CN112672749A - Novel Bifidobacterium longum strain or Lactobacillus rhamnosus strain having obesity preventing or treating effect and use thereof - Google Patents

Abstract

The present invention provides a novel strain of bifidobacterium longum or lactobacillus rhamnosus having an obesity preventing or treating effect and uses thereof. Treatment with the lactic acid bacteria bifidobacterium longum strain and lactobacillus rhamnosus strain causes browning of white adipocytes. In particular, the treatment significantly increased the expression of genes specific for beige and brown adipocytes compared to the untreated control. Furthermore, based on the results of the high-fat diet-induced obese mouse experiment, it was confirmed that each strain showed significant inhibition of body weight gain compared to the negative control, and increased expression of thermogenic specific genes in white adipocytes of mice. Therefore, each of the lactic acid bacterium bifidobacterium longum strain and lactobacillus rhamnosus strain exhibits an anti-obesity effect, and thus can be effectively used as a food, medicine or feed for preventing or treating obesity, and is very useful in the related industries.

Description

Novel Bifidobacterium longum strain or Lactobacillus rhamnosus strain having obesity preventing or treating effect and use thereof

[ technical field ] A method for producing a semiconductor device

The present disclosure relates to a novel Bifidobacterium longum (Bifidobacterium longum) strain or Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain having an obesity preventing or treating effect and uses thereof.

[ background of the invention ]

With the recent economic modernization, the living standard is improved, the intake of fat and sugar is increased, the intake of fiber is reduced, and particularly, the phenomenon that high-protein diet is favored due to the establishment of the culture of going out for eating is remarkable. In contrast, the reality is that the obese population is rapidly increasing due to the lifestyle of modern people with little physical activity. Obesity, particularly abdominal obesity, is becoming a serious social problem as intake of high calorie food increases and intake of fiber decreases. Obesity (obesity) refers to a state of excessive increase in adipose tissue, and the weight increase due to obesity is mainly due to fat increase. The mechanism for achieving the obese state is that, when excessive sugar is ingested, the sugar contained in the food is digested into monosaccharide, which is in turn absorbed into the body through the small intestine, so that the blood sugar level rises, and thus insulin secretion is stimulated, wherein insulin acts on fat cells to take in the monosaccharide in the blood and convert them into fat.

The risk of obesity is not only due to the compression of the abdomen by adipose tissue, often causing constipation, dyspepsia, gastrointestinal disorders, but also a risk factor for many adult diseases. Obesity is known to be directly associated with diabetes, hypertension, coronary artery disease and cancer, and WHO defines obesity as a chronic disease in the 21 st century. Its prevalence in korea is expected to increase to over 50% in 2030, and thus, the economic burden of countries and individuals is expected to increase significantly. Therefore, much attention and investment are being invested in the research for preventing and treating obesity in korea and abroad.

Mouse adipocytes 3T3-L1 are cells that have differentiated into white adipocytes only, and are the most widely used cell line in obesity studies. In the present disclosure, the effect of lactic acid bacteria medium on Browning of lactic acid bacteria medium was studied (Browning:, the process by which white adipocytes that accumulate energy consume energy and convert to brown adipocytes that maintain exothermic reactions and thermal homeostasis). Furthermore, analysis of the expression patterns of brown adipokine and beige adipokine specific genes in mouse C3H10T1/2 mesenchymal stem cells allows the development of compositions for the prevention and treatment of obesity under new paradigms.

In this regard, korean patent No. 1778734 discloses "an ESBP isolated from bifidobacterium longum KACC 91563 and an antiallergic composition using the same". Korean patent No. 1401530 discloses "a strain of bifidobacterium longum producing conjugated linoleic acid and its use". However, the above documents do not disclose "a bifidobacterium longum strain or a lactobacillus rhamnosus strain having an effect of preventing or treating obesity by inducing the formation of beige-colored adipocytes and brown-colored adipocytes and uses thereof" according to the present disclosure.

[ summary of the invention ]

[ problem ] to provide a method for producing a semiconductor device

The present disclosure is based on the above-mentioned needs. Unlike conventional methods for identifying anti-obesity effects in terms of reducing the number of white adipocytes, according to the present disclosure, precursor adipocytes 3T3-L1, which matured to be the most widely used for obesity research white adipocytes and have been differentiated, were treated with each of the culture media of bifidobacterium longum DS0956 strain and lactobacillus rhamnosus DS0508 strain to investigate the effect of the treatment on Browning of 3T3-L1 adipocytes (Browning): energy-accumulating white adipocytes consume energy and are converted into brown adipocytes that maintain exothermic reactions and homeostasis).

As a result, treatment with each of the culture media of the lactobacillus bifidobacterium longum DS0956 strain and lactobacillus rhamnosus DS0508 strain according to the present disclosure promoted the expression of beige-colored adipocytes and brown-colored adipocytes-specific genes in the white adipocytes 3T 3-L1. In addition, it was also demonstrated that this treatment promoted the expression of the beige adipocyte-specific gene and the expression of the brown adipocyte-specific gene in the mouse C3H10T1/2 mesenchymal stem cells.

In addition, based on the results of studying the inhibitory or ameliorating effect of obesity by administering the bifidobacterium longum and lactobacillus rhamnosus strains or the culture medium thereof according to the present disclosure to high-fat diet-induced obese mice, the effect of inhibiting significant weight gain compared to negative controls, as well as the increased expression level and the induced effect of thermogenic specific genes or brown adipocytes/beige adipocytes specific genes in white adipocytes of mice were confirmed. In addition, the lowering of total cholesterol and Low Density Lipoprotein (LDL) was also demonstrated, suggesting an overall improvement in lipid metabolism characteristics in an obese mouse model.

Based on the above facts, it was confirmed that the Bifidobacterium longum strain and the Lactobacillus rhamnosus strain oxidize body fat by activating gene expression associated with body fat cell oxidation, and greatly reduce body fat cell metabolism and fat cell accumulation, and significantly improve lipid metabolism characteristics of animals to which the strains are administered. Thus, the present disclosure has been completed.

[ technical solution ] A

To achieve these objects, the present disclosure provides a novel Bifidobacterium longum (Bifidobacterium longum) strain or Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain.

Further, the present disclosure provides a pharmaceutical composition for preventing or treating obesity, the composition comprising at least one selected from the group consisting of a strain, a culture medium of the strain, a concentrate of the culture medium, a dried material of the culture medium, and an extract of the culture medium as an active ingredient.

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

Further, the present invention provides a feed composition for preventing or improving obesity, the composition comprising at least one selected from the group consisting of a strain, a culture medium of the strain, a concentrate of the culture medium, a dried product of the culture medium, and an extract of the culture medium as an active ingredient.

