KR102570993B1 - Non-viable Lactobacillus johnsonii JNU3402 strain and composition for preventing or treating obesity comprising the same - Google Patents
Non-viable Lactobacillus johnsonii JNU3402 strain and composition for preventing or treating obesity comprising the same Download PDFInfo
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Abstract
본 발명은 락토바실러스 존스니 JNU3402 균주의 사균체 또는 상기 사균체의 배양여액을 포함하는 대사성 질환 예방 또는 치료용 조성물에 관한 것으로, 보다 상세하게는 락토바실러스 존스니(Lactobacillus johnsonii) JNU3402(수탁번호 KCCM12892P)의 사균체 또는 이의 배양여액을 포함함으로써 체중, 간 조직에서의 중성지방의 함량, 지방조직에서의 중성지방의 함량 및 지방세포의 지질축적을 감소시키며, 고지방식이에 의한 혈액내 포도당 및 인슐린 증가를 억제하고, 지질축적에 관여하는 유전자의 발현을 감소시키고, 지방산 베타 산화에 관여하는 유전자 또는 지방세포 갈색화에 관여하는 유전자의 발현을 증가시키며, 지방세포 내 에너지 대사를 주로 담당하는 미토콘드리아의 활성을 증가시킴으로써 비만, 당뇨, 고지혈증 및 지방간 등 대사성 질환의 예방 또는 치료 효과를 나타낸다.The present invention relates to a composition for preventing or treating metabolic diseases comprising dead cells of strain Lactobacillus johnsonii JNU3402 or a culture filtrate of the dead cells, and more particularly, to a composition for preventing or treating Lactobacillus johnsonii JNU3402 (accession number KCCM12892P). ) of dead cells or its culture filtrate, thereby reducing body weight, neutral fat content in liver tissue, neutral fat content in adipose tissue, and lipid accumulation in fat cells, and blood glucose and insulin by high-fat diet It suppresses the increase in lipid accumulation, decreases the expression of genes involved in lipid accumulation, increases the expression of genes involved in fatty acid beta oxidation or browning of fat cells, and activates mitochondria, which is mainly responsible for energy metabolism in adipocytes. By increasing, it shows the effect of preventing or treating metabolic diseases such as obesity, diabetes, hyperlipidemia and fatty liver.
Description
본 발명은 락토바실러스 존스니 JNU3402 균주의 사균체 및 이를 포함하는 비만 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a dead cell of Lactobacillus johnny JNU3402 strain and a composition for preventing or treating obesity comprising the same.
비만은 대부분의 국가에서 주요한 보건 상의 문제가 되고 있다. 세계보건 기구(WHO)는 전 세계적으로 10억 명 이상의 성인은 과체중이고, 이들 중 3억 명은 임상적으로 비만인 것으로 추정하고 있다. 이러한 비만은 심혈관 질환, 뇌졸중, 인슐린 내성, 당뇨병, 간 질환, 신경퇴행성 질환, 호흡기 질환 및 기타 중증 질환 등에 대한 주요한 위험 인자이고, 유방암 및 결장암을 포함한 일부 암에 대한 위험인자인 것으로 확인되었다. 또한, 신체 건강에 대한 영향 이외에, 비만은 우울증, 식이장애 및 낮은 자긍심을 초래하여 삶의 질 및 정신 건강에 유해한 영향을 미친다.Obesity is a major health problem in most countries. The World Health Organization (WHO) estimates that more than one billion adults worldwide are overweight, of which 300 million are clinically obese. Obesity is a major risk factor for cardiovascular disease, stroke, insulin resistance, diabetes, liver disease, neurodegenerative disease, respiratory disease and other severe diseases, and has been confirmed to be a risk factor for some cancers including breast and colon cancer. In addition to its effects on physical health, obesity also has detrimental effects on quality of life and mental health by leading to depression, eating disorders and low self-esteem.
이러한 가운데, 높은 에너지 부하를 갖는 식품 섭취의 꾸준한 증가 및 적은 운동과 연관된 사회적 변화들은 비만 및 비만으로 야기된 대사성 질환의 발생을 증가시키고 있다. 그러나, 저칼로리 다이어트와 운동에 기초한 전통적인 치료는 비만 제어에 있어서 그다지 효과를 나타내지 못하고, 일시적인 체중 손실을 초래할 뿐이다. 이에, 우수한 항 비만 효과를 갖는 약물의 개발이 필요한 실정이다.Among these, social changes associated with a steady increase in food intake with a high energy load and little exercise are increasing the occurrence of obesity and metabolic diseases caused by obesity. However, traditional treatments based on a low-calorie diet and exercise do not show much effect in controlling obesity and only result in temporary weight loss. Accordingly, it is necessary to develop drugs having excellent anti-obesity effects.
본 발명은 락토바실러스 존스니 균주의 사균체를 제공하는 것을 목적으로 한다.An object of the present invention is to provide dead cells of Lactobacillus johnny strain.
본 발명은 락토바실러스 존스니 균주의 사균체를 포함하는 대사성 질환의 예방 또는 치료용 약학 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic diseases comprising dead cells of Lactobacillus johnny strain.
본 발명은 락토바실러스 존스니 균주의 사균체를 포함하는 체지방 감소, 혈당 조절, 또는 혈중중성지방 개선용 건강기능식품을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a health functional food for reducing body fat, controlling blood sugar, or improving triglycerides in the blood, including dead cells of Lactobacillus johnny strain.
1. 락토바실러스 존스니(Lactobacillus johnsonii) JNU3402(수탁번호 KCCM12892P)의 사균체.1. Dead cells of Lactobacillus johnsonii JNU3402 (accession number KCCM12892P).
2. 락토바실러스 존스니(Lactobacillus johnsonii) JNU3402(수탁번호 KCCM12892P)의 사균체 또는 상기 사균체의 배양여액을 유효성분으로 포함하는 비만, 당뇨, 고지혈증 및 지방간으로 이루어진 군에서 선택된 적어도 하나의 대사성 질환 예방 또는 치료용 약학 조성물.2. Prevention of at least one metabolic disease selected from the group consisting of obesity, diabetes, hyperlipidemia, and fatty liver containing dead cells of Lactobacillus johnsonii JNU3402 (accession number KCCM12892P) or a culture filtrate of the dead cells as an active ingredient or a pharmaceutical composition for treatment.
3. 위 2에 있어서, 상기 조성물은 혈당을 감소시키는 것을 특징으로 하는, 대사성 질환 예방 또는 치료용 약학 조성물.3. The pharmaceutical composition for preventing or treating metabolic diseases according to 2 above, wherein the composition reduces blood sugar.
4. 위 2에 있어서, 상기 조성물은 fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) 및 sterol regulatory element-binding protein-1c (SREBP1c) 중 적어도 하나의 발현을 감소시키거나, acetyl-CoA oxidase (ACOX), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), uncoupling protein 1 (UCP1), PPARγ 및 Cidea 중 적어도 하나의 발현을 증가시키는 것을 특징으로 하는, 대사성 질환 예방 또는 치료용 약학 조성물.4. The method of 2 above, wherein the composition reduces the expression of at least one of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP1c), or acetyl-CoA oxidase Metabolic disease characterized by increased expression of at least one of (ACOX), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), uncoupling protein 1 (UCP1), PPARγ and Cidea A pharmaceutical composition for prevention or treatment.
5. 락토바실러스 존스니(Lactobacillus johnsonii) JNU3402(수탁번호 KCCM12892P)의 사균체 또는 상기 사균체의 배양여액을 유효성분으로 포함하는 체지방 감소, 혈당 조절, 또는 혈중중성지방 개선용 건강기능식품.5. Lactobacillus johnsonii JNU3402 (Accession No. KCCM12892P) dead cell body or a culture filtrate of the dead cell body as an active ingredient A health functional food for reducing body fat, controlling blood sugar, or improving triglycerides in blood.
6. 위 5에 있어서, 상기 건강기능식품은 혈당을 감소시키는 것을 특징으로 하는, 건강기능식품.6. The functional health food according to 5 above, characterized in that it reduces blood sugar.
7. 위 5에 있어서, 상기 건강기능식품은 fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) 및 sterol regulatory element-binding protein-1c (SREBP1c) 중 적어도 하나의 발현을 감소시키거나, acetyl-CoA oxidase (ACOX), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), uncoupling protein 1 (UCP1), PPARγ 및 Cidea 중 적어도 하나의 발현을 증가시키는 것을 특징으로 하는, 건강기능식품.7. The method of 5 above, wherein the health functional food reduces the expression of at least one of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP1c), or acetyl-CoA carboxylase (ACC). Characterized by increasing the expression of at least one of CoA oxidase (ACOX), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), uncoupling protein 1 (UCP1), PPARγ and Cidea, health functional food.
본 발명 락토바실러스 존스니 JNU3402의 사균체 또는 상기 사균체의 배양여액은 체중, 간 조직에서의 중성지방의 함량, 지방조직에서의 중성지방의 함량 및 지방세포의 지질축적을 감소시키며, 고지방식이에 의한 혈액내 포도당 및 인슐린 증가를 억제하고, 지질축적에 관여하는 유전자의 발현을 감소시키고, 지방산 베타 산화에 관여하는 유전자 또는 지방세포 갈색화에 관여하는 유전자의 발현을 증가시키며, 지방세포 내 에너지 대사를 주로 담당하는 미토콘드리아의 활성을 증가시키는 효과를 나타낸다.The dead cells of Lactobacillus johnny JNU3402 or the culture filtrate of the dead cells of the present invention reduce body weight, neutral fat content in liver tissue, neutral fat content in adipose tissue, and lipid accumulation in adipocytes, and can be used as a high-fat diet inhibits the increase in glucose and insulin in the blood, reduces the expression of genes involved in lipid accumulation, increases the expression of genes involved in fatty acid beta oxidation or browning of adipocytes, and increases energy metabolism in adipocytes shows the effect of increasing the activity of mitochondria, which is mainly responsible for
도 1은 락토바실러스 존스니 JNU3402의 계통학적 분류를 보여주는 계통도를 나타낸다.
도 2는 락토바실러스 존스니 JNU3402의 장내 부착성을 확인한 결과를 나타낸다.
도 3은 식이 종류 또는 락토바실러스 존스니 JNU3402 사균체의 투여 유무에 따라 식이 섭취량에 차이가 없는 것을 확인한 결과를 나타낸다.
