Novel Bifidobacterium longum strain and its uses

Abstract

The present invention relates to a novel Bifidobacterium longum strain and uses thereof, and more particularly to a novel Bifidobacterium longum subsp. Infantis YB0411 strain having anti-obesity activity, and a pharmaceutical composition, a health functional food, a cosmetic composition, a feed composition, a reagent composition, etc. for anti-obesity use, which contains a culture of the strain. The culture of Bifidobacterium longum subsp. Infantis YB0411 strain having the deposit number KCTC14393BP of the present invention has the effect of suppressing the maturation process in which preadipocytes become adipocytes, and therefore can be used for anti-obesity pharmaceutical compositions, health functional foods, cosmetic compositions, feed compositions, reagent compositions, and the like. Furthermore, when preadipocytes are treated with the culture of the strain of the present invention, maturation into adipocytes can be suppressed, and therefore it is expected that it can be actively used in the development and research of therapeutic agents related to obesity, etc.

Description

Technical Field

[0001] The present invention relates to a novel Bifidobacterium longum strain and its uses. More specifically, the present invention relates to a novel Bifidobacterium longum subsp. Infantis YB0411 strain having anti-obesity activity, and pharmaceutical compositions, health functional foods, cosmetic compositions, feed compositions, reagent compositions, etc. for anti-obesity uses containing a culture of the above strain.

Background Art

[0002] Adipose tissue is a central tissue for maintaining systemic homeostasis, and excessive accumulation of white adipose tissue causes obesity. The process of conversion from immature pre-adipocytes to adipocytes is called adipogenesis, and such adipogenesis is regulated by various factors including hormonal signals and various transcriptional factors.

[0003] In the initial stage of adipogenesis, adipogenic hormonal stimulation is necessary for inducing the expression of early transcriptional factors such as CCAAT / enhancer-binding protein (C / EBP)-β and -δ, along with the suppression of Wnt / β-catenin signaling. As a result, after the expression of such early genes, mid-stage adipogenic stimulation activates master regulators peroxisome proliferator-activated receptor γ (PPARγ) and C / EBPα, leading to the expression of adiponectin and fatty acid-binding protein 4 (FABP4) required for final differentiation and adipocyte formation.

[0004] On the other hand, Korean Patent Publication No. 10-2019-0118985 discloses the anti-obesity effect of Bifidobacterium longum strains as the inhibitory activity of adipocyte differentiation. However, it also discloses that the treatment of strain cultures significantly increases the expression of genes specific to beige adipocytes and brown adipocytes compared to non-treated controls (control groups), which is related to the browning of white adipocytes.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] The problem to be solved by the present invention is to provide a novel Bifidobacterium longum strain that suppresses the maturation of preadipocytes into adipocytes.

[0007] Another problem to be solved by the present invention is to provide a pharmaceutical composition, a health functional food, a cosmetic composition, a feed composition, a reagent composition, etc. for anti-obesity use containing the strain culture.

[0008] Furthermore, the problem to be solved by the present invention is to provide a method for suppressing the maturation of separated preadipocytes into adipocytes by treating them with the strain culture.

Means for Solving the Problems

[0009] To solve the above-mentioned various problems, the present invention provides a Bifidobacterium longum subsp. Infantis YB0411 strain with the deposit number KCTC14393BP.

[0010] In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity, comprising a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with deposit number KCTC14393BP as an active ingredient.

[0011] Preferably, the culture is selected from the group consisting of a culture solution, a culture concentrate, a dried culture solution, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and an extract of the culture supernatant.

[0012] Furthermore, the present invention provides a health functional food for preventing or improving obesity, comprising a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with deposit number KCTC14393BP.

[0013] Preferably, the culture is selected from the group consisting of a culture solution, a culture concentrate, a dried culture solution, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and an extract of the culture supernatant.

[0014] Moreover, the present invention provides a cosmetic composition for slimming, comprising a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with deposit number KCTC14393BP.

[0015] Preferably, the culture is selected from the group consisting of a culture solution, a culture concentrate, a dried culture solution, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and an extract of the culture supernatant.