[ advantageous effects of the invention ]

Treatment of each of the lactic acid bacteria bifidobacterium longum strain and lactobacillus rhamnosus strain according to the present disclosure caused browning of 3T3-L1 which should only mature into white adipocytes. In particular, we demonstrated that this treatment had the effect of significantly increasing the expression of genes specific to beige adipocytes and brown adipocytes in white adipocytes 3T3-L1 and mouse mesenchymal stem cells C3H10T1/2 cells, as compared to untreated controls.

Furthermore, we confirmed that when the lactic acid bacteria bifidobacterium longum and lactobacillus rhamnosus strains or culture media according to the present disclosure were administered to subjects, weight gain due to intake of high fat diet was inhibited and expression levels of genes related to thermogenesis and brown fat/beige adipocyte-specific genes were increased, thereby reducing the amount of lipid components such as cholesterol and LDL.

Therefore, both the lactic acid bacterium bifidobacterium longum strain and the lactobacillus rhamnosus strain show anti-obesity effects, and thus can be used as food, medicine or feed for preventing or treating obesity, improving lipid metabolism and related industries.

[ description of the drawings ]

FIG. 1a shows a selection scheme for selecting a strain having anti-obesity efficacy from 55 lactic acid bacteria using 3T3-L1 cells which are preadipocytes.

FIG. 1b shows the selection results after first treatment with 1, 5 and 10. mu.l of lactic acid bacteria medium, quantified using TG (triglyceride). A, 1 mul treatment group of lactobacillus culture medium; b, 5 mul of lactobacillus culture medium is used for treating the group; c, 10 mul of lactobacillus culture medium treatment group; t, excluded from the second screening due to cytotoxicity. PA, negative control, preadipocytes, untreated with MDI differentiation medium; MDI (M: methyl-isobutyl-xanthine D: dexamethasone, I: insulin) adipocyte differentiation medium-treated control; the positive control was treated with Rosi (rosiglitazone). Rosi is a PPAR-gamma agonist.

FIG. 2a is a comparative graph showing the relative accumulation of Triglycerides (TG) in selected strain media (#30 and #51 strains) compared to controls. PA, MDI and Rosi are the same as described in FIG. 1 b.

FIG. 2b is a microscopic image (5,000 magnification) of cells observed by TEM when 3T3-L1 cells were treated with the selected strain medium. White arrows indicate Lipid Droplets (LD).

FIG. 2c shows ORO staining (oil red O stain) of selected strain media (#30 and #51 strains). 1, 1 mul treatment group of lactobacillus culture medium; 2, treatment group of 5. mu.l of lactic acid bacteria culture medium; 3, treatment group with 10. mu.l of lactic acid bacteria culture medium; (-) negative control not treated with MDI differentiation medium; (+), MDI differentiation medium treated group; positive control treated with Rosi (rosiglitazone). Rosi is a PPAR-gamma agonist. #30 and #51 refer to treatments with the selected lactic acid bacteria medium.

FIG. 3a shows the effect of selected lactic acid bacteria media (strain #30, strain #51) on brown adipocyte-specific gene expression in mouse preadipocytes 3T3-L1 and shows a comparison of relative expression levels of mRNA. FIG. 3b shows the effect of selected lactic acid bacteria media (strains #30 and #51) on brown adipocyte-specific expressed genes in mouse mesenchymal stem cells C3H10T1/2 cells and shows a comparison of relative expression levels of gene mRNA. Negative control treated with MDI (M: methyl-isobutyl-xanthine, D: dexamethasone, I: insulin) adipocyte differentiation medium; positive control treated with Rosi (rosiglitazone). Rosi is a PPAR-gamma agonist. #30 and #51 refer to treatments with the selected lactic acid bacteria medium.

FIG. 4a shows the effect of selected lactic acid bacteria media (strain #30, strain #51) on the expression of beige adipocyte-specific genes in mouse preadipocytes 3T3-L1 and shows a comparison of the relative expression levels of mRNA. FIG. 4b shows the effect of selected lactic acid bacteria media (strains #30 and #51) on beige adipocyte-specific expressed genes in mouse mesenchymal stem cells C3H10T1/2 cells and shows a comparison of relative expression levels of gene mRNA. Negative control treated with MDI (M: methyl-isobutyl-xanthine, D: dexamethasone, I: insulin) adipocyte differentiation medium; positive control treated with Rosi (rosiglitazone). Rosi is a PPAR-gamma agonist. #30 and #51 refer to treatments with the selected lactic acid bacteria medium.

FIG. 5a is a comparative graph of the expression levels of lipolysis-related genes measured based on the relative amount of mRNA when 3T3-L1 cells were treated with selected lactic acid bacteria medium (strains #30 and # 51). FIG. 5b is a graph comparing the expression levels of beta oxidation-related genes measured based on the relative amounts of mRNA.

FIG. 6 demonstrates whether PKA signaling is activated when the lactic acid bacteria medium of strain #30 and strain #51 was applied to 3T3-L1 cells. A confirms whether PKA is phosphorylated. B shows the results of treatment with H89 as a PKA phosphorylation inhibitor. C is a graph of the expression level of thermogenesis-associated genes resulting from H89 treatment based on relative amounts of mRNA measured. D to F are graphs comparing expression levels of thermogenic-related genes and adipocyte differentiation-related genes based on the amount of mRNA and protein measured using siPKA cat a 1.

FIG. 7 demonstrates the changes in gene expression associated with lipolytic enzyme when 3T3-L1 cells were treated with lactobacillus media #30 and #51 (left panel, HSL S-660 and HSL S-563 refer to Ser respectively⁵⁶³And Ser⁶⁶⁰HSL phosphorylation) and confirmed the presence or absence of AMPK phosphorylation and activation of the transcriptional regulator CREB (middle panel), and confirmed the changes in the presence or absence of lipolytic enzyme-related genes and CREB phosphorylation according to PKA inhibitor H89 treatment (right panel).

FIG. 8 shows the body weight changes of obese mice induced by a high fat diet measured 12 weeks after administration of lactic acid bacterial strains or medium. (G1, high-fat diet non-treated group; G2, high-fat diet administered group; G3, high-fat diet and microorganism medium administered group; G4, high-fat diet and Lactobacillus rhamnosus GG bacterium administered group; G5, high-fat diet and #30 strain medium administered group; G6, high-fat diet and #51 strain medium administered group; G7, high-fat diet and #30 bacterium administered group; G8, high-fat diet and #51 bacterium administered group; others are the same as above)

Fig. 9 shows the results of H & E (hematoxylin and eosin) staining of white adipocytes in high fat diet-induced obese mice after 12 weeks of strain or medium administration.

