KR101426275B1 - The composition containing combination of 5 probiotics which have efficacy preventing fatty acid synthesis and promoting fatty acid oxidation as an effective factor - Google Patents
The composition containing combination of 5 probiotics which have efficacy preventing fatty acid synthesis and promoting fatty acid oxidation as an effective factor Download PDFInfo
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- KR101426275B1 KR101426275B1 KR1020140040710A KR20140040710A KR101426275B1 KR 101426275 B1 KR101426275 B1 KR 101426275B1 KR 1020140040710 A KR1020140040710 A KR 1020140040710A KR 20140040710 A KR20140040710 A KR 20140040710A KR 101426275 B1 KR101426275 B1 KR 101426275B1
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Abstract
본 발명은 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 지방합성 억제 및 지방산화 촉진에 의한 체지방 감소 효능을 갖는 5종의 유산균 복합균주를 유효성분으로 함유하는 조성물에 관한 것으로서, 본 발명에 따른 5종의 유산균 복합균주는 3T3-L1 지방세포 분화 억제에 의해 지방세포내 지방축적을 억제하고, 지방합성에 관련된 유전자 발현 억제 및 지방산화에 관련된 유전자 발현 촉진에 의해 체내 지방축적을 억제함으로써 체지방 감소 효과가 매우 뛰어나므로 5종의 유산균 복합균주를 이용한 지방 대사에 영향을 미치는 기능성 식품, 약학적 조성물 등으로 이용될 수 있다.The present invention relates to a pharmaceutical composition comprising Bifidobacterium bifidum HY8308, Lactobacillus acidophilus HY7038, Streptococcus thermophilus HY9008, Lactobacillus reuteri HY7508 and Lactobacillus carotovora The present invention relates to a composition comprising 5 kinds of lactic acid bacterial strain having Lactobacillus casei HY7208 inhibiting lipid synthesis and fat reduction effect promoting fat oxidation as effective ingredients, Since 3T3-L1 inhibits adipocyte differentiation and inhibits fat accumulation in adipocytes, suppresses gene expression related to lipogenesis and promotes gene expression related to lipid oxidation, Effects of Lactic Acid Bacteria on Lipid Metabolism It can be used in foods, pharmaceutical compositions and the like.
Description
본 발명은 체지방 감소 효능을 갖는 5종의 유산균 복합균주를 유효성분으로 함유하는 조성물에 관한 것으로서, 보다 상세하게는 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 지방합성 억제 및 지방산화 촉진에 의한 체지방 감소 효능을 갖는 5종의 유산균 복합균주를 유효성분으로 함유하는 조성물에 관한 것이다.
The present invention relates to a composition containing 5 kinds of lactic acid bacterial strains having an effect of reducing body fat as an active ingredient, and more particularly to a composition containing Bifidobacterium bifidum HY8308, Lactobacillus acidophilus ( Lactobacillus acidophilus) ) HY7038, Streptococcus thermophilus HY9008, Lactobacillus reuteri HY7508, and Lactobacillus casei HY7208, which inhibit lipid synthesis and promote fat oxidation, Of a lactic acid bacterium complex strain as an effective ingredient.
비만의 치료방법은 크게 식이-운동요법, 수술 요법 및 약물 요법이 있다. 식이-운동 요법은 저칼로리-저지방 섭취와 산소를 소비하는 육체의 활동을 통한 치료 방법인데, 이는 인내심을 가지고 반복적· 지속적으로 수행되어야 하기 때문에 대중적인 효과를 보기는 어려운 것으로 인식되고 있다. 수술요법은 외과적 수술을 통해 체지방을 물리적으로 제거하는 방법으로서 단기간에 효과를 볼 수 있는 장점이 있지만, 수술을 해야 하는 점, 효과의 지속성이 없다는 점, 비용이 많이 든다는 점 등 때문에 제한적으로 활용되고 있다. 약물 요법은 식욕 감소를 유발하거나 지방 흡수를 억제하는 약제들을 이용해서 비만을 치료 또는 예방하고자 하는 방법이다. 현재까지 개발된 비만 예방 및 치료를 위한 약제는 다양한 생리학적 메카니즘을 가지고 있다.Treatment methods for obesity are diet - exercise, surgery and drug therapy. Dietary-exercise therapy is a method of treatment through low-calorie-low-fat intake and physical activity that consumes oxygen, which is perceived to be difficult to see because of the need to be patient, repetitive, and continuous. Surgery is a method of physically removing body fat through surgical operations, which is advantageous in that it can be effective in a short period of time. However, it is limited due to the necessity of surgery, lack of persistence of effect, and cost . Pharmacotherapy is a method to treat or prevent obesity by using drugs that induce appetite reduction or inhibit fat absorption. So far, the drugs for prevention and treatment of obesity have various physiological mechanisms.
지방의 흡수 억제 약물로서는 췌장 지방 분해 효소 방해제(Orlistat)가 알려져 있는데, 췌장 지방분해 효소의 작용을 억제함으로 지방의 흡수를 감소시키는 약제이나 지용성 비타민의 흡수를 방해하며, 유방암 유발의 가능성이 있다.Pancreatic lipolytic enzyme inhibitor (Orlistat) is known as a fat absorption inhibitory drug. It inhibits the action of pancreatic lipase and inhibits the absorption of fat-soluble vitamin and absorption of fat-soluble vitamin. .
식욕을 저해하는 약제는 주로 뇌에 존재하는 신경전달물질(catecholamines)에 작용하여 식욕을 저하시킨다. 그러나 덱스펜플루르아민, 펜플루르아민은 신경독성, 판막성 심장질환의 부작용이 있고, 시부트라민은 심박수와 혈압을 상승시키는 부작용이 있다.Drugs that inhibit appetite usually act on neurotransmitters (catecholamines) that are present in the brain, reducing appetite. However, dexpenfluramine and phenfluramine have side effects of neurotoxicity, valvular heart disease, and sibutramine has side effects that increase heart rate and blood pressure.
체내 섭취열량의 증가와 소모열량 감소에 의해 생기는 대사의 불균형으로 과도한 지방 축적이 유발되는데, 이는 지방세포의 크기 및 수의 증가에 의해 나타난다. 3T3-L1 지방세포는 마우스 배아 섬유아세포(mouse embryonic fibroblast)로서 지방 조직의 생물학적 연구에 많이 사용되는 세포 주(cell line)이다. 3T3-L1 마우스 배아 섬유아세포내 지방합성 신호 경로가 활성화되면서 세포 내 지방량이 증가하고 세포의 모양이 원형으로 변한다. 이처럼 세포 내 신호 경로와 세포의 모양이 변하는 과정을 분화(differentiation)라고 하고 분화가 진행 중인 3T3-L1 세포를 '성숙 중인 3T3-L1 지방전구세포(maturing 3T3-L1 pre-adipocyte)', 분화가 완료된 3T3-L1 세포를 '성숙된 3T3-L1 지방세포(mature 3T3-L1 adipocyte)'라고 한다. 이러한 3T3-L1 세포의 지방분화를 억제하는 물질을 탐색하는 연구가 각국에서 진행되고 있다. 예를 들어, 녹차 폴리페놀 성분인 epigallocatechin gallate(Obesity res. 2005;13:982-90), 적포도주 성분인 resveratrol(Phytother Res. 2008;22:1367-71), 마늘 성분인 ajoene(Phytother Res. 2009;23:513-8) 등이 3T3-L1 세포의 지방분화 억제 효능을 가지고 있다는 연구결과가 보고된 바 있다. 3T3-L1 세포의 지방분화를 억제 즉, 지방세포내 지방합성을 억제하는 물질은 항비만 효능을 가진다고 볼 수 있기 때문에 식품, 의약 등의 분야에서 활용도가 매우 크다.The metabolic imbalance caused by the increase in body calorie intake and the decrease in caloric expenditure leads to excessive fat accumulation, which is caused by an increase in the size and number of fat cells. 3T3-L1 adipocytes are mouse embryonic fibroblasts, a cell line commonly used in biological studies of adipose tissue. In the 3T3-L1 mouse embryonic fibroblast, activation of the fat synthesis signal pathway increases intracellular fat mass and changes the shape of the cells to a circular shape. The differentiation process of 3T3-L1 cells, called 3T3-L1 pre-adipocyte, is known as "3T3-L1 pre-adipocyte" The completed 3T3-L1 cells are called " mature 3T3-L1 adipocytes ". Researches are underway in various countries to search for substances that inhibit lipid differentiation of 3T3-L1 cells. For example, green tea polyphenol component epigallocatechin gallate (Obesity res. 2005; 13: 982-90), red wine ingredient resveratrol (Phytother Res. 2008; 22: 1367-71), garlic ingredient ajoene (Phytother Res. ; 23: 513-8) have been shown to inhibit the lipid differentiation of 3T3-L1 cells. Inhibition of lipid differentiation of 3T3-L1 cells, that is, a substance inhibiting lipid synthesis in adipocytes, is considered to have an anti-obesity effect, and thus it is very useful in fields such as foods and medicines.
체내 지방축적을 감소시키는 기전으로 크게 3가지가 있다. 첫 번째는 식이로부터 섭취되는 지방의 흡수를 억제함으로써 체지방의 축적을 억제하는 기전이다. 이러한 지방 흡수 억제는 소장에서 중성지방을 분해하는 지방분해효소의 작용을 저해하든지, 섭취한 지방과 흡착하여 변으로의 지방 배설을 증진시키는 작용을 통해 가능하다. 둘째는 식이로부터 섭취된 지방이나 체내 축적된 체지방의 산화를 촉진시킴으로서 체지방을 조절하는 기전이다. 지방산 산화에 관여하는 효소의 활성을 증가시켜 지방산이 미토콘드리아로 이동하는 것을 증진시키고, 따라서 미토콘드리아 내에서의 β-산화를 촉진시킴으로써 궁극적으로는 지방으로부터의 에너지 생산을 증가시키는 것이다. 세 번째는 지방합성 관련 효소인 Fatty acid synthase 및 Citrate lyase 등의 활성을 저하시킴으로써 acetyl CoA로부터의 지방산 합성을 저해시키는 것이다. There are three major mechanisms to reduce the accumulation of fat in the body. The first is to inhibit the accumulation of body fat by inhibiting the absorption of fat from the diet. Such inhibition of lipid absorption is possible by either inhibiting the action of lipolytic enzymes that break down triglycerides in the small intestine, or by adsorbing the fat that is ingested to promote fat excretion into the stool. The second is the mechanism of controlling body fat by promoting oxidation of fat from the diet or body fat accumulated in the body. Increases the activity of enzymes involved in fatty acid oxidation and thereby promotes the transfer of fatty acids to mitochondria and thus promotes [beta] -oxidation in mitochondria, ultimately increasing energy production from fat. The third is to inhibit fatty acid synthesis from acetyl CoA by lowering the activities of fatty acid synthase and citrate lyase.
특히, 체내 에너지 소비에 있어서 축적된 지방을 직접 분해하거나 지방을 산화해서 에너지와 열을 생성하는 연소과정이 중요한데, 여기에는 많은 효소 및 유전자들이 관여하고 있다. 호르몬 민감성 지방분해효소(hormone sensitive lipase)는 지방조직에서 중성지방을 분해하는 단계에서 가장 중요한 효소이며, 지방산이 분해되어 에너지로 쓰이기 위해서는 에너지 생성 장소인 미토콘드리아로 이동해야 한다. 세포질에서 활성화된 지방산(acyl CoA)이 미토콘드리아 내부로 이동하기 위해서는 미토콘드리아의 막(외막과 내막)을 통과해야 하는데, 이 과정을 촉매하는 효소가 Carnitine palmitoyltransferase(CPT)로 CPTⅠ와 CPTⅡ가 있다. 그리고 Acyl-CoA oxidase는 퍼옥시좀(peroxisome)에 위치한 효소로서, acyl CoA와 O2로부터 trans-2,3-dehydroacyl-CoA와 H2O2를 생산하는데, 지방산 β-산화의 가장 첫 단계에서 작용하는 속도제한효소(rate-limiting enzyme)이다. 미토콘드리아의 탈공역단백질(uncoupling protein, UCP)은 갈색지방, 백색지방, 그리고 근육세포에서 발견된다. UCP들은 해당세포 내 미토콘드리아에서 산화적 인산화 과정을 탈공역(uncoupling)함으로서 ATP 생성을 감소시키고 열을 발생시킨다. 따라서 UCP 발현 정도가 에너지 소비량에 영향을 미쳐 비만 발생과 연관이 있는 것으로 보고되고 있으며, 실제로 비만한 사람의 경우 UCP 발현이 적은 경향을 보인다는 연구 결과들이 있다.