도 4는 락토바실러스 존스니 JNU3402 사균체의 투여에 따른 체중 감소 효과를 나타낸다.
도 5는 락토바실러스 존스니 JNU3402 사균체의 투여에 따라 지방 조직 및 간 조직의 무게가 감소하는 결과를 나타낸다.
도 6은 락토바실러스 존스니 JNU3402 사균체의 투여에 따라 혈액 및 조직 내 중성지방함량이 감소하는 결과를 나타낸다.
도 7은 락토바실러스 존스니 JNU3402 사균체의 투여에 따라 혈당이 감소하는 결과를 나타낸다.
도 8은 락토바실러스 존스니 JNU3402 사균체의 투여에 따라 혈액내 인슐린 함량이 감소하는 결과를 나타낸다.
도 9는 락토바실러스 존스니 JNU3402 사균체가 제거된 락토바실러스 존스니 JNU3402 사균체 배양 조건배지의 처리에 따라 3T3-L1 지방세포의 지질축적이 감소되는 결과를 나타낸다.
도 10은 락토바실러스 존스니 JNU3402 사균체의 투여에 따라 부고환 지방조직(eWAT)과 서혜부 백색지방(iWAT)에서 지질축적에 관여하는 유전자의 발현이 감소하고, 지방산 베타 산화에 관여하는 유전자와 지방세포 갈색화에 관여하는 유전자의 발현이 증가한 경과를 나타낸다.
도 11은 락토바실러스 존스니 JNU3402 사균체 조건배지의 처리에 따라 3T3-L1 지방세포에서 지질축적에 관여하는 유전자의 발현이 감소하고, 지방산 베타 산화에 관여하는 유전자와 지방세포 갈색화에 관여하는 유전자의 발현이 증가한 결과를 나타낸다.
도 12는 락토바실러스 존스니 JNU3402 사균체 조건배지의 처리에 따라 PPARγ의 전사 활성이 증가하고, UCP1 promoter와 PPARγ promoter의 활성이 증가한 결과를 나타낸다.
도 13은 락토바실러스 존스니 JNU3402 사균체 조건배지에 의한 에너지 대사 관련 유전자의 발현은 PPARγ 경로를 통해 조절되는 것을 확인한 결과를 나타낸다.
도 14는 락토바실러스 존스니 JNU3402 사균체 또는 이의 조건배지의 처리에 따라 지방조직이나 지방세포에서 미토콘드리아 DNA copy number와 CS 활성이 증가한 결과를 나타낸다.
도 15는 락토바실러스 존스니 JNU3402 사균체 처리에 의한 쥐의 직장 온도가 증가한 결과를 나타낸다.Figure 1 shows a phylogenetic diagram showing the phylogenetic classification of Lactobacillus johnny JNU3402.
Figure 2 shows the results of confirming the intestinal adhesion of Lactobacillus johnny JNU3402.
Figure 3 shows the results confirming that there is no difference in dietary intake depending on the type of diet or the administration of dead cells of Lactobacillus johnny JNU3402.
Figure 4 shows the weight loss effect according to the administration of Lactobacillus johnny JNU3402 dead cells.
Figure 5 shows the results of the decrease in the weight of fat tissue and liver tissue according to the administration of dead cells of Lactobacillus johnny JNU3402.
Figure 6 shows the results of reducing the neutral fat content in blood and tissues according to the administration of dead cells of Lactobacillus johnny JNU3402.
Figure 7 shows the results of blood glucose reduction according to the administration of dead cells of Lactobacillus johnny JNU3402.
Fig. 8 shows the result of the decrease in blood insulin content according to the administration of dead cells of Lactobacillus johnny JNU3402.
Fig. 9 shows the results of the reduction of lipid accumulation in 3T3-L1 adipocytes according to the treatment of the culture medium of the dead Lactobacillus johnny JNU3402 cells from which the dead cells of Lactobacillus johnny JNU3402 were removed.
10 shows a decrease in the expression of genes involved in lipid accumulation in epididymal adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) according to the administration of dead cells of Lactobacillus johnny JNU3402, genes involved in fatty acid beta oxidation and adipocytes It shows the process of increasing the expression of genes involved in browning.
11 shows the decrease in the expression of genes involved in lipid accumulation in 3T3-L1 adipocytes according to the treatment of the conditioned medium of Lactobacillus johnny JNU3402 dead cells, and the expression of genes involved in fatty acid beta oxidation and genes involved in browning of adipocytes. It shows the result of increased expression.
12 shows the results of the increased transcriptional activity of PPARγ and the increased activity of the UCP1 promoter and PPARγ promoter according to the treatment of the Lactobacillus johnny JNU3402 dead cell conditioned medium.
Figure 13 shows the results confirming that the expression of energy metabolism-related genes by the Lactobacillus johnny JNU3402 dead cell conditioned medium is regulated through the PPARγ pathway.
Figure 14 shows the results of mitochondrial DNA copy number and CS activity increased in adipose tissue or adipocytes according to the treatment of dead cells of Lactobacillus johnny JNU3402 or its conditioned medium.
Fig. 15 shows the results of the increase in rectal temperature of rats by treatment with dead cells of Lactobacillus johnny JNU3402.
본 발명은 락토바실러스 존스니(Lactobacillus johnsonii) JNU3402 균주의 사균체를 제공한다.The present invention provides dead cells of Lactobacillus johnsonii JNU3402 strain.
락토바실러스 존스니(Lactobacillus johnsonii) JNU3402 균주는 서열번호 1로 기재되는 16S rRNA 염기서열을 갖는다.Lactobacillus johnsonii JNU3402 strain has a 16S rRNA nucleotide sequence represented by SEQ ID NO: 1.
락토바실러스 존스니 JNU3402 균주는 한국미생물보존센터에 기탁되어 수탁번호 KCCM12892P로 등록된 것이다.The Lactobacillus johnny JNU3402 strain was deposited with the Korea Microorganism Conservation Center and registered under the accession number KCCM12892P.
락토바실러스 존스니 JNU3402 균주는 유아의 분변에서 분리된 것일 수 있다.Lactobacillus johnny JNU3402 strain may be isolated from feces of infants.
락토바실러스 존스니 JNU3402 균주는 우수한 내산성 및 내담즙성(담즙산 내성)을 나타낸다.The Lactobacillus johnny JNU3402 strain exhibits excellent acid resistance and cholelithiasis (bile acid tolerance).
락토바실러스 존스니 JNU3402 균주는 내산성 및 내담즙성에 대해 우수한 내성을 가져 위를 통과할 때 사멸되지 않고 장내 도달이 가능하다.Lactobacillus johnny JNU3402 strain has excellent resistance to acid resistance and bile resistance, so it is possible to reach the intestine without dying when passing through the stomach.
락토바실러스 존스니 JNU3402 균주는 장관점막에서 일시적으로 집락을 형성하여 병원성 세균의 생장을 억제하고 장의 투과성을 안정화시킬 수 있다.Lactobacillus johnny JNU3402 strain can temporarily colonize the intestinal mucosa to suppress the growth of pathogenic bacteria and stabilize intestinal permeability.
락토바실러스 존스니 JNU3402 균주는 우수한 장부착성을 나타낼 수 있다.Lactobacillus johnny JNU3402 strain can exhibit excellent intestinal adhesion.
락토바실러스 존스니 JNU3402의 사균체는 가열처리를 통해 사멸시킨 균체이다.The dead cells of Lactobacillus johnny JNU3402 are cells killed by heat treatment.
락토바실러스 존스니 JNU3402의 사균체는 생균의 건조온도보다 높은 온도에서 가열하여 제조한 것일 수 있다. 예를 들어, 락토바실러스 존스니(Lactobacillus johnsonii) JNU3402 균주의 사균체는 50 ℃ 내지 110 ℃, 60 ℃ 내지 100 ℃, 또는 70 ℃ 내지 90 ℃에서 가열하여 제조된 것일 수 있다.The dead cells of Lactobacillus johnny JNU3402 may be prepared by heating at a temperature higher than the drying temperature of live cells. For example, dead cells of Lactobacillus johnsonii strain JNU3402 may be prepared by heating at 50 °C to 110 °C, 60 °C to 100 °C, or 70 °C to 90 °C.
본 발명 락토바실러스 존스니 JNU3402의 사균체는 체중을 감소시키고, 지방 조직 및 간 조직의 무게를 감소시키는 효과를 나타낸다. 락토바실러스 존스니 JNU3402의 사균체는 조직 및 혈액 내 중성지방함량을 감소시키는 효과를 나타낸다. 락토바실러스 존스니 JNU3402의 사균체는 혈액 내 포도당을 감소시키고, 인슐린 함량을 감소시키는 효과를 나타낸다. 락토바실러스 존스니 JNU3402의 사균체는 지방세포의 지질축적을 억제하는 효과를 나타낸다. 락토바실러스 존스니 JNU3402의 사균체는 지질축적에 관여하는 유전자(예컨대, FAS, ACC, SREBP1c)의 발현을 억제하고, 지방산 베타 산화에 관여하는 유전자(예컨대, ACOX, CPT1, PGC1α)와 지방세포 갈색화에 관여하는 유전자(예컨대, UCP1, PPARγ, Cidea)의 발현을 증가시키는 효과를 나타낸다. 락토바실러스 존스니 JNU3402의 사균체는 미토콘드리아의 DNA copy number와 citrate synthase 활성을 증가시키는 효과를 나타낸다. 락토바실러스 존스니 JNU3402의 사균체는 직장의 온도를 높이는 효과를 나타낸다.The dead cell body of Lactobacillus johnny JNU3402 of the present invention shows an effect of reducing body weight and reducing the weight of adipose tissue and liver tissue. The dead cells of Lactobacillus johnny JNU3402 show the effect of reducing the triglyceride content in tissues and blood. The dead cells of Lactobacillus johnny JNU3402 show the effect of reducing blood glucose and insulin content. The dead cells of Lactobacillus johnny JNU3402 show the effect of inhibiting lipid accumulation in adipocytes. The dead cells of Lactobacillus johnny JNU3402 suppress the expression of genes involved in lipid accumulation (e.g., FAS, ACC, SREBP1c), genes involved in fatty acid beta oxidation (e.g., ACOX, CPT1, PGC1α) and browning of adipocytes. shows the effect of increasing the expression of genes involved in (eg, UCP1, PPARγ, Cidea). The dead cells of Lactobacillus johnny JNU3402 show the effect of increasing mitochondrial DNA copy number and citrate synthase activity. The dead cells of Lactobacillus johnny JNU3402 show the effect of increasing the temperature of the rectum.