Fig. 10 shows that after administering a lactic acid bacterium strain or a culture medium to a subject as a mouse induced obesity by a high-fat diet for 12 weeks, the high-fat diet induced changes in the levels of glucose, total cholesterol (T-chol), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in the blood of the obese mouse.

Fig. 11 shows changes in thermogenic specific gene expression in White Adipocytes (WAT), gonadal white adipocytes (gonadal WAT), peritoneal white adipocytes (peritoneal WAT), and mesenteric white adipocytes (mesenteric WAT) of mice with high-fat diet-induced obesity after 12 weeks of administration of the lactic acid bacteria strain or culture medium.

FIG. 12a is a graph comparing the relative mRNA expression levels of genes comprising M1 macrophage inflammation-associated cytokine in mice with high fat diet-induced obesity 12 weeks after administration of a lactic acid bacterial strain or culture medium.

FIG. 12b is a graph showing a comparison of the relative mRNA expression levels of the M2 macrophage specific gene in mice with high fat diet induced obesity after 12 weeks of administration of the lactic acid bacterial strain or culture medium.

[ detailed description ] embodiments

Hereinafter, the present disclosure will be described in detail.

Bifidobacterium longum strain and lactobacillus rhamnosus strain

According to one aspect of the present disclosure there is provided a novel strain of bifidobacterium longum or lactobacillus rhamnosus.

The bifidobacterium longum strain may be bifidobacterium longum DS0956, preferably bifidobacterium longum DS0956 strain deposited under accession number KCTC13505BP, but is not limited thereto. The strain Bifidobacterium longum DS0956 was deposited at Korea institute of bioscience and Biotechnology under accession No. KCTC13505BP at 3/26/2018.

The lactobacillus rhamnosus strain may be lactobacillus rhamnosus DS0508, preferably, lactobacillus rhamnosus DS0508 strain deposited as accession number KCTC13504BP, but is not limited thereto. The lactobacillus rhamnosus strain DS0508 was deposited at korean institute of bioscience and biotechnology under accession No. KCTC13504BP at 26/3/2018.

In a strain according to one embodiment of the present disclosure, the bifidobacterium longum strain or the lactobacillus rhamnosus strain is intended to induce the formation of beige adipocytes and brown adipocytes in order to promote an anti-obesity effect. Preferably, the bifidobacterium longum strain or the lactobacillus rhamnosus strain increases the expression of thermogenesis-associated genes and brown adipocyte-associated genes in 3T3-L1 adipocytes and mouse mesenchymal stem cells C3H10T1/2 in a specific manner, thereby inducing the formation of beige adipocytes and brown adipocytes. More preferably, bifidobacterium longum strain or lactobacillus rhamnosus strain increases the expression of 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, fnf 21 (fibroblast growth factor 21), P2RX5 (purinergic receptor P2X 5) and Tbx1(T-box 1) genes to induce the formation of beige and brown adipocytes. Most preferably, the bifidobacterium longum strain or the lactobacillus rhamnosus strain increases the expression of thermogenesis associated genes Ucp1, Pgc1a, Prdm16 and brown adipocyte associated CD137 and Fgf21 genes in 3T3-L1 adipocytes and mouse mesenchymal stem cells C3H10T1/2 in a specific manner, thereby inducing the formation of beige adipocytes and brown adipocytes. However, the present disclosure is not limited thereto. The CD137 gene is also known as TNFRSF9(TNF receptor superfamily member 9). Furthermore, the expression level of the Past1, Resistin or Sarpina3k gene specifically expressed in white adipocytes can be reduced upon treatment with a Bifidobacterium longum strain or a Lactobacillus rhamnosus strain according to the present disclosure.

The strain according to the present disclosure increases expression of brown adipocyte-or beige adipocyte-specific genes in white adipocytes that have differentiated, and can convert white adipocytes into brown or beige adipocytes. Since brown adipocytes and beige adipocytes are characterized by promoting lipolysis to generate energy, the strain according to the present disclosure has an effect of inhibiting or improving obesity.

In addition, the bifidobacterium longum strain or lactobacillus rhamnosus strain increases the expression level of the gene Atgl, HSL, Pnin1 or Pnin5, which is associated with fat degradation, or also increases the expression level of the gene LCAD, MCAD, LCPT or Abhd5, which is associated with β -oxidation of lipids. The fat degradation-related gene or the beta-oxidation-related gene may promote the decomposition and removal of accumulated fat. Therefore, the strain of the present invention has the effect of inhibiting or improving obesity by reducing the accumulation of fat and inhibiting weight gain.

Furthermore, bifidobacterium longum strains or lactobacillus rhamnosus strains can activate PKA signaling. The bifidobacterium longum strain or lactobacillus rhamnosus strain can activate the PKA signaling process to increase the amount of phosphorylated PKA, phosphorylated AMPK, and phosphorylated transcription regulator CREB, thereby increasing the expression of genes related to the differentiation of thermogenic adipocytes, i.e., Ucp1, Pgc1a, Pparg, or Ceba genes, and thus achieve the effect of suppressing or improving obesity by inducing the browning of white adipocytes.

The bifidobacterium longum strain or the lactobacillus rhamnosus strain according to the present disclosure has the effect of improving or treating obesity by improving lipid metabolism characteristics when administered to an obese subject. To demonstrate the effect of improving lipid metabolism characteristics according to the present disclosure, in one particular embodiment of the present disclosure, a strain or culture medium of bifidobacterium longum or lactobacillus rhamnosus is administered to mice with high-fat diet-induced obesity to observe any changes. The results show that the strains and culture media according to the present disclosure have the effects of inhibiting weight gain and increasing the expression levels of thermogenesis-related genes, brown adipocytes and beige adipocyte-specific genes in white adipocytes of mice. The increase in the gene expression level as described above can promote the white adipocytes to be transdifferentiated (transdifferentiation) into brown adipocytes or beige adipocytes in the subject, thereby suppressing or improving obesity. In addition, by reducing the amount of lipid components such as cholesterol and LDL in a subject administered the strain or medium, lipid metabolism can be improved.

Furthermore, in another specific embodiment of the present invention, based on the results of studying gene expression levels when strains or media of bifidobacterium longum or lactobacillus rhamnosus are administered to obese subjects, it was confirmed that the expression level of each of the inflammation promoting M1 macrophage markers CD11c, CD68, IL-1b, Mcp1 and TNF-a genes is decreased, while the expression level of the anti-inflammatory M2 macrophage markers, i.e., Arg1 and CD206 genes, is increased in white adipocytes of the subjects. Thus, when the strain or culture medium of the present disclosure is administered to a subject with obesity, transformation occurs in which the amount of M1 macrophages is reduced and the amount of M2 macrophages is increased. Therefore, it is suggested that administration of the strain or the medium to an obese subject can promote the effect of inhibiting or improving obesity.