In particular, in the energy consumption of the body, it is important to directly burn fat accumulated or to oxidize fat to generate energy and heat, and many enzymes and genes are involved. Hormone sensitive lipase is the most important enzyme in decomposing triglycerides in adipose tissue. In order to decompose fatty acids and use them as energy, they must move to mitochondria, the energy producing place. In order for the acyl-CoA to migrate inside the mitochondria, it has to pass through the membranes of the mitochondria (outer membrane and inner membrane). Carnitine palmitoyltransferase (CPT) is the enzyme that catalyzes this process. Acyl-CoA oxidase is an enzyme located in the peroxisome, which produces trans-2,3-dehydroacyl-CoA and H 2 O 2 from acyl CoA and O 2. In the first step of fatty acid β-oxidation Is a rate-limiting enzyme that acts. The uncoupling protein (UCP) of mitochondria is found in brown fat, white fats, and muscle cells. UCPs reduce ATP production and generate heat by uncoupling the oxidative phosphorylation process in the mitochondria of the cell. Therefore, it has been reported that the degree of UCP expression affects energy expenditure and is associated with obesity. In fact, there are studies showing that obese people tend to show less UCP expression.
한편, 최근에 안전한 미생물로 여겨져 온 유산균을 이용하여 비만을 예방 또는 치료하고자 하는 노력 역시 많이 이루어지고 있다. 유산균은 장내 정상균총의 유지, 장내 균총의 개선, 항당뇨 및 항고지혈증 효과, 발암 억제, 대장염 억제, 그리고 숙주의 면역체계의 비특이적 활성 등의 효과를 나타낸다고 보고되고 있다.
On the other hand, efforts to prevent or treat obesity using lactic acid bacteria, which have recently been regarded as safe microorganisms, have been made. Lactic acid bacteria have been reported to exhibit effects such as maintenance of intestinal flora, improvement of intestinal flora, antidiabetic and anti-hyperlipidemic effect, inhibition of carcinogenesis, suppression of colitis, and nonspecific activity of host immune system.
한편, 대한민국 특허등록 제0913405호(발명의 명칭: Th2-매개 면역질환의 예방 또는 치료용 조성물) 및 제0913406호(발명의 명칭: Th1-매개 면역질환의 예방 또는 치료용 조성물)에 비피도박테리움 비피덤(Bifidobacterium bifidum), 락토바실러스 애시도필러스(Lactobacillus acidophilus), 스트렙토코커스 써모필러스 (Streptococcus thermophilus), 락토바실러스 루테리(Lactobacillus reuteri) 및 락토바실러스 카제이(Lactobacillus casei)의 복합 프로바이오틱스가 Th1-매개 또는 Th2-매개 면역질환의 예방 또는 치료 효능이 있음이 개시되어 있을 뿐으로 본 발명자들은 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 5종의 유산균 복합균주가 3T3-L1 지방세포 내 지방축적을 억제하고 궁극적으로 지방합성에 관련된 유전자 발현 억제 및 지방산화에 관련된 유전자 발현 촉진에 의해 체내 지방축적을 억제함으로써 체지방 감소 효능을 갖는다는 사실을 발견함으로써 본 발명을 완성하게 되었다.
On the other hand, Korean Patent Registration No. 0913405 entitled "Composition for the Prevention or Treatment of Th2-Mediated Immunological Diseases" and No. 0913406 entitled "Composition for the Prevention or Treatment of Th1- The complex probiotics of Bifidobacterium bifidum , Lactobacillus acidophilus , Streptococcus thermophilus , Lactobacillus reuteri and Lactobacillus casei are Th1 Mediated or Th2-mediated immune diseases, the present inventors have found that Bifidobacterium ( Bifidobacterium < RTI ID = 0.0 > bifidum ) HY8308, Lactobacillus ( Lactobacillus acidophilus) HY7038, Streptococcus Thermo filler's (Streptococcus thermophilus) HY9008, Lactobacillus ruteri (Lactobacillus reuteri) HY7508 and Lactobacillus casei (Lactobacillus casei) 5 jong Lactobacillus composite strain 3T3-L1 adipocytes in consisting HY7208 fat accumulation And ultimately suppresses gene expression associated with lipid synthesis and promotes gene expression related to lipid oxidation. Thus, the present invention has been completed.
본 발명은 상기와 같은 문제점을 해결하기 위하여 안출된 것으로서, 지방합성 억제 및 지방산화 촉진에 의한 체지방 감소 효능을 갖는 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 5종의 유산균 복합균주를 유효성분으로 함유하는 약학적 조성물, 발효유, 음료, 건강기능식품을 제공하는 것을 목적으로 한다.
Disclosure of the Invention The present invention has been conceived in order to solve the above-mentioned problems, and it is an object of the present invention to provide a method for inhibiting lipid peroxidation and inhibiting lipid peroxidation by inhibiting lipid peroxidation, Bifidobacterium bifidum HY8308, Lactobacillus acidophilus A pharmaceutical composition comprising as an active ingredient five hybrid strains of lactic acid bacteria consisting of HY7038, Streptococcus thermophilus HY9008, Lactobacillus reuteri HY7508 and Lactobacillus casei HY7208, Fermented milk, beverage, and health functional food.
상기한 목적을 달성하기 위하여, 본 발명은 지방합성 억제 및 지방산화 촉진에 의한 체지방 감소 효능을 갖는 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 5종의 유산균 복합균주를 유효성분으로 함유하는 약학적 조성물, 발효유, 음료, 건강기능식품을 제공하는 것을 특징으로 한다.
In order to accomplish the above object, the present invention provides a method for inhibiting lipid peroxidation, comprising the steps of: Bifidobacterium bifidum HY8308, Lactobacillus acidophilus HY7038, A pharmaceutical composition comprising as an active ingredient five complex strains of lactic acid bacteria consisting of Streptococcus thermophilus HY9008, Lactobacillus reuteri HY7508 and Lactobacillus casei HY7208, a fermented milk, a beverage, And a health functional food.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308(기탁번호:KCTC 12563BP) 및 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038(기탁번호:KCTC 12560BP)은 성인 또는 유아 분변으로부터 분리 및 동정된 신규한 균주로서 생명공학연구원 유전자은행에 각각 단일균주로 기탁하였다. Bifidobacterium bifidum HY8308 (accession number: KCTC 12563BP) and Lactobacillus acidophilus HY7038 (accession number: KCTC 12560BP) of the present invention are isolated and identified from adult or infant feces As a new strain, each strain was deposited as a single strain in the genetic bank of the Institute of Biotechnology.
또한, 본 발명의 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008(기탁번호:KCTC 12564BP)은 유제품에서, 본 발명의 락토바실러스 루테리(Lactobacillus reuteri) HY7508(기탁번호:KCTC 12562BP)은 성인 또는 유아 분변, 모유에서 분리 및 동정된 신규한 균주로서 생명공학연구원 유전자은행에 각각 단일균주로 기탁하였다. In addition, in the case of Streptococcus thermophilus HY9008 (accession number: KCTC 12564BP) of the present invention, in the dairy product, Lactobacillus reuteri HY7508 of the present invention (accession number: KCTC 12562BP) As a new strain isolated and identified in breast milk, it was deposited as a single strain in each of the genetic banks of the Institute of Biotechnology.
또한, 본 발명의 락토바실러스 카제이(Lactobacillus casei) HY7208(기탁번호:KCTC 12561BP)은 성인 또는 유아 분변, 유제품, 과실, 채소에서 분리 및 동정된 신규한 균주로서 생명공학연구원 유전자은행에 단일균주로 기탁하였다.The Lactobacillus casei HY7208 of the present invention is a novel strain isolated and identified in adult or infant feces, dairy products, fruits and vegetables, and is a single strain in the genetic bank of the Institute of Biotechnology. ≪ / RTI >
또한, 본 발명의 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 5종의 유산균 복합균주를 'IRT5'로 명명하기로 한다.
Also, Bifidobacterium bifidum HY8308, Lactobacillus acidophilus HY7038, Streptococcus thermophilus HY9008, Lactobacillus reuteri HY7508, and Lactobacillus reuteri of the present invention The five bacterial strains of lactic acid bacteria consisting of Lactobacillus casei HY7208 will be referred to as " IRT5 ".
한편, 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 5종의 유산균 복합균주(IRT5)를 유효성분으로 함유하는 것을 특징으로 하는 체지방 감소를 통한 비만 또는 비만 관련 질환 치료 또는 예방용 약학적 조성물은 단독 또는 약제학적으로 사용되는 부형제들과 함께 약제학적으로 통상으로 사용되는 방법에 따라 정제, 캡슐제 등과 같은 제재형태로 제재화하여 사용될 수 있다.On the other hand, Bifidobacterium bifidum HY8308, Lactobacillus acidophilus HY7038, Streptococcus thermophilus HY9008, Lactobacillus reuteri HY7508 and Lactobacillus casei (IRT5) comprising Lactobacillus casei HY7208 as an active ingredient. The pharmaceutical composition for the treatment or prevention of obesity or obesity-related diseases through body fat reduction can be used alone or in a pharmaceutical use May be formulated into preparations such as tablets, capsules and the like according to a method commonly used in pharmaceuticals together with excipients.
사람의 경우, 통상적인 1일 투여량은 1~30㎎/㎏ 체중의 범위일 수 있고, 1회 또는 수회로 나누어 투여할 수 있다. 그러나 실제 투여량은 투여경로, 환자의 연령, 성별, 체중, 건강상태 및 질환의 중증도 등의 여러 관련 인자에 비추어 결정되어야 한다.In the case of humans, a typical daily dose may range from 1 to 30 mg / kg body weight, and may be administered in single or divided doses. However, the actual dosage should be determined in light of various relevant factors such as route of administration, age, sex, weight, health status and severity of the disease.
물론, 본 발명의 상기 약학적 조성물은 독성 및 부작용은 거의 없으므로 장기간 복용시에도 안심하고 사용할 수 있는 약제이다.
Of course, since the pharmaceutical composition of the present invention has little toxicity and side effects, it can be safely used even when taken for a long time.
또한, 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 5종의 유산균 복합균주(IRT5)를 유효성분으로 함유하는 것을 특징으로 하는 체지방 감소를 통한 비만 개선 또는 예방용 식품 조성물인 발효유는 상기 IRT5, 유산균 배양액 및 혼합과즙시럽을 일정비율로 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 용기에 포장하여 제조한다.
In addition, Bifidobacterium bifidum HY8308, Lactobacillus acidophilus HY7038, Streptococcus thermophilus HY9008, Lactobacillus reuteri HY7508, and Lactobacillus casei ( Lactobacillus casei ) HY7208 (IRT5) as an active ingredient. The fermented milk, which is a food composition for improving or preventing obesity through reduction of body fat, comprises the IRT5, a lactic acid bacterium culture liquid and a mixed juice syrup They are homogenized at a constant ratio of 150 bar, cooled to below 10 ° C and packaged in a container.
또한, 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 5종의 유산균 복합균주(IRT5)를 유효성분으로 함유하는 것을 특징으로 하는 체지방 감소를 통한 비만 개선 또는 예방용 식품 조성물인 기능성 음료는 상기 IRT5, 혼합과즙시럽 및 물을 일정한 비율로 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 유리병, 패트병 등 소포장 용기에 포장하여 제조한다.
In addition, Bifidobacterium bifidum HY8308, Lactobacillus acidophilus HY7038, Streptococcus thermophilus HY9008, Lactobacillus reuteri HY7508, and Lactobacillus casei (IRT5) comprising Lactobacillus casei HY7208 as an active ingredient. The functional beverage, which is a food composition for improving or preventing obesity through reduction of body fat, comprises the IRT5, a mixed juice syrup and water They are homogenized at a constant ratio of 150 bar, cooled to below 10 ° C, and packaged in glass bottles, plastic bottles, and other small containers.