[0016] Preferably, the cosmetic composition is in a dosage form selected from the group consisting of a lotion, a toner, a cream, an essence, a foam, and a pack.

[0017] Furthermore, the present invention provides a feed composition for preventing or improving obesity, which comprises a culture of Bifidobacterium longum subsp. Infantis strain YB0411 having a deposit number of KCTC14393BP.

[0018] The culture is preferably selected from the group consisting of a culture broth, a culture concentrate, a dried culture broth, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and a culture supernatant extract.

[0019] Furthermore, the present invention provides a reagent composition for inhibiting the maturation of pre-adipocytes into adipocytes, which comprises a culture of Bifidobacterium longum subsp. Infantis strain YB0411 having a deposit number of KCTC14393BP.

[0020] The culture is preferably selected from the group consisting of a culture broth, a culture concentrate, a dried culture broth, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and a culture supernatant extract.

[0021] In addition to these, the present invention provides a method for inhibiting the maturation of pre-adipocytes into adipocytes, which comprises treating the isolated pre-adipocytes with a culture of Bifidobacterium longum subsp. Infantis strain YB0411 having a deposit number of KCTC14393BP.

Advantages of the Invention

[0022] The culture of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP of the present invention has the effect of suppressing the maturation process in which preadipocytes differentiate into adipocytes. Therefore, it can be applied to uses such as anti-obesity pharmaceutical compositions, health functional foods, cosmetic compositions, feed compositions, and reagent compositions. Furthermore, when the strain culture of the present invention is treated with preadipocytes, the maturation into adipocytes can be suppressed, and thus it is expected to be actively utilized in the development and research of related therapeutic agents for obesity and the like.

Brief Description of the Drawings

[0023]

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Mode for Carrying Out the Invention

[0024] Hereinafter, the present invention will be described in detail.

[0025] The inventors of the present invention assayed the anti-obesity effect from various lactic acid bacteria culture solutions for the development of a novel anti-obesity agent, and confirmed that in the culture of Bifidobacterium longum subsp. Infantis YB0411 strain with the accession number KCTC14393BP, there is an inhibitory activity on the maturation of pre-adipocytes into adipocytes, and thus completed the present invention.

[0026] Accordingly, the present invention provides a Bifidobacterium longum subsp. Infantis YB0411 strain with the accession number KCTC14393BP.

[0027] The novel Bifidobacterium longum subsp. Infantis YB0411 strain of the present invention was deposited with the Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, on November 30, 2020, under the accession number KCTC14393BP.

[0028] The culture of Bifidobacterium longum subsp. Infantis YB0411 strain with the accession number KCTC14393BP of the present invention has an effect of suppressing the maturation process of pre-adipocytes into adipocytes, and thus is applicable for anti-obesity use.

[0029] Specifically, even when the culture (1 μl / ml or 5 μl / ml) of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP of the present invention was treated with preadipocytes over 24 to 72 hours, it did not show cytotoxicity.

[0030] In addition, when the culture (1 μl / ml or 5 μl / ml) of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP of the present invention was treated with preadipocytes simultaneously with MDI, it was confirmed that almost no accumulation of intracellular neutral fat occurred.

[0031] Furthermore, when the culture of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP of the present invention was treated with preadipocytes, it was confirmed that the mRNA expression of early and late stage markers of adipogenesis, Cebp / β, Cebp / α, Cebp / δ, Pparγ, aP2 and adiponectin, was significantly reduced.

[0032] Moreover, when the culture of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP of the present invention was treated with preadipocytes, it was confirmed that the protein expression of adipogenesis markers CEBP / α, CEBP / β, PPARγ, AP2 and SIRT1 was significantly reduced.

[0033] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating obesity, which contains a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with deposit number KCTC14393BP as an active ingredient.

[0034] Preferably, the culture is selected from the group consisting of a culture solution, a culture concentrate, a dried culture, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and an extract of the culture supernatant.

[0035] The pharmaceutical composition containing the culture of Bifidobacterium longum subsp. Infantis strain YB0411 with deposit number KCTC14393BP of the present invention can be formulated into various forms such as oral dosage forms including powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and injection agents such as sterile injection solutions according to ordinary methods according to the respective purposes of use, and can be administered orally, or can be administered via various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration, etc.

