KR102454241B1 - Effect of Lactobacillus plantarum LRCC5282 on alleviates obesity and its application - Google Patents
Effect of Lactobacillus plantarum LRCC5282 on alleviates obesity and its application Download PDFInfo
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- KR102454241B1 KR102454241B1 KR1020210067514A KR20210067514A KR102454241B1 KR 102454241 B1 KR102454241 B1 KR 102454241B1 KR 1020210067514 A KR1020210067514 A KR 1020210067514A KR 20210067514 A KR20210067514 A KR 20210067514A KR 102454241 B1 KR102454241 B1 KR 102454241B1
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/332—Promoters of weight control and weight loss
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- A23Y2220/67—
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
Abstract
Description
본 발명은 체지방 감소 효과를 가지는 락토바실러스 플랜타럼 균주 LRCC5282 및 이의 용도에 관한 것이다.The present invention relates to Lactobacillus plantarum strain LRCC5282 having a body fat reduction effect and uses thereof.
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 메커니즘을 가지고 있다.The treatment methods for obesity include diet-exercise therapy, surgery therapy, and drug therapy. Diet-exercise therapy is a treatment method through low-calorie-low-fat intake and physical activity that consumes oxygen, and it is recognized that it is difficult to see a popular effect because it must be performed repeatedly and continuously with patience. Surgical therapy is a method of physically removing body fat through surgical operation, which has the advantage of being effective in a short period of time, but has limited use due to the need for surgery, lack of continuity of effect, and high cost. is becoming Drug therapy is a method for treating or preventing obesity using drugs that induce a decrease in appetite or inhibit fat absorption. Drugs for the prevention and treatment of obesity developed so far 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 inhibitors (Orlistat) are known as fat absorption inhibitory drugs, which inhibit the action of pancreatic lipolytic enzymes to reduce fat absorption or interfere with the absorption of fat-soluble vitamins, and may cause breast cancer. .
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 suppress appetite mainly act on neurotransmitters (catecholamines) present in the brain to decrease appetite. However, dexfenfluramine and fenfluramine have side effects of neurotoxicity and valvular heart disease, and sibutramine has side effects of increasing 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.Excessive fat accumulation is caused by an imbalance in metabolism caused by an increase in the amount of calories consumed in the body and a decrease in the amount of energy consumed, which is indicated by an increase in the size and number of fat cells. 3T3-L1 adipocytes are mouse embryonic fibroblasts, a cell line widely used for biological studies of adipose tissue. As the liposynthesis signaling pathway in 3T3-L1 mouse embryonic fibroblasts is activated, the amount of intracellular fat increases and the cell shape changes to a circular shape. This process of changing intracellular signaling pathways and cell shape is called differentiation, and 3T3-L1 cells undergoing differentiation are referred to as 'maturing 3T3-L1 pre-adipocytes'. The completed 3T3-L1 cells are referred to as 'mature 3T3-L1 adipocytes'. Researches to search for substances that inhibit the adipogenesis of these 3T3-L1 cells are being conducted in each country. For example, green tea polyphenol component epigallocatechin gallate (Obesity res. 2005;13:982-90), red wine component resveratrol (Phytother Res. 2008; 22:1367-71), garlic component ajoene (Phytother Res. 2009) ;23:513-8) and others have been reported to have the effect of inhibiting adipogenesis in 3T3-L1 cells. Substances that inhibit the differentiation of 3T3-L1 cells, that is, the synthesis of fat in adipocytes, can be considered to have anti-obesity effects, so they are very useful in the fields of food and medicine.
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 β산화를 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 for reducing fat accumulation in the body. The first is a mechanism to inhibit the accumulation of body fat by inhibiting the absorption of fat ingested from the diet. This inhibition of fat absorption is possible either by inhibiting the action of lipolytic enzymes that break down triglycerides in the small intestine, or by adsorbing ingested fat and enhancing the excretion of fat in the feces. The second is the mechanism of controlling body fat by accelerating oxidation of ingested fat or body fat accumulated in the body. By increasing the activity of enzymes involved in fatty acid oxidation, the movement of fatty acids to the mitochondria is promoted, and thus β oxidation in the mitochondria is promoted, thereby 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, which are enzymes related to fat synthesis.
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 β산화의 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 consumption of energy in the body, the combustion process that directly decomposes the accumulated fat or oxidizes the fat to generate energy and heat is important, and many enzymes and genes are involved in this. Hormone sensitive lipase is the most important enzyme in the step of decomposing triglycerides in adipose tissue, and in order for fatty acids to be decomposed and used as energy, they must move to the mitochondria, a place of energy generation. In order for the fatty acid (acyl CoA) activated in the cytoplasm to move into the mitochondria, it must pass through the mitochondrial membrane (outer and inner membranes). The enzyme that catalyzes this process is Carnitine palmitoyltransferase (CPT), and there are CPT I and CPT II. And 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 , acting in the first step of fatty acid β oxidation. It is a rate-limiting enzyme. Mitochondrial uncoupling protein (UCP) is found in brown fat, white fat, and muscle cells. UCPs reduce ATP production and generate heat by uncoupling the oxidative phosphorylation process in the mitochondria of the corresponding cell. Therefore, it has been reported that the level of UCP expression affects energy consumption and is associated with obesity. In fact, there are studies showing that obese people tend to have less UCP expression.
지방조직의 분화는 근육세포 분화나 신경세포 분화와는 달리 여러 호르몬과 다양한 전사인자들의 상호작용을 통하여 매우 복잡하게 이루어진다. 지방전구세포가 지방세포로 분화해 나가는 과정에는 세포의 형태적 변화와 유전자 발현양상의 변화 등이 함께 일어난다. 대부분의 경우 이러한 변화는 전사단계에서 각 유전자의 발현양의 변화를 나타내는데, 지방세포의 분화조절은 C/EBP, PPAR, r/RXR, ADD1/SREBP 1C라고 불리는 전사인자 (transcription factor)가 중추적인 역할을 담당하고 있다. 이들 전사인자는 지방세포 분화 과정 중 각기 다른 시점에서 발현이 유도되며 서로 상호작용을 통하여 여러 지방세포 특이유전자들의 발현을 조절하고, 지방대사의 활성화와 지방세포 분화를 점진적으로 유도해 나간다Differentiation of adipose tissue is very complicated through the interaction of various hormones and various transcription factors, unlike muscle cell differentiation or nerve cell differentiation. In the process of differentiation of preadipocytes into adipocytes, cell morphological changes and gene expression changes occur together. In most cases, these changes indicate changes in the expression level of each gene at the transcriptional stage, and the regulation of differentiation of adipocytes is driven by transcription factors called C/EBP, PPAR, r/RXR, and ADD1/SREBP 1C. is playing a role. Expression of these transcription factors is induced at different times during the adipocyte differentiation process, and through interaction with each other, they control the expression of various adipocyte-specific genes, and gradually induce fat metabolism and adipocyte differentiation.
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, recent efforts to prevent or treat obesity using lactic acid bacteria, which have been considered safe microorganisms, are also being made. It is reported that lactic acid bacteria exhibit effects such as maintenance of normal intestinal flora, improvement of intestinal flora, anti-diabetic and anti-hyperlipidemic effects, suppression of carcinogenesis, suppression of colitis, and non-specific activity of the host's immune system.
상기와 같은 배경하에서, 본 발명자들은 하기와 같은 신규 미생물 및 이의 용도를 제공하고자 한다. Under the above background, the present inventors intend to provide the following novel microorganisms and uses thereof.
