KR20200084817A - Novel Lactobacillus rhamnosus strain for preventing or treating obesity and the use thereof - Google Patents

Abstract

The present invention relates to a novel Bifidobacterium longum strain or Lactobacillus rhamnosus strain effective for preventing or treating obesity. According to the present invention, treatment with each of the Bifidobacterium longum strain or Lactobacillus rhamnosus strain causes conversion of white fat cells into brown fat, and particularly shows an effect of significantly increasing expression of genes specific to beige fat cells and brown fat cells as compared to the non-treated control. In addition, after carrying out a test in a high-fat diet-induced obesity mouse, each strain shows an effect of significantly inhibiting an increase in body weight as compared to the negative control, and an effect of increasing expression of calorific specific genes in the mouse white fat cells. Therefore, each of the Bifidobacterium longum strain or Lactobacillus rhamnosus strain shows an anti-obesity effect, can be used advantageously for foods, pharmaceuticals or feeds for preventing or treating obesity, and shows high industrial applicability in the related industrial fields.

Description

Novel Lactobacillus rhamnosus strain for preventing or treating obesity and the use thereof

The present invention relates to a novel Bifidobacterium longum strain or Lactobacillus rhamnosus strain having a prophylactic or therapeutic effect on obesity and uses thereof.

With the recent improvement of the standard of living due to the modernization of the economy, the intake of fat and sugar increases, and the intake of fiber decreases. The reality is that the obese population is rapidly increasing due to the lifestyle of few modern people. Obesity, especially abdominal obesity, has emerged as a serious social problem as the consumption of high-calorie foods increases and fiber intake decreases. Obesity refers to a condition in which fat tissue is excessively increased. Most of the weight gain due to obesity is due to the increase in fat. The mechanism that leads to obesity is due to the ingestion of excess sugar, so that the sugar contained in food is digested to become monosaccharides, absorbed into the body through the small intestine, and the blood sugar rises and insulin secreted by its stimulation acts on fat cells to release the monosaccharides in the blood. Fat cells are accepted and converted into fat.

The harmfulness of obesity itself is often caused by constipation, indigestion, gastrointestinal obstruction, etc. due to the pressure of the abdomen by fat tissue itself, and there is a problem that it becomes a risk factor for many adult diseases. Obesity is known to have a direct relationship with diabetes, hypertension, coronary artery disease, and cancer, and WHO defines it as a chronic disease of the 21st century. It is estimated that the prevalence of domestic adults will increase to more than 50% in 2030, and that the national and personal economic burden will increase significantly. Accordingly, much attention and investment has been made in research to prevent and treat obesity at home and abroad.

Mouse adipocyte 3T3-L1 is a cell line that has been used for obesity research as it has already undergone differentiation so that it matures only as white adipocyte. In the present invention, using the fat cell line to investigate the effect of lactic acid bacteria culture on the brown adipose (Browning: the process of converting white fat cells that store energy into brown fat cells that consume energy to exothermic reaction, maintain thermal homeostasis) In addition, the composition for the prevention and treatment of obesity of the new paradigm is revealed through analysis of the specific gene expression patterns of brown adipocyte factor and beige adipocyte factor in mouse mesenchymal stem cells C3H10T1/2 cells.

On the other hand, Korean Registered Patent No. 1778734 discloses'ESBP separated from Bifidobacterium longum KACC 91563 and anti-allergic composition using the same', and Korean Registered Patent No. 1401530'Bifidobacterium producing conjugated linoleic acid Long gum strain and its use are disclosed, but as in the present invention,'Bifidobacterium long gum strain or Lactobacillus rhamnosus having an effect of preventing or treating obesity by inducing the formation of beige adipocytes and brown adipocytes The strain and its use' has not been revealed at all.

The present invention was derived by the above-mentioned needs, and in the present invention, unlike the general method of confirming the anti-obesity effect in terms of reducing the number of white adipocytes, in the present invention, it is most often used for obesity studies in which differentiation has already occurred. Browning of adipocyte 3T3-L1 by treatment of each of the culture medium of Bifidobacterium longum DS0956 and Lactobacillus rhamnosus DS0508 using progenitor adipocytes 3T3-L1 that mature into white adipocytes being used (Browning: Energy It was confirmed that the effect of the white adipocytes storing the energy on the exothermic reaction, the process of changing to brown adipocytes maintaining thermal homeostasis).

As a result, treatment of each of the lactic acid bacteria Bifidobacterium longum DS0956 strain and Lactobacillus rhamnosus DS0508 strain culture medium according to the present invention promoted the expression of beige adipocyte and brown adipocyte specific genes in white adipocyte 3T3-L1. In addition, it was confirmed that the expression of beige adipocyte specific genes and expression of brown adipocyte specific genes in mouse mesenchymal stem cells C3H10T1/2 cells was promoted.

In addition, as a result of confirming the suppression or improvement effect of obesity by administering the strain or culture solution of the Bifidobacterium longum, Lactobacillus rhamnosus of the present invention through a high-fat diet, a significant increase in weight compared to the negative control The inhibitory effect of the fever-specific gene or brown adipocyte/beige-colored adipocyte-specific gene in the white adipocytes was confirmed, and the effect of inducing and increasing the expression level was confirmed, and the lowering effect of total cholesterol and low density lipoprotein (LDL) was confirmed. Thus, it was confirmed that it has the effect of improving the lipid metabolism profile of obese mice.

Through the above fact, the Bifidobacterium longum strain and the Lactobacillus rhamnosus strain oxidize fat in the body, and also have an excellent effect of reducing fat metabolism and fat accumulation in the body through the activity of gene expression related to fat cell oxidation in the body, as well as the strain The present invention was completed by confirming that the effect of improving the lipid metabolism profile in the animal to which it was administered is excellent.

In order to solve the above problems, the present invention provides a novel Bifidobacterium longum strain or Lactobacillus rhamnosus strain.

In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity containing as an active ingredient one or more selected from the group consisting of the strain, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium and the extract of the culture medium. do.

In addition, the present invention is a health functional food composition for preventing or improving obesity containing at least one selected from the group consisting of the strain, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium and the extract of the culture medium as an active ingredient. Gives

In addition, the present invention provides a feed composition for preventing or improving obesity containing as an active ingredient one or more selected from the group consisting of the strain, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium and the extract of the culture medium. do.

Treatment of each of the lactic acid bacteria Bifidobacterium longum strain and Lactobacillus rhamnosus strain of the present invention caused brown fat localization of 3T3-L1, which should be matured only with white adipocytes, especially white adipocytes The effect of 3T3-L1 and mouse mesenchymal stem cell C3H10T1/2 cell expression of beige adipocyte and brown adipocyte specific genes was significantly increased compared to untreated control.

In addition, when the lactic acid bacteria Bifidobacterium longum and Lactobacillus rhamnosus strains or cultures of the present invention are administered to an individual, weight gain due to intake of a high-fat diet is suppressed and a fever-related gene, brown fat/beige fat cell specific It was confirmed that the expression level of the enemy gene increased and the amount of lipid components such as cholesterol and LDL decreased.