또한, 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208로 이루어진 5종의 유산균 복합균주(IRT5)를 유효성분으로 함유하는 것을 특징으로 하는 체지방 감소를 통한 비만 개선 또는 예방용 식품 조성물인 건강기능식품은 상기 IRT5를 포함하는 것 이외에 영양보조성분으로서 비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드, 올리고당 등이 첨가될 수 있으며 여타의 식품 첨가물이 첨가되어도 무방하다.
In addition, Bifidobacterium bifidum HY8308, Lactobacillus acidophilus HY7038, Streptococcus thermophilus HY9008, Lactobacillus reuteri HY7508, and Lactobacillus casei (IRT5) comprising Lactobacillus casei HY7208 as an active ingredient. The health functional food, which is a food composition for improving or preventing obesity through reduction of body fat, Vitamin B 1 , B 2 , B 5 , B 6 , E and acetic acid ester, nicotinic acid amide, oligosaccharide, etc. may be added as auxiliary components, and other food additives may be added.
이상에서와 같이, 본 발명은 체지방 감소 효능을 갖는 IRT5를 유효성분으로 함유하는 약학 조성물, 식품조성물, 발효유, 음료, 건강기능식품 등 지방 대사에 영향을 미치는 기능성 식품 및 약학 조성물 등으로 이용될 수 있다.
As described above, the present invention can be applied to pharmaceutical compositions, food compositions, functional foods and pharmaceutical compositions that affect fat metabolism such as fermented milk, beverages, and health functional foods, which contain IRT5 having an effect of reducing body fat have.
도 1은 IRT5에 의한 3T3-L1 세포내 지방 축적 억제 효과를 AdipoRed assay 방법으로 측정한 결과를 나타낸 그래프이다.
도 2는 고지방식이에 의해 비만이 유도된 실험동물에서 IRT5 투여에 의한 총지방량 증가 억제를 나타낸 그래프이다.
도 3은 고지방식이에 의해 비만이 유도된 실험동물에서 IRT5 투여에 의한 부고환지방량 증가 억제를 나타낸 그래프이다.
도 4는 고지방식이에 의해 비만이 유도된 실험동물에서 IRT5 투여에 의한 피하지방량 증가 억제를 나타낸 그래프이다.
도 5는 고지방식이에 의해 비만이 유도된 실험동물에서 IRT5 투여에 의한 장간막지방량 증가 억제를 나타낸 그래프이다.
도 6은 고지방식이에 의해 비만이 유도된 실험동물에서 IRT5 투여에 의한 혈중 렙틴 증가 억제를 나타낸 그래프이다.
도 7은 고지방식이에 의해 비만이 유도된 실험동물에서 IRT5 투여에 의한 간조직내 지방합성 및 지방산화 관련 유전자의 변화를 나타낸 그래프이다.
도 8은 고지방식이에 의해 비만이 유도된 실험동물에서 IRT5 투여에 의한 부고환지방 조직내 지방합성 및 지방산화 관련 유전자의 변화를 나타낸 그래프이다.
도 9는 고지방식이에 의해 비만이 유도된 실험동물에서 IRT5 유산균 투여에 의한 견갑골갈색지방 조직내 지방산화 및 열생성 관련 유전자의 변화를 나타낸 그래프이다.FIG. 1 is a graph showing the results of measuring the inhibitory effect of fat accumulation in 3T3-L1 cells by IRT5 by the AdipoRed assay method.
FIG. 2 is a graph showing the inhibition of total fat gain by administration of IRT5 in an experimental animal in which obesity was induced by a high fat diet.
FIG. 3 is a graph showing inhibition of epididymal fat mass increase by administration of IRT5 in an experimental animal in which obesity was induced by a high fat diet method.
FIG. 4 is a graph showing inhibition of subcutaneous fat administration by IRT5 administration in an experimental animal in which obesity was induced by a high fat diet.
FIG. 5 is a graph showing the suppression of the increase of the fat mass in the mesentery by administration of IRT5 in an experimental animal in which obesity was induced by the high fat diet method.
FIG. 6 is a graph showing the inhibition of blood leptin increase by IRT5 administration in an experimental animal in which obesity was induced by high fat diet.
FIG. 7 is a graph showing changes in lipid synthesis and lipid oxidation-related genes in liver tissues by administration of IRT5 in obesity-induced experimental animals by the high fat diet method.
FIG. 8 is a graph showing changes in lipid synthesis and lipid oxidation-related genes in epididymal adipose tissue by administration of IRT5 in an experimental animal in which obesity was induced by high fat diet.
FIG. 9 is a graph showing changes in the lipid oxidation and heat production-related genes in scapula brown fat adipose tissue by administration of IRT5 lactic acid bacteria in an obese-induced experimental animal by high fat diet method.
이하, 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나, 다음의 실시예는 본 발명의 범위를 한정하는 것은 아니며, 본 발명의 기술적 사상의 범위 내에서 당업자에 의한 통상적인 변화가 가능하다.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following embodiments are not intended to limit the scope of the present invention, and ordinary variations by those skilled in the art are possible within the scope of the technical idea of the present invention.
<실시예 1>≪ Example 1 >
IRT5의 분리 및 동정Isolation and identification of IRT5
1-1. 비피도박테리움 비피덤( Bifidobacterium bifidum ) HY8308의 분리 및 동정 1-1. Bifidobacterium Bifidobacterium bonus (Bifidobacterium bifidum) Separation and Identification of HY8308
본 발명에 따른 신균주를 분리하기 위하여 성인 또는 유아의 분변 0.1g을 혐기 희석액에 십진 희석하여 1.5% 한천(agar)이 포함된 BL 고체배지에 도말하여 37℃의 혐기조건에서 48시간 배양한 후 콜로니(colony)를 백금이(loop)로 취하여 새로운 BL 고체배지에 접종하고 순수 분리하였다. 순수 분리된 콜로니를 BL 액체배지에 접종하여 37℃에서 20시간 배양하였다. 이렇게 분리한 신균주를 Crystal violet 염색을 한 후 현미경을 통해 관찰하였고, DNA를 분리하여 Bifidobacteria 16S rRNA 분석(Im3, Im26)을 통한 Blast search한 결과(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_ SPEC=MicrobialGenomes)를 하기의 표 1에 나타내었다.
In order to isolate the new strain according to the present invention, 0.1 g of the feces of an adult or an infant was decanted into anaerobic dilution, and the mixture was plated on a BL solid medium containing 1.5% agar and incubated for 48 hours in an anaerobic condition at 37 ° C A colony was taken as a loop and inoculated into fresh BL solid medium and purified. The purely isolated colonies were inoculated into BL liquid medium and cultured at 37 占 폚 for 20 hours. The newly isolated strains were stained with Crystal violet and then observed under a microscope. The DNA was isolated and blasted by Bifidobacteria 16S rRNA analysis (Im3, Im26) (http: //blast.ncbi.nlm.nih. gov / Blast.cgi? PAGE_TYPE = BlastSearch & BLAST_SPEC = MicrobialGenomes) are shown in Table 1 below.
상기의 표 1에서 확인할 수 있는 바와 같이, 본 발명의 신균주의 16S rRNA 균주 동정결과 비피도박테리움 비피덤(Bifidobacterium bifidum) LCR9와 가장 높은 상동성을 나타내어 비피도박테리움 비피덤(Bifidobacterium bifidum)과 가장 높은 분자계통학적 유연관계를 보여 본 발명자들은 이를 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308로 명명하고, 2014년 3월 4일자로 한국생명공연구원 유전자은행(기탁번호: KCTC 12563BP)에 기탁하였다.
As can be seen from Table 1, 16S rRNA strains result of identification of the novel strain of the invention Bifidobacterium bipyridinium bushes (Bifidobacterium bifidum) exhibits a LCR9 with the highest homology to Bifidobacterium bipyridinium bushes (Bifidobacterium bifidum) The present inventors named Bifidobacterium bifidum HY8308 as the highest molecular phylogenetically related strain and named it HY8308 on March 4, 2014 and deposited the Korean Life Performance Gene Bank (Accession No .: KCTC 12563BP) ≪ / RTI >
본 발명에 따른 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308의 특성은 다음과 같다.The characteristics of Bifidobacterium bifidum HY8308 according to the present invention are as follows.
1)균의 형태1) Types of bacteria
비엘(BL) 한천평판배지에서 37℃, 2일간 혐기 조건에서 배양했을 때 균의 특성The characteristics of the bacteria when cultured under anaerobic conditions for 2 days at 37 ° C in Biel (BL) agar plate medium
①세포의 형태: 부정형의 간상형 균① Cell shape: amorphous hepatoblastic
②운동성: 없음② Mobility: None
③포자형성능: 없음③ Spore forming ability: None
④그람(Gram) 염색: 양성
④ Gram staining: positive
2)생리적 성질2) Physiological properties
①생육온도: 생장가능 생육온도 20~45℃① Growth temperature: Growth
최적 생장온도 37℃ Optimum growth temperature 37 ℃
②생육 pH: 생장가능 생육 pH 5.0~8.0② Growth pH: Growable growth pH 5.0 ~ 8.0
최적 pH 6.0~7.0 Optimal pH 6.0 ~ 7.0
③산소에 대한 영향: 혐기성
③ Effect on oxygen: anaerobic
3)카탈라제: -3) Catalase: -
4)가스형성여부: -4) Whether gas is formed: -
5)15℃에서 생육: -5) Growth at 15 ℃: -
6)45℃에서 생육: +6) Growth at 45 ° C: +
7)인돌생산: -7) Indole production: -
8)젖산생산: +8) Lactic acid production: +
9)Biomerieux 사의 혐기성 세균 동정용 API 20 A kit를 이용한 실험9) Experiment using Biomerieux API 20 A kit for anaerobic bacteria identification
1-2. 락토바실러스 애시도필러스( Lactobacillus acidophilus ) HY7038의 분리 및 동정 1-2. Lactobacillus ash FIG filler's (Lactobacillus acidophilus) separation and identification of the HY7038
본 발명에 따른 신균주를 분리하기 위하여 성인 또는 유아의 분변 0.1g을 호기 희석액에 십진 희석하여 1.5% 한천(agar)이 포함된 엠알에스(MRS) 고체배지에 도말하여 37℃에서 48시간 배양한 후 콜로니(colony)를 백금이(loop)로 취하여 새로운 MRS 고체배지에 접종하고 순수 분리하였다. 순수 분리된 콜로니를 MRS 액체배지에 접종하여 37℃에서 20시간 배양한 후 Crystal violet 염색을 한 후 현미경을 통해 관찰하였고, 이렇게 분리한 신균주를 동정하고자 Biomerieux 사의 API 50 CH kit를 이용하여 당 발효 실험을 하였다.
In order to isolate the new strain according to the present invention, 0.1 g of the feces of an adult or infant was decanted into an aerobic dilution solution, and the mixture was applied to an MRS solid medium containing 1.5% agar and cultured at 37 ° C for 48 hours Colony was taken as a loop and inoculated onto fresh MRS solid medium and purified. The purified colony was inoculated on MRS liquid medium and incubated at 37 ° C for 20 hours. The cells were stained with Crystal violet and observed with a microscope. To identify the new strains isolated, the
본 발명의 신균주의 당발효 실험 결과 락토바실러스 애시도필러스(Lactobacillus acidophilus)의 당발효 실험과 일치하는 것으로 확인되어 본 발명자들은 이를 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038로 명명하고, 2014년 3월 4일자로 한국생명공연구원 유전자은행(기탁번호: KCTC 12560BP)에 기탁하였다.
As a result of the sugar fermentation experiment of the novel strain of the present invention, it was confirmed that it was consistent with the sugar fermentation experiment of Lactobacillus acidophilus , and the present inventors named it Lactobacillus acidophilus HY7038, (KCTC 12560BP) on March 4, 2009. The results of this study are as follows.
본 발명에 따른 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038의 특성은 다음과 같다.The Lactobacillus acidophilus ( Lactobacillus < RTI ID = 0.0 > acidophilus ) HY7038 has the following characteristics.