본 발명의 일측면은 락토바실러스 속의 균주로 체지방 합성 억제 및 분해 촉진을 통해 항비만 효과를 나타내는 신규한 균주 및 이의 제제를 제공하고자 한다. 일 구현예에서 상기 균주는 인 비트로 및 동물 수준에서 체지방 감소 효능을 보유한 균주일 수 있다. 일 구현예에서 상기 균주는 사균체로서 제공될 수 있다. 일 구현예에서 마이크로바이옴 조절력을 보유한 SCFAs 생성력이 우수한 신규 균주일 수 있다.One aspect of the present invention is to provide a novel strain of the genus Lactobacillus, which exhibits an anti-obesity effect by inhibiting body fat synthesis and promoting decomposition, and a preparation thereof. In one embodiment, the strain may be a strain having the effect of reducing body fat both in vitro and at the animal level. In one embodiment, the strain may be provided as a dead cell. In one embodiment, it may be a novel strain with excellent SCFAs-producing ability with microbiome regulatory power.
본 발명의 목적은 기탁번호 KCCM 12896P로 기탁된 락토바실러스 플랜타럼 LRCC5282 (Lactobacillus plantarum LRCC5282) 균주를 제공하기 위한 것이다.It is an object of the present invention to provide a Lactobacillus plantarum LRCC5282 ( Lactobacillus plantarum LRCC5282) strain deposited with accession number KCCM 12896P.
본 발명의 다른 목적은 상기 락토바실러스 플랜타럼 LRCC5282 균주; 이의 파쇄물; 이의 배양물; 이의 발효물; 및 상기 균주, 파쇄물, 배양액 또는 발효물의 추출물; 중 하나 이상을 포함하는 미생물 제제를 제공하고자 한다.Another object of the present invention is the Lactobacillus plantarum LRCC5282 strain; lysate thereof; its culture; its ferment; and extracts of the strains, lysates, cultures or fermented products; It is intended to provide a microbial preparation comprising at least one of
본 발명의 또 다른 목적은 상기 미생물 제제를 포함하는 약학 조성물을 제공하고자 한다.Another object of the present invention is to provide a pharmaceutical composition comprising the microbial agent.
본 발명의 또 하나의 다른 목적은 상기 미생물 제제를 포함하는 건강기능식품 조성물을 제공하고자 한다. 일 구현예에서 상기 식품 조성물은 체지방 감소 음료일 수 있다. Another object of the present invention is to provide a health functional food composition comprising the microbial agent. In one embodiment, the food composition may be a body fat reduction beverage.
본 발명의 목적은 이상에서 언급한 목적으로 제한되지 않는다. 본 발명의 목적은 이하의 설명으로 보다 분명해 질 것이며, 특허청구범위에 기재된 수단 및 그 조합으로 실현될 것이다.The object of the present invention is not limited to the object mentioned above. The objects of the present invention will become more apparent from the following description, and will be realized by means and combinations thereof described in the claims.
상기 목적을 달성하기 위하여, 하기의 해결 수단을 제공한다.In order to achieve the above object, the following solutions are provided.
본 발명의 일측면은 지방세포 분화 억제 효과를 갖는 락토바실러스 플랜타럼 LRCC5282 (Lactobacillus plantarum LRCC5282) 균주를 제공한다.One aspect of the present invention provides a Lactobacillus plantarum LRCC5282 ( Lactobacillus plantarum LRCC5282) strain having an adipocyte differentiation inhibitory effect.
본 발명의 일 측면에 있어서, 상기 균주는 대사증후군의 예방 또는 치료용인, 균주를 제공한다.In one aspect of the present invention, the strain provides a strain for the prevention or treatment of metabolic syndrome.
본 발명의 일 측면에 있어서, 상기 균주는 체지방 감소 효과가 있는, 균주를 제공한다.In one aspect of the present invention, the strain provides a strain having a body fat reduction effect.
본 발명의 일 측면에 있어서, 상기 균주는 서열번호 1의 염기서열을 포함하는 16S rDNA를 갖는, 균주를 제공한다.In one aspect of the present invention, the strain provides a strain having 16S rDNA comprising the nucleotide sequence of SEQ ID NO: 1.
본 발명의 일 측면에 있어서, 상기 균주는 락토바실러스 플랜타럼 LRCC5282 균주를 제공한다.In one aspect of the present invention, the strain provides a Lactobacillus plantarum LRCC5282 strain.
본 발명의 일 측면에 있어서, 상기 균주는 기탁번호가 KCCM 12896P인, 균주를 제공한다.In one aspect of the present invention, the strain provides an accession number KCCM 12896P, the strain.
본 발명의 다른 측면은 상기 본 발명의 일측면 중 어느 하나에 따른 균주; 이의 파쇄물; 이의 배양물; 이의 발효물; 및 상기 균주, 파쇄물, 배양액 또는 발효물의 추출물; 중 하나 이상을 포함하는 미생물 제제를 제공한다.Another aspect of the present invention is the strain according to any one of the aspects of the present invention; lysate thereof; its culture; its ferment; and extracts of the strains, lysates, cultures or fermented products; It provides a microbial preparation comprising one or more of.
본 발명의 다른 측면에 있어서, 상기 균주는 사균체인, 미생물 제제를 제공한다.In another aspect of the present invention, the strain provides a dead cell, microbial preparation.
본 발명의 또 다른 측면은 상기 미생물 제제를 포함하는 대사증후군의 예방, 개선 또는 치료용 약학 조성물을 제공한다.Another aspect of the present invention provides a pharmaceutical composition for preventing, improving or treating metabolic syndrome comprising the microbial agent.
일 구현예에 있어서, 상기 대사증후군은 비만, 이상지질혈증, 고지혈증 및 인슐린 저항성 증후군으로 이루어진 군에서 선택된 어느 하나이다.In one embodiment, the metabolic syndrome is any one selected from the group consisting of obesity, dyslipidemia, hyperlipidemia and insulin resistance syndrome.
본 발명의 또 다른 측면은 상기 미생물 제제를 포함하는 대사증후군의 예방 또는 개선용 식품 조성물을 제공한다.Another aspect of the present invention provides a food composition for preventing or improving metabolic syndrome comprising the microbial agent.
일 구현예에 있어서, 상기 식품 조성물은 항비만 효과를 나타내는 식품 조성물이다. 일 구현예에 있어서, 상기 식품 조성물은 음료인, 바람직하게 체지방 감소 음료이다.In one embodiment, the food composition is a food composition exhibiting an anti-obesity effect. According to one embodiment, the food composition is a beverage, preferably a body fat reducing beverage.
본 발명의 일측면에 따른 균주, 상기 균주등을 포함하는 미생물제제 및 이를 포함하는 약학 조성물 또는 식품 조성물은 체지방 감소 효과가 우수하다.The strain according to one aspect of the present invention, a microbial agent comprising the strain, and a pharmaceutical composition or food composition comprising the same are excellent in reducing body fat.
본 발명의 일측면에 따른 균주, 상기 균주등을 포함하는 미생물제제 및 이를 포함하는 약학 조성물 또는 식품 조성물은 SCFAs 생성력이 우수하다.The strain according to one aspect of the present invention, a microbial agent comprising the strain, and a pharmaceutical or food composition comprising the same have excellent SCFAs production ability.
본 발명의 일측면에 따른 균주, 상기 균주등을 포함하는 미생물제제 및 이를 포함하는 약학 조성물 또는 식품 조성물은 체외 및 동물 수준에서 체지방 감소 효능이 우수하다.The strain according to one aspect of the present invention, a microbial agent comprising the strain, and a pharmaceutical or food composition comprising the same are excellent in body fat reduction efficacy in vitro and at the animal level.