Therefore, each of the Lactobacillus Bifidobacterium longum strain and Lactobacillus rhamnosus strain exhibits anti-obesity effect, and thus can be usefully used as food, medicine, or feed for prevention or treatment of obesity, and improvement of lipid metabolism. Very useful for.

1A shows a selection flow chart for selecting a strain having anti-obesity efficacy from 55 lactic acid bacteria using 3T3-L1 cells, adipocytes.
Figure 1b is the result of screening after treatment with primary 1, 5, 10 μl of lactic acid bacteria culture using quantification of TG (Triglyceride). A, lactic acid bacteria culture solution 1 μl treatment group; B, lactic acid bacteria culture solution 5 μl treatment group; C, 10 μl treatment group of lactic acid bacteria culture; t, excluded from the second screening due to cytotoxicity. PA; Negative control without pre-adipocyte treatment with MDI differentiation medium; MDI (M: methyl-isobutyl-xanthine D: dexamethasone, I: insulin) adipocyte differentiation medium treatment control group; Positive control, Rosi (Rosiglitazone) treatment. Rosi is a PPAR gamma agonist.
Figure 2a is a graph showing the comparison of the relative accumulation of triglycerides (TG) compared to the control of the selected strain culture medium (30, 51 strains). PA, MDI and Rosi are the same as described in FIG. 1B.
Figure 2b is a microscopic photo of cells observed through TEM by processing the strain culture medium selected on 3T3-L1 cells (5,000X magnification). White arrows indicate lipid droplets (LD).
Figure 2c shows the ORO stain (Oil Red O dye) of the selected strain culture medium (30, 51 strains). 1st, lactic acid bacteria culture solution 1 μl treatment group; 2nd, lactic acid bacteria culture solution 5 μl treatment group; 3rd, lactic acid bacteria culture solution 10 μl treatment group; (-), negative control not treated with MDI differentiation medium; (+), group treated with MDI differentiation medium; Positive control, Rosi (Rosiglitazone) treatment. Rosi is a PPAR gamma agonist. 30 and 51 are the culture medium of the selected lactic acid bacteria.
Figure 3a shows the effect of the selected lactic acid bacteria culture medium (strain 30, 51) on the expression of brown adipocyte specific genes in 3T3-L1 adipocytes of mice, comparing the relative mRNA expression of the genes 3B shows the effect of the selected lactic acid bacteria culture medium (strains 30 and 51) on brown adipocyte specific expression genes in mouse mesenchymal stem cells C3H10T1/2 cells. It is a graph showing comparison of the relative mRNA expression levels. MDI (M: methyl-isobutyl-xanthine, D: dexamethasone, I: insulin) adipocyte differentiation medium treatment negative control; Positive control, Rosi (Rosiglitazone) treatment. Rosi is a PPAR gamma agonist. 30 and 51 are the culture medium of the selected lactic acid bacteria.
Figure 4a shows the effect of the selected lactic acid bacteria culture medium (strains 30 and 51) on the expression of beige adipocyte specific genes in adipocytes 3T3-L1 of mice, showing the relative mRNA expression of the genes It is a graph shown in comparison, Figure 4b shows the effect of the selected lactic acid bacteria culture medium (strain 30, 51) on beige adipocyte specific expression genes in mouse mesenchymal stem cells C3H10T1/2 cells, the gene It is a graph showing the relative expression level of mRNA. MDI (M: methyl-isobutyl-xanthine, D: dexamethasone, I: insulin) adipocyte differentiation medium treatment negative control; Positive control, Rosi (Rosiglitazone) treatment. Rosi is a PPAR gamma agonist. 30 and 51 are the culture medium of the selected lactic acid bacteria.
5A is a graph comparing and comparing the expression level of a lipolysis-related gene with a relative amount of mRNA when treated with lactic acid bacteria culture medium selected from 3T3-L1 cells (30 and 51 strains), and FIG. It is a graph comparing the expression level of β-oxidation-related genes by measuring the relative amount of mRNA.
FIG. 6 shows whether PKA signaling is activated when 3T3-L1 cells are treated with lactic acid bacteria cultures of strains 30 and 51. A confirms whether PKA is phosphorylated, and B indicates the result when H89, a PKA phosphorylation inhibitor, is treated. C is a graph measuring the amount of expression of the fever-related gene by H89 treatment as a relative mRNA amount, and D to F are siPKA cat a1 to express the amount of expression of the fever-related gene and adipocyte differentiation-related gene with the amount of mRNA. It is a graph measured and compared with the amount of protein.
Figure 7 is treated with lactic acid bacteria culture medium 30, 51 times in 3T3-L1 cells, to confirm the expression change of the lipolytic enzyme-related genes (left picture, HSL S-660 and HSL S-563 are Ser ^{563 of} HSL, respectively) , Phosphorylated to Ser ⁶⁶⁰ ), to confirm the activation of AMPK phosphorylation and transcription regulator CREB (center figure), and to confirm changes in the lipolytic enzyme-related gene and CREB phosphorylation according to the PKA inhibitor H89 treatment (Picture on the right).
Figure 8 shows the change in body weight of the measured mice after 12 weeks of administration of the lactic acid bacteria strain or culture medium, targeting the mice inducing obesity through a high-fat diet. (G1, high-fat diet-free group; G2, high-fat diet-treated group; G3, high-fat diet and microbial medium-administered group; G4, high-fat diet and Lactobacillus rhamnosus GG cell-treated group; G5, high-fat diet and 30 strain culture medium Administration group; G6, high fat diet and 51 strain culture group; G7, high fat diet and 30 cell administration group; G8, high fat diet and 51 cell administration group.
Figure 9 shows the results of H&E (Hematoxylin and Eosin) staining of the measured white fat of mice after 12 weeks of administration of the lactic acid bacteria strain or culture medium to mice that induce obesity through a high-fat diet.
FIG. 10 is for mice inducing obesity through a high-fat diet, and after administering the lactic acid bacteria strain or culture solution for 12 weeks, glucose, blood cholesterol, total cholesterol (T-chol), and high-density lipoprotein in the measured mice (HDL) and low density lipoprotein (LDL).
FIG. 11 is a target of mice inducing obesity through a high-fat diet. After administration of the lactic acid bacteria strain or culture solution for 12 weeks, white adipocytes (WAT), gonad white adipocytes (Gonadal WAT), peritoneal white adipocytes ( Peritoneal WAT) and mesenteric WAT.
Figure 12a is a graph comparing the relative mRNA expression level of a gene containing M1 macrophage inflammation-related cytokines in mice after 12 weeks of administration of the lactic acid bacteria strain or culture medium for mice inducing obesity through a high-fat diet. .
Figure 12b is a graph for comparing and measuring the relative mRNA expression of M2 macrophage-specific genes in mice after 12 weeks of administration of the lactic acid bacteria strain or culture medium, targeting mice inducing obesity through a high-fat diet.

Hereinafter, the present invention will be described in detail.

One. Bifidobacterium long gum strain and Lactobacillus rhamnosus strain

One aspect of the present invention provides a new Bifidobacterium longum strain or Lactobacillus rhamnosus strain.