2. Composition comprising lactic acid bacteria

According to another aspect of the present disclosure there is provided a composition comprising a lactic acid bacterium.

The lactic acid bacteria include Bifidobacterium longum or Lactobacillus rhamnosus.

The composition comprising lactic acid bacteria contains at least one selected from the group consisting of a strain, a culture medium of the strain, a concentrate of the culture medium, a dried product of the culture medium, and an extract of the culture medium as an effective ingredient.

The compositions comprising lactic acid bacteria according to the present disclosure may be prepared in unit dosage form, or may be formulated into multi-dose containers using carriers, excipients and/or additives, using methods that can be readily performed by one of ordinary skill in the art to which the present disclosure pertains. In this regard, the formulations may be in the form of solutions, suspensions or emulsions in oily or aqueous media, or in the form of extracts, powders, granules, tablets, capsules, gels (e.g., hydrogels) or lyophilized formulations. Additives, such as dispersants, stabilizers or cryoprotectants, may additionally be included.

In particular, when the additive is a cryoprotectant, the strain may be lyophilized with the cryoprotectant and may be used in powder form. The cryoprotectant may be skim milk powder, maltodextrin, dextrin, trehalose, maltose, lactose, mannitol, cyclodextrin, glycerol and/or honey. Further, the composition may be mixed with a storage vehicle, and the mixture may be absorbed and dried and cured for use. The storage carrier may be diatomaceous earth, activated carbon, and/or defatted rice bran.

The composition comprising lactic acid bacteria according to the present disclosure may be prepared by mixing at least one selected from the group consisting of a strain, a culture medium of a strain, a concentrate of a culture medium, a dried product of a culture medium, and an extract of a culture medium, with any one of a carrier, an excipient, or an additive.

The description of the strains, carriers, excipients and additives is as described above. When a cryoprotectant is used as an additive, the composition comprising lactic acid bacteria may be prepared in the form of lyophilized powder, a mixture is prepared by mixing the strain and the cryoprotectant with each other, and is frozen at-45 ℃ to-30 ℃, dried at 30 ℃ to 40 ℃, and the mixture is ground with a mixer to obtain lyophilized powder. Specifically, the freezing process may be a process of vacuum-freezing the mixture at a temperature condition of-45 ℃ to-30 ℃ and a pressure condition of 5 to 50 mtorr for 65 to 75 hours.

3. Use of a composition comprising lactic acid bacteria for preventing, treating or ameliorating obesity

According to another aspect of the present disclosure, there is provided a use of a composition comprising lactic acid bacteria for preventing, treating or ameliorating obesity.

The composition comprising lactic acid bacteria may be a pharmaceutical product, a food product or a feed. When the composition comprising lactic acid bacteria is a pharmaceutical product, the composition may be a pharmaceutical composition for preventing or treating obesity. When the composition comprising lactic acid bacteria is a food, the composition may be a health functional food composition for preventing or improving obesity. When the composition containing lactic acid bacteria is a feed, the composition may be a feed composition for preventing or improving obesity.

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

The strains as described above, in one embodiment according to the present disclosure, the pharmaceutical composition may be prepared in unit dosage form, or may be introduced into a multi-dose container by formulation using a method that can be easily performed by one of ordinary skill in the art to which the present disclosure pertains, using pharmaceutically acceptable carriers and/or excipients. In this regard, the formulations may be in the form of solutions, suspensions or emulsions in oily or aqueous media, or in the form of extracts, powders, granules, tablets, capsules or gels (e.g. hydrogels). The formulation may additionally comprise a dispersant or stabilizer.

Furthermore, the strains comprised in the pharmaceutical composition may be carried on a pharmaceutically acceptable carrier, such as colloidal suspensions, powders, physiological saline, lipids, liposomes, microspheres (microspheres) or nanosphere particles. They may be complexed with (tethered) or linked to (tethered) carriers and may be delivered in vivo using delivery systems known in the art, such as lipids, liposomes, microspheres, gold, nanoparticles, polymers, condensing agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids.

In addition, the pharmaceutically acceptable carrier may include lactose, glucose (dextrose), sucrose, sorbitol, mannitol, starch, gum arabic, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, which are generally used in formulations. The present disclosure is not so limited. In addition, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like may be contained therein. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences,19th ed., 1995.

The pharmaceutical composition of the present invention may be administered orally or parenterally at the time of clinical administration, and may be used in the form of a general pharmaceutical preparation. That is, the pharmaceutical composition according to the present disclosure may be administered in various oral and parenteral dosage forms during actual clinical administration. The formulations may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules and the like. These solid formulations can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the herbal extract or the fermented herbal product. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid formulations for oral administration may include suspensions, liquid solutions, emulsions and syrups. In addition to water and liquid paraffin, which are generally used as simple diluents, various excipients, such as wetting agents, sweeteners, aromatics, preservatives, and the like, may be contained therein. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable esters (such as ethyl oleate) can be used as non-aqueous solvents and suspensions. Suppository bases may include Witepsol, Macrogol, tween 61, cocoa butter, laurin, glycerol, gelatin, and the like.

The pharmaceutical composition according to the present disclosure may be used alone or in combination with surgery, radiotherapy, hormonal therapy, chemotherapy and methods of using biological response modifiers for the inhibition and treatment of obesity.

The concentration of the active ingredient contained in the composition according to the present disclosure may be determined in consideration of the treatment purpose, the condition of the patient, the required time, etc., and is not limited to a specific concentration range. The pharmaceutical composition according to the present disclosure is administered in a pharmaceutically effective amount. In the present disclosure, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment. The effective dosage level may depend on factors including the type of disease, its severity, pharmaceutical activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, concurrent drug use and other factors well known in the medical arts. The pharmaceutical composition according to the present disclosure may be administered as a sole therapeutic agent, or may be administered in combination with a therapeutic agent for diseases caused by other contaminants, or an agent for improving skin aging, or may be administered simultaneously, separately or sequentially with a conventional therapeutic agent, or may be administered in a single or multiple times. When all of the above factors are considered, it is important to administer the composition in an amount that achieves maximum effect in a minimum amount without side effects. Such amounts can be readily determined by one skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present disclosure may vary according to the age, sex, condition, body weight, absorption of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and drug used in combination therewith of a patient, or may be increased or decreased according to the administration route, severity of obesity, sex, body weight, age, and the like. For example, a composition according to the invention may be administered in an amount of from about 0.0001 μ g to 500mg, for example from 0.01 μ g to 100mg per day per 1kg body weight of the patient. Furthermore, the compositions may be administered separately several times a day, e.g. 2 to 3 times a day at regular time intervals, at the discretion of a physician or pharmacist.