본 발명의 일측면에 따른 균주, 상기 균주등을 포함하는 미생물제제 및 이를 포함하는 약학 조성물 또는 식품 조성물은 사균체로 투여되어도 체지방 감소 효과가 우수하다.The strain according to an aspect of the present invention, a microbial agent comprising the strain, and a pharmaceutical composition or food composition comprising the same are excellent in reducing body fat even when administered as dead cells.
본 발명의 일측면에 따른 균주, 상기 균주등을 포함하는 미생물제제 및 이를 포함하는 약학 조성물 또는 식품 조성물은 지방세포 합성인자의 발현 억제 효과가 우수하다.The strain according to one aspect of the present invention, a microbial preparation comprising the strain, and a pharmaceutical or food composition comprising the same are excellent in the effect of inhibiting the expression of adipocyte synthesizing factors.
본 발명의 효과는 이상에서 언급한 효과로 한정되지 않는다. 본 발명의 효과는 이하의 설명에서 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 할 것이다.The effects of the present invention are not limited to the above-mentioned effects. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.
도 1 및 도 2는 본원발명 실시예 2에 따른 LRCC5282를 다양한 탄소원에서 배양 시 젖산 및 SCFAs 생성능 확인 결과이다. 도 1은 젖산 및 acetic acid의 결과이며, 도 2는 formic acid, propanoic acid 및 butyric acid의 결과이다. 도면에서 Blank: 탄소원 없음; Glu: glucose; Sor: sorbitol; Inu: inulin; Gal: galacto-oligosaccharides; Fru: fructo-oligosaccharides; Iso: isomalto-oligosaccharides 을 나타낸다.
도 3 및 도 4는 본원발명 실시예 3의 본원발명 균주의 당 이용성 분석 결과이다.
도 5는 실시예 5에서 지방분화도 및 중성지방량에 대한 결과이다.
도 6은 실시예 5에서 지방합성 관련 인자에 대한 결과이다.
도 7은 실시예 6-1 비만 실험의 개요이다.
도 8은 실시예 6-1의 혈액학적 분석 결과이며, 도 8a는 ALT, 도 8b는 AST, 도 8c는 총콜레스테롤, 도 8d는 HDL 콜레스테롤, 도 8e는 LDL 콜레스테롤 및 도 8f는 중성지방의 혈액학적 분석 결과이다.
도 9는 실시예 6-1의 BMI 분석 결과이다.
도 10은 실시예 6-1의 조직학적 분석 결과이며, 도 10a는 목부위의 지방조직, 도 10b는 피하 지방조직, 도 10c는 부고환의 지방조직에 대한 분석결과이다.
도 11은 실시예 6-1의 지방분화 관련인자 분석 결과이다. 도 11a는 FAS, 도 11b는 Leptin, 도 11c는 CEBP/α, 도 11d는 SREBP α1, 도 11e는 PPAR/γ의 분석 결과이다.
도 12는 실시예 6-1의 면역학적 분석 결과이다. 도 12a는 IL-6, 도 12b는 CCL2, 도 12c는 IL-10에 대한 분석 결과이다.
도 13은 실시예 6-2 비만 실험의 개요이다.
도 14은 실시예 6-2의 혈액학적 분석 결과이며, 도 14a는 ALT, 도 14b는 AST, 도 14c는 총콜레스테롤, 도 14d는 HDL 콜레스테롤, 도 14e는 LDL 콜레스테롤 및 도 14f는 중성지방의 혈액학적 분석 결과이다.
도 15는 실시예 6-2의 BMI 분석 결과이다.
도 16은 실시예 6-2의 조직학적 분석 결과이며, 도 16a는 목부위의 지방조직, 도 16b는 피하 지방조직, 도 16c는 부고환의 지방조직에 대한 분석결과이다.
도 17은 실시예 6-2의 지방분화 관련인자 분석 결과이다. 도 17a는 FAS, 도 17b는 Leptin, 도 17c는 CEBP/α, 도 17d는 SREBP α1, 도 17e는 PPAR/γ의 분석 결과이다.
도 18는 실시예 6-2의 면역학적 분석 결과이다. 도 18a는 TNF- α, 도 18b는 IL-6, 도 18c는 CCL2, 도 18d는 IL-10에 대한 분석 결과이다.1 and 2 are results of confirming the ability to produce lactic acid and SCFAs when LRCC5282 according to Example 2 of the present invention is cultured in various carbon sources. Figure 1 is the result of lactic acid and acetic acid, Figure 2 is the result of formic acid, propanoic acid and butyric acid. Blank in the drawing: no carbon source; Glu: glucose; Sor: sorbitol; Inu: inulin; Gal: galacto-oligosaccharides; Fru: fructo-oligosaccharides; Iso: Represents isomalto-oligosaccharides.
3 and 4 are results of analysis of sugar availability of the strain of the present invention of Example 3 of the present invention.
5 is a result for the degree of fat differentiation and the amount of triglycerides in Example 5.
6 is a result for lipogenesis-related factors in Example 5.
7 is an outline of Example 6-1 obesity experiment.
Fig. 8 is a result of hematological analysis of Example 6-1, Fig. 8a is ALT, Fig. 8b is AST, Fig. 8c is total cholesterol, Fig. 8d is HDL cholesterol, Fig. 8e is LDL cholesterol, and Fig. 8f is blood of triglycerides. It is the result of scientific analysis.
9 is a BMI analysis result of Example 6-1.
10 is a histological analysis result of Example 6-1, FIG. 10a is an analysis result of adipose tissue of the neck region, FIG. 10b is a subcutaneous adipose tissue, and FIG. 10c is an analysis result of adipose tissue of the epididymis.
11 is a result of analysis of factors related to fat differentiation in Example 6-1. Fig. 11a is the analysis result of FAS, Fig. 11b is Leptin, Fig. 11c is CEBP/α, Fig. 11d is SREBP α1, and Fig. 11e is the analysis result of PPAR/γ.
12 is an immunological analysis result of Example 6-1. Fig. 12a is an analysis result for IL-6, Fig. 12b is CCL2, and Fig. 12c is an analysis result for IL-10.
13 is an outline of Example 6-2 obesity experiment.
14 is a result of hematological analysis of Example 6-2, FIG. 14a is ALT, FIG. 14b is AST, FIG. 14c is total cholesterol, FIG. 14d is HDL cholesterol, FIG. 14e is LDL cholesterol, and FIG. 14f is blood of triglycerides. It is the result of scientific analysis.
15 is a BMI analysis result of Example 6-2.
Fig. 16 is a histological analysis result of Example 6-2, Fig. 16a is an analysis result of adipose tissue of the neck region, Fig. 16b is a subcutaneous adipose tissue, and Fig. 16c is an analysis result of adipose tissue of the epididymis.
17 is a result of analysis of factors related to fat differentiation in Example 6-2. Fig. 17a is FAS, Fig. 17b is Leptin, Fig. 17c is CEBP/α, Fig. 17d is SREBP α1, and Fig. 17e is analysis result of PPAR/γ.
18 is an immunological analysis result of Example 6-2. Fig. 18a is an analysis result for TNF-α, Fig. 18b is IL-6, Fig. 18c is CCL2, and Fig. 18d is an analysis result for IL-10.
이상의 본 발명의 목적들, 다른 목적들, 특징들 및 이점들은 첨부된 도면과 관련된 이하의 바람직한 실시예들을 통해서 쉽게 이해될 것이다. 그러나 본 발명은 여기서 설명되는 실시예들에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 실시예들은 개시된 내용이 철저하고 완전해질 수 있도록 그리고 통상의 기술자에게 본 발명의 사상이 충분히 전달될 수 있도록 하기 위해 제공되는 것이다.The above objects, other objects, features and advantages of the present invention will be easily understood through the following preferred embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosed subject matter may be thorough and complete, and that the spirit of the present invention may be sufficiently conveyed to those skilled in the art.