The Bifidobacterium long gum strain may be a Bifidobacterium long gum DS0956, preferably a Bifidobacterium long gum DS0956 strain having a deposit number of KCTC13505BP, but is not limited thereto. The Bifidobacterium long sword DS0956 strain was deposited on March 26, 2018 with a deposit number of KCTC13505BP to the Korea Research Institute of Bioscience and Biotechnology.

The Lactobacillus rhamnosus strain may be Lactobacillus rhamnosus DS0508, preferably a Lactobacillus rhamnosus DS0508 strain having a deposit number of KCTC13504BP, but is not limited thereto. The Lactobacillus rhamnosus DS0508 strain was deposited on March 26, 2018 with a deposit number of KCTC13504BP to the Korea Research Institute of Bioscience and Biotechnology.

In the strain according to an embodiment of the present invention, the Bifidobacterium longum strain or Lactobacillus rhamnosus strain induces the formation of beige adipocytes and brown adipocytes to induce anti-obesity effects. Preferably, the Bifidobacterium longum strain or Lactobacillus rhamnosus strain specifically increases heat-generating genes and gene expression related to brown adipocytes in 3T3-L1 adipocytes and mouse mesenchymal stem cells C3H10T1/2. It may be to induce the formation of beige adipocytes and brown adipocytes, more preferably Ucp1 (uncoupling protein 1), Pgc1a (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), Prdm16 (PR /SET domain 16), Pparg (peroxisome proliferator activated receptor gamma), CD137, Fgf21 (fibroblast growth factor 21), P2RX5 (purinergic receptor P2X 5) and Tbx1 (T-box 1) gene expression increase to increase beige beige cells (beige adipocyte) and brown adipocytes. Most preferably, 3T3-L1 adipocytes and mouse mesenchymal stem cells C3H10T1/2 specifically increase the gene expression of heat-related genes Ucp1, Pgc1a, Prdm16 and CD137, Fgf21 associated with brown adipocytes, and beige fat. Cell (beige adipocyte) and may be to induce the formation of brown adipocytes, but is not limited thereto. The CD137 gene is also called TNFRSF9 (TNF receptor superfamily member 9). In addition, by treating the Bifidobacterium longum strain or Lactobacillus rhamnosus strain of the present invention, the expression amount of Past1, Resistin or Sarpina3k genes , which are genes specifically expressed in white adipocytes, may be reduced.

The strain of the present invention can increase the expression of a specific gene specific to brown adipocytes or beige adipocytes in white adipocytes that have already been differentiated, thereby converting the white adipocytes into brown adipocytes or beige adipocytes. Brown adipocytes and beige adipocytes are characterized by promoting the action of decomposition of fat for energy generation, so the strain of the present invention has the effect of suppressing or improving obesity.

In addition, the Bifidobacterium longum strain or the Lactobacillus rhamnosus strain increases the expression level of Atgl, HSL, Plin1 or Plin5 genes , genes involved in the degradation of fat , or β-oxidation of lipids (β-oxidation) It may be to increase the expression level of genes related to LCAD , MCAD, LCPT or Abhd5 . Since the fat-decomposition-related gene or β-oxidation-related gene can promote the action of decomposing and removing accumulated fat, the strain of the present invention reduces or reduces fat accumulation and suppresses or improves obesity. It has the effect.

Furthermore, the Bifidobacterium longum strain or Lactobacillus rhamnosus strain may be to activate PKA signaling. By activating the PKA signaling process, the amount of phosphorylated PKA, phosphorylated AMPK, and phosphorylated transcription regulator CREB is increased, and accordingly, the genes related to the differentiation of fever-related genes or adipocytes Ucp1, Pgc1a, Pparg or Ceba genes By increasing the expression of, it has the effect of suppressing or improving obesity through the induction of brown adipose of white adipocytes.

The Bifidobacterium longum strain or Lactobacillus rhamnosus strain of the present invention has an effect of improving or treating obesity by improving the profile of lipid metabolism when administered to an obese person. In order to confirm the effect of improving the profile of lipid metabolism of the present invention, in a specific embodiment of the present invention, a strain or culture of Bifidobacterium longum or Lactobacillus rhamnosus is administered to a mouse inducing obesity through a high-fat diet. As a result of confirming the change, the strain and the culture solution of the present invention have an effect of suppressing an increase in body weight, and the expression level of a fever-related gene, a brown adipocyte, or a beige adipocyte specific gene in white adipocytes of the mouse increases. Was confirmed. By increasing the expression level of the gene as described above, the process of differentiating the white adipocytes of the individual into brown adipocytes or beige adipocytes can be promoted to suppress or improve obesity, and cholesterol of the individual who administered the strain or culture solution , LDL can improve lipid metabolism by reducing the amount of lipid components.

In addition, in another specific embodiment of the present invention, the strain or culture solution of the Bifidobacterium longum or Lactobacillus rhamnosus was administered to an obese individual to confirm the expression level of the gene, and inflammation in the white adipocytes of the individual- It was confirmed that the expression levels of the accelerated M1 macrophage markers CD11c, CD68, IL-1b, Mcp1, and TNF-a genes were decreased, and that the expression levels of the anti-inflammatory M2 macrophage markers Arg1 and CD206 genes were increased. Therefore, when the strain or the culture solution of the present invention is administered to an obese individual, the amount of the M1 macrophage is reduced and the amount of the M2 macrophage is converted in an increasing direction, so when the strain or the culture solution is administered to an obese individual It can be confirmed that there is an effect to be suppressed or improved.

2. Composition containing lactic acid bacteria

Another aspect of the present invention provides a composition comprising lactic acid bacteria.

The lactic acid bacteria include the Bifidobacterium longum strain or Lactobacillus rhamnosus strain.

The composition containing the lactic acid bacteria may include at least one selected from the group consisting of the strain, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium and the extract of the culture medium as an active ingredient.

The composition comprising the lactic acid bacteria of the present invention is prepared in a unit dose form by formulating using a carrier, excipient and/or additive according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. Or it can be manufactured by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an ex-agent, powder, granule, tablet, capsule, gel (e.g., hydrogel) or lyophilizer, dispersant as the additive, Stabilizers or cryoprotectants may additionally be included.

Specifically, in the case of the lyophilizer, the strain is lyophilized together with a cryoprotectant to be used in the form of a powder, and the cryoprotectant is skim milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, mannitol, cyclo Dextrin, glycerol and/or honey. In addition, it is mixed with a storage carrier, adsorbed, dried, and solidified to be used. The storage carrier may be diatomaceous earth, activated carbon, and/or degreased steel.

The composition comprising the lactic acid bacteria of the present invention is one or more selected from the group consisting of strains, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium and the extract of the culture medium, any one of the carrier, excipient or additive It can be prepared through a mixing step.

The strains, carriers, excipients and additives are described above. When a cryoprotectant is used as the additive, the composition containing the lactic acid bacteria is mixed with the strain and the cryoprotectant, and the mixture is frozen at -45 °C to -30 °C, then dried at 30 °C to 40 °C. By grinding with a blender may be prepared in the form of a lyophilized powder. Specifically, the freezing process may be a process of vacuum freezing for 65 to 75 hours under a temperature condition of -45°C to -30°C and a pressure condition of 5 to 50 mTorr.