또한, 본 발명은 락토바실러스 존스니 JNU3402의 사균체 또는 상기 사균체의 배양여액을 유효성분으로 포함하는 대사성 질환의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating metabolic diseases comprising dead cells of Lactobacillus johnny JNU3402 or a culture filtrate of the dead cells as an active ingredient.
용어 "예방"은 질환을 억제시키거나 또는 지연시키는 모든 행위를 의미한다.The term "prevention" refers to any action that suppresses or delays a disease.
용어 "치료"는 질환 의심 및 발병 개체의 증상이 호전 되거나 이롭게 변경되는 모든 행위를 의미한다.The term "treatment" refers to all activities that improve or beneficially change symptoms of suspected or affected individuals.
본 발명 조성물은 락토바실러스 존스니 JNU3402의 사균체를 포함할 수 있다. 예컨대, 본 발명 조성물은 락토바실러스 존스니 JNU3402의 사균체, 락토바실러스 존스니 JNU3402의 사균체를 포함하는 배양액, 농축 배양액, 또는 배양액 건조물을 포함할 수 있다.The composition of the present invention may include dead cells of Lactobacillus johnny JNU3402. For example, the composition of the present invention may include dead cells of Lactobacillus johnny JNU3402, a culture solution containing dead cells of Lactobacillus johnny JNU3402, a concentrated culture solution, or a dried culture solution.
본 발명 조성물은 락토바실러스 존스니 JNU3402의 사균체가 제거된 배양여액을 포함할 수 있다. 예컨대, 본 발명 조성물은 락토바실러스 존스니 JNU3402의 사균체의 배양 여과액, 농축 배양 여과액, 또는 배양 여과액의 건조물을 포함할 수 있다.The composition of the present invention may include a culture filtrate from which dead cells of Lactobacillus johnny JNU3402 are removed. For example, the composition of the present invention may include a culture filtrate of dead cells of Lactobacillus johnny JNU3402, a concentrated culture filtrate, or a dried culture filtrate.
락토바실러스 존스니 JNU3402의 사균체가 제거된 배양여액은 락토바실러스 존스니 사균체를 배양한 배양액에서 사균체를 제거한 여액일 수 있다. The culture filtrate from which dead cells of Lactobacillus johnny JNU3402 are removed may be a filtrate obtained by removing dead cells from a culture medium in which dead cells of Lactobacillus johnny are cultured.
락토바실러스 존스니 JNU3402의 사균체가 제거된 배양여액은 락토바실러스 존스니 균주를 배양 배지에서 배양한 배양액에 열을 가해 사균화시킨 배양액에서 사균체를 제거한 여액일 수 있다.The culture filtrate from which dead cell bodies of Lactobacillus johnny JNU3402 are removed may be a filtrate obtained by removing dead cells from a culture medium in which the Lactobacillus johnny strain is cultured in a culture medium and killed by applying heat.
대사성 질환은 비만, 당뇨, 고지혈증, 지방간, 간염, 간경화, 동맥경화, 고혈압 및 심혈관질환으로부터 선택된 것일 수 있다.The metabolic disease may be one selected from obesity, diabetes, hyperlipidemia, fatty liver, hepatitis, liver cirrhosis, arteriosclerosis, hypertension and cardiovascular disease.
대사성 질환은 비만에 의해 유발되는 대사성 질환일 수 있다.The metabolic disease may be a metabolic disease caused by obesity.
본 발명 락토바실러스 존스니 JNU3402의 사균체 또는 이의 배양여액은 체중 감소, 지방 조직 및 간 조직의 무게 감소 효과를 나타내며, 조직 및 혈액 내 중성지방함량 감소 효과, 혈액 내 포도당 및 인슐린 함량 감소 효과를 나타내고, 지방세포의 지질축적을 억제하는 효과, 지질축적에 관여하는 유전자(예컨대, FAS, ACC, SREBP1c)의 발현 억제 효과, 지방산 베타 산화에 관여하는 유전자(예컨대, ACOX, CPT1, PGC1α)와 지방세포 갈색화에 관여하는 유전자(예컨대, UCP1, PPARγ, Cidea)의 발현 증가 효과를 나타내며, 미토콘드리아의 DNA copy number와 citrate synthase 활성 증가 효과, 및 직장의 온도 상승 효과를 나타낸다.The dead cell body of Lactobacillus johnny JNU3402 or its culture filtrate of the present invention shows the effect of reducing the weight of adipose tissue and liver tissue, reducing the triglyceride content in tissue and blood, and reducing the glucose and insulin content in blood. , effect of inhibiting lipid accumulation in adipocytes, expression of genes involved in lipid accumulation (eg, FAS, ACC, SREBP1c), genes involved in fatty acid beta oxidation (eg, ACOX, CPT1, PGC1α) and adipocytes It shows the effect of increasing the expression of genes involved in browning (eg, UCP1, PPARγ, Cidea), increasing the mitochondrial DNA copy number and citrate synthase activity, and increasing the temperature of the rectum.
이에, 본 발명 락토바실러스 존스니 JNU3402의 사균체 또는 이의 배양여액은 대사성 질환, 예컨대, 비만, 당뇨병(예컨대 제2형 당뇨병), 고지혈증, 고혈압 등의 예방 또는 치료 용도로 사용될 수 있다. 나아가 혈중 지질 이상, 공복혈당장애, 내당능장애, 고혈당 등의 대사 이상 증상의 완화 및 개선의 용도로 사용될 수 있다.Thus, the dead cells of Lactobacillus johnny JNU3402 or its culture filtrate according to the present invention can be used for prevention or treatment of metabolic diseases such as obesity, diabetes (eg, type 2 diabetes), hyperlipidemia, and hypertension. Furthermore, it can be used for the purpose of alleviating and improving symptoms of metabolic abnormalities such as abnormal blood lipid, impaired fasting glucose, impaired glucose tolerance, and hyperglycemia.
또한, 본 발명의 락토바실러스 존스니 JNU3402 사균체 또는 이의 배양여액은 고지방식이에 따른 간 조직의 중성지방함량을 감소시키는 효과가 있는바, 지방간의 예방 또는 치료에 효과적일 수 있다. 지방간은 지방의 과도한 섭취나 내인적 간내 지방합성 증가 또는 그 배출이 감소되는 등의 원인으로 간 조직 내의 지방이 과도하게 축적되는 것이면 제한되지 않으나, 구체적으로 지방의 과도한 섭취에 의한 비알콜성 지방간일 수 있다.In addition, since the dead Lactobacillus johnny JNU3402 cell body or its culture filtrate of the present invention has an effect of reducing the neutral fat content of liver tissue according to a high-fat diet, it may be effective in preventing or treating fatty liver. Fatty liver is not limited as long as fat is excessively accumulated in the liver tissue due to causes such as excessive intake of fat or increased or decreased excretion of endogenous fat in the liver. Specifically, non-alcoholic fatty liver disease caused by excessive intake of fat can
약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태일 수 있다.The pharmaceutical composition may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages.
약학 조성물은 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 주사제의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and injections.
약학 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용 가능한 담체는 경구 투여 시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등일 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소투여용의 경우는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다.A pharmaceutical composition may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may be binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration, and buffers, preservatives, analgesics, A solubilizer, an isotonic agent, a stabilizer, etc. may be mixed and used, and in the case of topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used.
약학 조성물은 경구 또는 비경구로 투여될 수 있으며, 비경구 투여 시 피부 외용 또는 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식이 선택될 수 있다.The pharmaceutical composition may be administered orally or parenterally, and in the case of parenteral administration, external skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method may be selected.
아울러, 본 발명은 락토바실러스 존스니 JNU3402의 사균체 또는 상기 사균체의 배양여액을 유효성분으로 포함하는 체지방 감소, 혈당 조절, 또는 혈중중성지방 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for reducing body fat, controlling blood sugar, or improving triglyceride in blood, comprising dead cells of Lactobacillus johnny JNU3402 or a culture filtrate of the dead cells as an active ingredient.
락토바실러스 존스니 JNU3402 사균체, 상기 사균체의 배양여액 및 대사성 질환은 전술한 바 있어 구체적인 설명은 생략한다.Since the dead cells of Lactobacillus johnny JNU3402, the culture filtrate of the dead cells, and metabolic diseases have been described above, a detailed description thereof will be omitted.
건강기능식품은 드링크제, 육류, 소시지, 빵, 비스킷, 떡, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종스프, 음료수, 알코올 음료 및 비타민 복합제, 유제품 및 유가공 제품에서 선택될 수 있으며, 가공 식품 및 건강 기능 식품을 모두 포함할 수 있다.Health functional foods include drinks, meat, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, alcoholic beverages, and vitamin complexes. , can be selected from dairy products and milk-processed products, and can include both processed foods and health functional foods.
건강기능식품이 음료인 경우 통상의 음료와 같이 여러가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 천연 탄수화물은 예를 들어, 포도당, 과당, 말토스, 슈크로스, 덱스트린, 시클로덱스트린, 자일리톨, 소르비톨 및 에리트리톨에서 선택된 것일 수 있다.If the health functional food is a beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The natural carbohydrate may be selected from, for example, glucose, fructose, maltose, sucrose, dextrin, cyclodextrin, xylitol, sorbitol and erythritol.
건강기능식품은 식품 첨가제를 포함할 수 있다. 예컨대, 식품 첨가제는 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 및 탄산음료에 사용되는 탄산화제에서 선택될 수 있다.Health functional food may contain food additives. For example, food additives include nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages.
이하, 실시예를 들어 본 발명의 구성 및 효과를 보다 구체적으로 설명한다. 그러나 아래 실시예는 본 발명에 대한 이해를 돕기 위해 예시의 목적으로만 제공된 것일 뿐 본 발명의 범주 및 범위가 그에 의해 제한되는 것은 아니다.Hereinafter, the configuration and effects of the present invention will be described in more detail by way of examples. However, the following examples are provided only for illustrative purposes to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.