본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부분품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.In the present specification, terms such as “comprise” or “have” are intended to designate that a feature, number, step, operation, component, part, or combination thereof described in the specification exists, but one or more other features It is to be understood that it does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.
본 명세서에 있어서, 범위가 변수에 대해 기재되는 경우, 상기 변수는 상기 범위의 기재된 종료점들을 포함하는 기재된 범위 내의 모든 값들을 포함하는 것으로 이해될 것이다. 예를 들면, "5 내지 10"의 범위는 5, 6, 7, 8, 9, 및 10의 값들뿐만 아니라 6 내지 10, 7 내지 10, 6 내지 9, 7 내지 9 등의 임의의 하위 범위를 포함하고, 5.5, 6.5, 7.5, 5.5 내지 8.5 및 6.5 내지 9 등과 같은 기재된 범위의 범주에 타당한 정수들 사이의 임의의 값도 포함하는 것으로 이해될 것이다. 또한 예를 들면, "10% 내지 30%"의 범위는 10%, 11%, 12%, 13% 등의 값들과 30%까지를 포함하는 모든 정수들뿐만 아니라 10% 내지 15%, 12% 내지 18%, 20% 내지 30% 등의 임의의 하위 범위를 포함하고, 10.5%, 15.5%, 25.5% 등과 같이 기재된 범위의 범주 내의 타당한 정수들 사이의 임의의 값도 포함하는 것으로 이해될 것이다. In this specification, when a range is described for a variable, the variable will be understood to include all values within the stated range including the stated endpoints of the range. For example, a range of “5 to 10” includes the values of 5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc. It will be understood to include any value between integers that are appropriate for the scope of the recited range, such as 5.5, 6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, and the like. Also for example, a range of “10% to 30%” includes values such as 10%, 11%, 12%, 13%, and all integers up to and including 30%, as well as 10% to 15%, 12% to It will be understood to include any subrange, such as 18%, 20% to 30%, etc., as well as any value between reasonable integers within the scope of the recited range, such as 10.5%, 15.5%, 25.5%, and the like.
이하, 본 발명을 구체적으로 설명한다. Hereinafter, the present invention will be specifically described.
본 발명은 장내 마이크로바이옴 조절을 통한 체지방 감소능 우수 유산균 락토바실러스 플랜타럼 LRCC5282 (Lactobacillus plantarum LRCC5282) 균주의 사균체, 이를 함유한 음료에 대한 것이다. 본 발명은 김치로부터 분리된 유산균 L. plantarum LRCC 5282 및 올리고당에 대한 우수한 이용성을 제공하고자 한다. 본 발명은 체지방 합성 억제 및 분해 촉진을 통해 항비만 효과를 나타내는 유산균 사균체를 제공하고자 하며, 일 구현예에서 락토바실러스 플랜타럼 LRCC5282를 이용한 체지방 감소 음료를 제공하고자 한다.The present invention relates to a dead cell of the Lactobacillus plantarum LRCC5282 strain , which is excellent in reducing body fat through the regulation of the intestinal microbiome, and a beverage containing the same. An object of the present invention is to provide excellent usability for lactic acid bacteria
본 발명은 상기 배경 기술에서 기재한 바와 같이 비만 인구 증가에 따른 항비만에 대한 관심이 커지고 있었고, 기존 항비만 의약품 등의 부작용이 문제되고 있었으며, 이에 따른 대체 소재의 필요성이 증가하게 된 배경에서 발명되었다. 본 발명은 장내 마이크로바이옴과 비만 연관성 및 유산균에 의한 마이크로바이옴 조절을 특징으로 한다. 본 발명은 체외 및 동물 수준에서 체지방 감소 효능을 보유한 유산균 LRCC 5282 사균체의 제공하고자 한다. 본 발명은 마이크로바이옴 조절력을 보유한 SCFAs(short chain fatty acids) 생성력이 우수한 유산균을 선별하여 이를 제공하고자 한다. 본 발명은 유산균 락토바실러스 플랜타럼 LRCC5282사균체를 포함한 체지방 감소 기능성 음료의 제조방법을 제공한다.As described in the background art, the present invention was invented against the background of increasing interest in anti-obesity due to an increase in the obese population, and the side effects of existing anti-obesity drugs, and thus the need for alternative materials increased. became The present invention is characterized by the association between the intestinal microbiome and obesity and the regulation of the microbiome by lactic acid bacteria. An object of the present invention is to provide a dead cell of lactic
이하, 본 발명의 다양한 측면에 대하여 설명한다.Hereinafter, various aspects of the present invention will be described.
본 발명의 일측면은 지방세포 분화 억제 효과를 갖는 락토바실러스 플랜타럼 LRCC5282 (Lactobacillus plantarum LRCC5282) 균주를 제공한다.One aspect of the present invention provides a Lactobacillus plantarum LRCC5282 ( Lactobacillus plantarum LRCC5282) strain having an adipocyte differentiation inhibitory effect.
본 발명의 일 측면에 있어서, 상기 균주는 대사증후군의 예방 또는 치료용인, 균주를 제공한다.In one aspect of the present invention, the strain provides a strain for the prevention or treatment of metabolic syndrome.
본 발명의 일 측면에 있어서, 상기 균주는 체지방 감소 효과가 있는, 균주를 제공한다.In one aspect of the present invention, the strain provides a strain having a body fat reduction effect.
본 발명의 일 측면에 있어서, 상기 균주는 서열번호 1의 염기서열을 포함하는 16S rDNA를 갖는, 균주를 제공한다.In one aspect of the present invention, the strain provides a strain having 16S rDNA comprising the nucleotide sequence of SEQ ID NO: 1.
본 발명의 일 측면에 있어서, 상기 균주는 락토바실러스 플랜타럼 LRCC5282인 균주를 제공한다.In one aspect of the present invention, the strain provides a strain of Lactobacillus plantarum LRCC5282.
본 발명의 일 측면에 있어서, 상기 균주는 기탁번호가 KCCM 12896P인 균주를 제공한다.In one aspect of the present invention, the strain provides a strain whose accession number is KCCM 12896P.
본 발명의 다른 측면은 상기 본 발명의 일측면 중 어느 하나에 따른 균주; 이의 파쇄물; 이의 배양물; 이의 발효물; 및 상기 균주, 파쇄물, 배양액 또는 발효물의 추출물; 중 하나 이상을 포함하는 미생물 제제를 제공한다.Another aspect of the present invention is the strain according to any one of the aspects of the present invention; lysate thereof; its culture; its ferment; and extracts of the strains, lysates, cultures or fermented products; It provides a microbial preparation comprising one or more of.
본 발명의 다른 측면에 있어서, 상기 균주는 사균체인, 미생물 제제를 제공한다.In another aspect of the present invention, the strain provides a dead cell, microbial preparation.
본 발명의 또 다른 측면은 상기 미생물 제제를 포함하는 대사증후군의 예방, 개선 또는 치료용 약학 조성물을 제공한다.Another aspect of the present invention provides a pharmaceutical composition for preventing, improving or treating metabolic syndrome comprising the microbial agent.
일 구현예에 있어서, 상기 대사증후군은 비만, 이상지질혈증, 고지혈증 및 인슐린 저항성 증후군으로 이루어진 군에서 선택된 어느 하나이다.In one embodiment, the metabolic syndrome is any one selected from the group consisting of obesity, dyslipidemia, hyperlipidemia and insulin resistance syndrome.