3. Prevention, treatment or improvement of obesity of a composition containing lactic acid bacteria

Another aspect of the present invention provides a use for preventing, treating or improving obesity of a composition comprising the lactic acid bacteria.

The composition containing the lactic acid bacteria may be a medicine, food or feed. The composition containing the lactic acid bacteria may be a pharmaceutical composition for preventing or treating obesity when the composition is a medicine, a health functional food for preventing or improving obesity when the composition is a food, and when the composition is a feed , It may be a feed composition for preventing or improving obesity.

The present invention provides a pharmaceutical composition for preventing or treating obesity containing as an active ingredient one or more selected from the group consisting of the strain, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium and the extract of the culture medium.

The strain is as described above, and in one embodiment of the present invention, the pharmaceutical composition is pharmaceutically acceptable according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. It can be produced in unit dose form by formulating with a carrier and/or excipient, or it can be prepared by incorporating it into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an ex-agent, powder, granule, tablet, capsule or gel (eg, hydrogel), and may additionally include a dispersant or stabilizer. have.

In addition, the strain contained in the pharmaceutical composition may be carried in a pharmaceutically acceptable carrier such as colloidal suspension, powder, saline, lipids, liposomes, microspheres, or nanospherical particles. These can be complexed or associated with transport vehicles, and can be used in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be transported in vivo using known transport systems.

In addition to this, pharmaceutically acceptable carriers are lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinyl pyridone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, but is not limited thereto. In addition, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, preservatives, etc. may be further included in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

The pharmaceutical composition according to the present invention can be administered orally or parenterally during clinical administration and may be used in the form of a general pharmaceutical preparation. That is, the pharmaceutical composition of the present invention may be administered in various dosage forms, oral and parenteral, during actual clinical administration. When formulated, diluents such as fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, etc. It is prepared using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc.These solid preparations include at least one excipient in herbal extracts or herbal fermentations, such as starch, calcium carbonate, sucrose or It is prepared by mixing lactose and gelatin. In addition, lubricants such as magnesium stearate talc are used in addition to simple excipients. Liquid preparations for oral administration include suspending agents, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used as diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As a non-aqueous solvent and a suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the suppression and treatment of obesity.

The concentration of the active ingredient contained in the composition of the present invention can be determined in consideration of the purpose of treatment, the patient's condition, and the required period, and is not limited to a specific range of concentration. The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention,'a pharmacologically effective amount' means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, drug activity, drug Sensitivity to, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with a therapeutic agent for diseases caused by other contaminants or a therapeutic agent for improving skin aging, and simultaneously, separately or sequentially with a conventional therapeutic agent. And can be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of active ingredients in the body, inactivation rate, excretion rate, disease type, and drugs used in combination, administration route, obesity It can be increased or decreased depending on the severity, sex, weight, age, etc., for example, the peptide of the present invention can be administered about 0.0001 μg to 500 mg per kg of patient body weight per day, such as 0.01 μg to 100 mg. In addition, it may be administered several times a day, for example, divided into 2 to 3 times a day at regular time intervals according to the judgment of the doctor or pharmacist.

The present invention provides a method for preventing or treating obesity, comprising administering the pharmaceutical composition to a subject.

The object may be a human or an animal other than a human, and may be in a non-obesity state or an obese state. When the subject is in a non-obesity state, the pharmaceutical composition can be administered to the subject in a pharmaceutically effective amount to prevent obesity, and when the subject is in an obese state, the pharmaceutical composition is administered to the subject in a pharmaceutically effective amount. It can be administered to treat obesity.

The formulation of the pharmaceutical composition, method of administration, dosage and concentration of the active ingredient contained in the composition are as described above.

In addition, the present invention is a health functional food composition for preventing or improving obesity containing at least one selected from the group consisting of the strain, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium and the extract of the culture medium as an active ingredient. Gives

In the dietary supplement composition according to an embodiment of the present invention, the dietary supplement composition may suppress an increase in weight or accumulation of fat.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition may be added as it is or used with other foods or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient may be appropriately used depending on the purpose of use (prevention or improvement). In general, the health functional food composition of the present invention in the manufacture of food or beverage is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less with respect to the raw material. However, when ingested for a long period of time for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There are no particular restrictions on the type of the health functional food. Examples of foods to which the health functional food composition can be added are meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum products, dairy products including ice cream, various soups, beverages, tea Drinks, alcoholic beverages, and vitamin complexes are included, and all foods in the ordinary sense are included.

In addition, the health functional food composition of the present invention may be prepared as a food, particularly a functional food. The functional food of the present invention includes ingredients that are commonly added in food preparation, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink agent, natural carbohydrates or flavoring agents may be included as additional components in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g. glucose, fructose, etc.), disaccharides (e.g. maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g. dextrins, cyclodextrins, etc.) or sugar alcohols (e.g. , Xylitol, sorbitol, erythritol, and the like). The flavoring agent may be a natural flavoring agent (eg, tau martin, stevia extract, etc.) and a synthetic flavoring agent (eg, saccharin, aspartame, etc.).

Various nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid, in addition to the above-mentioned health functional food composition It may further contain a carbonation agent used in beverages. The proportion of the components to be added is not particularly important, but it is generally selected from the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the health functional food composition of the present invention.

In addition, the present invention provides a feed composition for preventing or improving obesity containing as an active ingredient one or more selected from the group consisting of the strain, the culture medium of the strain, the concentrate of the culture medium, the dried product of the culture medium and the extract of the culture medium. do.

The strain is as described above, and may be added as a feed additive composition for the purpose of preventing or improving obesity. The feed additive of the present invention corresponds to the supplementary feed under the feed management law.

In the present invention, the term "feed" may mean any natural or artificial diet, one meal or the like, or a component of the one meal, for the animal to eat, eat, and digest. The type of the feed is not particularly limited, and a feed commonly used in the art may be used. Non-limiting examples of the feed, vegetable feed, such as grains, muscles, food processing by-products, algae, fiber, pharmaceutical by-products, fats and oils, starches, peels or grain by-products; And animal feeds such as proteins, inorganics, oils and fats, oils and fats, single cell proteins, animal planktons and food. These may be used alone or in combination of two or more.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only intended to specifically illustrate the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

Reagents used

Dexamethasone, IBMX (isobutyl-1-metylxanthine), insulin (insulin), rosiglitazone (Rosiglitazone, Rosi), Oil Red O dye, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide) and 4% formaldehyde were purchased from Sigma Aldrich (St. Louis, MO, USA).

Dulbecco Modified Eagle's Media (DMEM), newborn calf serum (NBCS) and recombinant human BMP4 were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum was purchased from Atlas Biologics (Fort Collins, CO, USA). Penicillin-streptomycin solution was purchased from Hyclone Laboratories, Inc. (South Logan, NY, USA).

Lactobacillus separation

The separation of various lactic acid bacteria used MRS medium under absolute anaerobic conditions, and sterilized after removing oxygen present in the medium using N2 gas for anaerobic conditions. 0.1 g of the collected fecal sample was suspended in 10 ml MRS medium, diluted in steps, and plated 100 μl in MRS plate medium or blood agar medium for 2 days at 37° C. under anaerobic conditions. As a result, the single colonies produced were subcultured, purified, and stored for a long time.