1)균의 형태1) Types of bacteria
엠알에스(MRS) 한천평판배지에서 37℃, 2일간 배양했을 때 균의 특성The characteristics of the bacteria when cultured on an MRS agar plate medium at 37 ° C for 2 days
①세포의 형태: 간균① Cell shape: Bacillus
②운동성: 없음② Mobility: None
③포자형성능: 없음③ Spore forming ability: None
④그람(Gram) 염색: 양성
④ Gram staining: positive
2)균락의 형태2) Morphology
엠알에스(MRS) 한천평판배지에서 37℃, 2일간 배양했을 때 균락의 형태When cultured on MRS agar plate medium at 37 ° C for 2 days,
①형상: 원형① Shape: Circular
②융기: 볼록② Bump: convex
③표면: 매끄러움(Smooth)
③ Surface: Smooth
3)생리적 성질3) Physiological properties
①생육온도: 생장가능 생육온도 15~45℃① Growth temperature: Growth possible Growth temperature 15 ~ 45 ℃
최적 생장온도 37℃ Optimum growth temperature 37 ℃
②생육 pH: 생장가능 생육 pH 4.0~7.5② Growth pH: Growable Growth pH 4.0 ~ 7.5
최적 pH 6.0~6.5 Optimum pH 6.0 to 6.5
③산소에 대한 영향: 통성혐기성
③ Effect on oxygen: Tumor anaerobic
4)카탈라제: -4) Catalase: -
5)가스형성여부: -5) Whether gas is formed: -
6)15℃에서 생육: -6) Growth at 15 ℃: -
7)45℃에서 생육: +7) Growth at 45 ° C: +
8)인돌생산: -8) Indole production: -
9)젖산생산: +9) Lactic acid production: +
10) Biomerieux 사의 유산균 동정용 API 50 CH kit를 이용한 실험10) Experiment with
1-3. 스트렙토코커스 써모필러스 ( Streptococcus thermophilus ) HY9008 의 분리 및 동정 1-3. Streptococcus Thermo's filler (Streptococcus thermophilus) Isolation and Identification of HY9008
본 발명에 따른 균주를 분리하기 위하여 유제품을 호기 희석액에 십진 희석하여 1.5% 한천(agar)이 포함된 M17 고체배지에 도말하여 42℃에서 24시간 배양한 후 콜로니(colony)를 백금이(loop)로 취하여 새로운 M17 고체배지에 접종하고 순수 분리하였다. 순수 분리된 콜로니를 M17 액체배지에 접종하여 42℃에서 16시간 배양한 후 Crystal violet 염색을 한 후 현미경을 통해 관찰하였고, 균주의 DNA를 분리하여 16S rRNA 분석(27F, 1492R)을 통한 Blast search한 결과(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE= BlastIn order to isolate the strain according to the present invention, the dairy product was decanted into an aerobic dilution solution and then streaked on a M17 solid medium containing 1.5% agar and cultured at 42 ° C for 24 hours. The colony was plated with a loop, , Inoculated onto fresh M17 solid medium and purely isolated. The purified colonies were inoculated on M17 liquid medium and incubated at 42 ° C for 16 hours. The cells were stained with Crystal violet and observed under a microscope. The DNA of the strain was isolated and blasted through 16S rRNA analysis (27F, 1492R) Results (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE= Blast
Search&BLAST_SPEC=MicrobialGenomes)를 하기의 표 2에 나타내었다.
Search & BLAST_SPEC = Microbial Genomes) are shown in Table 2 below.
상기의 표 2에서 확인할 수 있는 바와 같이, 본 발명의 신균주의 16S rRNA 균주 동정결과 스트렙토코커스 써모필러스(Streptococcus thermophilus) M4와 가장 높은 상동성을 나타내어 스트렙토코커스 써모필러스(Streptococcus thermophilus)와 가장 높은 분자계통학적 유연관계를 보여 본 발명자들은 이를 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008로 명명하고, 2014년 3월 4일자로 한국생명공연구원 유전자은행(기탁번호: KCTC 12564BP)에 기탁하였다.
As can be seen from the above Table 2, the 16S rRNA strain of the new strain of the present invention has the highest homology with Streptococcus thermophilus M4 and has the highest homology with Streptococcus thermophilus and Streptococcus thermophilus The present inventors named Streptococcus thermophilus HY9008 as a high molecular phylogenetic relationship and donated to Korea Life Performance Salvage Bank (Accession No .: KCTC 12564BP) on March 4, 2014.
본 발명에 따른 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008의 특성은 다음과 같다.The characteristics of Streptococcus thermophilus HY9008 according to the present invention are as follows.
1)균의 형태1) Types of bacteria
엠십칠(M17) 한천평판배지에서 37℃, 2일간 배양했을 때 균의 특성The characteristics of the bacterium when cultured for 2 days at 37 ° C on an M10 (M17) agar plate medium
①세포의 형태: 구균① Cell morphology:
②운동성: 없음② Mobility: None
③포자형성능: 없음③ Spore forming ability: None
④그람(Gram) 염색: 양성
④ Gram staining: positive
2)균락의 형태2) Morphology
엠십칠(M17) 한천평판배지에서 37℃, 2일간 배양했을 때 균락의 형태When cultured for 2 days at 37 ° C on M13 agar plate medium,
①형상: 원형① Shape: Circular
②융기: 볼록② Bump: convex
③표면: 거침(Rough)
③ Surface: Rough
3)생리적 성질3) Physiological properties
①생육온도: 생장가능 생육온도 25~45℃① Growth temperature: Growth possible Growth temperature 25 ~ 45 ℃
최적 생장온도 37~40℃ Optimum growth temperature 37 ~ 40 ℃
②생육 pH: 생장가능 생육 pH 4.0~7.5② Growth pH: Growable Growth pH 4.0 ~ 7.5
최적 pH 6.0~6.5 Optimum pH 6.0 to 6.5
③산소에 대한 영향: 통성혐기성
③ Effect on oxygen: Tumor anaerobic
4)카탈라제: -4) Catalase: -
5)가스형성여부: -5) Whether gas is formed: -
6)15℃에서 생육: -6) Growth at 15 ℃: -
7)45℃에서 생육: +7) Growth at 45 ° C: +
8)인돌생산: -8) Indole production: -
9)젖산생산: +9) Lactic acid production: +
10)Biomerieux 사의 연쇄상구균 동정용 API 20 Step kit를 이용한 실험10)
1-4. 락토바실러스 루테리( Lactobacillus reuteri ) HY7508의 분리 및 동정 1-4. Ruteri Lactobacillus (Lactobacillus reuteri) Isolation and Identification of HY7508
본 발명에 따른 신균주를 분리하기 위하여 성인 또는 유아 분변, 모유를 1.5% 한천(agar)이 포함된 엠알에스(MRS) 고체배지에 도말하고 37℃에서 48시간 동안 배양한 후 콜로니(colony)를 백금이(loop)로 취하여 새로운 MRS 액체배지에 접종하고 37℃에서 20시간 배양한 후 Crystal violet 염색을 한 후 현미경을 통해 관찰하였고, 균주의 DNA를 분리하여 16S rRNA 분석(27F, 1492R)을 통한 Blast search한 결과(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch &BLAST_SPEC=MicrobialGenomes)를 하기의 표 3에 나타내었다.
In order to separate the new strain according to the present invention, adult or infant feces and breast milk were plated on an MRS solid medium containing 1.5% agar, cultured at 37 ° C for 48 hours, and colony Platinum was inoculated into a new MRS liquid medium and incubated at 37 ° C for 20 hours. The cells were stained with Crystal violet and observed under a microscope. The DNA of the strain was isolated and analyzed by 16S rRNA analysis (27F, 1492R) Blast search results (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch & BLAST_SPEC = MicrobialGenomes) are shown in Table 3 below.
상기의 표 3에서 확인할 수 있는 바와 같이, 본 발명의 신균주의 16S rRNA 균주 동정결과 락토바실러스 루테리(Lactobacillus reuteri) KLDS와 가장 높은 상동성을 나타내어 락토바실러스 루테리(Lactobacillus reuteri)와 가장 높은 분자계통학적 유연관계를 보여 본 발명자들은 이를 락토바실러스 루테리(Lactobacillus reuteri) HY7508로 명명하고, 2014년 3월 4일자로 한국생명공연구원 유전자은행(기탁번호: KCTC 12562BP)에 기탁하였다.
As can be seen from the above Table 3, the 16S rRNA strain of the novel strain of the present invention has the highest homology with Lactobacillus reuteri KLDS and is the most homologous to Lactobacillus reuteri and the highest molecular phylogenetics The present inventors named this mutant Lactobacillus reuteri HY7508, and deposited it on the Korea Life Performance Gene Bank (Accession No .: KCTC 12562BP) on March 4, 2014.
본 발명에 따른 락토바실러스 루테리(Lactobacillus reuteri) HY7508의 특성은 다음과 같다.The characteristics of Lactobacillus reuteri HY7508 according to the present invention are as follows.
1)균의 형태1) Types of bacteria
엠알에스(MRS) 한천평판배지에서 37℃, 2일간 배양했을 때 균의 특성The characteristics of the bacteria when cultured on an MRS agar plate medium at 37 ° C for 2 days
①세포의 형태: 간균① Cell shape: Bacillus
②운동성: 없음② Mobility: None
③포자형성능: 없음③ Spore forming ability: None
④그람(Gram) 염색: 양성
④ Gram staining: positive
2)균락의 형태2) Morphology
엠알에스(MRS) 한천평판배지에서 37℃, 2일간 배양했을 때 균락의 형태When cultured on MRS agar plate medium at 37 ° C for 2 days,
①형상: 원형① Shape: Circular
②융기: 볼록② Bump: convex
③표면: 매끄러움(Smooth)
③ Surface: Smooth
3)생리적 성질3) Physiological properties
①생육온도: 생장가능 생육온도 20~45℃① Growth temperature: Growth
최적 생장온도 37℃ Optimum growth temperature 37 ℃
②생육 pH: 생장가능 생육 pH 4.0~7.5② Growth pH: Growable Growth pH 4.0 ~ 7.5
최적 pH 6.0~6.5 Optimum pH 6.0 to 6.5
③산소에 대한 영향: 통성혐기성
③ Effect on oxygen: Tumor anaerobic
4)카탈라제: -4) Catalase: -
5)가스형성여부: -5) Whether gas is formed: -
6)15℃에서 생육: -6) Growth at 15 ℃: -
7)45℃에서 생육: +7) Growth at 45 ° C: +
8)인돌생산: -8) Indole production: -
9)젖산생산: +9) Lactic acid production: +
10) Biomerieux 사의 유산균 동정용 API 50 CH kit를 이용한 실험10) Experiment with
1-5. 락토바실러스 카제이( Lactobacillus casei ) HY7208의 분리 및 동정 1-5. Lactobacillus casei (Lactobacillus casei) separation and identification of the HY7208
본 발명에 따른 신균주를 분리하기 위하여 성인 또는 유아 분변, 유제품, 과실 채소를 호기 희석액에 십진 희석하여 1.5% 한천(agar)이 포함된 엠알에스(MRS) 고체배지에 도말하여 37℃에서 48시간 배양한 후 콜로니(colony)를 백금이(loop)로 취하여 새로운 MRS 고체배지에 접종하고 순수 분리하였다. 순수 분리된 콜로니를 MRS 액체배지에 접종하여 37℃에서 20시간 배양한 후 Crystal violet 염색을 한 후 현미경을 관찰하였고, 균주의 DNA를 분리하여 16S rRNA 분석(27F, 1492R)을 통한 Blast search한 결과(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch &BLAST_SPEC=MicrobialGenomes)를 하기의 표 4에 나타내었다.
In order to isolate the new strain according to the present invention, adult or infant feces, dairy products, and fruit vegetables were decanted into an aerosol dilution solution and plated on an MRS solid medium containing 1.5% agar. After culturing, the colony was taken as a loop and inoculated on fresh MRS solid medium and purified. The purified colonies were inoculated on the MRS liquid medium and cultured at 37 ° C for 20 hours. The cells were stained with Crystal violet and observed under a microscope. The DNA of the strain was isolated and blasted by 16S rRNA analysis (27F, 1492R) (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch & BLAST_SPEC = MicrobialGenomes) are shown in Table 4 below.
상기의 표 4에서 확인할 수 있는 바와 같이, 본 발명의 신균주의 16S rRNA 균주 동정결과 락토바실러스 카제이(Lactobacillus casei) LRSL2와 가장 높은 상동성을 나타내어 락토바실러스 카제이(Lactobacillus casei)와 가장 높은 분자계통학적 유연관계를 보여 본 발명자들은 이를 락토바실러스 카제이(Lactobacillus casei) HY7208로 명명하고, 2014년 3월 4일자로 한국생명공연구원 유전자은행(기탁번호: KCTC 12561BP)에 기탁하였다.