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

The subject may be a human or non-human animal, and may be in a non-obese state or in an obese state. Obesity may be prevented by administering the pharmaceutical composition to a subject in a pharmaceutically effective amount when the subject is not in an obese state. When the subject is in an obese state, obesity can be treated by administering to the subject the pharmaceutical composition in a pharmaceutically effective amount.

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

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

In the health-promoting functional food composition according to one embodiment of the present disclosure, the health-promoting functional food composition may inhibit weight gain or fat accumulation.

When the health-promoting functional food composition according to the present disclosure is used as a food additive, the health-promoting functional food composition may be added in an unaltered manner or may be used together 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 determined depending on the purpose of use (prevention or improvement). Generally, in preparing foods or beverages, the health functional food composition according to the present disclosure may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the weight of the raw materials. However, for long-term consumption for health purposes, the amount may be less than the above range. Since there is no problem in safety, the active ingredient may be used in an amount higher than this range.

The kind of the health functional food is not particularly limited. Examples of the food to which the health functional food composition may be added may include meat, sausage, bread, chocolate, candy (candy), snack, candy (confection), pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complex, and the like. The food may include all kinds of health foods in the general sense.

Further, the health functional food composition according to the present disclosure may be prepared as a food, particularly a functional food. Functional food products according to the present disclosure comprise ingredients that are typically added during food preparation. Examples thereof may include proteins, carbohydrates, fats, nutrients, and seasonings. For example, when the food is prepared as a beverage, natural carbohydrates or flavoring agents may be included therein as additional ingredients in addition to the active ingredient. Natural carbohydrates may 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, etc.). The flavoring agent may be a natural flavoring agent (e.g., thaumatin (tamarind), stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame (asparatame), etc.).

In addition to the health functional food composition, the food may further comprise various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salt, alginic acid and its salt, organic acids, protective colloid thickener, pH regulator, stabilizer, preservative, glycerin, alcohol, carbonating agent for carbonated beverage, and the like. Although the content of the additional ingredient is not very important, the content may be generally selected from the range of 0.01 to 0.1 parts by weight for 100 parts by weight of the health functional food composition according to the present disclosure.

Further, the present invention provides a feed composition for preventing or improving obesity, the composition comprising at least one selected from the group consisting of a strain, a culture medium of the strain, a concentrate of the culture medium, a dried product of the culture medium, and an extract of the culture medium as an active ingredient.

The strain as described above, which can be added as a feed additive composition for preventing or improving obesity. The feed additive according to the present disclosure may be an auxiliary feed under the feed management act.

In the present disclosure, the term "feed" may refer to any natural or artificial diet, meal or meal component, consumed, ingested and digested or otherwise suitable by an animal. The kind of the feed is not particularly limited. Feeds commonly used in the art may be used. Non-limiting examples of feeds can include plant feeds such as grains, roots, food processing byproducts, algae, fibers, pharmaceutical byproducts, oils and fats, starch, melons (gourd), or grain byproducts; and animal-based feeds such as proteins, minerals, oils, minerals, single cell proteins, zooplankton, food, and the like. These may be used alone or in combination of two or more.

Hereinafter, the present disclosure will be described in detail based on embodiments. However, the following embodiments are only intended to specifically exemplify the present disclosure, and the content according to the present disclosure is not limited to the following embodiments.

Materials and methods

Reagents used

Dexamethasone, IBMX (isobutyl-1-methylxanthine), insulin, rosiglitazone (Rosi), oil red O dye, MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazole bromide) and 4% formaldehyde, purchased from Sigma Aldrich (st louis, missouri).

DMEM (Dulbecco Modified Eagle's Media), newborn bovine serum (NBCS) and recombinant human BMP4 were purchased from Gibco (Glandeland, N.Y., USA). Fetal bovine serum was purchased from atlas biologics (corinsburg, colorado, usa). Penicillin-streptomycin solution was purchased from Hyclone Laboratories, Inc (southern roots, n.y., usa).

Lactic acid bacteria isolation

For isolation of the various lactic acid bacteria MRS medium was used under absolutely anaerobic conditions. For anaerobic conditions, N is used₂The gas removes oxygen present in the medium, which is then sterilized. 0.1g of the collected fecal sample was suspended in 10ml of MRS medium, gradually diluted, and plated in 100. mu.l on MRS plate medium or blood agar medium, and cultured at 37 ℃ for 2 days under anaerobic conditions. As a result, the obtained single colonies were subcultured, purified and isolated, and stored for a long period of time.

Identification of isolated lactic acid bacteria

For molecular biological identification of 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 ACG ACT T-3': SEQ ID NO:4) were used as universal primers targeting the 16S rRNA gene. Nucleotide sequence analysis of the 16S rRNA gene was performed. The nucleotide sequence obtained by the analysis was identified by an identification search in EZbiocloud (http:// www.ezbiocloud.net /).

Study of anti-obesity Activity

To investigate anti-obesity activity, 5% CO was used₂Box, 3T3-L1 cells were cultured in DMEM Glutamax containing 10% NBCS and 1% penicillin-streptomycin therein at 37 ℃. Cells were seeded into 48-well plates when the cell concentration of 3T3-L1 cells was in the range of 70 to 80%. When the cell isWhen the concentration reaches 100%, the medium is replaced by adipocyte differentiation medium MDI (insulin, dexamethasone, isobutyl-1-methylxanthine (IBMX)). On day 2, each of insulin, a combination of insulin and ROSI, and a combination of insulin and the sample (strain medium) was added thereto. On day 4, only insulin was added thereto. Then, on day 6, the cells were fixed without sample treatment. At this time, for the anti-obesity effect of the lactic acid bacteria solution, 1, 5 and 10. mu.l of samples were added to the medium on days 0 and 2, respectively, and MDI was replaced with new MDI every 2 days during cell differentiation. The day when the cell concentration became 100% was defined as day 0. On day 0, each of MDI, a combination of ROSI and MDI, and a combination of the sample and MDI was added thereto. In this experiment, three independent tests were performed on each sample. To visualize the cells, 3T3-L1 cells were cultured in 24-well plates for 6 days, fixed, and stained with oil red o (oro) stain. To briefly explain the experimental procedure, cells were first washed once with 1 × PBS and then fixed with 10% formalin for 1 hour at room temperature. Then, the staining was performed for 20 minutes at room temperature using a 0.3% ORO solution, and then washed 4 times with distilled water. After washing, the altered phenotype was observed and photographed using an Axiovert-25 microscope. Then, the stained cells were dissolved in 100% isopropanol, and then the amount of ORO therein was measured based on absorbance at 520nm using victor tmx 3.