[0036] Such a pharmaceutical composition may further contain a carrier, an excipient, a diluent, etc. Examples of suitable carriers, excipients or diluents that may be included are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, etc. Further, the pharmaceutical composition of the present invention may further contain a filler, an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, etc.

[0037] As a preferred embodiment, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations are formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., into the pharmaceutical composition. In addition to mere excipients, lubricants such as magnesium stearate and talc can also be used.

[0038] As a preferred embodiment, liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are mere diluents frequently used, various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc., can be included.

[0039] As a preferred embodiment, preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, etc. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Injectable preparations can include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.

[0040] The culture of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective volume level can be determined by factors such as the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route and excretion rate, the treatment period, drugs used simultaneously, and other well-known factors in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent, in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

[0041] As a preferred embodiment, in the pharmaceutical composition of the present invention, the effective amount of the culture of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP may vary depending on the age, sex, and weight of the patient. Generally, it may be administered at 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight daily or every other day, and may be administered in 1 to 3 divided doses per day. However, since it can be increased or decreased depending on the administration route, severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0042] The pharmaceutical composition of the present invention can be administered to a subject via various routes. Although all modes of administration are predictable, for example, it can be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine epidural, or intracerebroventricular injection.

[0043] In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention can be administered orally or parenterally via any normal route as long as it can reach the target tissue. Further, the composition of the present invention may be administered using any device capable of delivering the active ingredient to the target cells.

[0044] In the present invention, the "subject" is not particularly limited, and includes, for example, humans, monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs, and preferably mammals, more preferably humans.

[0045] The present invention also provides a health functional food for preventing or improving obesity, which contains a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP.

[0046] The culture is preferably selected from the group consisting of a culture solution, a culture concentrate, a dried culture solution, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and a culture supernatant extract.

[0047] The health functional food of the present invention can be variously used in foods and beverages effective for preventing or improving obesity. The health functional food of the present invention contains a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP. Examples of such foods include various foods, beverages, gums, teas, vitamin complexes, health supplements, etc., and can be used in the form of powders, granules, tablets, capsules or beverages.

[0048] The culture of the strain Bifidobacterium longum subsp. Infantis YB0411 with the deposit number KCTC14393BP of the present invention can generally be added at 0.01 to 15% by weight of the total weight of the food, and the healthy beverage composition can be added at a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml.

[0049] In addition to the health functional food of the present invention containing the compound as an essential component at the indicated ratio, it may contain food auxiliaries acceptable in food science, such as natural carbohydrates and various flavoring agents, as additional components.

[0050] Examples of the natural carbohydrates include ordinary sugars such as monosaccharides like glucose and fructose, disaccharides like maltose and sucrose, and polysaccharides like dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0051] As the flavoring agent, natural flavoring agents such as stevia like thaumatin, rebaudioside A or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame can be used. The ratio of the natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, minerals, flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition to these, the health functional food of the present invention may contain pulp for the production of natural fruit juices, fruit juice beverages, vegetable beverages, etc. Such components can be used independently or in combination. The ratio of such additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the delphinidin and its pharmaceutically acceptable salts of the present invention.

[0052] In addition, the present invention provides a slimming cosmetic composition containing a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP.

[0053] The culture is preferably selected from the group consisting of a culture solution, a culture concentrate, a dried culture solution, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and a culture supernatant extract.

[0054] The "slimming" means a phenomenon in which the volume of the whole body or a specific part of the body decreases, and for example, it may be the removal or reduction of fat in the whole body or a specific part of the body.

[0055] The active ingredient of the cosmetic composition of the present invention may be contained in an amount of 0.01 to 50% by weight, preferably 0.1 to 20% by weight, more preferably 1 to 10% by weight, based on the total weight of the composition. Appropriate dosage form stability can be ensured within such a content range, and the desired slimming effect can be expected.