As can be seen from Table 4 above, the 16S rRNA strain of the new strain of the present invention has the highest homology with Lactobacillus casei LRSL2 and has the highest homology with Lactobacillus casei and the highest molecule the present inventors show phylogenetic relationships are flexible Lactobacillus casei (Lactobacillus this casei ) HY7208, and deposited on March 4, 2014 with the Korean Life Performance Salvage Gene Bank (Accession No .: KCTC 12561BP).
본 발명에 따른 락토바실러스 카제이(Lactobacillus casei) HY7208의 특성은 다음과 같다.The characteristics of Lactobacillus casei HY7208 according to the present invention are as follows.
1)균의 형태1) Types of bacteria
엠알에스(MRS) 한천평판배지에서 37℃, 2일간 배양했을 때 균의 특성The characteristics of the bacteria when cultured on an MRS agar plate medium at 37 ° C for 2 days
①세포의 형태: 간균① Cell shape: Bacillus
②운동성: 없음② Mobility: None
③포자형성능: 없음③ Spore forming ability: None
④그람(Gram) 염색: 양성
④ Gram staining: positive
2)균락의 형태2) Morphology
엠알에스(MRS) 한천평판배지에서 37℃, 2일간 배양했을 때 균락의 형태When cultured on MRS agar plate medium at 37 ° C for 2 days,
①형상: 원형① Shape: Circular
②융기: 볼록② Bump: convex
③표면: 매끄러움(Smooth)
③ Surface: Smooth
3)생리적 성질3) Physiological properties
①생육온도: 생장가능 생육온도 15~40℃① Growth temperature: Growth possible Growth temperature 15 ~ 40 ℃
최적 생장온도 37℃ Optimum growth temperature 37 ℃
②생육 pH: 생장가능 생육 pH 4.0~7.5② Growth pH: Growable Growth pH 4.0 ~ 7.5
최적 pH 6.0~6.5 Optimum pH 6.0 to 6.5
③산소에 대한 영향: 통성혐기성
③ Effect on oxygen: Tumor anaerobic
4)카탈라제: -4) Catalase: -
5)가스형성여부: -5) Whether gas is formed: -
6)15℃에서 생육: +6) Growth at 15 ° C: +
7)45℃에서 생육: -7) Growth at 45 ℃: -
8)인돌생산: -8) Indole production: -
9)젖산생산: +9) Lactic acid production: +
10) Biomerieux 사의 유산균 동정용 API 50 CH kit를 이용한 실험10) Experiment with
<실시예 2>≪ Example 2 >
IRT5를 포함한 동결건조 분말제조Preparation of freeze-dried powder containing IRT5
2-1. 비피도박테리움 비피덤 ( Bifidobacterium bifidum ) HY8308 을 포함한 동결건조 분말 2-1. Bifidobacterium BP bonus (Bifidobacterium bifidum ) Freeze-dried powder containing HY8308
본 발명의 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308 1 X 109cfu/㎖의 균수를 BL 액체배지에 0.1% 접종하고, 배양기에서 혐기조건 하에서 37℃에서 20시간 동안 정치배양(batch culture)하였다. 이것을 4000rpm에서 15분간 원심분리하여 상층액을 제거하고, 인산완충식염수(phosphate buffered saline; PBS)로 균체를 세척한 후 다시 4000rpm에서 15분간 원심분리하여 상층액을 제거하였다. 수거한 균체는 보존제인 탈지분유 분말과 1:1의 중량비로 섞어 동결건조하여 분말을 제조하였다.
Bifidobacterium bifidum HY8308 of the present invention 1 x 10 9 cfu / ml bacterium was inoculated 0.1% in BL liquid medium and incubated in an incubator for 20 hours at 37 ° C under anaerobic conditions. Respectively. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.
2-2. 락토바실러스 애시도필러스( Lactobacillus acidophilus ) HY7038을 포함한 동결건조 분말 2-2. Lactobacillus ash FIG filler's (Lactobacillus acidophilus) the freeze-dried powder containing the HY7038
본 발명의 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038 1 X 109cfu/㎖의 균수를 MRS 액체배지(Difco)에 0.1% 접종하고 배양기에서 37℃에서 20시간 동안 정치배양(batch culture)하였다. 이것을 4000rpm에서 15분간 원심분리하여 상층액을 제거하고, 인산완충식염수(phosphate buffered saline; PBS)로 균체를 세척한 후 다시 4000rpm에서 15분간 원심분리하여 상층액을 제거하였다. 수거한 균체는 보존제인 탈지분유 분말과 1:1의 중량비로 섞어 동결건조하여 분말을 제조하였다.
Lactobacillus acidophilus HY7038 of the present invention 1 x 10 9 cfu / ml bacterium was inoculated 0.1% in MRS liquid medium (Difco) and batch cultured in an incubator at 37 ° C for 20 hours . The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.
2-3. 스트렙토코커스 써모필러스( Streptococcus thermophilus ) HY9008을 포함한 동결건조 분말 2-3. Streptococcus Thermo filler's (Streptococcus thermophilus) freeze-dried powder containing the HY9008
본 발명의 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008 1 X 109cfu/㎖의 균수를 M17 액체배지(Difco)에 0.1% 접종하고 배양기에서 42℃에서 16시간 동안 정치배양(batch culture)하였다. 이것을 4000rpm에서 15분간 원심분리하여 상층액을 제거하고, 인산완충식염수(phosphate buffered saline; PBS)로 균체를 세척한 후 다시 4000rpm에서 15분간 원심분리하여 상층액을 제거하였다. 수거한 균체는 보존제인 탈지분유 분말과 1:1의 중량비로 섞어 동결건조하여 분말을 제조하였다.
Streptococcus thermophilus HY9008 of the present invention 1 x 10 9 cfu / ml bacterium was inoculated 0.1% in M17 liquid medium (Difco) and batch cultured in an incubator at 42 ° C for 16 hours. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.
2-4. 락토바실러스 루테리 ( Lactobacillus reuteri ) HY7508 을 포함한 동결건조 분말 2-4. Lactobacillus Ruteri (Lactobacillus freeze-dried powder containing reuteri) HY7508
본 발명의 락토바실러스 루테리(Lactobacillus reuteri) HY7508 1 X 109cfu/㎖의 균수를 MRS 액체배지(Difco)에 0.1% 접종하고 배양기에서 37℃에서 20시간 동안 정치배양(batch culture)하였다. 이것을 4000rpm에서 15분간 원심분리하여 상층액을 제거하고, 인산완충식염수(phosphate buffered saline; PBS)로 균체를 세척한 후 다시 4000rpm에서 15분간 원심분리하여 상층액을 제거하였다. 수거한 균체는 보존제인 탈지분유 분말과 1:1의 중량비로 섞어 동결건조하여 분말을 제조하였다.
Lactobacillus reuteri HY7508 of the present invention 1 x 10 9 cfu / ml bacterium was inoculated 0.1% in MRS liquid medium (Difco) and batch cultured in an incubator at 37 ° C for 20 hours. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.
2-5. 락토바실러스 카제이 ( Lactobacillus casei ) HY7208 을 포함한 동결건조 분말 2-5. Lactobacillus Casei (Lactobacillus casei ) freeze-dried powder containing HY7208
본 발명의 락토바실러스 카제이(Lactobacillus casei) HY7208 1 X 109cfu/㎖의 균수를 MRS 액체배지(Difco)에 0.1% 접종하고 배양기에서 37℃에서 20시간 동안 정치배양(batch culture)하였다. 이것을 4000rpm에서 15분간 원심분리하여 상층액을 제거하고, 인산완충식염수(phosphate buffered saline; PBS)로 균체를 세척한 후 다시 4000rpm에서 15분간 원심분리하여 상층액을 제거하였다. 수거한 균체는 보존제인 탈지분유 분말과 1:1의 중량비로 섞어 동결건조하여 분말을 제조하였다.
Lactobacillus casei HY7208 of the present invention 1 x 10 9 cfu / ml bacterium was inoculated 0.1% in MRS liquid medium (Difco) and batch cultured in an incubator at 37 ° C for 20 hours. The supernatant was removed by centrifugation at 4000 rpm for 15 minutes, washed with phosphate buffered saline (PBS), and centrifuged at 4000 rpm for 15 minutes to remove the supernatant. The collected cells were mixed with a skim milk powder as a preservative at a weight ratio of 1: 1 and lyophilized to prepare a powder.
2-6. IRT5를 포함한 동결건조분말의 제조 2-6. Preparation of freeze-dried powder containing IRT5
상기 실시예 2-1 내지 2-5의 각각의 균주를 포함한 동결건조분말을 각각 동일한 중량비(1:1:1:1:1:1:1)로 혼합하여 본 발명의 IRT5를 포함한 총 균수 5 X 109cfu/㎖의 동결건조분말을 제조하였다.The lyophilized powders containing the strains of Examples 2-1 to 2-5 were mixed at the same weight ratios (1: 1: 1: 1: 1: 1: 1) A lyophilized powder of X 10 9 cfu / ml was prepared.
한편, 본 발명의 IRT5는 상기와 같이 동결 건조된 분말 형태 또는 배양물 형태로 제공될 수 있다.
Meanwhile, the IRT5 of the present invention may be provided in the form of a lyophilized powder or a culture as described above.
<실시예 3>≪ Example 3 >
IRT5IRT5 를 유효성분으로 함유하는 약학적 조성물의 제조Preparation of a pharmaceutical composition containing as an active ingredient
본 발명의 IRT5를 유효성분으로 함유하는 약학적 조성물의 제제예를 설명하나, 본 발명은 이를 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.
The preparation examples of the pharmaceutical composition containing the IRT5 of the present invention as an active ingredient will be described below, but the present invention is not intended to be limited thereto but is specifically described.
정제의 제조Manufacture of tablets
상기 실시예 2-6의 IRT5를 포함한 동결건조분말 100㎎, 옥수수전분 100㎎, 유당 100㎎ 및 폴리비닐피롤리돈 97㎎을 균질하게 혼합하여 습식과립법으로 과립화하고 스테아린산 마그네슘 2㎎을 가하여 혼합한 후 1정이 400㎎이 되도록 타정하였다.
100 mg of the lyophilized powder containing IRT5 of Example 2-6, 100 mg of corn starch, 100 mg of lactose and 97 mg of polyvinylpyrrolidone were homogeneously mixed and granulated by the wet granulation method, and 2 mg of magnesium stearate was added After mixing, one tablet was compressed to 400 mg.
캡슐제의 제조Preparation of capsules
상기 실시예 2-6의 IRT5를 포함한 동결건조분말 100㎎, 옥수수 전분 100㎎, 유당 100㎎, 스테아린산 마그네슘 2㎎을 완전히 혼합한 후 통상의 캡슐제의 제조방법에 따라서 경질 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.
100 mg of the lyophilized powder containing IRT 5 of Example 2-6, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were thoroughly mixed and filled into hard gelatine capsules according to a conventional preparation method of capsules, .
<실시예 4><Example 4>
IRT5IRT5 를 유효성분으로 함유하는 발효유의 제조Production of fermented milk containing as an active ingredient
본 발명의 IRT5를 유효성분으로 함유하는 발효유를 제조하는 방법은 다음과 같다.A method for producing a fermented milk containing the IRT5 of the present invention as an active ingredient is as follows.