In addition, C3H10T1/2 mouse mesenchymal stem cells were purchased from Korean cell line Bank (KCLB-10226) and used 5% CO at 37 ℃₂The incubator was cultured in high concentration glucose DMEM medium containing 10% NBCS and 1% penicillin-streptomycin. To induce differentiation (commitment), C3H10T1/2 cells were seeded therein at a cell concentration of 20 to 30%. In order to differentiate it into adipocytes, the cells were treated with 50ng/mL of human recombinant BMP4 until the cell concentration became 100%. Thereafter, the medium was replaced with fresh medium every 2 to 3 days. The day within 48 hours after the cell concentration became 100% was defined as day 0. Culture was replaced with DMEM containing 10. mu.g/ml insulin (MDI), 10% FBS, 0.5mM IBMX, and 1. mu.M dexamethasone under conditions in which cells were treated with either Rosi or lactic acid bacteria medium at predetermined concentrationsThereby inducing differentiation thereof. Differentiated cells were exposed to 500 μ M dibutyryl-cAMP 4 hours to stimulate the thermogenic process.

qRT-PCR analysis

To find the expression of genes in cells treated with the lactic acid bacteria medium, total RNA was extracted therefrom using an RNA extraction kit (Qiagen, valencia, ca, usa) according to the manufacturer's instructions. The concentration was measured using a Scandrop Analytik Jena AG spectrometer (Jena, Germany). Mu.g of RNA was synthesized into cDNA using the Maxime RT Premix kit (Korea iNtRON Biotechnology). The PCR reactions were performed in a Veriti 96-well thermal cycler (Singapore Applied Biosystems). Using iQ^TMSYBR Green Supermix kit (Singapore Bio-Rad) in CFX96^TMQuantitative real-time PCR was performed on a real-time PCR detection system (Singapore Bio-Rad). The sequences of the primers are listed in table 1. Expression levels were quantified using Gapdh (glyceraldehyde 3-phosphate dehydrogenase) (Yoon D, Imran KM, Kim YS.2018Toxicol Appl Pharmacol.Feb 1; 340: 9-20).

[ TABLE 1 ]

Primer set used in this experiment

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

Study of resistance by administration of mice induced obesity on a high fat dietEfficacy of obesity

After inducing an obesity model by allowing the mice to ingest a high-fat diet, the intestinal microorganism medium or the microorganism cells were administered to the mice for 12 weeks. The efficacy was examined. The mice used in this study were 3-week-old C57BL/6 SPF male mice. Among mice acclimated for 7 days, only healthy animals were used for the test. Mice were fed a high fat Diet for 12 weeks using 45% kcal high fat Diet D12451(Research Diet) to establish a Diet Induced Obesity (DIO) mouse model. The compositions of the groups used in the experiments are illustrated in table 2 below. The amount of lactobacillus administered is 10⁹Cells/kg. 1ml of the medium was freeze-dried per animal, then dissolved in 150. mu.l of distilled water, and administered once a day.

[ TABLE 2 ]

For all animals, general symptoms were observed once daily until necropsy day, and body weight and food amount were measured five times a week during the test. After the end of the test period, anesthesia was performed in a respiratory anesthesia mode and blood was collected therefrom by cardiac blood collection. Fat fractions were excised from 2 mice per group and stored in 4% paraformaldehyde solution or RNA storage solution (ThermoFisher). The blood cell analysis was performed using a hemocytometer (Beckman Coulter). Then, the sample subjected to the blood cell analysis is centrifuged to obtain plasma. The samples were then analyzed for total cholesterol, HDL, LDL and glucose using plasma. Adipose tissue contained in a 4% PFA solution was used for paraffin block preparation.

Statistical analysis

All data for this experiment are presented as mean ± Standard Deviation (SD) of three or more independent experiments. Unless otherwise indicated, MDI treated sample groups were used to identify changes in data compared to control groups. Significant differences between the data of the control and other treatment groups were calculated using student's t-test. 0.05, 0.01, 0.001. P values less than 0.05 are considered statistically significant.

Example 1 search for anti-obesity active strains

To observe the possibility of using 55 lactobacillus strains to convert to beige and brown adipocytes in 3T3-L1 preadipocytes, each lactobacillus strain was cultured in MRS medium for 48 hours, and then centrifuged to obtain a supernatant. The resulting supernatant was freeze-dried, and then sterile distilled water of an initial volume of 1/10 volumes was added to the supernatant to prepare a concentrate thereof. 1, 5 and 10 μ L of each of the concentrates were applied to 3T3-L1 cells, where the accumulation of triglycerides was quantified by oil red O staining. Therefore, an active candidate group (diagonal filled bars) causing browning and beige adipocytes was selected in the lactic acid bacteria medium. Furthermore, a candidate group (dotted bars) that inhibited adipocyte formation was selected from among them (fig. 1B).

In the group in which proliferation of adipocytes treated with each of 1, 5, and 10. mu.l of lactic acid bacterium concentrates was increased by 10 to 20% (using Rosi as a control), the activity candidate group that produced browned adipocytes was first selected from the group having a relatively high treatment concentration of 10. mu.l. For #51 having the effect of inhibiting adipocyte formation, the activity candidate group was selected from the group having a relatively high concentration of 10 μ l in the three concentration tests. After the first screening based on the amount of triglycerides accumulated as described above, the selected candidate group was further screened through the second and third screening processes based on whether UCP1, one of brown adipocyte-specific genes, was expressed, thereby selecting four candidates. Then, lactic acid bacterial strains #30 and #51 were finally selected from the four candidate strains.

To determine the effect of the finally selected #30 and #51 on adipocytes, treatment was performed with each of 1, 5 and 10. mu.l thereof. The amount of triglycerides was then investigated by oil red O staining. Lipid Droplets (LDs) present in adipocytes were identified (fig. 2A to 2C). As a result, when treated with the lactic acid bacterium strains #30 and #51, the accumulation of triglyceride was increased, and lipid droplets were not bound to each other, and their sizes were small. Therefore, selected #30 and #51 were found to be strains expected to have effects of inhibiting the maturation of 3T3-L1 cells into white adipocytes and enhancing their conversion into brown adipocytes.

Example 2 expression of genes specific to mouse Brown and Rice-colored adipocytes in Lactobacillus Medium Influence of

Beige adipocytes can express the UCP1 (uncoupling protein 1) gene that is not expressed in white adipose tissue. It is known that the expression of this gene in white adipocytes indicates that transdifferentiation from white adipocytes to beige adipocytes or brown adipocytes has occurred, and that transdifferentiation of cells reversibly occurs according to feeding or the external environment. Thus, in this experiment, the effect of the culture medium of the selected strain on the expression of genes specific to beige-colored and brown-colored adipocytes was investigated.