[0056] In addition to the active ingredient of the present invention, the composition of the present invention can be used by further including known natural product extracts and other components for the effects of antioxidant, anti-aging, moisturizing, and promoting skin regeneration as long as the active activity of the present invention is not impaired.

[0057] In addition, the composition can be formed into a specific dosage form by including components usually added to cosmetic compositions, such as fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering agents and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators, and other auxiliaries and carriers commonly used in the fields of cosmetics or dermatology.

[0058] The cosmetic composition of the present invention can be manufactured into any dosage form that is usually manufactured in the industry. For example, it can be formulated into solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, packs, soaps, surfactant-containing cleansings, oils, and sprays, etc., but is not limited thereto. More specifically, it can be manufactured into dosage forms such as lotions, toners, softening toners, nourishing toners, creams, nourishing creams, massage creams, essences, eye creams, powder foundations, emulsion foundations, wax foundations, cleansing creams, foams, cleansing foams, cleansing waters, packs, sprays, powders, compacts, lip glosses, lipsticks, shadows, shampoos, and rinses.

[0059] When the dosage form of the present invention is a paste, cream or gel, as the carrier component, animal oils, vegetable oils, waxes, paraffins, starches, tragacanths, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talcs or zinc oxides, etc. can be used.

[0060] When the dosage form of the present invention is a powder or spray, as the carrier component, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used. In particular, when it is a spray, it may further contain a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0061] When the dosage form of the present invention is a solution or emulsion, as the carrier component, solvents, solubilizers or emulsifiers can be used. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol fatty acid esters, polyethylene glycol or sorbitan fatty acid esters can be mentioned.

[0062] When the dosage form of the present invention is a suspension, as carrier components, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth can be used.

[0063] When the dosage form of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester can be used.

[0064] The present invention also provides a feed composition for preventing or improving obesity, which contains a culture of Bifidobacterium longum subsp. Infantis YB0411 strain with the deposit number KCTC14393BP.

[0065] The culture is preferably selected from the group consisting of culture broth, culture concentrate, dried culture broth, culture extract, culture supernatant, concentrated culture supernatant, dried culture supernatant and culture supernatant extract.

[0066] In the present invention, the term "feed" can mean any natural or artificial formulated diet, such as a single meal or the components of said single meal, for animals to eat, ingest, and digest, or suitable for these. The type of the feed is not particularly limited, and feeds commonly used in the art can be used. Non-limiting examples of the feed include vegetable feeds such as grains, root vegetables, food processing by-products, algae, fibrous substances, pharmaceutical by-products, oils and fats, starches, oil cakes or cereal by-products, and animal feeds such as proteins, inorganic substances, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food and drink. These can be used alone or in combination of two or more kinds.

[0067] Furthermore, the present invention provides a reagent composition for inhibiting the maturation of pre-adipocytes into adipocytes, which contains a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with the deposit number KCTC14393BP.

[0068] The culture is preferably selected from the group consisting of a culture broth, a culture concentrate, a dried culture broth, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and an extract of the culture supernatant.

[0069] The reagent composition can be applied, for example, as a reagent composition for research. Usually, the reagent composition of the present invention can be administered to cells or experimental animals under laboratory conditions, and specific administration methods and safety rules are preferably carried out in accordance with the internal compliance regulations of each laboratory of the experimenter.

[0070] Furthermore, the present invention provides a method for suppressing the maturation of pre-adipocytes into adipocytes, which comprises treating isolated pre-adipocytes with a culture of Bifidobacterium longum subsp. Infantis strain YB0411 having the deposit number KCTC14393BP.

[0071] The pre-adipocytes are those isolated from an individual by natural or artificial methods. The cells isolated from an individual may be cultured for several days to several weeks under appropriate culture conditions, and in some cases, may be cryopreserved.

[0072] The method of administering the active ingredient of the present invention to the isolated cells is obvious to an ordinary technician and preferably should be in accordance with the description in the following examples in this specification.

[0073] Hereinafter, the present invention will be described in more detail with specific examples. It is obvious that the following examples only describe a desirable aspect of the present invention, and the scope of the rights of the present invention should not be construed as limited by the matters described in the following examples.