1.One. Lactobacillus johnsonii Lactobacillus johnsonii JNU3402의 분리 및 동정 Isolation and identification of JNU3402
생후 2개월 이내 유아분변을 시료로 사용하여 유산균을 분리하였다. 구체적으로, 상기 시료를 0.004%의 브로모크레졸 퍼플(bromocresol purple)이 함유된 MRS agar 배지(Difco, 미국)에 도말평판법(streak plate)으로 도말한 후 혐기상태(이산화탄소 5 ~ 15%)에서 37℃, 48시간 동안 배양하였다. 상기 배양된 균주 중에서 노란색 집락을 띄는 균주들을 분리하고 그람염색, 카탈라아제 및 옥시다아제 테스트, 가스 형성, 당 이용능 등을 조사하여 우수한 균주를 선별하였다. 선별된 균주를 Lactobacillus johnsonii JNU3402로 명명하였다.Lactobacillus was isolated using infant feces within 2 months of age as a sample. Specifically, the sample was smeared on MRS agar medium (Difco, USA) containing 0.004% of bromocresol purple by a streak plate, and then in anaerobic conditions (5 to 15% carbon dioxide) at 37 ℃, incubated for 48 hours. Among the cultured strains, strains showing yellow colonies were isolated, and excellent strains were selected by examining Gram staining, catalase and oxidase tests, gas formation, sugar availability, and the like. The selected strain was named Lactobacillus johnsonii JNU3402.
Lactobacillus johnsonii JNU3402의 배지에서의 형태학적 및 생리학적 특징을 아래 표 1에 나타내었다.The morphological and physiological characteristics of the medium of Lactobacillus johnsonii JNU3402 are shown in Table 1 below.
또한, 선별된 균주를 동정하기 위해 16S rDNA 염기서열 분석을 실시하였으며 그 결과는 서열번호 1로 나타내었다. 염기서열 분석은 바이오닉스에 의뢰 분석하였으며, NCBI blast search를 통해서 서열분석 결과를 확인하였다. 서열분석 결과 본 발명의 균주 JNU3402는 락토바실러스 존스니의 16S rDNA와 99%의 서열 상동성을 보였고, 이를 통해 분리된 균주 JNU3402는 락토바실러스 존스니라는 것을 확인할 수 있었다. 본 발명자들은 위와 같이 분리된 균주를 2020년 12월 09일자로 국제기탁기관인 한국미생물보존센터에 수탁번호 KCCM12892P로 기탁하였다.In addition, 16S rDNA sequencing was performed to identify the selected strain, and the results are shown in SEQ ID NO: 1. The sequencing analysis was requested to Bionics, and the sequencing results were confirmed through NCBI blast search. As a result of sequencing, the strain JNU3402 of the present invention showed 99% sequence homology with the 16S rDNA of Lactobacillus johnny, and through this, it was confirmed that the isolated strain JNU3402 was Lactobacillus johnny. The present inventors deposited the strain isolated as above with accession number KCCM12892P to the Korea Center for Microorganism Conservation, an international depositary institution, on December 09, 2020.
2.2. Lactobacillus johnsonii Lactobacillus johnsonii JNU3402의 내산성 및 내담즙성 평가 Evaluation of acid resistance and bile resistance of JNU3402
Lactobacillus johnsonii JNU3402(수탁번호 KCCM12892P) 균주를 MRS broth에 18시간 2차 계대 배양한 후 원심분리(2,000 rpm, 20분) 한 다음 멸균 생리식염수로 3회 반복해서 수세하여 본 평가에 사용하였다. The Lactobacillus johnsonii JNU3402 (accession number KCCM12892P) strain was subcultured in MRS broth for 18 hours, centrifuged (2,000 rpm, 20 minutes), and washed three times with sterile physiological saline and used for this evaluation.
내산성 평가를 위해 0.05 M 인산나트륨 완충용액을 제조하여 HCl로 pH를 2.0와 2.5로 각각 조정한 MRS broth에 pepsin(Sigma Co., USA)을 1,000 unit/mL 이 되도록 첨가하여 내산성 측정용 배지로 사용하였다. 초기 균수가 107 cfu/ml 정도가 되도록 0.05M 인산나트륨 완충용액에 현탁시킨 후 배지에 접종하였다. 37℃에서 2시간 진탕배양기에서 배양시킨 후 집락수를 계산하여 내산성을 평가하였다. 내산성 평가 결과 본 발명의 락토바실러스 존스니 JNU3402균주는 pH 2.5에서 1시간 처리했을 때에도 107 수준을 유지하여 우수한 내산성을 갖는 것으로 확인되었다(표 2 참조).To evaluate acid resistance, a 0.05 M sodium phosphate buffer solution was prepared, and pepsin (Sigma Co., USA) was added to MRS broth adjusted to pH 2.0 and 2.5 with HCl to a concentration of 1,000 units/mL to be used as a medium for measuring acid resistance. did After suspending in 0.05M sodium phosphate buffer so that the initial number of bacteria is about 10 7 cfu/ml, the culture medium was inoculated. Acid resistance was evaluated by counting the number of colonies after culturing in a shaking incubator at 37°C for 2 hours. As a result of acid resistance evaluation, it was confirmed that the Lactobacillus johnny JNU3402 strain of the present invention had excellent acid resistance by maintaining the 10 7 level even when treated at pH 2.5 for 1 hour (see Table 2).
또한, 내담즙성 평가를 위해 MRS 배지에 여과 제균된 oxgall(Difco, USA) 용액을 oxgall 함량으로 0.5% (w/v)가 되도록 첨가하여 준비하였다. 내산성 평가에서 사용한 것과 동일한 유산균 현탁액을 배지에 접종하여 37°C에서 24시간 배양한 후 생균수를 평가하였다. 평가 결과 0.5% oxgall 농도에서도 생육을 할 수 있고, 높은 증식률을 나타내는 것을 확인하였다. 구체적으로, 접종 후 24시간이 경과한 때의 균수는 초기 접종시 균수 대비 200% 이상 높은 것을 확인하였다(표 3 참조).In addition, for the evaluation of bile resistance, a filtered oxgall (Difco, USA) solution was added to the MRS medium so that the oxgall content was 0.5% (w/v) and prepared. The same lactic acid bacteria suspension used in the acid resistance evaluation was inoculated into the medium and cultured at 37 ° C for 24 hours, and then the number of viable cells was evaluated. As a result of the evaluation, it was confirmed that growth was possible even at 0.5% oxgall concentration and a high growth rate was observed. Specifically, it was confirmed that the number of bacteria at 24 hours after inoculation was higher than the number of bacteria at the time of initial inoculation by 200% or more (see Table 3).
(CFU/mL)Viable cell count after 0.5% oxgall treatment
(CFU/mL)
3.3. Lactobacillus johnsonii Lactobacillus johnsonii JNU3402의 장내 부착성 평가 Intestinal adhesion evaluation of JNU3402
Monolayer를 형성한 장 상피세포(HT-29)를 PBS 버퍼로 5회 세척하고 항생제가 첨가되지 않은 RPMI 1640 배지를 첨가하였다. JNU3402 균주를 1~2 X 1010 CFU/mL 의 농도가 되도록 RPMI 에 현탁한 다음 well-plate에 접종하고 5% CO2 존재 하에 37℃에서 2시간 배양하였다. 배양이 완료된 후 부착되지 않은 유산균의 제거와 세척을 위하여 3분씩 200rpm 속도로 교반하면서 PBS 버퍼를 사용하여 3회 세척을 실시하였다. 세척이 완료된 후 0.2% Trypsin-EDTA를 분주하여 부착되어 있는 세포를 떼어내고 peptone 수를 이용하여 연속희석법으로 MRS 고체배지에 도말하고 37에서 24시간 배양하여 생균수를 측정하였다.Intestinal epithelial cells (HT-29) forming a monolayer were washed 5 times with PBS buffer, and RPMI 1640 medium without antibiotics was added. The JNU3402 strain was suspended in RPMI to a concentration of 1 to 2 X 10 10 CFU/mL, then inoculated into a well-plate and cultured at 37°C for 2 hours in the presence of 5% CO 2 . After the culture was completed, washing was performed three times using PBS buffer while stirring at a speed of 200 rpm for 3 minutes each to remove and wash non-adhered lactic acid bacteria. After washing was completed, 0.2% Trypsin-EDTA was dispensed to remove attached cells, and the number of viable cells was measured by incubating at 37°C for 24 hours.
실험 대조구로 장내 부착활성이 높은 Lactobacillus GG 균을 사용하였으며 실험결과 JNU3402 균주는 Lactobacillus GG 균과 유사한 장상피세포 부착활성을 나타냈었다. 그러나 김치에서 분리한 Lactobacillus plantarum 은 장내 부착이 낮은 것으로 나타났다(도 2 참조).As a control, Lactobacillus GG bacteria with high intestinal adhesion activity was used. As a result of the experiment, JNU3402 strain showed intestinal epithelial cell adhesion activity similar to that of Lactobacillus GG bacteria. However, Lactobacillus plantarum isolated from kimchi showed low intestinal adhesion (see FIG. 2).
4. 4. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체 및 그의 배양여액 제조 Preparation of dead cells of JNU3402 and their culture filtrate
1) One) Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체(NV-LJ3402) 제조 Manufacturing of dead cells of JNU3402 (NV-LJ3402)
Lactobacillus johnsonii JNU3402 (수탁번호 KCCM12892P) 균주를 MRS broth (BD, Difco Laboratories, Detroit, MI, USA)에 접종하고 37℃에서 24시간 동안 배양하여 배양액을 OD 600 = 1.0 x 108 cfu/mL로 조정하였다. 조정된 배양액을 80 ℃에서 15 분간 가열하여 사균화시켰다. MRS 한천 플레이트(BD, Difco Laboratories, Detroit, MI, USA)를 이용해 박테리아 콜로니가 사멸화된 것을 확인하였다. Lactobacillus johnsonii JNU3402 (accession number KCCM12892P) strain was inoculated into MRS broth (BD, Difco Laboratories, Detroit, MI, USA) and cultured at 37 ° C for 24 hours to adjust the culture medium to OD 600 = 1.0 x 10 8 cfu / mL. . The conditioned culture was killed by heating at 80° C. for 15 minutes. It was confirmed that the bacterial colonies were killed using an MRS agar plate (BD, Difco Laboratories, Detroit, MI, USA).