The effect of lactobacillus culture medium on the expression of genes specific to mouse brown adipocytes and beige adipocytes was studied. As shown in fig. 3A, the expression of uncoupling protein 1(UCP1) gene and genes associated with thermogenesis, which are called brown adipocyte-specific genes, were significantly increased in 3T3-L1 adipocytes treated with #30 lactic acid bacteria culture medium. Increased expression of other brown adipocyte-specific genes, Pgc1a (peroxisome proliferator activated receptor gamma coactivator 1-alpha) and Prdm16(PR/SET domain 16), were identified. In addition, it has been found that the expression of peroxisome proliferator activated receptor γ (Pparg), which is important for the differentiation of general adipocytes, is increased. It was identified that treatment with Lactobacillus culture medium #51 did not significantly increase expression of brown adipocyte-specific genes, but increased expression of the Prdm16 gene.

In addition, in order to verify the effect of the lactic acid bacteria medium again, when C3H10T1/2 cells, which are mouse mesenchymal stem cells, were treated with the strain medium, the expression levels of thermogenesis-related and brown adipocyte-related genes Ucp1, Pgc1a and Prdm16 were identified. From the identification results, it was confirmed that the treatment with the culture media of lactic acid bacteria #30 and #51 of the present disclosure increased the expression level of brown adipocyte-specific genes (fig. 3B).

Then, we identified the effect of #30 on the transdifferentiation of white adipocytes to beige adipocytes. Thus, it was characterized that expression of several beige adipocyte-specific markers, such as fibroblast growth factor 21(Fgf21), Tbx1, P2RX5, CD137, cytochrome c oxidase subunit II (Cox2), was significantly increased in 3T3-L1 adipocytes treated with lactobacillus culture medium # 30. In addition, it was confirmed that the expression of CD137 and Fgf21, which are genes important for expression of beige adipocytes, was significantly increased in the cells treated with the #51 medium (fig. 4A).

In addition, to again demonstrate the effect of the selected lactic acid bacterial strain or culture medium, the strain culture medium was applied to mouse C3H10T1/2 mesenchymal stem cells. Then, the expression levels of the beige adipocyte-specific genes Fgf21, P2RX5, CD137 and Tbx1(T-box 1) were identified. The results showed that the expression level of the beige adipocyte-specific gene was increased in the cells treated with the lactic acid bacteria medium #30 and #51 of the present invention (fig. 4B).

Example 3 expression of genes associated with fat degradation and beta-Oxidation and PKA signalling in Lactobacillus culture Medium Influence of activation of a process

To further identify the effect of the lactic acid bacteria medium according to the present disclosure, the lactic acid bacteria medium was applied to 3T3-L1 cells, and then expression levels of the genes Atgl, HSL, Plin1, Plin5 associated with lipolysis were identified. The expression levels of the genes LCAD, MCAD, LCPT and Abhd5 associated with beta oxidation of lipids were identified.

As a result, the expression levels of Atgl, HSL, Plin1, Plin5 gene and LCAD, MCAD, LCPT and Abhd5 gene were higher in 3T3-L1 cells treated with the lactic acid bacteria medium #30 and #51 according to the present disclosure than in the control. (FIG. 5A and FIG. 5B). Therefore, it was confirmed that the treatment had the effect of inhibiting fat accumulation and weight gain by inducing the action of decomposing fat or oxidizing lipid components.

In addition, the culture medium for lactic acid bacteria according to the present invention was applied to 3T3-L1 cells, and then activation of PKA signaling was measured. Whether phosphorylated PKA increased was identified by Western blot (fig. 6A). The cells were then treated with 10mM H89 as PKA inhibitor (fig. 6B). The phosphorylated PKA is then re-identified. Thus, it has been determined that treatment of cells with the lactic acid bacteria culture media #30 and #51 of the present invention has the effect of activating the PKA signaling process based on an increase in phosphorylated PKA. After treatment of cells with PKA inhibitor H89, changes in the expression levels of thermogenesis-associated genes Ucp1 and Pgc1a gene (C in fig. 6) were identified. Thus, it was again demonstrated that treatment with H89 reduced the expression levels of the Ucp1 and Pgc1a genes. Thus, it was again confirmed that the culture medium for lactic acid bacteria of the present invention induces an increase in the expression level of the thermogenic-associated gene by PKA activation. In addition, si-PKA cat a1 was used to identify the expression of thermogenesis-associated genes, adipocyte differentiation-associated genes, and changes in the expression levels of these proteins. Thus, inhibition of expression of Ucp1, Pgc1a, Pparg, and Ceba genes was identified, while the amount of Ucp1, Pparg, and Pgc1a proteins was also reduced. Thus, it was again confirmed that the lactic acid bacterium medium of the present invention induces an increase in the expression level of thermogenesis-associated genes by PKA activation (D to F in FIG. 6).

In addition, it has been determined that when the lactic acid bacteria #30 and #51 of the present invention are applied to 3T3-L1 cells, lipolytic enzyme, AMPK phosphorylation and CREB phosphorylation as transcription regulators are increased. The effect of the present invention was again determined based on the above-described results that inhibition of lipolytic enzyme and CREB phosphorylation was caused by treatment with H89 as a PKA inhibitor (see figure 7).

Example 4 study of anti-obesity efficacy due to administration of high fat diet-based induced obese mice

During the test, no general abnormal symptoms were observed in all groups. However, the body weight of mice fed with a high fat diet was significantly increased by 34.0% compared to the normal group. In particular, in the strain medium # 30-administered group (G5) and the strain cell # 51-administered group (G8), the body weight was reduced by 10.8% and 5.7%, respectively, on average, as compared with the negative control (G2). The average weight loss occurred in both experimental groups (fig. 8). Furthermore, based on the results of analyzing H & E pathology (area measurement) of each adipose cell tissue, a reduction in gonadal fat (19.6%, 18.9% respectively), a reduction in peritoneal fat (21.2%, 22.4% respectively), a reduction in mesenteric fat (33.9%, 24.4% respectively) and a reduction in white adipose tissue (26.2%, 23.4% respectively) in the G5 and G8 groups were identified compared to the negative control (G2) (fig. 9). Thus, strains #30 and #51 of the present invention or the medium thereof were identified to have the effect of reducing fat accumulation.

In addition, based on the results of performing biochemical analysis, it was determined that the levels of total cholesterol and LDL were significantly reduced in the G5 and G8 groups compared to the negative control (fig. 10). Therefore, it was confirmed that both the strain and the medium of the present invention had the effect of lowering cholesterol and LDL components. Based on the results of identifying the expression levels of the genes associated with thermogenesis or lipolytic enzymes, i.e., Ucp1, Pgc1, 1a and Prdm16, in each adipose tissue, it was identified that the expression levels thereof in the four adipose tissues of the mouse were significantly increased in the group to which the strain or culture medium of the present invention was administered (fig. 11).