Identification of isolated lactic acid bacteria

For molecular biological identification of the isolated lactic acid bacteria, 27F (5'-AGA GTT TGA TCM TGG CTC A-3': SEQ ID NO: 3) and 1492R (5'-TAC GGY TAC CTT GTT) are universal primers targeting the 16S rRNA gene. ACG ACT T-3': SEQ ID NO: 4) was used, and sequencing of the 16S rRNA gene was performed. The nucleotide sequences obtained by analysis were identified through identifiable searches in EZbiocloud (http://www.ezbiocloud.net/).

Anti-obesity activity investigation

To investigate anti-obesity activity, 3T3-L1 cells were mixed with 10% NBCS and 1% penicillin-streptomycin in DMEM glutamax and cultured at 37°C in a 5% CO2 incubator. When the cell concentration of 3T3-L1 cells is 70-80%, inoculate into a 48 well plate. When the cell concentration was 100%, the adipocyte differentiation medium MDI (Insulin, Dexamethasone, Isobutyl-1-methylxanthine (IBMX)) was changed. On Day 2, insulin, insulin and ROSI, and insulin (sample culture medium) were treated, and on Day 4, only insulin was treated. And on day 6, cells were fixed without sample treatment. At this time, for anti-obesity effect of the lactic acid bacteria solution, 1, 5, and 10 µl were added to Day 0 and 2, respectively, to the medium. This MDI was performed by changing every other day during cell differentiation. The day when the cell concentration was 100% was designated as Day 0, and on Day 0, MDI, ROSI and MDI, and sample and MDI were treated as such. In this experiment, three independent repeat experiments were performed for each sample. To observe the cells, 3T3-L1 cells were cultured in a 24-well plate for 6 days, and then stained using a fixed, Oil Red O (ORO) staining reagent. To briefly describe this experimental method, first, after washing the cells once with 1X PBS, the cells were fixed at room temperature for 1 hour with 10% formalin. Then, the mixture was dyed at room temperature for 20 minutes using a 0.3% ORO solution and washed 4 times with distilled water. After washing, the phenotype changed through an Axiovert-25 microscope was observed under a microscope and photographed. Thereafter, the stained cells were dissolved in 100% isopropanol, and the amount of ORO was measured with an absorbance of 520 nm in Victor ^TM X3.

In addition, C3H10T1/2 mouse mesenchymal stem cells were purchased from Korean Cell Line Bank (KCLB-10226), and 5% CO2 in high concentration glucose DMEM medium containing 10% NBCS and 1% penicillin-streptomycin. The incubator was cultured at 37°C. To induce differentiation, C3H10T1/2 cells with a cell concentration of 20-30% were inoculated. For differentiation into adipocytes, cells were treated with 50 ng/mL of human recombinant BMP4 until the cell concentration was 100%. Thereafter, the medium was changed to a new medium on the 2-3rd day. 48 hours after 100% concentration of cells is referred to as 0 days, the medium is treated with 10% FBS, 0.5mM IBMX, 1μM dexamethasone and 10μg/ml in conditions treated with Rosiglitazone (Rosi) or lactic acid bacteria culture solution at the specified concentration. Differentiation was induced by changing to DMEM containing insulin (MDI). Differentiated cells were exposed to 500 μM dibutyryl-cAMP for 4 hours to stimulate the heat generation program.

qRT-PCR analysis

Total RNA was extracted using lactic acid bacteria culture treated cells using RNA extraction kit (Valencia, CA, USA, Qiagen) according to the manufacturer's instructions, and Scandrop Analytik jena AG spectrometer (Jena, Germany) The concentration was measured using. 1 μg of RNA was synthesized cDNA with Maxime RT PreMix kit (Intron Biotechnology, Seoul, Korea), and PCR reaction was performed in a Veriti 96-well thermal cycler Applied Biosystems, Singapore). Quantitative real-time PCR was performed on a CFX96TM real-time PCR detection system (Bio-Rad, Singapore) using the iQTM SYBR Green Supermix kit (Bio-Rad, Singapore). The sequences for the primers are listed in Table 1. The amount of expression was quantified by Gapdh (Glyceraldehyde 3-phosphate dehydrogenase) (Yoon D, Imran KM, Kim YS. 2018 Toxicol Appl Pharmacol. Feb 1;340:9-20).

Primer set used in this experiment Gene Name Forward (5' → 3') (SEQ ID NO) Reverse (5' → 3') (SEQ ID NO) Ucp1 GGCATTCAGAGGCAAATCAGCT(5) CAATGAACACTGCCACACCTC(6) Pgc1a ACAGCTTTCTGGGTGGATT(7) TGAGGACCGCTAGCAAGTTT(8) Prdm16 CAGCACGGTGAAGCCATTC(9) GCGTGCATCCGCTTGTG(10) Tbx1 GGCAGGCAGACGAATGTTC(11) TTGTCATCTACGGGCACAAAG(12) Fgf21 AGATCAGGGAGGATGGAACA(13) TCAAAGTGAGGCGATCCATA(14) CD137 CGTGCAGAACTCCTGTGATAAC(15) GTCCACCTATGCTGGAGAAGG(16) Cox2 GACTGGGCCATGGAGTGG(17) CACCTCTCCACCAATGACC(18) P2RX5 CTGCAGCTCACCATCCTGT(19) CACTCTGCAGGGAAGTGTCA(20) Gapdh GACATGCCGCCTGGAGAAAC(21) AGCCCAGGATGCCCTTTAGT(22)

Anti-obesity efficacy investigation by administration of high-fat diet-induced obesity mouse

While ingesting a high-fat diet to a mouse to induce an obesity model, the efficacy was compared by administering an intestinal microbial culture or cell to the mouse for 12 weeks. The mice used in this study were C57BL/6, SPF male mice obtained at 3 weeks of age and were used for testing only healthy animals after 7 days of purification. The high-fat diet was fed for 12 weeks using a 45% kcal high fat diet, D12451 (Research Diet) to establish a diets induced obesity (DIO) obesity model. The group composition used in the experiment was exemplified in Table 2 below, and the administered lactic acid bacteria were 10 ⁹ cell/kg, and the culture medium was lyophilized in 1 ml culture medium per horse, then dissolved in 150 μl of distilled water and administered once daily.

Group Animal number Sex Treat Dose concent. (Vol) G1 (Saline, Non-45% kcal HFD) 6 male 150 μl/Head G2 (Saline, 45% kcal HFD) 6 150 μl/Head G3 (MRS broth, 45% kcal HFD) 6 150 μl/Head G4 (LGG, 45% kcal HFD) 6 150 μl/Head G5 (30 strain culture, 45% kcal HFD) 6 150 μl/Head G6 (Strain culture No. 51, 45% kcal HFD) 6 150 μl/Head G7 (strain cell 30, 45% kcal HFD) 6 150 μl/Head G8 (Strain 51, 45% kcal HFD) 6 150 μl/Head

All animals were observed for general symptoms once a day until the necropsy day, and body weight and diet were measured 5 times a week during the examination period. After the end of the test period, anesthesia was performed using respiratory anesthesia, blood was collected through cardiac blood collection, and fat areas were extracted 2 times per group, stored in 4% Paraformaldehyde solution or stored in RNA Storage solution (ThermoFisher). The blood cell analysis was performed using a blood cell analysis equipment (Beckman coulter). After completing the blood cell analysis, total cholesterol, HDL, LDL, and glucose were analyzed by centrifugation and plasma. Adipose tissue in 4% PFA solution was used to prepare paraffin blocks.