2) 2) Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체를 포함하지 않는 사균체 배양여액 (락토바실러스 존스니 JNU3402 사균체 조건배지, Killed cell culture filtrate not containing JNU3402 killed cells (Lactobacillus johnny JNU3402 killed cell conditioned medium, NV-LJ3402-CM) 제조NV-LJ3402-CM) Manufacturing
전술한 조정된 배양액을 80 ℃에서 15 분간 가열하여 락토바실러스 존스니 JNU3402 균주를 사균화시킨 후, 4,000 g의 속도로 15 분간 원심분리하여 사균체가 없는 상층배양액만 분리하여 락토바실러스 존스니 JNU3402 사균체 조건배지를 제조하였다.The above-described adjusted culture solution was heated at 80 ° C. for 15 minutes to kill the Lactobacillus johnny JNU3402 strain, and then centrifuged at 4,000 g for 15 minutes to separate only the supernatant culture without dead cells to obtain Lactobacillus johnny JNU3402 strain. Cell conditioned medium was prepared.
5. 5. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체에 의한 체중 변화 분석 Weight change analysis by JNU3402 dead cells
생후 6주 된 C57BL/6J 실험 쥐 21 마리(무게 19-20 g, 중앙 동물 연구소, 대전, 대한민국)를 1주간 적응시킨 후 7주부터 일반식이(Normal diet, ND) (16% of total calories from fat, LabDiet, St. Louis, MO, USA) 또는 고지방식이(High-fat diet, HFD) (45% of total calories from fat, Research Diets Inc., New Brunswick, NJ, USA)을 섭취하도록 하여 비만 질환을 유도하였다(표 4 참조).Twenty-one 6-week-old C57BL/6J experimental mice (weight 19-20 g, JoongAng Animal Research Center, Daejeon, Korea) were acclimatized for 1 week and then fed to normal diet (Normal diet, ND) from 7 weeks. (16% of total calories from fat, LabDiet, St. Louis, MO, USA) or High-fat diet (HFD) (45% of total calories from fat, Research Diets Inc., New Brunswick, NJ, USA) was induced to induce obesity (see Table 4).
그 후, 200μl의 락토바실러스 존스니 JNU3402 사균체 또는 PBS를 14주 동안 매일 마우스에게 경구투여하였다. 락토바실러스 존스니 JNU3402 사균체나 PBS를 투여하는 14주 동안 각 대조군과 실험군의 식이 섭취량을 0, 8, 10, 14 주차에 주 3회 측정하였다. 대조군과 실험군의 식이 섭취량은 유의미한 차이를 나타내지 않았다(도 3, ND: 대조군1, HFD: 대조군2, HFD+NV-LJ3402: 실험군1)Thereafter, 200 μl of dead cells of Lactobacillus johnny JNU3402 or PBS was orally administered to mice every day for 14 weeks. During 14 weeks of administration of dead cells of Lactobacillus johnny JNU3402 or PBS, food intake of each control group and experimental group was measured three times a week at 0, 8, 10, and 14 weeks. There was no significant difference in food intake between the control group and the experimental group (Fig. 3, ND: control group 1, HFD: control group 2, HFD+NV-LJ3402: experimental group 1)
대조군과 실험군의 체중을 2주에 1회 측정한 결과, 일반식이 그룹인 대조군 1에 비해 고지방식이 그룹인 대조군 2의 체중은 49% 증가하였다. 한편, 고지방식이를 섭취하고 락토바실러스 존스니 JNU3402 사균체를 경구투여 한 그룹인 실험군 1의 체중은 고지방식이를 섭취한 대조군 2에 비해 약 10% 감소하였다(도 4, ND: 대조군1, HFD: 대조군2, HFD+NV-LJ3402: 실험군1).As a result of measuring the body weight of the control group and the experimental group once every 2 weeks, the body weight of the control group 2, a high-fat diet group, increased by 49% compared to the control group 1, a normal diet group. On the other hand, the body weight of experimental group 1, which is a group that consumed a high-fat diet and orally administered dead cells of Lactobacillus johnny JNU3402, decreased by about 10% compared to the control group 2 that consumed a high-fat diet (Fig. 4, ND: control group 1, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).
6. 6. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체에 의한 지방 및 간 조직 무게 변화 Fat and liver tissue weight changes by JNU3402 dead cells
락토바실러스 존스니 JNU3402 사균체를 경구투여 한 실험군 1의 간, 서혜부 백색지방(iWAT), 부고환 백색지방(eWAT), 갈색지방조직(BAT)의 무게가 고지방식이 그룹인 대조군 2에 비해 각각 16%, 29%, 26%, 30% 감소하는 것을 확인하였다(도 5, ND: 대조군1, HFD: 대조군2, HFD+NV-LJ3402: 실험군1).The weights of liver, inguinal white fat (iWAT), epididymal white fat (eWAT), and brown adipose tissue (BAT) of Experimental Group 1 orally administered with dead cells of Lactobacillus johnny JNU3402 were 16, respectively, compared to the high-fat diet group, Control Group 2. It was confirmed that %, 29%, 26%, and 30% decrease (FIG. 5, ND: Control 1, HFD: Control 2, HFD+NV-LJ3402: Experimental Group 1).
7. 7. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체에 의한 조직 및 혈액 내 중성지방의 변화 Changes in triglycerides in tissues and blood caused by dead cells of JNU3402
락토바실러스 존스니 JNU3402 사균체가 혈액 및 조직 내 중성지방함량에 미치는 영향을 조사하기 위해 TG quantification kit (SCG Biomax, Seoul, Korea)를 사용하여 각 조직에서 중성지방을 측정하였다. 그 결과, 고지방식이 그룹인 대조군 2에 비해 락토바실러스 존스니 JNU3402 사균체를 경구투여 한 그룹인 실험군 1의 혈액에서 중성지방함량이 49% 감소하였다. 또한 간 조직과 지방 조직에서 중성지방함량을 측정한 결과, 고지방식이 그룹인 대조군 2에 비해 락토바실러스 존스니 JNU3402 사균체를 경구투여 한 그룹인 실험군 1에서 중성지방함량이 각각 38%, 23% 감소하였다(도 6, ND: 대조군1, HFD: 대조군2, HFD+NV-LJ3402: 실험군1). Triglyceride levels were measured in each tissue using a TG quantification kit (SCG Biomax, Seoul, Korea) to investigate the effect of dead cells of Lactobacillus johnny JNU3402 on blood and tissue triglyceride levels. As a result, compared to the control group 2, which is a high-fat diet group, the neutral fat content in the blood of experimental group 1, which was orally administered with dead cells of Lactobacillus johnny JNU3402, decreased by 49%. In addition, as a result of measuring the triglyceride content in liver and adipose tissue, compared to the control group 2, which is a high-fat diet group, the triglyceride content was 38% and 23%, respectively, in experimental group 1, which was orally administered with dead cells of Lactobacillus johnny JNU3402. decreased (FIG. 6, ND: control group 1, HFD: control group 2, HFD + NV-LJ3402: experimental group 1).
8. 8. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체에 의한 혈액 내 포도당 및 인슐린의 변화 Changes in blood glucose and insulin by dead cells of JNU3402
고지방식이에 의한 혈액내 포도당 및 인슐린 증가에 대한 락토바실러스 존스니 JNU3402 사균체의 영향을 알아보기 위해 혈당측정기를 사용하여 혈액 내 포도당 수치를 측정하였으며, 인슐린 ELISA kit (ALPCO, Windham, NH, USA)를 사용하여 혈액 내 인슐린 수치를 측정하였다. 공복 혈당을 측정하기 위해 대조군 및 실험군의 쥐는 12 시간 공복 상태를 유지하였다. 고지방식이를 섭취한 그룹인 대조군 2에 비해 락토바실러스 존스니 JNU3402 사균체를 경구투여 한 그룹인 실험군 1의 쥐에서 공복혈당과 식후혈당이 각각 43%, 27% 감소하였다(도 7, ND: 대조군1, HFD: 대조군2, HFD+NV-LJ3402: 실험군1). 또한 혈액 내 인슐린 함량은 락토바실러스 존스니 JNU3402 사균체를 경구투여 한 그룹인 실험예 1의 쥐가 고지방식이를 섭취한 그룹인 대조군 2의 쥐 대비 51% 낮은 것으로 나타났다(도 8, ND: 대조군1, HFD: 대조군2, HFD+NV-LJ3402: 실험군1).To investigate the effect of dead cells of Lactobacillus johnny JNU3402 on the increase in blood glucose and insulin by high-fat diet, blood glucose levels were measured using a glucometer and insulin ELISA kit (ALPCO, Windham, NH, USA ) was used to measure insulin levels in the blood. To measure fasting blood sugar, rats in the control and experimental groups were kept in a fasting state for 12 hours. Fasting blood glucose and postprandial blood sugar decreased by 43% and 27%, respectively, in the rats of experimental group 1, the group orally administered with dead cells of Lactobacillus johnny JNU3402, compared to the control group 2, which was fed a high-fat diet (Fig. 7, ND: Control group 1, HFD: Control group 2, HFD+NV-LJ3402: Experimental group 1). In addition, the insulin content in the blood was found to be 51% lower than that of the rats of Experimental Example 1, the group orally administered with dead cells of Lactobacillus johnny JNU3402, compared to the rats of the control group 2, the group consuming a high-fat diet (Fig. 8, ND: control group 1, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).
9. 9. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체 JNU3402 dead cells 조건배지의 처리에 따른 3T3-L1 지방세포의 지질축적 변화Changes in Lipid Accumulation in 3T3-L1 Adipocytes by Treatment with Conditioned Medium
지방세포 내 지질축적에 락토바실러스 존스니 JNU3402 사균체 배양 조건배지가 미치는 영향을 확인하고자 3T3-L1 지방세포에서 Oil-Red O 염색을 실시하였다. 가열한 MRS broth를 대조군 배지로 사용하여 처리한 조건(con)에 비교하여 락토바실러스 존스니 JNU3402 사균체 조건배지(NV-LJ3402-CM)를 24, 36, 48 시간 처리하였을 때 3T3-L1 지방세포의 지질 축적은 각 22%, 46%, 61% 감소하였다(도 9). 지방세포의 지질축적의 통계적 차이는 Students' t-test를 사용하여 분석하였다.Oil-Red O staining was performed on 3T3-L1 adipocytes to confirm the effect of Lactobacillus johnny JNU3402 dead cell culture medium on lipid accumulation in adipocytes. 3T3-L1 adipocytes when treated with Lactobacillus johnny JNU3402 killed cell conditioned medium (NV-LJ3402-CM) for 24, 36, and 48 hours compared to the condition (con) treated using heated MRS broth as a control medium Lipid accumulation was reduced by 22%, 46%, and 61%, respectively (FIG. 9). Statistical differences in lipid accumulation in adipocytes were analyzed using Students' t-test.