In addition, the effect of the lactic acid bacterial strain or culture medium according to the present disclosure on inflammation-associated macrophage polarization of white adipose tissue in a group of mice to which the strain or culture medium was administered was identified. We identified changes in the expression levels of the pro-inflammatory M1 macrophage markers CD11c, CD68, IL-1B, Mcp1, and TNF-a genes (fig. 12A) and the anti-inflammatory M2 macrophage markers Arg1 and CD206 genes (fig. 12B). It has been determined that in adipocytes of subjects in obese states, the inflammatory response is promoted, a transformation from M2 macrophages to M1 macrophages occurs, inducing a change in macrophage polarity. Therefore, we confirmed that the lactic acid bacterium strain or culture medium according to the present disclosure has an effect of inhibiting or improving obesity. As a result, the expression level of M1 macrophage marker gene was decreased and the expression level of M2 macrophage marker gene was increased in mice administered with the lactic acid bacterium strain or the medium of the present invention. Therefore, it was confirmed that the strains #30 and #51 of the present invention or the culture medium thereof had the effect of inhibiting or improving obesity.

Example 5 identification of selected strains

As a result of the first screening, two strains (#30 and #51) having excellent anti-obesity activity were selected. According to the results of 16S rRNA gene analyses (SEQ ID Nos: 1 and 2), these two strains were identified as Bifidobacterium longum subspecies (Bifidobacterium longum spp. longum) and Lactobacillus rhamnosus (Lactobacillus rhamnosus), respectively. The two strains were named Bifidobacterium longum DS0956 (99.86% homology of 16S rRNA to Bifidobacterium longum JCM 1217T) and Lactobacillus rhamnosus DS0508 (100% homology to Lactobacillus rhamnosus JCM 1136T), respectively. These two strains were then deposited in a proprietary manner under accession numbers KCTC13505BP and KCTC13504BP, respectively. From the results of their genome analysis, it was found that Bifidobacterium longum DS0956 and Lactobacillus rhamnosus DS0508 had genome sizes of 2.43Mbp and 3.01Mbp, respectively, on one chromosome thereof, and had no plasmid.

The present disclosure has been described in detail above with reference to only the embodiments. However, it is apparent to those skilled in the art that various modifications and variations thereof are possible within the scope of the technical idea according to the present disclosure. Such modifications and variations are intended to fall within the scope of the appended claims.

[ DENSATION NUMBER ]

The name of the depository institution: korean institute of Life engineering

Accession number: KCTC13505BP

The preservation date is as follows: 26/3/2018

The name of the depository institution: korean institute of Life engineering

Accession number: KCTC13504BP

The preservation date is as follows: 26/3/2018

<110> Korean Institute of Bioscience and Biotechnology

School production Cooperation group of the Tianxiang University (Soonchun University Industry collaboration Foundation)

<120> novel Bifidobacterium longum strain or Lactobacillus rhamnosus strain having obesity preventing or treating effect and use thereof

<130> 2019OPA2513

<150> KR 10-2018-0041917

<151> 2018-04-11

<160> 22

<170> KoPatentIn 3.0

<210> 1

<211> 1448

<212> DNA

<213> Bifidobacterium longum (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

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acaacgggat gcgacgcggc gacgcggagc ggatccctga aaaccggtct cagttcggat 1260

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cacccgaagc cggtggccta accccttgtg ggatggagcc gtctaaggtg aggctcgtga 1440

ttgggact 1448

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<211> 1491

<212> DNA

<213> Lactobacillus rhamnosus (Lactobacillus rhamnosus)

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atgaacgctg gcggcgtgcc taatacatgc aagtcgaacg agttctgatt attgaaaggt 60

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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

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ggtcgtaaaa ctctgttgtt ggagaagaat ggtcggcaga gtaactgttg tcggcgtgac 480

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gcaagcgtta tccggattta ttgggcgtaa agcgagcgca ggcggttttt taagtctgat 600

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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

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<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> 27F primers

<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 (14)

1. A strain of bifidobacterium longum DS0956 deposited under accession number KCTC13505 BP.

2. The strain of claim 1, wherein the Bifidobacterium longum DS0956 strain induces white adipocytes to form beige adipocytes or brown adipocytes.

3. The strain of claim 2, wherein the strain increases expression of each of Ucp1 (uncoupling protein 1), Pgc1a (peroxisome proliferator-activated receptor gamma coactivator 1- α), Prdm16(PR/SET domain 16), Pparg (peroxisome proliferator-activated receptor gamma), CD137, fnf 21 (fibroblast growth factor 21), P2RX5 (purinergic receptor P2X 5), and Tbx1(T-box 1) genes in adipocytes, thereby inducing formation of beige adipocytes or brown.

4. The strain of claim 1, wherein the strain promotes degradation of fat or β -oxidation of lipids in adipocytes.

5. The strain of claim 1, wherein the strain reduces the accumulation of cholesterol or Low Density Lipoprotein (LDL) in a subject suffering from obesity.

6. A Lactobacillus rhamnosus DS0508 strain deposited under accession No. KCTC13504 BP.

7. The strain of claim 6, wherein the strain induces the formation of beige adipocytes or brown adipocytes.

8. The strain of claim 7, wherein the strain increases expression of each of Ucp1 (uncoupling protein 1), Pgc1a (peroxisome proliferator-activated receptor gamma coactivator 1- α), 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) genes in adipocytes, thereby inducing formation of beige adipocytes or brown.

9. The strain of claim 6, wherein the strain promotes degradation of fat or β -oxidation of lipids in adipocytes.

10. The strain of claim 6, wherein the strain reduces the accumulation of cholesterol or Low Density Lipoprotein (LDL) in a subject suffering from obesity.

11. A pharmaceutical composition for preventing or treating obesity, the composition comprising at least one selected from the group consisting of the strain of any one of claims 1 to 10, a culture medium of the strain, a concentrate of the culture medium, a dried product of the culture medium, and an extract of the culture medium as an active ingredient.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is prepared as a formulation selected from the group consisting of a capsule, a powder, a granule, a tablet, a pill, or a lyophilized formulation.

13. A health functional food composition for preventing or improving obesity, comprising at least one selected from the group consisting of the strain of any one of claims 1 to 10, a culture medium of the strain, a concentrate of the culture medium, a dried product of the culture medium, and an extract of the culture medium as an active ingredient.

14. A feed composition for preventing or improving obesity, the composition comprising at least one selected from the group consisting of the strain of any one of claims 1 to 10, a culture medium of the strain, a concentrate of the culture medium, a dried substance of the culture medium, and an extract of the culture medium as an active ingredient.

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