Statistical analysis

All data from this experiment were expressed as the mean±standard deviation (SD) of three or more independent experiments. Unless otherwise specified, using the sample group treated with MDI, it was confirmed how much change was made compared to the control group. Significant differences between control and other treatment groups were calculated using Student's t-test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. P values less than 0.05 were considered statistically significant.

Example 1. Exploration of anti-obesity active strains

To observe the possibility of conversion from 3T3-L1 adipocytes to beige and brown adipocytes using 55 lactic acid bacteria, each lactic acid bacteria strain was cultured in MRS medium for 48 hours and centrifuged to obtain a supernatant. The secured supernatant was lyophilized and then concentrated to prepare a concentrate by adding 1/10 volume of sterile distilled water to the 3T3-L1 cells with 1, 5, and 10 μl of each to triglyceride through Oil Red O staining. The amount of accumulation was quantified. Accordingly, an active candidate group (green bar) for activating brown adipocytes and beige adipocytes in lactic acid bacteria culture was selected. In addition, candidate groups that inhibited the formation of adipocytes were selected (red bars) (FIG. 1B).

For the treatment of brown adipocytes, in groups treated with relatively high concentrations of 10 µl, among those that increase proliferation 10 to 20% in adipocytes treated with 1, 5, and 10 µl of lactic acid bacteria concentrate individually, as in the case of treatment with Rosi as a control It was selected primarily. In the case of No. 51, which showed an inhibitory effect on adipocyte formation, it was selected from the group treated with 10 µl, which is a relatively high concentration in three individual concentration experiments. After the first screening with the accumulation amount of triglyceride as described above, the selected candidate groups were screened again through the 2nd to 3rd screening process through the experiment to confirm whether or not the expression of ucp1 , one of the brown adipocyte specific genes, was expressed again . Lactic acid bacteria strains 30 and 51 were finally selected.

To determine the effect on adipocyte production by individually treating 1, 5, and 10 µl for the final screening Nos. 30 and 51, the amount of triglyceride was examined through Oil red O staining, and lipid droplets (LD) present in the adipocytes (LD , lipid droplets) (FIGS. 2A to 2C ). As a result, when the lactic acid bacteria strains 30 and 51 are treated, the accumulation amount of triglycerides appears to increase, and it is confirmed that the lipid droplets are formed smaller in a non-combined form, and the selected 30 and 51 are white of 3T3-L1 cells. It was found that the strain is expected to inhibit the maturation to adipocytes and increase the conversion to brown adipocytes.

Example 2. Effect of lactic acid bacteria culture solution on mouse fat cell brown adipocyte and beige adipocyte specific gene expression

The expression of UCP1 (Uncoupling protein 1) gene that is not expressed in white adipose tissue is called beige adipocyte. The expression of this gene in white adipocytes suggests that transdifferentiation of white adipocytes to beige adipocytes or brown adipocytes has occurred, and that the heteromorphic changes in these cells can be reversible depending on feeding or external environment. Is known. Therefore, in this experiment, the effect of the selected strain culture medium on the expression of beige adipocyte and brown adipocyte genes was investigated.

The effect of lactic acid bacteria culture on the expression of specific genes in mouse adipocytes, brown adipocytes and beige adipocytes was investigated. As shown in Figure 3a, it was confirmed that the expression of the Ucp1 (Uncoupling protein 1) gene, known as the heatogenesis- related gene and the brown adipocyte specific gene, is significantly increased in 3T3-L1 adipocytes treated with the lactic acid bacteria culture solution 30, It was confirmed that the expression of other brown fat specific genes Pgc1a (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and Prdm16 (PR/SET domain 16) was increased. In addition, it was confirmed that the expression of peroxisome proliferator activated receptor gamma ( Pparg ), which is important for general adipocyte differentiation, increases. When the lactic acid bacteria culture solution 51 was treated, the expression of the brown adipocyte specific gene was not significantly increased, but it was confirmed that the expression of the Prdm16 gene was increased.

In addition, in order to verify the effect of the lactic acid bacteria culture solution once again, the strain culture solution was treated with C3H10T1/2 cells, which are mouse mesenchymal stem cells, to confirm the heat generation and specific brown adipocyte factors Ucp1, Pgc1a , and Prdm16 expression levels. When processing the culture medium of the lactic acid bacteria No. 30 and 51 of the present invention, it was confirmed that the expression level of the brown adipocyte specific genes increased (FIG. 3B).

Then, in order to confirm the effect of heterozygous transformation from 30 white adipocytes to beige adipocytes, fibroblast growth factor 21 ( Fgf21 ), Tbx1, P2RX5, CD137 , and citrome c oxidation in 3T3-L1 adipocytes treated with lactic acid bacteria culture solution It was characteristically observed that the expression of specific markers of several beige adipocytes, such as the enzyme subunit II ( Cox2 ), increased significantly. In addition, it was confirmed that the expression of CD137 and Fgf21 , genes important for the expression of beige adipocytes, was significantly increased in cells treated with the culture solution of strain 51 (FIG. 4A).

Again, to verify the effect of the selected lactic acid bacteria strain culture solution, the strain culture solution is treated in mouse mesenchymal stem cells C3H10T1/2 cells, and beige adipocyte specific genes Fgf21, P2RX5, CD137 and Tbx1 (T-box 1) As a result of confirming the expression level of the present invention, it was confirmed that the expression level of the beige adipocyte-specific gene increased when the culture medium of lactic acid bacteria No. 30 and No. 51 of the present invention was treated (FIG. 4B).

Example 3. Effects of Lactic Acid Bacteria Culture on Lipid Decomposition, β-oxidation Related Gene Expression, and Effects on Activation of PKA Signaling Process

In order to further confirm the effect of the lactic acid bacteria culture solution of the present invention , the expression level of Atgl, HSL, Plin1, and Plin5 genes related to lipolysis is confirmed by treating the lactic acid bacteria culture solution targeting 3T3-L1 cells, and β of lipid -The expression levels of the genes related to oxidation ( LCAD, MCAD, LCPT, and Abhd5 ) were confirmed.

As a result, the expression levels of the Atgl, HSL, Plin1, and Plin5 genes and the LCAD, MCAD, LCPT, and Abhd5 genes in the 3T3-L1 cells treated with the lactic acid bacteria culture medium No. 30 and 51 of the present invention were measured to be higher than the control group (FIG. 5A). And Figure 5b), it was confirmed that the effect of inhibiting the accumulation of fat and weight gain by inducing the action to decompose fat or oxidize lipid components.