10. 10. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체 또는 이의 조건배지에 의한 유전자 발현 조절 Regulation of gene expression by killed cells of JNU3402 or its conditioned medium
1) One) Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체에 의한 유전자 발현 조절 Regulation of gene expression by dead cells of JNU3402
고지방식이 쥐에 락토바실러스 존스니 JNU3402 사균체를 경구투여 했을 때 지방 조직 내 지질축적 및 에너지 대사에 관여하는 유전자의 발현 조절 여부를 알아보기 위해 실험쥐의 부고환과 서혜부 백색지방에서 RT-qPCR을 실시하였다.RT-qPCR was performed in the epididymis and inguinal white fat of mice to investigate whether or not the expression of genes involved in lipid accumulation and energy metabolism in adipose tissue was regulated when Lactobacillus johnny JNU3402 killed cells were orally administered to mice on a high-fat diet. conducted.
구체적으로, Trizol 시약 (Invitrogen, Waltham, MA, USA)을 사용하여 3T3-L1 세포 및 지방조직(WAT)에서 총 RNA를 분리하고, Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Promega, Madison WIS, USA)를 사용하여 총 RNA 1g에서 cDNA를 합성하였다. RT-qPCR은 공지된 방법으로 수행하였고. 그 결과는 36B4 mRNA 발현으로 정규화하였다. PCR 산물의 상대적인 정량화는2-△△Ct 방법을 사용하여 표적 유전자와 36B4 유전자 사이의 Ct 값의 차이로 계산하였다. 아래 표 5는 PCR용 프라이머 서열을 나타낸다.Specifically, total RNA was isolated from 3T3-L1 cells and adipose tissue (WAT) using Trizol reagent (Invitrogen, Waltham, MA, USA), and Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Promega, Madison WIS). , USA) was used to synthesize cDNA from 1 g of total RNA. RT-qPCR was performed by a known method. Results were normalized to 36B4 mRNA expression. Relative quantification of PCR products was calculated as the difference in Ct values between the target gene and the 36B4 gene using the 2 -ΔΔCt method. Table 5 below shows primer sequences for PCR.
(서열번호 2)AGATCCTGGAACGAGAACACGAT
(SEQ ID NO: 2)
(서열번호 3)GAGACGTGTCACTCCTGGACTTG
(SEQ ID NO: 3)
(서열번호 4)GTATGTTCGAAGGGCTTACATTG
(SEQ ID NO: 4)
(서열번호 5)TGGGCAGCATGAACTGAAATT
(SEQ ID NO: 5)
(서열번호 6)ACTGTGACCTCACAGGTCCA
(SEQ ID NO: 6)
(서열번호 7)GGCAGTTTGTCTGTGTCCACA
(SEQ ID NO: 7)
(서열번호 8)TCGAGGCTTGGAAACCACTG
(SEQ ID NO: 8)
(서열번호 9)TCGAGTGATGAGCTGAGCC
(SEQ ID NO: 9)
(서열번호 10)ACTCCTGGAAGAAGAAGTTC
(SEQ ID NO: 10)
(서열번호 11)TAGGGTCCGATTGATCTTTG
(SEQ ID NO: 11)
(서열번호 12)GAGACTTTGGAGGCCAGCA
(SEQ ID NO: 12)
(서열번호 13)CGCCATCCCTTAGTTCACTGG
(SEQ ID NO: 13)
(서열번호 14)GGAGGTGTGGCAGTGTTC
(SEQ ID NO: 14)
(서열번호 15)TCTGTGGTGGCTATAACTCTG
(SEQ ID NO: 15)
(서열번호 16)GAAGACCACTCGCATTCCTT
(SEQ ID NO: 16)
(서열번호 17)GAAGGTTCTTCATGAGGCCTG
(SEQ ID NO: 17)
(서열번호 18)ATCACAACTGGCCTGGTTACG
(SEQ ID NO: 18)
(서열번호 19)TACTACCCGGTGTCCATTTCT
(SEQ ID NO: 19)
(서열번호 20)AGATGCAGCAGATCCGCAT
(SEQ ID NO: 20)
(서열번호 21)ATATGAGGCAGCAGTTTCTCCAG
(SEQ ID NO: 21)
(서열번호 22)AATCTACCATCCTCCGTG
(SEQ ID NO: 22)
(서열번호 23)GACTAATGATTCTTCACCGT
(SEQ ID NO: 23)
(서열번호 24)GTTGTCTCCTGCGACTTCA
(SEQ ID NO: 24)
(서열번호 25)GGTGGTCCAGGGTTTCTTA
(SEQ ID NO: 25)
그 결과, 고지방식이 그룹인 대조군 2에 비해 락토바실러스 존스니 JNU3402 사균체를 경구투여한 그룹인 실험군 1의 부고환 지방조직(eWAT)에서 지질축적에 관여하는 유전자인 fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), sterol regulatory element-binding protein-1c (SREBP1c)의 발현이 각각 40%, 32%, 51% 낮은 값을 나타내었다. 이와 반대로 지방산 베타 산화에 관여하는 유전자인 acetyl-CoA oxidase (ACOX), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α)의 발현은 고지방식이 그룹인 대조군 2에 비해 락토바실러스 존스니 JNU3402 사균체를 경구투여한 그룹인 실험군 1에서 각각 1.8 배, 1.9 배, 2.3 배 높은 값을 나타내었다(도 10, HFD: 대조군2, HFD+NV-LJ3402: 실험군1).As a result, genes involved in lipid accumulation, fatty acid synthase (FAS) and acetyl, were found in epididymal adipose tissue (eWAT) of experimental group 1, which was orally administered with dead cells of Lactobacillus johnny JNU3402, compared to control group 2, which was a high-fat diet group. The expression of -CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP1c) was 40%, 32%, and 51% lower, respectively. Conversely, the expression of acetyl-CoA oxidase (ACOX), carnitine palmitoyltransferase 1 (CPT1), and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), which are genes involved in fatty acid beta oxidation, were higher than those in the control group 2, which is a high-fat diet. In experimental group 1, which is a group orally administered with dead cells of Lactobacillus johnny JNU3402, the values were 1.8 times, 1.9 times, and 2.3 times higher, respectively (FIG. 10, HFD: control 2, HFD+NV-LJ3402: experimental group 1).
또한, 고지방식이 그룹인 대조군 2와 비교하여 락토바실러스 존스니 JNU3402 사균체를 경구투여한 그룹인 실험군 1의 서혜부 백색지방(iWAT)에서 지방세포 갈색화에 관여하는 유전자인 uncoupling protein 1 (UCP1), PPARγ, Cidea의 발현이 각각 2.6 배, 4.5 배, 2.2 배 높은 값을 나타내었다(도 10, HFD: 대조군2, HFD+NV-LJ3402: 실험군1).In addition, uncoupling protein 1 (UCP1), a gene involved in browning of adipocytes, in inguinal white fat (iWAT) of experimental group 1, a group to which dead cells of Lactobacillus johnny JNU3402 were orally administered, compared to control group 2, a high-fat diet group. Expressions of PPARγ and Cidea were 2.6-fold, 4.5-fold, and 2.2-fold higher, respectively (FIG. 10, HFD: Control 2, HFD+NV-LJ3402: Experimental Group 1).
2) 사균체 조건배지 처리에 의한 유전자 발현 조절2) Regulation of gene expression by treatment with dead cells conditioned media
전술한 JNU3402 사균체에 의한 지방 조직 내 지질축적 및 에너지 대사에 관여하는 유전자의 발현 조절 결과와 마찬가지로, 3T3-L1 지방세포에 락토바실러스 존스니 JNU3402 사균체 조건배지(NV-LJ3402-CM)를 처리하지 않은 조건(con)에 비해, 락토바실러스 존스니 JNU3402 사균체 조건배지(NV-LJ3402-CM)를 처리하였을 때 지질축적에 관여하는 유전자인 FAS, ACC, SREBP1c의 발현이 각각 50%, 51%, 48% 감소하였으며, 지방산 베타 산화에 관여하는 유전자인 ACOX, CPT1, PGC1α의 발현은 3.4 배, 2 배, 2 배 증가하였다. 또한, 지방세포 갈색화에 관여하는 유전자인 UCP1, PPARγ, Cidea의 발현은 2.3 배, 1.7 배, 1.9 배 증가하였다(도 11). 유전자 발현의 통계적 차이는 Students’ t-test를 사용하여 분석하였다.Similar to the results of regulation of expression of genes involved in lipid accumulation and energy metabolism in adipose tissue by killed JNU3402 cells, 3T3-L1 adipocytes were treated with Lactobacillus johnny JNU3402 dead cell conditioned media (NV-LJ3402-CM). Compared to the untreated condition (con), the expression of FAS, ACC, and SREBP1c, which are genes involved in lipid accumulation, were increased by 50% and 51%, respectively, when Lactobacillus johnny JNU3402 killed cell conditioned medium (NV-LJ3402-CM) was treated. , decreased by 48%, and the expression of ACOX, CPT1, and PGC1α, which are genes involved in fatty acid beta oxidation, increased 3.4 times, 2 times, and 2 times. In addition, the expression of UCP1, PPARγ, and Cidea, which are genes involved in adipocyte browning, increased 2.3-fold, 1.7-fold, and 1.9-fold (FIG. 11). Statistical differences in gene expression were analyzed using Students' t-test.
11. 11. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체 조건배지에 의한 PPARγ의 전사활성 증가 Increased transcriptional activity of PPARγ by JNU3402 dead cells conditioned medium
PPARγ는 지방조직 내 지질대사의 주조절자로써 에너지 대사에 관여하는 유전자들의 활성과 발현을 조절하는 역할을 한다. 락토바실러스 존스니 JNU3402 사균체 조건배지의 PPARγ 전사 활성에 대한 영향을 조사하기 위해 HEK293T 세포에서 reporter gene assay를 실시하였다.PPARγ is a major regulator of lipid metabolism in adipose tissue and plays a role in regulating the activity and expression of genes involved in energy metabolism. To investigate the effect of Lactobacillus johnny JNU3402 killed cell conditioned medium on PPARγ transcriptional activity, a reporter gene assay was performed in HEK293T cells.
