac gggtggtaat gccggatgct ccagttgatc 180 gcatggtctt ctgggaaagc tttcgcggta tgggatgggg tcgcgtccta tcagcttgac 240 ggcggggtaa cggcccaccg tggcttcgac gggtagccgg cctgagaggg cgaccggcca 300 cattgggact gagatacggc ccagactcct acgggaggca gcagtgggga atattgcaca 360 atgggcgcaa gcctgatgca gcgacgccgc gtgagggatg gaggccttcg ggttgtaaac 420 ctcttttatc ggggagcaag cgtgagtgag tttacccgtt gaataagcac cggctaacta 480 cgtgccagca gccgcggtaa tacgtagggt gcaagcgtta tccggaatta ttgggcgtaa 540 agggctcgta ggcggttcgt cgcgtccggt gtgaaagtcc atcgcttaac ggtggatccg 600 cgccgggtac gggcgggctt gagtgcggta ggggagactg gaattcccgg tgtaacggtg 660 gaatgtgtag atatcgggaa gaacaccaat ggcgaaggca ggtctctggg ccgttactga 720 cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag tccacgccgt 780 aaacggtgga tgctggatgt ggggcccgtt ccacgggttc cgtgtcggag ctaacgcgtt 840 aagcatcccg cctggggagt acggccgcaa ggctaaaact caaagaaatt gacgggggcc 900 cgcacaagcg gcggagcatg cggattaatt cgatgcaacg cgaagaacct tacctgggct 960 tgacatgttc ccgacggtcg tagagatacg gcttcccttc ggggcgggtt cacaggtggt 1020 gcatggtcgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac 1080 cctcgccccg tgttgccagc ggattatgcc gggaactcac gggggaccgc cggggttaac 1140 tcggaggaag gtggggatga cgtcagatca tcatgcccct tacgtccagg gcttcacgca 1200 tgctacaatg gccggtacaa cgggatgcga cgcggcgacg cggagcggat ccctgaaaac 1260 cggtctcagt tcggatcgca gtctgcaact cgactgcgtg aaggcggagt cgctagtaat 1320 cgcgaatcag caacgtcgcg gtgaatgcgt tcccgggcct tgtacacacc gcccgtcaag 1380 tcatgaaagt gggcagcacc cgaagccggt ggcctaaccc cttgtgggat ggagccgtct 1440 aaggtgaggc tcgtgattgg gactaagtcg taacaaggta gccgtaccgg aaggtgcggc 1500 tggatcacct ccttt 1515
Claims (11)
상기 비피도박테리엄 롱검 아종 롱검 BCBL-583은 총 콜레스테롤 감소, LDL-콜레스테롤 감소 및 HDL-콜레스테롤을 증가시키며,
항염증성 사이토카인인 IL-4 및 IL-10의 분비를 증가시키며, 전염증성 사이토카인인 TNF-α 및 IL-6의 분비의 감소로 면역기능을 조절하는 것을 특징으로 하는 균주.As a novel Bifidobacterium longum subsp. longum BCBL-583 strain containing the nucleotide sequence of the 16S rRNA of SEQ ID NO: 1 deposited as accession number KCTC13859BP,
The Bifidobacterium longum subspecies Longum BCBL-583 decreases total cholesterol, decreases LDL-cholesterol, and increases HDL-cholesterol,
A strain characterized in that it increases the secretion of anti-inflammatory cytokines IL-4 and IL-10, and regulates immune function by decreasing the secretion of pro-inflammatory cytokines TNF-α and IL-6.
상기 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주는, 항염증성 사이토카인인 IL-4 및 IL-10의 분비를 증가시키며, 전염증성 사이토카인인 TNF-α 및 IL-6의 분비의 감소로 면역기능을 조절하는 것을 특징으로 하는 면역기능 개선용 건강기능식품 조성물.A health functional food for improving immune function comprising at least one of the novel Bifidobacterium longum subspecies Longum BCBL-583 strain, its culture, or extract containing the nucleotide sequence of 16S rRNA of SEQ ID NO: 1 with accession number KCTC13859BP As a composition,
The Bifidobacterium longum subspecies Longum BCBL-583 strain increases the secretion of anti-inflammatory cytokines IL-4 and IL-10, and is immune by a decrease in the secretion of pro-inflammatory cytokines TNF-α and IL-6. Health functional food composition for improving immune function, characterized in that to control the function.
상기 비피도박테리엄 롱검 아종 롱검 BCBL-583 균주는, 항염증성 사이토카인인 IL-4 및 IL-10의 분비를 증가시키며, 전염증성 사이토카인인 TNF-α 및 IL-6의 분비의 감소로 면역기능을 조절하는 것을 특징으로 하는 면역기능 개선용 일반 식품 조성물.A general food composition for improving immune function comprising at least one of the novel Bifidobacterium longum subspecies Longum BCBL-583 strain, its culture, or extract containing the nucleotide sequence of 16S rRNA of SEQ ID NO: 1 with accession number KCTC13859BP as,
The Bifidobacterium longum subspecies Longum BCBL-583 strain increases the secretion of anti-inflammatory cytokines IL-4 and IL-10, and is immune by a decrease in the secretion of pro-inflammatory cytokines TNF-α and IL-6. General food composition for improving immune function, characterized in that to regulate the function.
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| WO2023173181A1 (en) | 2022-03-18 | 2023-09-21 | Lb Bulgaricum Ead | Bifidobacterium longum ssp. longum strain and a bifidobacterial preparation having hypolipidemic, anti-inflammatory immunomodulating and insulin resistance decreasing effects |
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| KR102524102B1 (en) * | 2021-06-17 | 2023-04-19 | 경희대학교 산학협력단 | Composition for enhancing immunity comprising Curtobacterium proimmune K3 |
| CN116814501B (en) * | 2023-07-17 | 2024-02-09 | 微康益生菌(苏州)股份有限公司 | Bifidobacterium longum subspecies capable of relieving obesity and application thereof |
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| US7195906B2 (en) | 1999-01-15 | 2007-03-27 | Enterprise Ireland (Trading As Bioresearch Ireland) | Bifidobacterium in the treatment of inflammatory disease |
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| KR100472865B1 (en) | 2002-08-28 | 2005-03-10 | (주) 피엘바이오 | Korean Isolate Bifidobacterium infantis PL9506 with High Nutraceutical Activity |
| KR100930251B1 (en) | 2009-05-15 | 2009-12-09 | 삼육대학교산학협력단 | Bifidobacterium longum having the ability of reducing cholesterol in blood |
| KR101239806B1 (en) | 2011-04-01 | 2013-03-06 | 주식회사한국야쿠르트 | The new Lactobacillus plantarum HY7712 stimulate immunity |
| KR20170032815A (en) * | 2015-09-15 | 2017-03-23 | 경희대학교 산학협력단 | Novel lactic acid bacteria and composition for preventing, improving or treating neurodegenerative diseases or cognitive dysfunction |
| CA3006176C (en) * | 2015-12-11 | 2023-10-31 | Alimentary Health Limited | Bifidobacterium longum for treating obesity and associated metabolic disorders |
| KR101868517B1 (en) | 2016-07-29 | 2018-06-20 | (주) 피엘바이오 | Lactobacillus fermentum PL9119 with biofunctional activities and high heat stability as a probiotic without antibiotic resistance |
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| WO2023173181A1 (en) | 2022-03-18 | 2023-09-21 | Lb Bulgaricum Ead | Bifidobacterium longum ssp. longum strain and a bifidobacterial preparation having hypolipidemic, anti-inflammatory immunomodulating and insulin resistance decreasing effects |
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