In addition, the lactic acid bacteria culture solution of the present invention was treated to 3T3-L1 cells, and then the degree of activation of PKA signaling was measured. Whether phosphorylated PKA was increased was confirmed by Western blotting (A in FIG. 6), and PKA inhibitor H89 was treated with 10 mM to confirm phosphorylated PKA again (B in FIG. 6), No. 30 of the present invention, When the lactic acid bacteria culture solution 51 was processed, it was observed that phosphorylated PKA increased, and thus it was confirmed that there is an effect of activating the PKA signaling process. As a result of confirming the change in the expression level of the heat-related genes Ucp1 and Pgc1a genes after treatment with the PKA inhibitor H89 (C in FIG. 6), it was further confirmed that the expression levels of the Ucp1 and Pgc1a genes decreased during H89 treatment, PKA Through activation, it was confirmed once again that the lactic acid bacteria culture solution of the present invention induces an increase in the expression level of the fever-related gene. And as a result of confirming the expression of the fever gene, adipocyte differentiation related gene and the expression level of these proteins using si-PKA cat a1, the expressions of Ucp1, Pgc1a, Pparg, Ceba genes are suppressed, and the Ucp1, Pparg, Pgc1a proteins are suppressed. It was confirmed that the amount was also reduced, and it was confirmed once again that the lactic acid bacteria culture solution of the present invention induces an increase in the expression level of the fever-related gene through PKA activation (D to F in FIG. 6).

Further, it was confirmed that the phosphorylation of lipolytic enzyme, AMPK phosphorylation and CREB, which is an electron regulatory factor, was increased as the lactic acid bacteria No. 30 and No. 51 of the present invention were treated with 3T3-L1 cells. The effect of the present invention was confirmed once again through the inhibition of lipolytic enzyme and CREB phosphorylation (see FIG. 7 ).

Example 4. Anti-obesity efficacy investigation by high-fat diet-induced obesity mouse administration

During the test period, general abnormalities were not observed in all groups, but body weight increased significantly by 34.0% in the high-fat mice compared to the normal group. In particular, in the case of the administration group of strain 30 culture medium (G5) and the administration group of strain 51 cells (G8), the difference in mean weight compared to the negative control group (G2) was 10.8% and 5.7%, respectively, showing the average weight in the two experimental groups. Decrease occurred (Fig. 8). In addition, as a result of analyzing the H&E pathology of each adipocyte tissue (area measurement), Gonadal fat (19.6%, 18.9%, respectively), Peritoneal fat (21.2%, 22.4%, respectively) in the G5 and G8 administration groups compared to the negative control (G2), Mesenteric fat (33.9%, 24.4%, respectively), White adipose tissue (26.2%, 23.4%) shows the difference (FIG. 9), strains 30 and 51 of the present invention or its culture medium is effective in reducing the accumulation of fat It was confirmed that there is.

In addition, as a result of performing the biochemical analysis, it was confirmed that the levels of total cholesterol and LDL were significantly decreased in the G5 and G8 groups compared to the negative control group (FIG. 10 ), and the strain and culture solution of the present invention were administered to the animals together. In the case, it was confirmed that cholesterol and LDL components have an effect of being reduced. As a result of confirming the expression levels of Ucp1, Pgc1a, and Prdm16 genes , which are genes related to fever or lipolytic enzymes in each adipose tissue, the expression level was significantly increased in the group administered with the strain or culture solution of the present invention in 4 adipose tissues of mice. It was confirmed (Fig. 11).

Furthermore, the effect of the lactic acid bacteria strain or the culture medium of the present invention on the inflammation-related macrophage polarization of the white fat was confirmed in mice. The expression level of the pro-inflammatory M1 macrophage markers CD11c, CD68, IL-1b, Mcp1, and TNF-a genes was confirmed (FIG. 12A), and the anti-inflammatory M2 macrophage markers Arg1 and CD206 genes The expression level change was confirmed (Fig. 12B). In the fat cells of an obese individual, the inflammatory response is promoted, and the conversion from M2 macrophages to M1 macrophages is confirmed to confirm the change in polarity of the macrophages, thereby confirming the effect of suppressing or improving obesity. As a result, in the case of the mice to which the lactic acid bacteria strain of the present invention or the culture solution was administered, the expression level of the M1 macrophage marker gene was reduced, and the expression level of the M2 macrophage marker gene was confirmed to be increased. It was confirmed that the strain or the culture broth had an effect of suppressing or improving obesity.

Example 5. Identification of selected strains

As a result of performing the 16S rRNA gene analysis (SEQ ID NOs: 1 and 2) by selecting two strains (Nos. 30 and 51) showing excellent anti-obesity activity as a result of the first screening, Bifidobacterium longum spp. longum ) and Lactobacillus rhamnosus , and each strain was Bifidobacterium longum DS0956 (99.86% 16S rRNA homology with B. longum JCM1217T) and Lactobacillus rhamnosus DS0508 ( L. rhamnosus JCM 1136T) And 100% homology). Each strain was deposited with a patent number KCTC13505BP and KCTC13504BP, respectively. As a result of genomic analysis, Bifidobacterium long sword DS0956 and Lactobacillus rhamnosus DS0508 each showed a genomic size of 2.43 Mbp and 3.01 Mbp on one chromosome, and it was found to have no plasmid.

In the above, the present invention has been described in detail only with respect to the described embodiments, but it is apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and it is natural that such modifications and modifications belong to the appended claims.

Korea Research Institute of Bioscience and Biotechnology KCTC13505BP 20180326 Korea Research Institute of Bioscience and Biotechnology KCTC13504BP 20180326