[0074] [Examples] 1. Materials and Methods 1.1. Chemicals and Reagents Dexamethasone, isobutyl-1-methylxanthine (IBMX), insulin, rosiglitazone (ROSI), Oil Red O dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 4% formaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium (DMEM) and newborn calf serum (NBCS) were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum was purchased from Atlas Biologicals (Fort Collins, CO, USA). Penicillin-streptomycin solution was purchased from HyClone Laboratories (South Logan, UT, USA). Antibodies against C / EBPβ, C / EBPα, PPARγ, AP2, and SIRT1 were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against β-actin was purchased from Abcam (Cambridge, MA, USA). The BCA protein assay kit was purchased from Thermo Fisher Scientific (Rockford, IL, USA), and the protein loading buffer was purchased from Bio-Rad Laboratories (Hercules, CA, USA). The lipolysis assay kit was purchased from BioVision (Milpitas, CA, USA).

[0075] 1.2. Cell culture, differentiation, and treatment 3T3-L1 mouse embryonic fibroblasts were obtained from the Korean Cell Line Bank (KCLB-10092.1) and cultured in DMEM (high glucose) medium supplemented with 10% (v / v) heat-inactivated NCS (neocarcinostatin) and 1% (v / v) antibiotic-antifungal solution (100 U / ml penicillin, 100 U / ml streptomycin) in a 5% CO 2It was cultured in a humidified incubator at 37°C. Differentiation of 3T3-L1 preadipocytes was induced by replacing them into DMEM (MDI) containing 10% fetal bovine serum (FBS), 0.5 mM IBMX, 1 μM dexamethasone and 10 μg / ml insulin. After 2 days, the MDI medium was replaced with DMEM supplemented with 10% FBS and 10 μg / ml insulin. Approximately 90% of the preadipocytes were converted into mature adipocytes with a round shape 5 - 7 days after induction of differentiation. The insulin-supplemented medium was changed every 2 days.

[0076] 1.3. Cell viability assay 3T3-L1 preadipocytes were seeded and grown on a 96-well plate at a density of 1×10 4 cells / well, and then treated with various concentrations of the bacterial strain culture solution for the indicated time. After incubation was completed, 20 μl of 5 mg / ml MTT solution was added, and the cells were incubated at 37°C for 3 hours. The resulting formazan crystals were dissolved in 150 μl of dimethyl sulfoxide (DMSO), and the absorbance at 590 nm was measured using a Victor (trademark) X3 multilabel reader (manufactured by PerkinElmer, Inc., located in Waltham, Massachusetts, USA).

[0077] 1.4. Isolation and identification of lactic acid bacteria

[0078] Isolation of various lactic acid bacteria was performed using MRS medium (a medium developed by DE MAN, ROGOSA and SHARPE) under strictly anaerobic conditions. For anaerobic conditions, after removing the oxygen present in the medium using N 2 gas, it was sterilized. 0.1 g of the collected fecal sample was suspended in 10 ml of MRS medium, then serially diluted and 100 μl aliquots were spread on MRS plate medium or blood agar medium and cultured under anaerobic conditions at 37°C for 2 days. As a result, the resulting single colonies were subcultured for pure isolation and used while storing them long-term.

[0079] For the molecular biological identification of isolated lactic acid bacteria, the 16S rRNA gene was analyzed by sequencing using the universal primers 27F (5'-AGA GTT TGA TCM TGG CTC A-3': SEQ ID NO: 1) and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3': SEQ ID NO: 2) targeting the 16S rRNA gene. Confirmation of the obtained nucleotide sequence was performed using the identification search from EZbiocloud (http: / / www.ezbiocloud.net / ).