먼저, 유산균 배양액은 원유 95.36중량%와 탈지분유(또는 혼합분유) 4.6중량%를 교반하여 15℃에서의 비중은 1.0473~1.0475, 적정산도는 0.200~0.220%, pH는 6.65~6.70, 20℃에서의 브릭스(Brixo)는 16.3~16.5% 정도가 되도록 혼합하였다. 혼합 후에 이를 UHT 열처리(135℃에서 2초간 살균)하고 40℃로 냉각한 뒤, 실시예 2-3의 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008과 유당분해효소(Valley laboratory, USA)를 각기 0.02중량%씩 첨가하고 6시간 동안 배양하여 BCP 배지에서의 총 유산균수가 1.0 × 109cfu/㎖ 이상, 적정산도가 0.89~0.91%, pH는 4.55~4.65가 되도록 하여 제조하였다.The lactic acid bacteria culture solution was prepared by stirring 95.36 wt% of crude oil and 4.6 wt% of skim milk powder (or mixed powdered milk) and measuring specific gravity at 15 ° C from 1.0473 to 1.0475, a titratable acidity of 0.200 to 0.220%, pH of 6.65 to 6.70, of Brix (Brix o) were mixed such that the degree of 16.3 ~ 16.5%. Streptococcus Thermo filler's of this embodiment after mixture (2 seconds sterilized at 135 ℃) UHT heat treatment, and then cooled to 40 ℃, Example 2-3 (Streptococcus thermophilus) was added HY9008 and lactose (Valley laboratory, USA) for respectively by 0.02%, and cultured for six hours total number of lactic acid bacteria in the BCP medium 1.0 × 10 9 cfu / ㎖ above, the titratable acidity 0.89 ~ 0.91%, The pH was adjusted to 4.55 to 4.65.
그 다음, 혼합과즙시럽은 액상과당 13중량%, 백설탕 5중량%, 혼합과즙농축액 56Brixo 10.9중량%, 펙틴 1.0중량%, 후레쉬 후르츠 믹스 에센스 0.1중량% 및 정제수 70중량%를 30~35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.Next, the mixed fruit juice syrup was prepared by mixing 13 wt% of liquid fructose, 5 wt% of white sugar, 10.9 wt% of mixed juice concentrate 56Brix o , 1.0 wt% of pectin, 0.1 wt% of fresh fruit mix essence and 70 wt% of purified water at 30-35 캜 Mixed with stirring, heat-treated with UHT (sterilized at 135 ° C for 2 seconds), and cooled.
그런 다음, 상기 유산균배양액 69.5중량%와 실시예 2-1, 실시예 2-2, 실시예 2-4 및 실시예 2-5의 4종의 유산균 복합균주(중량비 1:1:1:1)를 포함한 동결건조분말 0.1중량% 및 상기 혼합과즙시럽 30.4중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각하여 IRT5를 유효성분으로 함유하는 발효유를 제조하였다.
Thereafter, four kinds of lactic acid bacterial strain (weight ratio 1: 1: 1: 1) of 69.5% by weight of the above culture medium of lactic acid bacteria and Example 4-1, Example 2-2, 2-4, , And 30.4% by weight of the mixed fruit juice syrup were homogenized at 150 bar, followed by cooling to 10 캜 or lower to prepare fermented milk containing IRT5 as an active ingredient.
<실시예 5>≪ Example 5 >
IRT5를 유효성분으로 함유하는 기능성 음료의 제조Preparation of a functional beverage containing IRT5 as an active ingredient
본 발명의 IRT5를 유효성분으로 함유하는 기능성 음료를 제조하는 방법은 다음과 같다.A method for producing a functional beverage containing the IRT5 of the present invention as an active ingredient is as follows.
먼저, 혼합과즙시럽은 액상과당 13중량%, 백설탕 2.5중량%, 갈색설탕 2.5중량%, 혼합과즙농축액 56Brixo 10.9중량%, 펙틴 1.0중량%, 후레쉬 후르츠 믹스 에센스 0.1중량% 및 정제수 70중량%를 30~35℃에서 교반하여 혼합한 후 UHT 열처리(135℃에서 2초간 살균)한 후 냉각하여 제조하였다.
First, a mixed juice syrup was 13% by weight of liquid fructose, white sugar, 2.5 wt%, brown sugar 2.5% by weight, mixed juice concentrate 56Brix o 10.9% by weight of pectin, 1.0% by weight, fresh 0.1% by weight fruit mix essence and purified water 70% The mixture was stirred at 30 to 35 ° C, mixed, and heat-treated by UHT (sterilized at 135 ° C for 2 seconds) and cooled.
그리고, 상기의 방법으로 제조된 혼합과즙시럽 30.4중량%와 실시예 2-6의 IRT5를 포함한 동결건조분말 0.1중량% 및 나머지 정제수 69.5중량%를 조합하여 150bar에서 균질한 후 10℃ 이하로 냉각한 후 이를 유리병, 패트병 등 소포장 용기에 포장하여 IRT5를 유효성분으로 함유하는 기능성 음료를 제조하였다.
Then, 30.4% by weight of the mixed juice syrup prepared by the above method, 0.1% by weight of freeze-dried powder containing IRT5 of Example 2-6 and 69.5% by weight of the remaining purified water were combined and homogenized at 150 bar, After that, it was packaged in glass bottles, plastic bottles, etc., and a functional beverage containing IRT5 as an active ingredient was prepared.
<실시예 6>≪ Example 6 >
IRT5를 유효성분으로 함유하는 건강기능식품의 제조Production of Health Functional Foods Containing IRT5 as Active Ingredient
상기 실시예 2-6의 IRT5를 포함한 동결건조분말 0.1중량%에 영양보조성분(비타민 B1, B2, B5, B6, E 및 초산에스테르, 니코틴산 아미드) 및 올리고당을 상기 실시예 2-6의 IRT5를 포함한 동결건조분말 100중량부에 대하여 10중량부가 되도록 첨가하여 고속회전 혼합기에서 혼합하였다. 상기 혼합물 100중량부에 대하여 멸균 정제수 10중량부를 첨가, 혼합하고 직경 1~2mm의 과립상으로 성형하였다. 상기 성형된 과립은 40~50℃의 진공건조기에서 건조시킨 후 12~14 메쉬(mesh)를 통과시켜 균일하게 과립을 제조하였다. 상기와 같이 제조된 과립은 적당량씩 압출 성형되어 정제 또는 분말로 되거나 경질캡슐에 충전되어 경질캡슐제품으로 제조하였다.
(Vitamin B 1 , B 2 , B 5 , B 6 , E and acetic acid ester, nicotinic amide) and oligosaccharide were added to 0.1 wt% of the lyophilized powder containing IRT 5 of Example 2-6, 6 was added in an amount of 10 parts by weight based on 100 parts by weight of the lyophilized powder containing IRT5 and mixed in a high-speed rotary mixer. To 100 parts by weight of the mixture, 10 parts by weight of sterilized purified water was added and mixed to form granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum drier at 40 to 50 DEG C and then passed through 12 to 14 mesh to prepare uniform granules. The granules thus prepared were extruded in suitable amounts to be purified or powdered or filled into hard capsules to prepare hard capsule products.
<시험예 1>≪ Test Example 1 >
IRT5IRT5 에 의한 3By 3 T3T3 -- L1L1 지방세포 분화억제능력 측정 Ability to inhibit adipocyte differentiation
1-1. 3T3-L1 지방세포 분화 유도 1-1. Induction of 3T3-L1 adipocyte differentiation
American Type Culture Collection(ATCC)에서 구입한 3T3-L1 mouse embryonic fibroblast를 10% fetal bovine serum(FBS)이 포함된 Dulbecco's modified Eagle's medium(DMEM)에서 배양하였다. 3T3-L1 mouse embryonic fibroblast를 지방 세포로 분화시키기 위한 절차를 나열하면 다음과 같다. 3T3-L1 mouse embryonic fibroblast가 confluent하게 자란 시점(day 0)으로부터 2일 후에 배양 배지를 10% FBS, 1㎍/㎖ insulin, 0.5mM isobutylmethylxanthine(IBMX) 및 1μM dexamethasone가 포함된 DMEM 배지(분화 배지)로 교환하여 배양하였다. 이로부터 2일 후(day 2)에 10% FBS와 1㎍/㎖ 인슐린(insulin)이 포함된 DMEM 배지로 교환하여 배양하였다. 이로부터 2일 후(day 4)에 10% FBS만 포함된 배지로 교환하여 배양하였다. 이로부터 4일 후(day 8)에 3T3-L1 세포의 90% 이상이 지방세포로 완전히 분화되었다고 볼 수 있다. 3T3-L1 mouse embryonic fibroblast가 지방세포로 완전히 분화되기 전(day 0 ~ day 8)까지를 성숙 중인 3T3-L1 지방전구세포(maturing 3T3-L1 pre-adipocyte)라고 명명하고, 지방세포로 완전히 분화된 후(day 8 이후)에는 성숙된 3T3-L1 지방세포(mature 3T3-L1 adipocyte)라고 명명한다. 세포 배양 조건은 5% CO2, 37℃이다.
3T3-L1 mouse embryonic fibroblasts purchased from the American Type Culture Collection (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). The procedure for differentiating 3T3-L1 mouse embryonic fibroblasts into adipocytes is as follows. After 2 days from the time of confluent growth of 3T3-L1 mouse embryonic fibroblast (day 0), the culture medium was inoculated into DMEM medium (differentiation medium) containing 10% FBS, 1 / / ㎖ insulin, 0.5 mM isobutylmethylxanthine (IBMX) and 1 MM dexamethasone, And cultured. After 2 days, the cells were cultured in DMEM medium supplemented with 10% FBS and 1 μg / ml insulin. After 2 days (day 4), the medium was replaced with a medium containing only 10% FBS. Four days later, on day 8, more than 90% of 3T3-L1 cells were completely differentiated into adipocytes. The 3T3-L1 mouse embryonic fibroblast was termed maturing 3T3-L1 pre-adipocyte (maturing 3T3-L1 pre-adipocyte) before completely differentiating into adipocytes (
1-2. IRT5의 세포 추출물 확보 1-2. Obtain cell extract of IRT5
①비피도박테리움 비피덤 ( Bifidobacterium bifdum ) HY8308 세포추출물 ① Bifidobacterium BP bonus (Bifidobacterium bifdum ) HY8308 cell extract
본 발명의 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308을 BL 액체배지에서 혐기조건 하에 37℃, 20시간 동안 배양하였다. 균체를 원심 분리한 후 PBS로 2~3번 세척한 후 남아있는 배지 성분을 제거하였다. 균체를 동결건조해서 분말화 시킨 후 연속희석법(serial dilution)의 과정을 거쳐 균수를 측정하였다. 균체의 농도가 1010cfu/㎖이 되도록 PBS에 리서스펜션(resuspension)한 후 소니케이션(sonication)하여 균체를 용균(lysis)하였다. 이를 원심분리한 후 상층액을 유산균 세포 추출물로 시험에 사용하였다.
Bifidobacterium bifidum HY8308 of the present invention was cultured in BL liquid medium under anaerobic conditions at 37 DEG C for 20 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 10 cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.
②락토바실러스 애시도필러스 ( Lactobacillus acidophilus ) HY7038 세포추출물 ② Lactobacillus Ash's also a filler (Lactobacillus acidophilus ) HY7038 cell extract
본 발명의 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038을 MRS 액체배지(Difco)에서 37℃, 20시간 동안 배양하였다. 균체를 원심 분리한 후 PBS로 2~3번 세척한 후 남아있는 배지 성분을 제거하였다. 균체를 동결건조해서 분말화 시킨 후 연속희석법(serial dilution)의 과정을 거쳐 균수를 측정하였다. 균체의 농도가 1010cfu/㎖이 되도록 PBS에 리서스펜션(resuspension)한 후 소니케이션(sonication)하여 균체를 용균(lysis)하였다. 이를 원심분리한 후 상층액을 유산균 세포 추출물로 시험에 사용하였다.
Lactobacillus acidophilus HY7038 of the present invention was cultured in an MRS liquid medium (Difco) at 37 DEG C for 20 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 10 cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.
③스트렙토코커스 써모필러스 ( Streptococcus thermophilus ) HY9008 세포추출물 ③ Streptococcus Thermo's filler (Streptococcus thermophilus ) HY9008 cell extract
본 발명의 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008을 M17 액체배지(Difco)에서 42℃, 16시간 동안 배양하였다. 균체를 원심 분리한 후 PBS로 2~3번 세척한 후 남아있는 배지 성분을 제거하였다. 균체를 동결건조해서 분말화 시킨 후 연속희석법(serial dilution)의 과정을 거쳐 균수를 측정하였다. 균체의 농도가 1010cfu/㎖이 되도록 PBS에 리서스펜션(resuspension)한 후 소니케이션(sonication)하여 균체를 용균(lysis)하였다. 이를 원심분리한 후 상층액을 유산균 세포 추출물로 시험에 사용하였다.
Streptococcus Lactococcus of the invention Thermo-pillar's (Streptococcus thermophilus HY9008 was cultured in M17 liquid medium (Difco) at 42 DEG C for 16 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 10 cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.