그 결과 PPARγ response element (PPRE)만 함유한 reporter에서 락토바실러스 존스니 JNU3402 사균체 조건배지를 처리했을 때, 락토바실러스 존스니 JNU3402 사균체 조건배지를 처리하지 않은 조건(도 12의 PPARγ)에 비해 PPARγ의 전사 활성이 약 2.3 배 증가하였으며, 표적유전자인 UCP1 promoter와 PPARγ promoter의 활성은 각각 2 배, 2.2 배 증가하였다(도 12).As a result, when the Lactobacillus johnny JNU3402 killed cell conditioned medium was treated in a reporter containing only the PPARγ response element (PPRE), compared to the condition in which the Lactobacillus johnny JNU3402 killed cell conditioned medium was not treated (PPARγ in FIG. 12), PPARγ The transcriptional activity of was increased by about 2.3 times, and the activities of the UCP1 promoter and PPARγ promoter, which are target genes, were increased by 2 times and 2.2 times, respectively (FIG. 12).
락토바실러스 존스니 JNU3402 사균체 조건배지에 의한 PPARγ의 표적유전자의 발현 증가가 PPARγ 활성증가를 통해 일어나는 것인지 조사하기 위해 3T3-L1 지방세포에 락토바실러스 존스니 JNU3402 사균체 조건배지와 함께 PPARγ antagonist인 GW9662를 처리하여 PPARγ 표적유전자 발현을 조사하였다. 그 결과 락토바실러스 존스니 JNU3402 사균체 조건배지를 처리하지 않은 경우(도 13 con)에 비해 락토바실러스 존스니 JNU3402 사균체 조건배지를 처리(도 13 NV LJ3402-CM)하였을 때 PPARγ, UCP1, ACOX의 발현이 각 약 2.2 배, 2.1 배, 3.1 배 증가하였다(도 13). 한편, 락토바실러스 존스니 JNU3402 사균체 조건배지를 PPARγ antagonist인 GW9662와 함께 처리한 조건(도 13 GW9662 + NV LJ3402-CM)에서는 세 유전자의 발현이 모두 감소하였다(도 13). 상기 결과에 따르면 락토바실러스 존스니 JNU3402 사균체 조건배지에 의한 에너지 대사 관련 유전자의 발현은 PPARγ 경로를 통해 조절되는 것으로 사료된다.In order to investigate whether the increased expression of PPARγ target genes by Lactobacillus johnny JNU3402 killed cell conditioned medium occurs through the increase in PPARγ activity, Lactobacillus johnny JNU3402 killed cell conditioned medium and GW9662, a PPARγ antagonist, were applied to 3T3-L1 adipocytes. was treated to investigate the expression of PPARγ target genes. As a result, when the Lactobacillus johnny JNU3402 dead cell conditioned medium was treated (Fig. 13 NV LJ3402-CM) compared to the case where the Lactobacillus johnny JNU3402 dead cell conditioned medium was not treated (Fig. 13 con), PPARγ, UCP1, and ACOX Expression increased about 2.2-fold, 2.1-fold, and 3.1-fold, respectively (FIG. 13). On the other hand, the expression of all three genes decreased under the condition that the Lactobacillus johnny JNU3402 dead cell conditioned medium was treated together with the PPARγ antagonist GW9662 (Fig. 13 GW9662 + NV LJ3402-CM) (Fig. 13). According to the above results, it is thought that the expression of genes related to energy metabolism by the dead cell conditioned medium of Lactobacillus johnny JNU3402 is regulated through the PPARγ pathway.
12. 12. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체 또는 이의 조건배지에 의한 미토콘드리아 함량 및 활성 변화 Changes in mitochondrial content and activity by killed cells of JNU3402 or its conditioned medium
지방세포 내 에너지대사는 주로 미토콘드리아에서 일어나며 미토콘드리아에서 열 생성 반응은 UCP1 발현 증가와 관련이 있다. 지방조직과 3T3-L1 지방세포에서 락토바실러스 존스니 JNU3402 사균체에 의한 미토콘드리아 copy number와 활성을 측정하기 위해, qPCR를 이용해 미토콘드리아 DNA 함량을 측정하였고, citrate synthase (CS) activity assay kit (BioVision, Milpitas, CA, USA)를 이용하여 미토콘드리아의 활성도를 측정하였다. 그 결과, 실험 쥐의 서혜부와 부고환 지방조직에서 미토콘드리아 DNA copy number는 고지방식이 그룹인 대조군 2에 비해 락토바실러스 존스니 JNU3402 사균체를 처리한 그룹인 실험군 1에서 각각 1.6 배, 1.3배 증가하였으며 CS 활성은 각각 2.9 배, 2.2 배 증가하였다(도 14, HFD: 대조군2, HFD+NV-LJ3402: 실험군1).Energy metabolism in adipocytes mainly occurs in mitochondria, and thermogenesis reaction in mitochondria is related to increased expression of UCP1. In order to measure mitochondrial copy number and activity by dead cells of Lactobacillus johnny JNU3402 in adipose tissue and 3T3-L1 adipocytes, mitochondrial DNA content was measured using qPCR, and citrate synthase (CS) activity assay kit (BioVision, Milpitas , CA, USA) was used to measure mitochondrial activity. As a result, the mitochondrial DNA copy number in the inguinal and epididymal adipose tissue of the experimental mice increased by 1.6 and 1.3 times, respectively, in Experimental Group 1, which was treated with dead cells of Lactobacillus johnny JNU3402, compared to Control Group 2, which was a high-fat diet group. CS The activity increased 2.9 times and 2.2 times, respectively (FIG. 14, HFD: control group 2, HFD+NV-LJ3402: experimental group 1).
또한, 3T3-L1 지방세포에 락토바실러스 존스니 JNU3402 사균체 조건배지를 처리(도 14 NV-LJ3402-CM)한 결과 대조군(도 14 con)과 비교하여 미토콘드리아 DNA copy number는 1.9 배 증가하였으며 CS 활성은 4.3 배 증가하였다(도 14).In addition, as a result of treating 3T3-L1 adipocytes with dead cell conditioned medium of Lactobacillus johnny JNU3402 (Fig. 14 NV-LJ3402-CM), the mitochondrial DNA copy number increased 1.9 times compared to the control group (Fig. 14 con), and CS activity increased by 4.3 times (FIG. 14).
13. 13. Lactobacillus johnsoniiLactobacillus johnsonii JNU3402 사균체에 의한 직장 온도 증가 Increased rectal temperature by killed cells of JNU3402
락토바실러스 존스니 JNU3402 사균체에 의한 미토콘드리아의 기능 및 UCP1 발현 증가로 인해 열 생성 반응이 유도되었는지를 확인하기 위해 실험 쥐의 직장 온도를 측정하였다. 고지방식이 쥐 그룹인 대조군2와 락토바실러스 존스니 JNU3402 사균체를 경구투여한 쥐 그룹인 실험군 1을 4 ℃ 에 노출시킨 후 일정 시간 간격으로 직장 온도를 측정한 결과, 저온 노출 4시간과 6시간 경과하였을 때 락토바실러스 존스니 JNU3402를 경구투여한 쥐의 직장 온도가 고지방식이 쥐의 직장 온도보다 더 높은 것을 확인하였다(도 15, HFD: 대조군2, HFD+NV-LJ3402: 실험군1).In order to confirm whether the thermogenesis was induced by the increased mitochondrial function and UCP1 expression by killed cells of Lactobacillus johnny JNU3402, the rectal temperature of the experimental mice was measured. Control group 2, a group of mice on a high-fat diet, and experimental group 1, a group of mice orally administered with dead cells of Lactobacillus johnny JNU3402, were exposed to 4 ℃ and rectal temperature was measured at regular intervals. After the passage of time, it was confirmed that the rectal temperature of the rats orally administered with Lactobacillus johnny JNU3402 was higher than that of the rats fed the high-fat diet (FIG. 15, HFD: Control 2, HFD+NV-LJ3402: Experimental Group 1).