[0080] 1.5. Anti-obesity activity evaluation To examine the anti-obesity activity, 3T3-L1 cells were cultured in DMEM GlutaMax® supplemented with 10% NBCS and 1% penicillin-streptomycin in a 5% CO 2 incubator at 37 °C. When the cell density of 3T3-L1 cells reached 70 - 80%, they were seeded in 48-well plates. When the cell density reached 100%, the cells were switched to the adipocyte differentiation medium MDI (insulin, dexamethasone, isobutyl-1-methylxanthine (IBMX)). On the second day, the cells were treated with insulin, insulin and ROSI, and insulin and the sample (bacterial culture solution). On the fourth day, the cells were treated with insulin only. Then, on the sixth day, 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 μl of the sample were added to the medium on days 0 and 2, respectively. This MDI was changed once every other day during cell differentiation. The day when the cell density reached 100% was designated as day 0, and on day 0, the cells were treated in the composition of MDI, ROSI and MDI, sample and MDI. This experiment was performed as three independent repeated experiments for each sample.

[0081] 1.6. Oil Red O staining Six days after differentiation, the cells were washed with 1×PBS, fixed with 10% formalin for at least 1 hour, and stained with 0.3% filtered Oil Red O solution for 15 minutes. The stained cells were thoroughly rinsed with distilled water, and the phenotypic changes of the fully differentiated cells were photographed with an Axiovert-25 microscope (Carl Zeiss, Jena, Germany). The overall image of the culture well was captured using a Canon 6D digital camera. To quantify the amount of Oil Red O, the stained cells were eluted with 100% isopropanol, and the absorbance was measured at 520 nm using a Victor (trademark) X3.

[0082] 1.7. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was extracted using an RNA extraction kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, and the concentration was measured using a scandrop (registered trademark) spectrophotometer (Analytik Jena AG, Jena, Germany). To apply total RNA (1 μg) to reverse transcription polymerase chain reaction (RT-PCR), cDNA was synthesized using a Maxime (registered trademark) RT PreMix kit (Intron Biotechnology, Seoul, Republic of Korea), and the reaction was performed in a Veriti (registered trademark) 96-Well Thermal Cycler (Applied Biosystems, Singapore). Quantitative real-time PCR was performed using an iQ (trademark) SYBR (registered trademark) Green Supermix kit (Bio-Rad, Singapore) in a CFX96 (trademark) real-time PCR detection system (Bio-Rad, Singapore). The primer sequences used in this study are shown in Table 1. The expression level of the target gene was normalized with Tbp.

[0083]

Table 1

[0084] 1.8. Preparation of whole cell extracts and Western blot analysis Before recovery, treated or untreated cells with lactic acid bacteria treatment were washed twice with ice-cold 1×PBS and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (manufactured by Santa Cruz Biotechnology, Inc.) supplemented with protease inhibitor cocktail, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM sodium orthovanadate. The lysate was scraped and collected, and the cells were incubated in an ice bath for 15 minutes. The samples were centrifuged at 10,000×g for 15 minutes at 4°C to obtain the supernatant. The protein concentration was determined using a BCA Protein Assay Kit (manufactured by Pierce, Rockford, IL, USA). The same amount of protein was loaded per sample and separated on a 4–20% sodium dodecyl sulfate–polyacrylamide gel (Mini-PROTEAN® Precast Gel, manufactured by Bio-Rad). After electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Trans-Blot® SD Semi-Dry Cell, manufactured by Bio-Rad) at 13 V for approximately 1.5 hours using a semi-dry transfer cell (manufactured by Bio-Rad). The membrane was blocked with 5% dry skim milk in 1× Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) on a shaker at room temperature for 1 hour and incubated with the primary antibody overnight at 4°C. After incubation with the primary antibody, the membrane was washed three times with 1×TBST for 5 minutes each. The membrane was incubated with a specific horseradish peroxidase (HRP)-conjugated secondary antibody on a shaker at room temperature for 1.5 hours and washed three times for approximately 5 minutes each. All washing steps were performed in 1×TBST. The immunoreactive protein signals were detected using a chemiluminescence electrochemiluminescence (ECL) assay and measured using molecular imaging software (manufactured by Bio-Rad). For better visualization of the data, the brightness and contrast were slightly adjusted, but the same changes were applied to the entire complete image panel to ensure no parts were lost from the image. Anti-β-actin was used as a control.Western blot data was quantified using ImageJ software (developed by the National Institutes of Health, USA).