본 발명의 또 다른 측면은 상기 미생물 제제를 포함하는 대사증후군의 예방 또는 개선용 식품 조성물을 제공한다.Another aspect of the present invention provides a food composition for preventing or improving metabolic syndrome comprising the microbial agent.
일 구현예에 있어서, 상기 식품 조성물은 항비만 효과를 나타내는 식품 조성물이다. 일 구현예에 있어서, 상기 식품 조성물은 음료인, 바람직하게 체지방 감소 음료이다.In one embodiment, the food composition is a food composition exhibiting an anti-obesity effect. According to one embodiment, the food composition is a beverage, preferably a body fat reducing beverage.
이하, 실시예, 제조예 및 실험예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 이들 실시예, 제조예 및 실험예는 오로지 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들 실시예, 제조예 및 실험예에 의해 한정되는 것은 아니다.Hereinafter, the configuration and effect of the present invention will be described in more detail through Examples, Preparation Examples and Experimental Examples. These Examples, Preparation Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention is not limited by these Examples, Preparation Examples and Experimental Examples.
실시예 1. 락토바실러스 플랜타럼 LRCC 5282 균주의 분리Example 1. Isolation of
전통 발효식품인 김치로부터 채취한 시료에 멸균 증류수를 첨가하여 희석액을 만든 후, 분쇄 균질기(Stomacher, Pro-Media SH-001, ELMEX)로 시료 내부를 균질화 하였다. 멸균 식염수를 이용하여 상기 희석액을 10배 희석법으로 단계별 희석 한 후 0.1㎖를 취하였다. 이를 0.002중량%의 BCP(Bromocresol purple)와 1.5중량%의 한천(agar)이 첨가된 MRS 고체배지에 도말하여 37℃ 배양기에서 48시간동안 배양하였다. 배양 후 노란색 환 형태의 콜로니(colony)를 선별하여 개체별로 동일한 배지에 2~3회 계대배양함으로써 각 균주를 순수분리하였다. 분리된 균주 개체별로 MRS 액체 배지에 접종하여 37℃에서 48시간동안 재배양 한 후 4℃ 냉장보관하며 사용하였다. Sterile distilled water was added to a sample collected from kimchi, a traditional fermented food, to make a diluted solution, and then the inside of the sample was homogenized with a grinding homogenizer (Stomacher, Pro-Media SH-001, ELMEX). Using sterile saline, the diluted solution was diluted stepwise by a 10-fold dilution method, and then 0.1 ml was taken. This was smeared on MRS solid medium to which 0.002% by weight of BCP (Bromocresol purple) and 1.5% by weight of agar were added, and cultured in an incubator at 37°C for 48 hours. After culturing, colonies in the form of yellow rings were selected and subcultured 2-3 times in the same medium for each individual to purely isolate each strain. Separated strains were inoculated into MRS liquid medium for each individual, cultured at 37°C for 48 hours, and stored at 4°C refrigerated before use.
실시예 2. 유산균 스크리닝Example 2. Lactobacillus screening
(1) 올리고당별 생장 시 발생하는 대사산물에 따른 pH 저하능 측정 (1) Measurement of pH lowering ability according to metabolites generated during growth for each oligosaccharide
- 하기 표 1의 조성에 따라 탄소원이 제외된 MRS broth에 7종류 탄소원 1%씩 투여- According to the composition of Table 1 below, 1% of 7 types of carbon sources are administered to MRS broth excluding carbon sources.
- 균주별 배양액 0.5%씩 (균주별x배지7종) 접종하고 배양- Inoculate and culture 0.5% of the culture medium for each strain (each strain x 7 media)
: 실시간 배양 분석기(Bio-lector) 이용하여 48시간 동안 배양하며 30분마다 pH 측정: Incubate for 48 hours using a real-time culture analyzer (Bio-lector) and measure pH every 30 minutes
(glucose, beef ext. 제외)Modified-MRS broth
(except glucose, beef ext.)
(2) pH 감소력 우수 유산균 확보 결과(2) Result of securing lactic acid bacteria with excellent pH reducing power
실험 결과는 하기 표 2와 같았다.The experimental results are shown in Table 2 below.
(3) SCFAs 조성 분석 결과(LRCC5282)(3) SCFAs composition analysis result (LRCC5282)
상기 균주를 다양한 탄소원에서 배양 시 젖산 및 SCFAs 생성능을 측정하였다.The ability to produce lactic acid and SCFAs was measured when the strain was cultured in various carbon sources.
실험결과는 도 1 및 도 2와 같았다.Experimental results were the same as in FIGS. 1 and 2 .
실시예 3. 균주의 동정 및 특성 파악Example 3. Identification and characterization of strains
(1) 당 이용성(API kit 분석), API 50 CHL(1) Sugar availability (API kit analysis),
락토바실러스 플랜타럼 LRCC5282 균주의 생화학적 특성을 분석하고자, API50CHB키트(BioMerieux사, France)를 이용하여 탄소원 이용성 여부를 측정하였다. To analyze the biochemical characteristics of the Lactobacillus plantarum LRCC5282 strain, the carbon source availability was measured using the API50CHB kit (BioMerieux, France).
구체적으로 락토바실러스 플랜타럼 LRCC5282 균주를 MRS 고체배지에서 배양한 후, 콜로니를 일정량 채취하여 API50CHL medium 5㎖에 현탁하였다. 락토바실러스 플랜타럼 LRCC5282 균주가 현탁된 API50CHL medium 배지를 API50CH 스트립의 튜브에 접종하고, 37℃ 배양기에서 48시간동안 배양한 후 락토바실러스 플랜타럼 LRCC5282 균주의 탄수화물 대사 유무 결과를 확인하였다. Specifically, after culturing the Lactobacillus plantarum LRCC5282 strain in MRS solid medium, a certain amount of colonies were collected and suspended in 5 ml of API50CHL medium. The API50CHL medium in which the Lactobacillus plantarum LRCC5282 strain was suspended was inoculated into the tube of the API50CH strip, and the result of carbohydrate metabolism of the Lactobacillus plantarum LRCC5282 strain was confirmed after culturing for 48 hours in an incubator at 37°C.
실험 결과는 도 3과 같았다. 이때, 하기 도 3에서 "+"는 탄소원 이용성이 양성인 경우를 나타내고, "-"는 탄소원 이용성이 음성인 경우를 나타내었다. The experimental results were as shown in FIG. 3 . In this case, in FIG. 3, "+" indicates a case in which the carbon source availability is positive, and "-" indicates a case in which the carbon source availability is negative.
API50CHL 키트를 이용하여 락토바실러스 플랜타럼 LRCC5282 균주의 탄수화물 대사를 분석해 본 결과, 하기 도 3에 나타낸 바와 같이, L-아라비노스(L-Arabinose), D-리보스(D-Ribose),D-갈락토스(D-Galactose), D-글루코스(D-Glucose), D-프럭토스(D-Fructose), D-만노스(D-Mannose), L-람노스(L-Rhamnose), D-만니톨(D-Mannitol), D-솔비톨(D-Sorbitol), 메틸- α D-글루코피라노스(Methyl-α D-Glucopyranoside), N-아세틸 글루코사민(N-Acethyl glucosamine), 아미그달린(Amygdalin), 알부틴(Arbutin), 살리신(Salicin), D-셀로비오스(D-Cellobiose), D-말토스(D-Maltose), D-락토스(D-Lactose), D-멜리바이오스(D-Melibiose), D-수크로스(D-Saccharose (sucrose)), D-트레할로오스(D-Trehalose), 이눌린(Inulin), D-멜레치토스(D-Melezitose), D-라피노스(D-Raffinose), 겐티오비오스(Gentiobiose), 포타슘글루코네이드(Potassium gluconate)를 대사하는 것, 즉, 상기 탄소원들을 이용하는 것으로 나타났다. As a result of analyzing the carbohydrate metabolism of the Lactobacillus plantarum LRCC5282 strain using the API50CHL kit, as shown in FIG. 3 below, L-arabinose (L-Arabinose), D-ribose (D-Ribose), D-galactose ( D-Galactose), D-Glucose, D-Fructose, D-Mannose, L-Rhamnose, D-Mannitol ), D-Sorbitol, Methyl-α D-Glucopyranoside, N-Acethyl glucosamine, Amygdalin, Arbutin, Salicin (Salicin), D-cellobiose, D-maltose, D-lactose, D-Melibiose, D-sucrose (D- Saccharose (sucrose)), D-trehalose (D-Trehalose), inulin, D-melezitose, D-raffinose, gentiobiose, potassium It has been shown to metabolize gluconate (Potassium gluconate), ie to utilize these carbon sources.