④락토바실러스 루테리 ( Lactobacillus reuteri ) HY7508 세포추출물 ④ Lactobacillus Ruteri (Lactobacillus reuteri ) HY7508 cell extract
본 발명의 락토바실러스 루테리(Lactobacillus reuteri) HY7508을 MRS 액체배지(Difco)에서 37℃, 20시간 동안 배양하였다. 균체를 원심 분리한 후 PBS로 2~3번 세척한 후 남아있는 배지 성분을 제거하였다. 균체를 동결건조해서 분말화 시킨 후 연속희석법(serial dilution)의 과정을 거쳐 균수를 측정하였다. 균체의 농도가 1010cfu/㎖이 되도록 PBS에 리서스펜션(resuspension)한 후 소니케이션(sonication)하여 균체를 용균(lysis)하였다. 이를 원심분리한 후 상층액을 유산균 세포 추출물로 시험에 사용하였다.
The Lactobacillus lutea of the present invention reuteri HY7508 was cultured in MRS liquid medium (Difco) at 37 캜 for 20 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 10 cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.
⑤락토바실러스 카제이 ( Lactobacillus casei ) HY7208 세포추출물 ⑤ Lactobacillus Casei (Lactobacillus casei ) HY7208 cell extract
본 발명의 락토바실러스 카제이(Lactobacillus casei) HY7208을 MRS 액체배지(Difco)에서 37℃, 20시간 동안 배양하였다. 균체를 원심 분리한 후 PBS로 2~3번 세척한 후 남아있는 배지 성분을 제거하였다. 균체를 동결건조해서 분말화 시킨 후 연속희석법(serial dilution)의 과정을 거쳐 균수를 측정하였다. 균체의 농도가 1010cfu/㎖이 되도록 PBS에 리서스펜션(resuspension)한 후 소니케이션(sonication)하여 균체를 용균(lysis)하였다. 이를 원심분리한 후 상층액을 유산균 세포 추출물로 시험에 사용하였다.
Lactobacillus casei HY7208 of the present invention was cultured in MRS liquid medium (Difco) at 37 DEG C for 20 hours. The cells were centrifuged, washed with PBS 2-3 times, and the remaining medium was removed. The cells were lyophilized and pulverized, and then the number of bacteria was measured by serial dilution. The cells were resuspended in PBS to a concentration of 10 10 cfu / ml and sonicated to lysis the cells. After centrifugation, the supernatant was used for the test as a lactic acid bacterium cell extract.
⑥IRT5의 세포 추출물 ⑥ Cell extract of IRT5
상기 ① 내지 ⑤의 각각의 유산균 세포추출물을 각각 동일한 중량비(1:1:1:1:1:1:1)로 혼합하여 본 발명의 IRT5의 세포추출물로 시험에 사용하였다.
Each of the lactic acid bacterial cell extracts of (1) to (5) was mixed at the same weight ratios (1: 1: 1: 1: 1: 1: 1) and used as a cell extract of IRT5 of the present invention.
1-3. 3T3-L1 지방세포내 지방축적 억제 능력 측정(AdipoRed assay) 1-3. 3T3-L1 AdipoRed assay for the ability to inhibit fat accumulation in adipocytes
(1)96-well plate에서 배양한 3T3-L1 지방세포에 분화 배지를 처리하는 시점(day 0)에 상기 시험예 1-2의 IRT5 세포 추출물을 1%(v/v)로 함께 처리하였다. 이로부터 6일 후(day 6) 생성된 성숙 중인 3T3-L1 지방전구세포(maturing 3T3-L1 pre-adipocyte)를 PBS로 헹궈서 배지 성분을 제거하고 200㎕ PBS와 5㎕ AdipoRed Assay Reagent(Lonza)를 처리하였다. AdipoRed 시약은 지방 덩어리에 끼어 들어가서 형광을 나타내기 때문에 세포의 지방량을 측정하기에 매우 유용하다. 10분 동안 실온에서 반응시킨 후 형광검출기로 형광을 측정(excitation 485nm, emission 572nm)하였다.
(1) 3T3-L1 adipocytes cultured on a 96-well plate were treated with 1% (v / v) of the IRT5 cell extract of Test Example 1-2 at the time of treatment of the differentiation medium (day 0). After 6 days, the mature 3T3-L1 preadipocyte produced by maturation was rinsed with PBS to remove media components and 200 μl PBS and 5 μl AdipoRed Assay Reagent (Lonza) Respectively. AdipoRed reagents are very useful for measuring the amount of fat in a cell because they enter the fat mass and display fluorescence. After incubation at room temperature for 10 minutes, the fluorescence was measured with a fluorescence detector (excitation 485 nm, emission 572 nm).
(2)상기 (1)의 IRT5 대신에 상기 시험예 1-2의 각각 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 및 락토바실러스 카제이(Lactobacillus casei) HY7208 세포 추출물을 사용한 것을 제외하고는 상기 (1)과 동일한 시험방법으로 시험을 하였다.(2) In above (1), respectively Bifidobacterium bipyridinium bushes (Bifidobacterium bifidum) HY8308, Lactobacillus ash of the Test Example 1-2 instead IRT5 FIG filler's (Lactobacillus acidophilus) of HY7038, Streptococcus Thermo filler's (Streptococcus The test was carried out in the same manner as in (1) above, except that thermophilus HY9008, Lactobacillus reuteri HY7508 and Lactobacillus casei HY7208 cell extracts were used.
그 결과를 도 1에 나타내었다.The results are shown in Fig.
'대조군'은 3T3-L1 지방세포에 아무것도 처리하지 않은 군을 나타내며, The " control group " represents a group in which 3T3-L1 adipocytes were not treated with anything,
'HY8308'은 3T3-L1 지방세포에 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308을 처리한 군을 나타내며,&Quot; HY8308 " is a 3T3-L1 adipocyte cell line that expresses Bifidobacterium bifidum ) treated with HY8308,
'HY7038'은 3T3-L1 지방세포에 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038을 처리한 군을 나타내고,&Quot; HY7038 " represents 3T3-L1 adipocytes treated with Lactobacillus acidophilus HY7038,
'HY9008'은 3T3-L1 지방세포에 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008을 처리한 군을 나타내며,'HY9008' represents 3T3-L1 adipocytes treated with Streptococcus thermophilus HY9008,
'HY7508'은 3T3-L1 지방세포에 락토바실러스 루테리(Lactobacillus reuteri) HY7508을 처리한 군을 나타내고,&Quot; HY7508 " is a 3T3-L1 adipocyte infected with Lactobacillus < RTI ID = 0.0 & reuteri ) HY7508 treated group,
'HY7208'은 3T3-L1 지방세포에 락토바실러스 카제이(Lactobacillus casei) HY7208을 처리한 군을 나타내며,'HY7208' represents 3T3-L1 adipocytes treated with Lactobacillus casei HY7208,
'IRT5군'은 3T3-L1 지방세포에 IRT5를 처리한 군을 나타낸다.
'IRT5 group' represents 3T3-L1 adipocytes treated with IRT5.
도 1에서 확인할 수 있는 바와 같이, 대조군에 비하여 비피도박테리움 비피덤(Bifidobacterium bifidum) HY8308 세포 추출물을 처리한 군(HY8308)은 16%, 락토바실러스 애시도필러스(Lactobacillus acidophilus) HY7038 세포 추출물을 처리한 군(HY7038)은 25%, 스트렙토코커스 써모필러스(Streptococcus thermophilus) HY9008 세포 추출물을 처리한 군(HY9008)은 11%, 락토바실러스 루테리(Lactobacillus reuteri) HY7508 세포 추출물을 처리한 군(HY7508)은 29%, 락토바실러스 카제이(Lactobacillus casei) HY7208 세포 추출물을 처리한 군(HY7208)은 16%의 지방축적 정도가 감소하였다. 그러나 5종의 유산균 복합균주 세포 추출물을 처리한 군(IRT5군)은 40%의 지방축적 정도가 감소하여 상기 각각의 유산균주 단독으로 처리를 하였을 때 보다 5종의 유산균를 복합으로 처리를 하였을 때 지방축적 정도 감소효과(지방세포 분화억제능력)가 더 크게 나타남을 알 수 있었다.
As can be seen from FIG. 1, 16% of the group treated with Bifidobacterium bifidum HY8308 cell extract (HY8308) and 16% of Lactobacillus acidophilus HY7038 cell extract treated group (HY7038) by 25%, Streptococcus Thermo filler's (Streptococcus thermophilus) HY9008 group treated cell extracts (HY9008) is 11%, Lactobacillus ruteri (Lactobacillus reuteri) group (HY7508) treated with HY7508 cell extract is 29%, Lactobacillus casei (Lactobacillus casei) HY7208 group treated cell extracts (HY7208) decreased the extent of 16% fat. However, in the group treated with 5 kinds of lactic acid bacterial cell extracts (IRT5 group), the degree of fat accumulation was decreased by 40%. When 5 kinds of lactic acid bacteria were treated with each of the lactic acid bacteria alone, (Ability to inhibit the differentiation of adipocytes).
<시험예 2>≪ Test Example 2 &
IRT5IRT5 에 의한 체지방 감소능력 측정Measurement of body fat reduction ability by
2-1. 체지방 감소 능력 측정 2-1. Body fat reduction ability measurement
실험동물은 5주령의 C57BL/6J 마우스를 SJC(Japan)로부터 구입하여 1주간 순치 후 이용하였다. 8주간 실험소재를 혼합한 식이 및 멸균수를 자유급식 시키며 매주 1회 식이량 및 체중을 측정하였다.Five-week-old C57BL / 6J mice were purchased from SJC (Japan) and used for 1 week. Diet and sterilized water mixed with experimental material for 8 weeks were fed free and diet and body weight were measured once a week.
대조군으로는 정상식이를 먹인 정상식이군(정상식이군, n=8), 고지방식이를 먹은 고지방식이군(고지방식이군, n=8)을 이용하였고, 실험군은 고지방식이에 가르시니아 캄보지아를 급여한 군(가르시니아 캄보지아군, n=8), 고지방식이에 5종의 유산균 복합균주를 급여한 군(IRT5군, n=8)으로 구성하였다.The control group was a normal diet (normal diet, n = 8), high fat diet (high fat diet, n = 8), and the experimental group was fed high fat diet (Egginae cambogia) (N = 8), and the high fat diet group (n = 8) were fed with five lactic acid bacterial strains (IRT5 group, n = 8).
실험동물의 사료는 표 5와 같은 조성으로 제조하였다. 정상식이군을 제외한, 고지방식이군, 가르시니아 캄보지아군, IRT5군에 대해서는 8주간 고지방식이를 급여하여 비만을 유도하였으며, 가르시니아 캄보지아군, IRT5군에 대해서는 각각 가르시니아 캄보지아(100mg/day)와 5종의 유산균 복합균주(5 × 109cfu/day)를 8주간 경구투여 하였다.
The experimental animal feeds were prepared as shown in Table 5. In the case of Garcinia cambogia and IRT5, the effects of garcinia cambogia (100 mg / day) and 5 kinds of insecticides were investigated in the high fat diet group, Garcinia cambogia group and IRT5 group for 8 weeks, (5 × 10 9 cfu / day) was orally administered for 8 weeks.
체중 및 식이량은 주 1회 측정하였으며 실험 기간이 종료된 후 실험동물은 12시간 절식시키고, zoletil 및 ethyl ether로 과량 마취하여 희생시켰다. 실험동물을 개복하여 간문맥에서 전혈을 채취하고 혈장을 분리하였으며, 지방조직을 적출하여 무게를 측정하였다. 혈장 및 조직 샘플은 사용시까지 영하 80℃에서 보관하였다.Body weight and dietary dose were measured once a week. After the end of the experimental period, the animals were fasted for 12 hours and sacrificed by anesthesia with zolethyl and ethyl ether. Whole blood was collected from the portal vein, plasma was separated, and adipose tissue was extracted and weighed. Plasma and tissue samples were stored at -80 ° C until use.
그 결과를 도 2(총지방량), 도 3(부고환지방량), 도 4(피하지방량) 및 도 5(장간만지방량)에 나타내었다.
The results are shown in Fig. 2 (total fat mass), Fig. 3 (epididymol fat mass), Fig. 4 (subcutaneous fat mass) and Fig.