[0085] 1.9. Evaluation of immunofluorescence staining To observe the regulation of protein expression of C / EBPβ and PPARγ, which are transcriptional regulatory factors for adipocyte differentiation by Lactobacillus casei YB0411, an immunofluorescence staining method using 3T3-L1 preadipocytes was utilized. In this experiment, rosiglitazone (Rosi), an antagonist of PPARγ, was used as a positive control, and the Lactobacillus culture solution was treated with 3T3-L1 preadipocytes at a concentration of 5 μl / ml. After reacting 3T3-L1 preadipocytes with ice-cold MetOH at -20 °C for 15 minutes, they were treated with 3% BSA containing 0.1% Triton X-100 for 15 minutes for permeabilization. Then, after treatment with a blocking solution (1% BSA, 5% goat serum, 0.3% Triton X-100), the primary antibodies, C / EBPβ (CST) and PPARγ (manufactured by Santa Cruz Biotechnology), were reacted at 4 °C for 1 day. Subsequently, Alexa Fluor® 594-conjugated anti-rabbit IgG and Alexa Fluor® 594-conjugated anti-mouse IgG secondary antibodies were each reacted for 1 hour. To stain DNA, the cells were reacted with 1 μg / ml DAPI (4’,6-diamidino-2-phenylindole) for 1 minute, washed 3 times with 1×PBST, and then mounted on mounting medium containing an anti-fading agent (manufactured by Dako, located in California, USA) and observed with a confocal microscope (manufactured by Olympus, located in Tokyo, Japan).

[0086] 1.10. Statistical analysis All data were shown as mean ± standard deviation (SD). A significant difference between different treatment groups was detected using Student's t-test and two-way ANOVA, followed by a post hoc Bonferroni test. A p-value < 0.05 was considered statistically significant.

[0087] 2. Results 2.1. Inhibition of intracellular triglyceride accumulation by lactic acid bacteria culture broth Before determining the function of lactic acid bacteria culture broth in the maturation of 3T3-L1 preadipocytes into adipocytes, the inventors of the present invention determined an appropriate solution of lactic acid bacteria culture broth for use in 3T3-L1 preadipocytes and differentiated 3T3-L1 cells using cell viability assessment. As shown in Figure 2, when 3T3-L1 preadipocytes were treated with lactic acid bacteria culture broth at concentrations of 1 μl / ml or 5 μl / ml for 24, 48, and 72 hours, no notable cytotoxicity was observed.

[0088] Next, to confirm the effect of lactic acid bacteria culture broth on intracellular triglyceride accumulation, 3T3-L1 preadipocytes after confluence were treated with MDI medium in the presence or absence of lactic acid bacteria culture broth at concentrations of 1 μl / ml or 5 μl / ml to induce differentiation into adipocytes. Microscopic observation after Oil Red O staining on day 6 showed that treatment with lactic acid bacteria culture broth had an effect of inhibiting intracellular triglyceride accumulation in a concentration-dependent manner compared to the control exposed to MDI medium alone (Figure 3).

[0089] 2.2. Regulation and inhibition of the expression of lipidogenic transcription factors by lactic acid bacteria culture broth in lipidogenesis The process of differentiation and maturation of preadipocytes into adipocytes is regulated by many transcriptional regulatory factors. In the initial differentiation process into adipocytes, C / EBPβ plays an important role, and in the later maturation process, the expression of PPARγ is very important. And C / EBPδ and C / EBPα play important roles in regulating the expression of C / EBPβ and PPARγ during adipocyte differentiation. AdipoQ and aP2 are target genes of PPARγ and are highly expressed during the maturation process of adipocytes. The inventors of the present invention investigated whether the lactic acid bacteria culture broth affects the expression of lipidogenic transcription factors in lipidogenesis. To induce the differentiation of preadipocytes, the 3T3-L1 culture medium was replaced with MDI medium. When treated with the lactic acid bacteria culture broth of the present invention, the mRNA expressions of Cebpβ, Cebpδ, Cebpα, PPARγ, AdipoQ and aP2 were significantly reduced (Figure 4). Also, the lactic acid bacteria culture broth significantly decreased the concentrations of C / EBPα, C / EBPβ, PPARγ and aP2 even at the translation stage (Figure 5). Such results suggest that the lactic acid bacteria culture broth can effectively inhibit important lipidogenic transcription factors.