<110> Korea Research Institute of Bioscience and Biotechnology Soonchunhyang University Industry Academy Cooperation Foundation <120> Bifidobacterium longum strain or Lactobacillus rhamnosus strain for preventing or treating obesity and the use thereof <130> 2020DPA3703D <150> KR 10-2018-0041917 <151> 2018-04-11 <160> 22 <170> KoPatentIn 3.0 <210> 1 <211> 1448 <212> DNA <213> Bifidobacterium longum <400> 1 gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac gggatccatc aggctttgct 60 tggtggtgag agtggcgaac gggtgagtaa tgcgtgaccg acctgcccca tacaccggaa 120 tagctcctgg aaacgggtgg taatgccgga tgctccagtt gatcgcatgg tcttctggga 180 aagctttcgc ggtatgggat ggggtcgcgt cctatcagct tgacggcggg gtaacggccc 240 accgtggctt cgacgggtag ccggcctgag agggcgaccg gccacattgg gactgagata 300 cggcccagac tcctacggga ggcagcagtg gggaatattg cacaatgggc gcaagcctga 360 tgcagcgacg ccgcgtgagg gatggaggcc ttcgggttgt aaacctcttt tatcggggag 420 caagcgagag tgagtttacc cgttgaataa gcaccggcta actacgtgcc agcagccgcg 480 gtaatacgta gggtgcaagc gttatccgga attattgggc gtaaagggct cgtaggcggt 540 tcgtcgcgtc cggtgtgaaa gtccatcgct taacggtgga tccgcgccgg gtacgggcgg 600 gcttgagtgc ggtaggggag actggaattc ccggtgtaac ggtggaatgt gtagatatcg 660 ggaagaacac caatggcgaa ggcaggtctc tgggccgtta ctgacgctga ggagcgaaag 720 cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacgg tggatgctgg 780 atgtggggcc cgttccacgg gttccgtgtc ggagctaacg cgttaagcat cccgcctggg 840 gagtacggcc gcaaggctaa aactcaaaga aattgacggg ggcccgcaca agcggcggag 900 catgcggatt aattcgatgc aacgcgaaga accttacctg ggcttgacat gttcccgacg 960 gtcgtagaga tacggcttcc cttcggggcg ggttcacagg tggtgcatgg tcgtcgtcag 1020 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caaccctcgc cccgtgttgc 1080 cagcggatta tgccgggaac tcacggggga ccgccggggt taactcggag gaaggtgggg 1140 atgacgtcag atcatcatgc cccttacgtc cagggcttca cgcatgctac aatggccggt 1200 acaacgggat gcgacgcggc gacgcggagc ggatccctga aaaccggtct cagttcggat 1260 cgcagtctgc aactcgactg cgtgaaggcg gagtcgctag taatcgcgaa tcagcaacgt 1320 cgcggtgaat gcgttcccgg gccttgtaca caccgcccgt caagtcatga aagtgggcag 1380 cacccgaagc cggtggccta accccttgtg ggatggagcc gtctaaggtg aggctcgtga 1440 ttgggact 1448 <210> 2 <211> 1491 <212> DNA <213> Lactobacillus rhamnosus <400> 2 atgaacgctg gcggcgtgcc taatacatgc aagtcgaacg agttctgatt attgaaaggt 60 gcttgcatct tgatttaatt ttgaacgagt ggcggacggg tgagtaacac gtgggtaacc 120 tgcccttaag tgggggataa catttggaaa cagatgctaa taccgcataa atccaagaac 180 cgcatggttc ttggctgaaa gatggcgtaa gctatcgctt ttggatggac ccgcggcgta 240 ttagctagtt ggtgaggtaa cggctcacca aggcaatgat acgtagccga actgagaggt 300 tgatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360 atcttccaca atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag aaggctttcg 420 ggtcgtaaaa ctctgttgtt ggagaagaat ggtcggcaga gtaactgttg tcggcgtgac 480 ggtatccaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540 gcaagcgtta tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600 gtgaaagccc tcggcttaac cgaggaagtg catcggaaac tgggaaactt gagtgcagaa 660 gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt 720 ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagcatg ggtagcgaac 780 aggattagat accctggtag tccatgccgt aaacgatgaa tgctaggtgt tggagggttt 840 ccgcccttca gtgccgcagc taacgcatta agcattccgc ctggggagta cgaccgcaag 900 gttgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960 gaagcaacgc gaagaacctt accaggtctt gacatctttt gatcacctga gagatcaggt 1020 ttccccttcg ggggcaaaat gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080 tgttgggtta agtcccgcaa cgagcgcaac ccttatgact agttgccagc atttagttgg 1140 gcactctagt aagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1200 atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agttgcgaga 1260 ccgcgaggtc aagctaatct cttaaagcca ttctcagttc ggactgtagg ctgcaactcg 1320 cctacacgaa gtcggaatcg ctagtaatcg cggatcagca cgccgcggtg aatacgttcc 1380 cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccga agccggtggc 1440 gtaacccttt tagggagcga gccgtctaag gtgggacaaa tgattagggt g 1491 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 3 agagtttgat cmtggctca 19 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 1492R primer <400> 4 tacggytacc ttgttacgac tt 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ucp1 forward primer <400> 5 ggcattcaga ggcaaatcag ct 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Ucp1 reverse primer <400> 6 caatgaacac tgccacacct c 21 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Pgc1a forward primer <400> 7 acagctttct gggtggatt 19 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Pgc1a reverse primer <400> 8 tgaggaccgc tagcaagttt 20 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Prdm16 forward primer <400> 9 cagcacggtg aagccattc 19 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Prdm16 reverse primer <400> 10 gcgtgcatcc gcttgtg 17 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tbx1 forward primer <400> 11 ggcaggcaga cgaatgttc 19 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tbx1 reverse primer <400> 12 ttgtcatcta cgggcacaaa g 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fgf21 forward primer <400> 13 agatcaggga ggatggaaca 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fgf21 reverse primer <400> 14 tcaaagtgag gcgatccata 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD137 forward primer <400> 15 cgtgcagaac tcctgtgata ac 22 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD137 reverse primer <400> 16 gtccacctat gctggagaag g 21 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Cox2 forward primer <400> 17 gactgggcca tggagtgg 18 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cox2 reverse primer <400> 18 cacctctcca ccaatgacc 19 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> P2RX5 forward primer <400> 19 ctgcagctca ccatcctgt 19 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P2RX5 reverse primer <400> 20 cactctgcag ggaagtgtca 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh forward primer <400> 21 gacatgccgc ctggagaaac 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Gapdh reverse primer <400> 22 agcccaggat gccctttagt 20

Claims (9)

Lactobacillus rhamnosus DS0508 strain deposited with accession number KCTC13504BP. Contains at least one selected from the group consisting of Lactobacillus rhamnosus DS0508 strain deposited with accession number KCTC13504BP, culture medium of the strain, concentrate of the culture medium, dried material of the culture medium and extract of the culture medium as an active ingredient Pharmaceutical composition for preventing or treating obesity. [3] The pharmaceutical composition for preventing or treating obesity according to claim 2, wherein the pharmaceutical composition is prepared in any one form selected from the group consisting of capsules, powders, granules, tablets, pills, or lyophilizers. The pharmaceutical composition for preventing or treating obesity according to claim 2, wherein the pharmaceutical composition induces formation of beige adipocytes or brown adipocytes from white adipocytes. The method of claim 2, wherein the pharmaceutical composition is Ucp1 (uncoupling protein 1), Pgc1a (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), Prdm16 (PR/SET domain 16), Pparg (peroxisome proliferator activated receptor gamma) in adipocytes. ), CD137, Fgf21 (fibroblast growth factor 21), increase the expression of P2RX5 (purinergic receptor P2X 5) and Tbx1 (T-box 1) to induce the formation of beige adipocytes or brown adipocytes A pharmaceutical composition for preventing or treating obesity. [3] The pharmaceutical composition for preventing or treating obesity according to claim 2, wherein the pharmaceutical composition promotes decomposition of fat in fat cells or β-oxidation of lipids. [3] The pharmaceutical composition for preventing or treating obesity according to claim 2, wherein the pharmaceutical composition reduces the accumulation of cholesterol or low density lipoprotein (LDL) in obese individuals. Contains at least one selected from the group consisting of Lactobacillus rhamnosus DS0508 strain deposited with accession number KCTC13504BP, culture medium of the strain, concentrate of the culture medium, dried material of the culture medium and extract of the culture medium as an active ingredient Health functional food composition for preventing or improving obesity. Contains at least one selected from the group consisting of Lactobacillus rhamnosus DS0508 strain deposited with accession number KCTC13504BP, culture medium of the strain, concentrate of the culture medium, dried material of the culture medium and extract of the culture medium as an active ingredient Feed composition for preventing or improving obesity.

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