도 3 및 도 4를 참조하면, 락토바실러스 플랜타럼 LRCC5282 균주는 표준균주 락토바실러스 플랜타럼(Lactobacillus plantarum) 1과 98.8 % 일치하지만, 에스쿨린(Esculin)에 대한 이용성은 99 % 다른 것으로 확인되었다. 또한 차순위 동정결과로 표준균주 락토바실러스 브레비스(Lactobacillus brevis) 1과 0.6%일치하는 것으로 나타났다3 and 4, the Lactobacillus plantarum LRCC5282 strain is 98.8% identical to the standard
(2) 유전동정 16S rDNA 분석(2) Genetic identification 16S rDNA analysis
16S rDNA 서열 분석을 통해 상기 실시예 1에서 분리한 균주를 동정하였다. The strain isolated in Example 1 was identified through 16S rDNA sequence analysis.
확인 결과, 16S rDNA유전자 염기서열은 서열번호 1과 같았다.As a result of confirmation, the nucleotide sequence of the 16S rDNA gene was the same as SEQ ID NO: 1.
염기서열 분석 결과 상기균주는 락토바실러스 속 균주와 최대 99% 상동성을 가지는 것으로 확인되었다. As a result of nucleotide sequence analysis, the strain was confirmed to have a maximum of 99% homology with the Lactobacillus sp.
(3) 미생물 기탁(3) Deposit of microorganisms
상기 동정된 신규 락토바실러스 플랜타럼 균주를 락토바실러스 플랜타럼 LRCC5282 (Lactobacillus plantarum LRCC5282)로 명명하고 한국미생물보존센터에 2020년 12월 15일자로 기탁하여 수탁번호 KCCM12896P를 부여받았다. The identified new Lactobacillus plantarum strain was named Lactobacillus plantarum LRCC5282 ( Lactobacillus plantarum LRCC5282) and deposited with the Korea Microorganism Conservation Center on December 15, 2020, and was given an accession number KCCM12896P.
실시예 4. 유사균 사균체 제조Example 4. Preparation of pseudo-bacterial cells
락토바실러스 플랜타럼 LRCC5282 균주를 MRS 고체배지에서 배양한 후, 콜로니를 일정량 채취하여 500ml MRS broth에 현탁하였다. 이를 37℃에서 48시간 배양 후 10,160 g로 10분 동안 원심분리를 거쳐 상층액을 제거하였다. 균주 pellet은 회수하여 동결건조물로 제조한 다음 생균수를 측정하였다. 적정 균체수 농도에 맞추어 PBS에 용해한 다음 열을 가하여 사균화 한다. 95℃ water-bath에서 30분간 열처리를 진행 후 사균체를 냉동고에 보관하며 이후 실험을 진행 하였다. After culturing the Lactobacillus plantarum LRCC5282 strain in MRS solid medium, a certain amount of colonies were collected and suspended in 500ml MRS broth. After incubation for 48 hours at 37° C., the supernatant was removed by centrifugation at 10,160 g for 10 minutes. The strain pellet was recovered and prepared as a freeze-dried product, and then the number of viable cells was measured. After dissolving in PBS according to the appropriate cell concentration, heat is applied to kill the cells. After heat treatment in 95℃ water-bath for 30 minutes, the dead cells were stored in a freezer and then the experiment was carried out.
실시예 5. 지방세포 합성 억제능 평가 Example 5. Evaluation of adipocyte synthesis inhibitory ability
락토바실러스 플랜타럼 LRCC5282 균주의 지방세포 합성 억제능을 평가하고자, 지방세포에 위 균주의 사균체를 처리하여 지방세포 합성 인자들의 발현량을 측정하였다. To evaluate the adipocyte synthesis inhibitory ability of the Lactobacillus plantarum LRCC5282 strain, adipocytes were treated with the dead cells of the above strain to measure the expression levels of adipocyte synthesis factors.
대조군으로는 동일속 동일종 균주의 KCCM12540P를 사용하였다.As a control, KCCM12540P of the same strain of the same genus was used.
(1) 지방 전구세포 분화 유도 및 treatment(1) Induction and treatment of adipocyte differentiation
- 지방 전구세포인 ATCC 3T3-L1 cell의 분화를 유도하며 하기 표 3과 같이 분화단계별로 사균체 1x10^8 CFU를 처리하였다.- Induced differentiation of ATCC 3T3-L1 cells, which are adipocytes, and treated with 1x10^8 CFU of dead cells for each differentiation stage as shown in Table 3 below.
(2) 지방세포 합성인자 발현량 측정(2) Measurement of expression level of adipocyte synthesis factor
지방세포의 합성인자 발현량 측정을 하기 표 4와 같은 측정법으로 측정하였다.The expression level of synthetic factors in adipocytes was measured by the measurement method shown in Table 4 below.
(3) 지방세포 합성인자 발현량 측정 결과(대조군 대비 발현율, %)(3) Adipocyte synthesis factor expression level measurement result (expression rate compared to control group, %)
상기 지방세포 발현량 측정 결과는 도 5 내지 도 6과 같았다.The measurement results of the adipocyte expression level were shown in FIGS. 5 to 6 .
도 5는 지방분화도 및 중성지방량에 대한 결과이며, 도 6은 지방합성 관련 인자에 대한 결과이다. Figure 5 is a result for the degree of fat differentiation and triglyceride amount, Figure 6 is a result for the fat synthesis-related factors.
실시예 6. 동물 실험Example 6. Animal Testing
비만 개선 및 예방 효과 검증, 유효 Dosage 설정 및 안전성 검증을 하였.Obesity improvement and prevention effect verification, effective dosage setting and safety verification were conducted.
대조군 및 실험군 설계는 하기 표 5와 같았다.The design of the control group and the experimental group is shown in Table 5 below.
양성 대조군으로 Lactobacillus gasseri BNR-17을 사용하였는데 이 균주는 체지방 감소에 도움을 줄 수 있는 개별인정형 원료로써 모유유산균인 Lactobacillus gasseri BNR-17은 모유에서 분리 배양한 유산균주로 각종 임상실험을 통해 체지방 감소 효과를 입증했으며, 지금까지 전 세계 SCI급 논문 304개 논문에서 인용됐다.As a positive control, Lactobacillus gasseri BNR-17 was used, and this strain is an individually recognized raw material that can help reduce body fat. It has proven its effectiveness and has been cited in 304 SCI-level papers worldwide so far.
실험군으로 본원발명의 균주인 LRCC5282와 대조군으로 동일속 동일종의 KCCM12540P를 사용하였다.As an experimental group, LRCC5282, the strain of the present invention, and KCCM12540P of the same genus and the same species as a control were used.