[0090] [Deposit Number]

[0091] Depositor: Korea Research Institute of Bioscience and Biotechnology

[0092] Deposit Number: KCTC14393BP

[0093] Date of Deposit: 20201130

[0094] [Correction according to Article 26, November 15, 2022]

[0095] [National Research and Development Project]

[0096] [Project Specific Number] 2015161030

[0097] [Project Number] 2015R1A6A1A03032522

[0098] [Department Name] Ministry of Education

[0099] [Name of Project Management Agency] National Research Foundation of Korea

[0100] [Name of Research Project] Project for Supporting Key Research Institutes

[0101] [Name of Research Topic] Development of Metal-Ceramic-Polymer Hybrid Biomaterials for Clinical Therapy and Research on Tissue Regeneration

[0102] [Contribution Rate] 1 / 2

[0103] [Name of Project Implementing Agency] Industry-Academia Collaboration Foundation, Soonchunhyang University

[0104] [Research Period] June 1, 2015 to May 31, 2024

[0105] [National Research and Development Project]

[0106] [Project Specific Number] 2017395051

[0107] [Project Number] NRF-2017M3A9A5051180

[0108] [Department Name] Ministry of Science and ICT

[0109] [Name of Project Management Agency] National Research Foundation of Korea

[0110] [Name of Research Project] Bio-Medical Technology Development Project

[0111] [Name of Research Topic] Securing, Managing, and Utilizing Research Results of Biological Resources

[0112] [Contribution Rate] 1 / 2

[0113] [Name of Project Implementing Agency] Korea Research Institute of Bioscience and Biotechnology

[0114] [Research Period] January 1, 2020 to December 31, 2020

Claims

1. Bifidobacterium longum subsp. Infantis strain YB0411 with deposit number KCTC14393BP.

2. A pharmaceutical composition for preventing or treating obesity, containing a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with deposit number KCTC14393BP as an active ingredient.

3. The pharmaceutical composition according to claim 2, characterized in that the culture is selected from the group consisting of a culture broth, a culture concentrate, a dried culture broth, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and a culture supernatant extract.

4. A health functional food for preventing or improving obesity, containing a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with deposit number KCTC14393BP.

5. The health functional food according to claim 4, characterized in that the culture is selected from the group consisting of a culture broth, a culture concentrate, a dried culture broth, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and a culture supernatant extract.

6. A cosmetic composition for slimming, containing a culture of Bifidobacterium longum subsp. Infantis strain YB0411 with deposit number KCTC14393BP.

7. The cosmetic composition according to claim 6, characterized in that the culture is selected from the group consisting of a culture broth, a culture concentrate, a dried culture broth, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and a culture supernatant extract.

8. The cosmetic composition according to claim 6, characterized in that the cosmetic composition is in a dosage form selected from the group consisting of a lotion, a toner, a cream, an essence, a foam, and a pack.

9. A feed composition for preventing or improving obesity, comprising a culture of Bifidobacterium longum subsp. Infantis YB0411 strain with deposit number KCTC14393BP.

10. The feed composition according to claim 9, characterized in that the culture is selected from the group consisting of a culture solution, a culture concentrate, a dried culture solution, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and a culture supernatant extract.

11. A reagent composition for inhibiting the maturation of pre-adipocytes into adipocytes, comprising a culture of Bifidobacterium longum subsp. Infantis YB0411 strain with deposit number KCTC14393BP.

12. The reagent composition according to claim 11, characterized in that the culture is selected from the group consisting of a culture solution, a culture concentrate, a dried culture solution, a culture extract, a culture supernatant, a concentrated culture supernatant, a dried culture supernatant, and a culture supernatant extract.

13. A method for inhibiting the maturation of pre-adipocytes into adipocytes, comprising treating isolated pre-adipocytes with a culture of Bifidobacterium longum subsp. Infantis YB0411 strain with deposit number KCTC14393BP.

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