<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Non-viable Lactobacillus johnsonii JNU3402 strain and composition for preventing or treating obesity comprising the same <130> 20P11046 <160> 25 <170> KoPatentIn 3.0 <210> 1 <211> 1443 <212> DNA <213> Artificial Sequence <220> <223> JNU3402 <400> 1 gcagtcgagc gagcttgcct agatgatttt ggtgcttgca ctaaatgaaa ctagatacaa 60 gcgagcggcg gacgggtgag taacacgtgg gtaacctgcc caagagactg ggataacacc 120 tggaaacaga tgctaatacc ggataacaac actagacgca tgtctagagt ttgaaagatg 180 gttctgctat cactcttgga tggacctgcg gtgcattagc tagttggtaa ggtaacggct 240 taccaaggca atgatgcata gccgagttga gagactgatc ggccacattg ggactgagac 300 acggcccaaa ctcctacggg aggcagcagt agggaatctt ccacaatgga cgaaagtctg 360 atggagcaac gccgcgtgag tgaagaaggg tttcggctcg taaagctctg ttggtagtga 420 agaaagatag aggtagtaac tggcctttat ttgacggtaa ttacttagaa agtcacggct 480 aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg atttattggg 540 cgtaaagcga gtgcaggcgg ttcaataagt ctgatgtgaa agccttcggc tcaaccggag 600 aattgcatca gaaactgttg aacttgagtg cagaagagga gagtggaact ccatgtgtag 660 cggtggaatg cgtagatata tggaagaaca ccagtggcga aggcggctct ctggtctgca 720 actgacgctg aggctcgaaa gcatgggtag cgaacaggat tagataccct ggtagtccat 780 gccgtaaacg atgagtgcta agtgttggga ggtttccgcc tctcagtgct gcagctaacg 840 cattaagcac tccgcctggg gagtacgacc gcaaggttga aactcaaagg aattgacggg 900 ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960 gtcttgacat ccagtgcaaa cctaagagat taggtgttcc cttcggggac gctgagacag 1020 gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080 gcaacccttg tcattagttg ccatcattaa gttgggcact ctaatgagac tgccggtgac 1140 aaaccggagg aaggtgggga tgacgtcaag tcatcatgcc ccttatgacc tgggctacac 1200 acgtgctaca atggacggta caacgagaag cgaacctgcg aaggcaagcg gatctcttaa 1260 agccgttctc agttcggact gtaggctgca actcgcctac acgaagctgg aatcgctagt 1320 aatcgcggat cagcacgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380 caccatgaga gtctgtaaca cccaaagccg gtgggataac ctttatagga gtcagccgtc 1440 taa 1443 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> fatty acid synthase sense primer <400> 2 agatcctgga acgagaacac gat 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> fatty acid synthase antisense primer <400> 3 gagacgtgtc actcctggac ttg 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> acetyl-CoA carboxylase sense primer <400> 4 gtatgttcga agggcttaca ttg 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> acetyl-CoA carboxylase antisense primer <400> 5 tgggcagcat gaactgaaat t 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sterol regulatory element-binding protein-1c sense primer <400> 6 actgtgacct cacaggtcca 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sterol regulatory element-binding protein-1c antisense primer <400> 7 ggcagtttgt ctgtgtccac a 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> acetyl-CoA oxidase sense primer <400> 8 tcgaggcttg gaaaccactg 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> acetyl-CoA oxidase antisense primer <400> 9 tcgagtgatg agctgagcc 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> carnitine palmitoyltransferase 1 sense primer <400> 10 actcctggaa gaagaagttc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> carnitine palmitoyltransferase 1 antisense primer <400> 11 tagggtccga ttgatctttg 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> peroxisome proliferator-activated receptor gamma coactivator 1-alpha sense primer <400> 12 gagactttgg aggccagca 19 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> peroxisome proliferator-activated receptor gamma coactivator 1-alpha antisense primer <400> 13 cgccatccct tagttcactg g 21 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> uncoupling protein 1 sense primer <400> 14 ggaggtgtgg cagtgttc 18 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> uncoupling protein 1 antisense primer <400> 15 tctgtggtgg ctataactct g 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PPAR gamma sense primer <400> 16 gaagaccact cgcattcctt 20 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PPAR gamma antisense primer <400> 17 gaaggttctt catgaggcct g 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cidea sense primer <400> 18 atcacaactg gcctggttac g 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Cidea antisense primer <400> 19 tactacccgg tgtccatttc t 21 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 36B4 sense primer <400> 20 agatgcagca gatccgcat 19 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 36B4 antisense primer <400> 21 atatgaggca gcagtttctc cag 23 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> D-loop sense primer <400> 22 aatctaccat cctccgtg 18 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> D-loop antisense primer <400> 23 gactaatgat tcttcaccgt 20 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH sense primer <400> 24 gttgtctcct gcgacttca 19 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GAPDH antisense primer <400> 25 ggtggtccag ggtttctta 19 <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Non-viable Lactobacillus johnsonii JNU3402 strain and composition for preventing or treating obesity comprising the same <130> 20P11046 <160> 25 <170> KoPatentIn 3.0 <210> 1 <211> 1443 <212> DNA <213> artificial sequence <220> <223> JNU3402 <400> 1 gcagtcgagc gagcttgcct agatgatttt ggtgcttgca ctaaatgaaa ctagatacaa 60 gcgagcggcg gacgggtgag taacacgtgg gtaacctgcc caagagactg ggataacacc 120 tggaaacaga tgctaatacc ggataacaac actagacgca tgtctagagt ttgaaagatg 180 gttctgctat cactcttgga tggacctgcg gtgcattagc tagttggtaa ggtaacggct 240 taccaaggca atgatgcata gccgagttga gagactgatc ggccacattg ggactgagac 300 acggcccaaa ctcctacggg aggcagcagt agggaatctt ccacaatgga cgaaagtctg 360 atggagcaac gccgcgtgag tgaagaaggg tttcggctcg taaagctctg ttggtagtga 420 agaaagatag aggtagtaac tggcctttat ttgacggtaa ttacttagaa agtcacggct 480 aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg atttatggg 540 cgtaaagcga gtgcaggcgg ttcaataagt ctgatgtgaa agccttcggc tcaaccggag 600 aattgcatca gaaactgttg aacttgagtg cagaagagga gagtggaact ccatgtgtag 660 cggtggaatg cgtagatata tggaagaaca ccagtggcga aggcggctct ctggtctgca 720 actgacgctg aggctcgaaa gcatgggtag cgaacaggat tagataccct ggtagtccat 780 gccgtaaacg atgagtgcta agtgttggga ggtttccgcc tctcagtgct gcagctaacg 840 cattaagcac tccgcctggg gagtacgacc gcaaggttga aactcaaagg aattgacggg 900 ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960 gtcttgacat ccagtgcaaa cctaagagat taggtgttcc cttcggggac gctgagacag 1020 gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc 1080 gcaacccttg tcattagttg ccatcattaa gttgggcact ctaatgagac tgccggtgac 1140 aaaccggagg aaggtgggga tgacgtcaag tcatcatgcc ccttatgacc tgggctacac 1200 acgtgctaca atggacggta caacgagaag cgaacctgcg aaggcaagcg gatctcttaa 1260 agccgttctc agttcggact gtaggctgca actcgcctac acgaagctgg aatcgctagt 1320 aatcgcggat cagcacgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380 caccatgaga gtctgtaaca cccaaagccg gtgggataac ctttatagga gtcagccgtc 1440 1443 taa <210> 2 <211> 23 <212> DNA <213> artificial sequence <220> <223> fatty acid synthase sense primer <400> 2 agatcctgga acgagaacac gat 23 <210> 3 <211> 23 <212> DNA <213> artificial sequence <220> <223> fatty acid synthase antisense primer <400> 3 gagacgtgtc actcctggac ttg 23 <210> 4 <211> 23 <212> DNA <213> artificial sequence <220> <223> acetyl-CoA carboxylase sense primer <400> 4 gtatgttcga agggcttaca ttg 23 <210> 5 <211> 21 <212> DNA <213> artificial sequence <220> <223> acetyl-CoA carboxylase antisense primer <400> 5 tgggcagcat gaactgaaat t 21 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> sterol regulatory element-binding protein-1c sense primer <400> 6 actgtgacct cacaggtcca 20 <210> 7 <211> 21 <212> DNA <213> artificial sequence <220> <223> sterol regulatory element-binding protein-1c antisense primer <400> 7 ggcagtttgt ctgtgtccac a 21 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> acetyl-CoA oxidase sense primer <400> 8 tcgaggcttg gaaaccactg 20 <210> 9 <211> 19 <212> DNA <213> artificial sequence <220> <223> acetyl-CoA oxidase antisense primer <400> 9 tcgagtgatg agctgagcc 19 <210> 10 <211> 20 <212> DNA <213> artificial sequence <220> <223> carnitine palmitoyltransferase 1 sense primer <400> 10 actcctgggaa gaagaagttc 20 <210> 11 <211> 20 <212> DNA <213> artificial sequence <220> <223> carnitine palmitoyltransferase 1 antisense primer <400> 11 tagggtccga ttgatctttg 20 <210> 12 <211> 19 <212> DNA <213> artificial sequence <220> <223> peroxisome proliferator-activated receptor gamma coactivator 1-alpha sense primer <400> 12 gagactttgg aggccagca 19 <210> 13 <211> 21 <212> DNA <213> artificial sequence <220> <223> peroxisome proliferator-activated receptor gamma coactivator 1-alpha antisense primer <400> 13 cgccatccct tagttcactg g 21 <210> 14 <211> 18 <212> DNA <213> artificial sequence <220> <223> uncoupling protein 1 sense primer <400> 14 ggaggtgtgg cagtgttc 18 <210> 15 <211> 21 <212> DNA <213> artificial sequence <220> <223> uncoupling protein 1 antisense primer <400> 15 tctgtggtgg ctataactct g 21 <210> 16 <211> 20 <212> DNA <213> artificial sequence <220> <223> PPAR gamma sense primer <400> 16 gaagaccact cgcattcctt 20 <210> 17 <211> 21 <212> DNA <213> artificial sequence <220> <223> PPAR gamma antisense primer <400> 17 gaaggttctt catgaggcct g 21 <210> 18 <211> 21 <212> DNA <213> artificial sequence <220> <223> Cidea sense primer <400> 18 atcacaactg gcctggttac g 21 <210> 19 <211> 21 <212> DNA <213> artificial sequence <220> <223> Cidea antisense primer <400> 19 tactacccgg tgtccatttc t 21 <210> 20 <211> 19 <212> DNA <213> artificial sequence <220> <223> 36B4 sense primer <400> 20 agatgcagca gatccgcat 19 <210> 21 <211> 23 <212> DNA <213> artificial sequence <220> <223> 36B4 antisense primer <400> 21 atatgaggca gcagtttctc cag 23 <210> 22 <211> 18 <212> DNA <213> artificial sequence <220> <223> D-loop sense primer <400> 22 aatctaccat cctccgtg 18 <210> 23 <211> 20 <212> DNA <213> artificial sequence <220> <223> D-loop antisense primer <400> 23 gactaatgat tcttcaccgt 20 <210> 24 <211> 19 <212> DNA <213> artificial sequence <220> <223> GAPDH sense primer <400> 24 gttgtctcct gcgacttca 19 <210> 25 <211> 19 <212> DNA <213> artificial sequence <220> <223> GAPDH antisense primer <400> 25 ggtggtccag ggtttctta 19
Claims (7)
Dead cells of Lactobacillus johnsonii JNU3402 (accession number KCCM12892P).
A pharmaceutical composition for preventing or treating at least one metabolic disease selected from the group consisting of obesity, diabetes, hyperlipidemia and fatty liver, comprising dead cells of Lactobacillus johnsonii JNU3402 (accession number KCCM12892P) as an active ingredient.
The pharmaceutical composition for preventing or treating metabolic diseases according to claim 2, wherein the composition reduces blood sugar.
The method according to claim 2, wherein the composition reduces the expression of at least one of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP1c), or acetyl-CoA oxidase (ACOX ), carnitine palmitoyltransferase 1 (CPT1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1α), uncoupling protein 1 (UCP1), PPARγ, and Cidea. Therapeutic pharmaceutical composition.
A health functional food for reducing body fat, controlling blood sugar, or improving triglycerides in blood, comprising dead cells of Lactobacillus johnsonii JNU3402 (accession number KCCM12892P) as an active ingredient.
The health functional food according to claim 5, characterized in that the health functional food reduces blood sugar.
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