실시예 6-1. 비만 유도 이후 LRCC5282의 비만 개선 실험Example 6-1. Obesity improvement experiment of LRCC5282 after obesity induction
비만 유도 이후에 실험물질을 투여하여 비만 개선 효과가 있는지를 실험하였다.After induction of obesity, it was tested whether the test substance was administered to improve obesity.
구체적인 비만 실험의 개요는 도 7과 같았다. The outline of the specific obesity experiment was shown in FIG. 7 .
8-12주 동안 비만을 유도하였고, 8주 이후에 본원발명 물질 및 양성대조군 및 비교군의 투여를 진행하였다. 비만관련 지표(사료섭취량과 비만도), 혈액학적 분석, 면역학적 분석 및 조직학적 분석을 시행하였다.Obesity was induced for 8-12 weeks, and administration of the inventive substance and the positive control group and the comparative group was performed after 8 weeks. Obesity-related indicators (feed intake and obesity), hematological analysis, immunological analysis, and histological analysis were performed.
혈액학적 분석 결과는 도 8과 같았다. The results of hematological analysis were shown in FIG. 8 .
도 8a는 ALT, 도 8b는 AST, 도 8c는 총콜레스테롤, 도 8d는 HDL 콜레스테롤, 도 8e는 LDL 콜레스테롤 및 도 8f는 중성지방의 혈액학적 분석 결과이다. 도 8에서 CNT는 Blank, P-CNT은 양성대조군인 L. gasseri BNR-17를, 12540는 비교군인 KCCM12540P을 5282는 본원발명 균주인 LRCC5282을 나타낸다.Fig. 8a is ALT, Fig. 8b is AST, Fig. 8c is total cholesterol, Fig. 8d is HDL cholesterol, Fig. 8e is LDL cholesterol, and Fig. 8f is the result of hematological analysis of triglycerides. In FIG. 8, CNT denotes Blank, P-CNT denotes a positive control L. gasseri BNR-17, 12540 denotes KCCM12540P, a comparative group, and 5282 denotes LRCC5282, a strain of the present invention.
체중 분석 결과는 하기 표 6과 같았다.The weight analysis results are shown in Table 6 below.
CNT: Blank; P-CNT: CNT: Blank; P-CNTs: L. gasseri L. gasseri BNR-17; 12540: KCCM12540P; 5282: LRCC5282BNR-17; 12540: KCCM12540P; 5282: LRCC5282
BMI 분석 결과는 하기 도 9와 같았다. The results of BMI analysis were shown in FIG. 9 below.
조직학적 분석 결과는 하기 도 10과 같았다. 도 10은 다양한 조직의 지방분화도에 대한 것으로, 도 10a는 목부위의 지방조직, 도 10b는 피하 지방조직, 도 10c는 부고환의 지방조직에 대한 분석결과이다.The histological analysis results were shown in FIG. 10 below. Figure 10 is for the degree of fat differentiation of various tissues, Figure 10a is the adipose tissue of the neck region, Figure 10b is the subcutaneous adipose tissue, Figure 10c is the analysis result of the adipose tissue of the epididymis.
도 11은 지방분화 관련인자 분석 결과이다. 도 11a는 FAS, 도 11b는 Leptin, 도 11c는 CEBP/α, 도 11d는 SREBP α1, 도 11e는 PPAR/γ 의 분석 결과이다.11 is an analysis result of fat differentiation-related factors. Fig. 11a is FAS, Fig. 11b is Leptin, Fig. 11c is CEBP/α, Fig. 11d is SREBP α1, and Fig. 11e is analysis result of PPAR/γ.
도 12는 면역학적 분석 결과로 염증 cytokine 분석 결과이다. 도 12a는 IL-6, 도 12b는 CCL2, 도 12c는 IL-10에 대한 분석 결과이다.12 is an inflammatory cytokine analysis result as an immunological analysis result. Fig. 12a is an analysis result for IL-6, Fig. 12b is CCL2, and Fig. 12c is an analysis result for IL-10.
실시예 6-2. 비만 유도 및 LRCC5282 동시 투여의 비만 예방 실험Example 6-2. Obesity induction and obesity prevention trial of simultaneous administration of LRCC5282
비만 유도와 동시에 실험물질을 투여하여 비만 예방 효과가 있는지를 실험하였다.It was tested whether there was an effect of preventing obesity by administering the test substance at the same time as inducing obesity.
구체적인 비만 실험의 개요는 도 13과 같았다. The outline of a specific obesity experiment was shown in FIG. 13 .
10주 동안 비만을 유도하였고, 비만 유도와 동시에 본원발명 물질 및 양성대조군 및 비교군의 투여를 진행하였다. 비만관련 지표(사료섭취량과 비만도), 혈액학적 분석, 면역학적 분석 및 조직학적 분석을 시행하였다.Obesity was induced for 10 weeks, and administration of the material of the present invention and a positive control group and a comparative group was carried out at the same time as obesity induction. Obesity-related indicators (feed intake and obesity), hematological analysis, immunological analysis and histological analysis were performed.
혈액학적 분석 결과는 도 14과 같았다. The results of hematological analysis were shown in FIG. 14 .
도 14a는 ALT, 도 14b는 AST, 도 14c는 총콜레스테롤, 도 14d는 HDL 콜레스테롤, 도 14e는 LDL 콜레스테롤 및 도 14f는 중성지방의 혈액학적 분석 결과이다. 도 14에서 CNT는 Blank, P-CNT은 양성대조군인 L. gasseri BNR-17를, 12540는 비교군인 KCCM12540P을 5282는 본원발명 균주인 LRCC5282을 나타낸다.Fig. 14a is ALT, Fig. 14b is AST, Fig. 14c is total cholesterol, Fig. 14d is HDL cholesterol, Fig. 14e is LDL cholesterol, and Fig. 14f is the result of hematological analysis of triglycerides. 14, CNT is Blank, P-CNT is L. gasseri BNR-17, a positive control, 12540 is KCCM12540P, a control, 5282 is LRCC5282, a strain of the present invention.
체중 분석 결과는 하기 표 7과 같았다.The weight analysis results are shown in Table 7 below.
CNT: Blank; P-CNT: CNT: Blank; P-CNTs: L. gasseri L. gasseri BNR-17; 12540: KCCM12540P; 5282: LRCC5282BNR-17; 12540: KCCM12540P; 5282: LRCC5282
BMI 분석 결과는 하기 도 15와 같았다. The results of BMI analysis were shown in FIG. 15 below.
조직학적 분석 결과는 하기 도 16과 같았다. 도 16은 다양한 조직의 지방분화도에 대한 것으로, 도 16a는 목부위의 지방조직, 도 16b는 피하 지방조직, 도 16c는 부고환의 지방조직에 대한 분석결과이다.The histological analysis results were shown in FIG. 16 below. Figure 16 is for the degree of fat differentiation of various tissues, Figure 16a is the adipose tissue of the neck region, Figure 16b is the subcutaneous adipose tissue, Figure 16c is the analysis result of the adipose tissue of the epididymis.
도 17은 지방분화 관련인자 분석 결과이다. 도 17a는 FAS, 도 17b는 Leptin, 도 17c는 CEBP/α, 도 17d는 SREBP α1, 도 17e는 PPAR/γ 의 분석 결과이다.17 is an analysis result of fat differentiation-related factors. Fig. 17a is the analysis result of FAS, Fig. 17b is Leptin, Fig. 17c is CEBP/α, Fig. 17d is SREBP α1, and Fig. 17e is the analysis result of PPAR/γ.