'a': AIN-76 mineral mixture, 'b': AIN-76 vitamin mixture
'a': AIN-76 mineral mixture, 'b': AIN-76 vitamin mixture
도 2에서 확인 할 수 있는 바와 같이, 총지방량은 고지방 식이군에서 16.37g이었으나, IRT5군에서는 13.55g으로 유의적으로 감소하였으며(p<0.01), 특히 가르시니아 캄보지아군의 14.68g의 지방량 보다 더 크게 감소하였다.
As can be seen from FIG. 2, the total fat content was 16.37 g in the high fat diet group but 13.55 g in the IRT 5 group (p <0.01), and was especially larger than the fat content of 14.68 g in the Garcinia cambogia group Respectively.
도 3에서 확인 할 수 있는 바와 같이, 부고환지방량은 고지방 식이군에서 10.37g이었으나, IRT5군에서는 7.98g으로 유의적으로 감소하였다(p<0.01).
As can be seen in FIG. 3, the epididymal fat content was 10.37 g in the high fat dietary group, but was significantly reduced to 7.98 g in the IRT 5 group (p <0.01).
도 4에서 확인 할 수 있는 바와 같이, 피하지방량은 고지방 식이군에서 3.91g이었으나, IRT5군에서는 2.84g으로 유의적으로 감소하였다(p<0.05).
As can be seen from FIG. 4, the subcutaneous fat content was 3.91 g in the high fat diet group, but significantly decreased to 2.84 g in the IRT 5 group (p <0.05).
도 5에서 확인 할 수 있는 바와 같이, 장간막지방량은 고지방 식이군에서 2.24g이었으나, IRT5군에서는 1.70g으로 유의적으로 감소하였다(p<0.05).
As shown in FIG. 5, the amount of mesentery fat was 2.24 g in the high fat dietary group, but it was 1.70 g in the IRT 5 group (p <0.05).
2-2. 혈중 렙틴 측정 2-2. Blood leptin measurement
렙틴은 비만유전자로 알려져 있으며, 주로 지방세포에서 발현된다. 지방조직에 중성지방이 축적되어 지방세포의 크기가 증가하면 렙틴 합성이 촉진된다. 따라서, IRT5에 의한 체지방 감소능력을 측정하기 위하여 혈중 렙틴 농도를 측정하였다. 렙틴 농도는 Mouse leptin ELISA(enzyme-linked immunosorbent assay) kit(Biovendor, Czech)을 이용하여 측정하였다.Leptin is known to be an obesity gene and is mainly expressed in adipocytes. The accumulation of triglycerides in adipose tissue increases the size of adipocytes and promotes leptin synthesis. Therefore, blood leptin concentration was measured to measure body fat reduction ability by IRT5. Leptin concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Biovendor, Czech).
그 결과를 도 6에 나타내었다.
The results are shown in Fig.
도 6에서 확인할 수 있는 바와 같이, 혈중 렙틴의 양은 고지방 식이군에서 15.88ng/㎖이었으나, IRT5군에서는 12.10ng/㎖으로 유의적으로 감소하였다(p<0.01).
As shown in FIG. 6, the amount of leptin in the blood was 15.88 ng / ml in the high fat diet group, but 12.10 ng / ml in the IRT5 group (p <0.01).
2-3. 마우스 간 조직 및 지방 조직에서 5종의 유산균 복합균주에 의한 마커 유전자 유의적 발현 차이 측정 2-3. Differential Expression of Marker Gene by Five Lactic Acid Microbial Strain in Mouse Liver and Adipose Tissue
상기 시험예 2-1의 마우스로부터 적출한 간조직의 지방합성 관련 유전자 FASN과 SCD1, 지방분해효소 관련 유전자 HSL, 지방산화관련 유전자 CPT2, 그리고 GAPDH(실험결과를 보정하기 위한 house-keeping 유전자) 유전자들의 발현 차이를 리얼타임(Real time) PCR을 통해 측정하였다.The gene for FASN and SCD1, the lipolytic enzyme-related gene HSL, the lipid oxidation-related gene CPT2, and the GAPDH (house-keeping gene for correcting the experimental results) genes derived from the mouse of Test Example 2-1 Were measured by real time PCR.
우선, RNA queous kit(Ambion, AM1912)를 이용하여 간조직으로부터 RNA를 추출하고, High Capacity RNA-to-cDNA kit(Applied Biosystems, 4387406)를 이용하여 RNA에 대한 상보적인 DNA(complimentary DNA, 이하 'cDNA'라 함)를 확보하였다. 하기의 표 6과 같이 구입한 택맨프로브(Taqman probes, Applied Biosystems 사에서 구입)와 상기 cDNA를 이용하여 리얼타임 RCR(Applied Biosystems, 7500 Real time PCR)로 RNA 발현양을 정량하였다. 부고환지방 조직에서는 지방합성 관련 유전자 FASN, 지방분해효소 관련 유전자 HSL, 지방산화 관련 유전자 CPT2, 열생성 관련 유전자 UCP1에 대해, 그리고 견갑골 갈색지방 조직에서는 지방산화 관련 유전자 PPARα, CPT1, CPT2 및 ACOX1, 열생성관련 유전자 UCP1 및 UCP2에 대해 간조직의 지방대사 관련 유전자 분석과 동일한 방법으로 리얼타임 PCR을 통해 측정하였다. First, RNA was extracted from liver tissues using an RNA queous kit (Ambion, AM1912) and complementary DNA (hereinafter referred to as "complement DNA") was extracted from the RNA using a High Capacity RNA-to-cDNA kit (Applied Biosystems, 4387406) quot; cDNA "). The amount of RNA expression was quantified by real-time RCR (Applied Biosystems, 7500 Real time PCR) using the purchased Taqman probes (purchased from Applied Biosystems) and the cDNAs as shown in Table 6 below. In epididymal adipose tissue, the fat synthesis related gene FASN, the lipolytic enzyme related gene HSL, the lipid oxidation related gene CPT2, the heat production related gene UCP1, and the fatty lipid oxidation genes PPARα, CPT1, CPT2 and ACOX1, Generation related genes UCP1 and UCP2 were measured by real time PCR in the same manner as the analysis of lipid metabolism related genes in liver tissues.
그 결과를 도 7(간 조직내 mRNA 발현), 도 8(부고환지방 조직내 mRNA 발현) 및 도 9(견갑골 갈색지방 조직내 mRNA 발현)에 나타내었다.
The results are shown in Fig. 7 (mRNA expression in liver tissue), Fig. 8 (mRNA expression in epididymal adipose tissue) and Fig. 9 (mRNA expression in scapular brown fat adipose tissue).
도 7에서 확인할 수 있는 바와 같이, 고지방식이군과 비교하여 IRT5군에서는 지방합성 관련된 FASN과 SCD1 유전자의 발현이 유의적으로 감소하였으며(p<0.05), 반대로 지방분해와 관련된 HSL 유전자와 지방산 산화와 관련된 CPT2 유전자의 발현은 유의적으로 증가하였다(p<0.05).
As shown in FIG. 7, the expression of FASN and SCD1 gene in the IRT5 group was significantly decreased (p <0.05) compared with that of the high fat diet group. On the contrary, the expression of HSL gene and fatty acid oxidation Expression of the related CPT2 gene was significantly increased (p <0.05).
도 8에서 확인할 수 있는 바와 같이, 고지방식이군과 비교하여 IRT5군에서는 지방합성 관련된 FASN 유전자의 발현이 유의적으로 감소하였으며(p<0.05), 반대로 지방분해와 관련된 HSL 유전자와 지방산 산화와 관련된 CPT2 유전자의 발현은 유의적으로 증가하였다(p<0.05). 또한 지방산 산화에 의한 열 발생과 관련된 UCP1 유전자의 발현이 유의적으로 증가하였다(p<0.05).
As can be seen in FIG. 8, the expression of FASN gene associated with lipogenesis was significantly decreased (p <0.05) in the IRT5 group compared with the high fat diet group, whereas the HSL gene related to lipolysis and CPT2 Expression of the gene was significantly increased (p <0.05). In addition, the expression of UCP1 gene related to heat generation by fatty acid oxidation was significantly increased (p <0.05).
도 9에서 확인할 수 있는 바와 같이, 고지방식이군과 비교하여 IRT5군에서는 견갑골 갈색지방 조직내 지방산화에 관련된 CPT1, CPT2 및 ACOX1 유전자의 발현이 증가하였으며(p<0.01), 지방산 산화에 의한 열발생과 관련된 UCP1과 UCP2 유전자의 발현이 유의적으로 증가하였다(p<0.05).
As shown in FIG. 9, the expression of CPT1, CPT2 and ACOX1 genes related to lipid oxidation in scapula brown fat tissue was increased (p <0.01) in the IRT5 group compared to the high fat diet group UCP1 and UCP2 gene expression were significantly increased (p <0.05).
이상의 시험예 2-3의 시험결과를 종합하여 보면, 5종의 유산균 복합균주를 섭취한 군(IRT5군)에서 고지방식이만을 섭취한 군(고지방식이군)과 비교하여 체내 지방합성과 관련된 유전자들(FASN과 SCD1 유전자)의 발현이 감소되고, 섭취된 지방의 분해 또는 산화와 관련된 유전자들(HSL, ACOX1, CPT1, CPT2 유전자)의 발현이 증가되어 체내 지방축적을 감소시키는 효과를 나타냄을 알 수 있었다. 또한 지방분해 및 산화 이외에도 체내 지방을 연소시켜 에너지 및 열을 발생시키는데 관여하는 유전자들(UCP1과 UCP2 유전자)의 발현을 증가시켜 체내 지방축적을 감소시키는 효과도 있는 것으로 확인되었다.
In summary, the test results of Test Example 2-3 were compared with those of the group fed only with the high fat diet (high fat diet group) in the group fed with the five kinds of lactic acid bacteria complex (IRT5 group) (HSL, ACOX1, CPT1, and CPT2 genes) associated with the degradation or oxidation of the fat that is consumed is decreased and the accumulation of fat in the body is reduced by decreasing the expression of FASN and SCD1 gene I could. In addition to lipid degradation and oxidation, it was also found to have an effect of reducing body fat accumulation by increasing the expression of genes (UCP1 and UCP2 gene) involved in the generation of energy and heat by burning fat in the body.
Claims (5)
Bifidobacterium bifidum HY8308 (Accession No .: KCTC 12563BP), Lactobacillus acidophilus HY7038 (Accession No .: KCTC 12560BP), Streptococcus thermophilus HY9008 (Deposit number: (KCTC 12564BP), Lactobacillus reuteri HY7508 (Accession No .: KCTC 12562BP), and Lactobacillus casei HY7208 (Accession No .: KCTC 12561BP) as active ingredients Or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for treating or preventing obesity through the reduction of body fat.
Bifidobacterium bifidum HY8308 (Accession No .: KCTC 12563BP), Lactobacillus acidophilus HY7038 (Accession No .: KCTC 12560BP), Streptococcus thermophilus HY9008 (Deposit number: (KCTC 12564BP), Lactobacillus reuteri HY7508 (Accession No .: KCTC 12562BP), and Lactobacillus casei HY7208 (Accession No .: KCTC 12561BP) as active ingredients Or a pharmaceutically acceptable salt thereof. The composition for improving or preventing obesity through reduction of body fat.
Bifidobacterium bifidum HY8308 (Accession No .: KCTC 12563BP), Lactobacillus acidophilus HY7038 (Accession No .: KCTC 12560BP), Streptococcus thermophilus HY9008 (Deposit number: (KCTC 12564BP), Lactobacillus reuteri HY7508 (Accession No .: KCTC 12562BP), and Lactobacillus casei HY7208 (Accession No .: KCTC 12561BP) as active ingredients Wherein the fermented milk is used for improving or preventing obesity through reduction of body fat.
Bifidobacterium bifidum HY8308 (Accession No .: KCTC 12563BP), Lactobacillus acidophilus HY7038 (Accession No .: KCTC 12560BP), Streptococcus thermophilus HY9008 (Deposit number: (KCTC 12564BP), Lactobacillus reuteri HY7508 (Accession No .: KCTC 12562BP), and Lactobacillus casei HY7208 (Accession No .: KCTC 12561BP) as active ingredients A functional drink for improving or preventing obesity through reduction of body fat.
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