도 18는 면역학적 분석 결과로 염증 cytokine 분석 결과이다. 도 18a는 TNF- α, 도 18b는 IL-6, 도 18c는 CCL2, 도 18d는 IL-10에 대한 분석 결과이다.18 is an inflammatory cytokine analysis result as an immunological analysis result. Fig. 18a is an analysis result for TNF-α, Fig. 18b is IL-6, Fig. 18c is CCL2, and Fig. 18d is an analysis result for IL-10.
실시예 7. 유산균 사균체 대량생산 및 표준화Example 7. Mass production and standardization of lactic acid bacteria dead cells
유산균 사균체의 대량생산 및 표준화를 위하여 ㈜락토메이슨에 위탁하여 락토바실러스 플랜타럼 LRCC5282 균의 사균체 시료를 생산하였다. 3회 반복 생산 시 사균수 기준 1.0 x 10^11 사균수/g 이상 확보 하였다.Lactobacillus plantarum LRCC5282 dead cell samples were produced by consigning to Lactomason Co., Ltd. for mass production and standardization of lactic acid bacteria dead cells. In the case of repeated production three times, more than 1.0 x 10^11 dead cells/g was secured based on the number of dead cells.
실시예 8. 유산균 사균체 포함 음료 제조 Example 8. Preparation of beverages containing lactic acid bacteria dead cells
상기의 LRCC5282 유산균체 및 오렌지 과즙을 이용하여 유산균 과채음료를 제조하였다. 본 발명의 락토바실러스 플랜타럼 LRCC5282은 ㈜락토메이슨(경상남도 진주, 한국)에 위탁하여 사균체 분말로 생산하였으며, 생산된 분말 내 유산균수는 1.0 x 10^11 CFU/g이었다.A lactic acid bacterium fruit and vegetable beverage was prepared using the above LRCC5282 lactic acid bacteria and orange juice. Lactobacillus plantarum LRCC5282 of the present invention was entrusted to Lactomason Co., Ltd. (Jinju, Gyeongsangnam-do, Korea) and produced as dead cell powder, and the number of lactic acid bacteria in the produced powder was 1.0 x 10^11 CFU/g.
(1) 유산균 오렌지 주스의 제조(pH 3.8)(1) Production of lactic acid bacteria orange juice (pH 3.8)
오렌지 농축액(65Brix 기준) 13.85 중량%(과즙함량으로 100 중량%), 천연착향료(오렌지향) 0.10 중량%, 아라비아 검 2 중량%, 정제수 83.95 중량%의 유산균 오렌지 주스 조성물을 만들었다.Orange juice composition containing 13.85% by weight of orange concentrate (based on 65Brix) (100% by weight as fruit juice content), 0.10% by weight of natural flavoring (orange flavor), 2% by weight of gum arabic, and 83.95% by weight of purified water was prepared.
pH Meter를 이용하여 상기에서 얻어진 제품의 pH를 측정한 결과 3.8로 적정 pH를 나타낸다. 해당 조성물을 95℃에서 30초간 살균한 후 10℃ 이하로 냉각하였다. 냉각된 조성물에 사균체 분말 0.1 중량%를 투입하여 유산균수 10^8 CFU/ml가 되도록 제조하였다. As a result of measuring the pH of the product obtained above using a pH meter, an appropriate pH is indicated as 3.8. The composition was sterilized at 95° C. for 30 seconds and then cooled to 10° C. or less. By adding 0.1 wt% of dead cell powder to the cooled composition, the number of lactic acid bacteria was prepared to be 10^8 CFU/ml.
<110> Lotte Chilsung Beverage Co., LTD.
<120> Effect of Lactobacillus plantarum LRCC5282 on alleviates obesity
and its application
<130> DP-2020-0721
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 1501
<212> DNA
<213> Artificial Sequence
<220>
<223> 16S rDNA of LRCC5282
<400> 1
gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaactctggt attgattggt 60
gcttgcatca tgatttacat ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc 120
tgcccagaag cgggggataa cacctggaaa cagatgctaa taccgcataa caacttggac 180
cgcatggtcc gagcttgaaa gatggcttcg gctatcactt ttggatggtc ccgcggcgta 240
ttagctagat ggtggggtaa cggctcacca tggcaatgat acgtagccga cctgagaggg 300
taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360
atcttccaca atggacgaaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg 420
gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc aggtattgac 480
ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540
gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600
gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 660
gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720
ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac 780
aggattagat accctggtag tccataccgt aaacgatgaa tgctaagtgt tggagggttt 840
ccgcccttca gtgctgcagc taacgcatta agcattccgc ctggggagta cggccgcaag 900
gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960
gaagctacgc gaagaacctt accaggtctt gacatactat gcaaatctaa gagattagac 1020
gttcccttcg gggacatgga tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080
tgttgggtta agtcccgcaa cgagcgcaac ccttattatc agttgccagc attaagttgg 1140
gcactctggt gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1200
atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaac 1260
tcgcgagagt aagctaatct cttaaagcca ttctcagttc ggattgtagg ctgcaactcg 1320
cctacatgaa gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380
cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa agtcggtggg 1440
gtaacctttt aggaaccagc cgcctaaggt ggacagatga ttagggtgaa gtcgtacaag 1500
g 1501
<110> Lotte Chilsung Beverage Co., LTD.
<120> Effect of Lactobacillus plantarum LRCC5282 on alleviates obesity
and its application
<130> DP-2020-0721
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 1501
<212> DNA
<213> Artificial Sequence
<220>
<223> 16S rDNA of LRCC5282
<400> 1
gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaactctggt attgattggt 60
gcttgcatca tgatttacat ttgagtgagt ggcgaactgg tgagtaacac gtgggaaacc 120
tgcccagaag cgggggataa cacctggaaa cagatgctaa taccgcataa caacttggac 180
cgcatggtcc gagcttgaaa gatggcttcg gctatcactt ttggatggtc ccgcggcgta 240
ttagctagat ggtggggtaa cggctcacca tggcaatgat acgtagccga cctgagaggg 300
taatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360
atcttccaca atggacgaaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg 420
gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc aggtattgac 480
ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540
gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600
gtgaaagcct tcggctcaac cgaagaagtg catcggaaac tgggaaactt gagtgcagaa 660
gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720
ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagtatg ggtagcaaac 780
aggattagat accctggtag tccataccgt aaacgatgaa tgctaagtgt tggagggttt 840
ccgcccttca gtgctgcagc taacgcatta agcattccgc ctggggagta cggccgcaag 900
gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960
gaagctacgc gaagaacctt accaggtctt gacatactat gcaaatctaa gagattagac 1020
gttcccttcg gggacatgga tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080
tgttgggtta agtcccgcaa cgagcgcaac ccttattatc agttgccagc attaagttgg 1140
gcactctggt gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1200
atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaac 1260
tcgcgagagt aagctaatct cttaaagcca ttctcagttc ggattgtagg ctgcaactcg 1320
cctacatgaa gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380
cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa agtcggtggg 1440
gtaacctttt aggaaccagc cgcctaaggt ggacagatga ttagggtgaa gtcgtacaag 1500
Claims (8)
상기 균주는 체지방 감소 효과가 있는, 균주.According to claim 1,
The strain has a body fat reduction effect, the strain.
상기 균주는 기탁번호가 KCCM 12896P인, 균주.According to claim 1,
The strain has an accession number KCCM 12896P, the strain.
상기 균주는 사균체인, 미생물 제제.6. The method of claim 5,
The strain is a dead cell, a microbial preparation.
상기 식품 조성물은 항비만 효과를 나타내는 식품 조성물.
8. The method of claim 7,
The food composition is a food composition exhibiting an anti-obesity effect.
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