Osteoporosis-inhibiting strain and use thereof

Abstract

The present invention relates to an osteoporosis-inhibiting strain and a use thereof. More specifically, the present invention relates to a mixed strain comprising Limosillactobacillus reuteri, Weissella cibaria, or mixed strains including Lymosillactobacillus reuteri and Weissella cibaria, a culture thereof, a pharmaceutical composition for preventing or treating osteoporosis, and a health functional food composition for preventing or improving osteoporosis, each comprising same as an active ingredient.

Description

Osteoporosis-inhibiting strain and its use

[0001] The present invention relates to an osteoporosis-inhibiting strain and its use, and relates to a mixed strain comprising Limosillactobacillus reuteri, Weissella cibaria or Limosillactobacillus reuteri and Weissella cibaria, a culture thereof, a pharmaceutical composition for preventing or treating osteoporosis comprising the same, and a health functional food composition for preventing or improving osteoporosis.

[0002] The global increase in osteoporosis due to an aging population is emerging as a public health issue. Osteoporosis is associated with an imbalance in bone metabolism, where bone resorption exceeds bone formation.

[0003] The main treatments for osteoporosis include calcium and vitamin D supplementation, hormone replacement therapy, and medication. Some treatments, including calcium and vitamin D supplementation, selective estrogen receptor modulators, and hormone replacement therapy, have been shown to effectively reduce bone loss and the risk of complex fractures in patients with osteoporosis.

[0004] Non-pharmacological treatments include nutritional diet, exercise, and surgical treatment for fractures, but recent studies have reported that probiotic strains, which are beneficial bacteria in humans, are effective in preventing osteoporosis (Microbiol Spectr. 2017 Aug; 5(4): 10.1128 / microbiolspec.BAD-0015-2016).

[0005] As of yet, there is no raw material that uses probiotic bacteria cells as they are and has the functionality to improve osteoporosis.

[0006] 'Probiotics' are live bacteria that have a beneficial effect when ingested into the human body. Although the consumption rate of osteoporosis-related products is high in Korea, there are no raw materials that have been approved for improving osteoporosis using probiotic bacteria.

[0007] Under these technical backgrounds, the inventors of the present application confirmed that Limosillactobacillus reuteri, Weissella cibaria, or a mixed strain including Limosillactobacillus reuteri and Weissella cibaria, or a culture thereof, exhibits an effect of preventing, treating, or improving osteoporosis, and completed the present invention.

[0008] The purpose of the present invention is to recognize the problems of the prior art mentioned above,

[0009] The purpose of the present invention is to provide a strain of the genus Limosyl Lactobacillus or a culture thereof.

[0010] The purpose of the present invention is to provide a strain of the genus Weissella or a culture thereof.

[0011] The purpose of the present invention is to provide a mixed strain or a culture thereof comprising a strain of the genus Limosyl Lactobacillus and a strain of the genus Weissella.

[0012] The purpose of the present invention is to provide a pharmaceutical or health functional food composition for preventing, treating or improving osteoporosis, comprising the mixed strain or a culture thereof.

[0013] To achieve the above purpose, the present invention provides a strain of Limosillactobacillus reuteri MGE 3301 having the deposit number KCTC 15398BP or a culture thereof.

[0014] The present invention also provides a Weissella cibaria MGE 3110 strain or a culture thereof, having the deposit number KCTC 15399BP.

[0015] The present invention also provides a mixed strain comprising a Limosillactobacillus reuteri MGE 3301 strain with a deposit number of KCTC 15398BP and a Weissella cibaria MGE 3110 strain with a deposit number of KCTC 15399BP.

[0016] The present invention also provides a culture of the mixed strain.

[0017] The present invention also provides a pharmaceutical composition for preventing or treating osteoporosis, comprising the mixed strain or a culture thereof.

[0018] The present invention also provides a health functional food composition for preventing or improving osteoporosis, comprising the mixed strain or a culture thereof.

[0019] Through the present invention, it was confirmed that probiotics containing Limosillactobacillus reuteri MGE 3301 strain isolated from the feces of a healthy infant and Weissella cibaria MGE 3110 strain isolated from kimchi or a mixed strain thereof exhibit an excellent osteoporosis improvement effect.

[0020] Figure 1. Effect of oral administration of lactic acid bacteria on bone volume (BV / TV, %) and bone density (BMD, g / cm) in a menopausal animal model. 2 ) changes. Sham, control; OVX, ovariectomized mice; LR, ovariectomized mice were orally administered Limosillactobacillus reuteri MGE 3301 (~10 9cells / 0.1g / day); WC, ovariectomized mice were orally administered Weissella cibariaMGE 3110 (~10 9 cells / 0.1g / day); M, oral administration of a mixture of two strains to ovariectomized mice (~10 9 cells / 0.1g / day).

[0021] Figure 2. Changes in trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, / mm), and trabecular separation (Tb.Sp, mm) by orally administered lactic acid bacteria in a menopausal animal model. Sham, control; OVX, ovariectomized mice; LR, ovariectomized mice orally administered Limosillactobacillus reuteri MGE 3301 (~10 9 cells / 0.1g / day); WC, ovariectomized mice were orally administered Weissella cibariaMGE 3110 (~10 9 cells / 0.1g / day); M, oral administration of a mixture of two strains to ovariectomized mice (~10 9 cells / 0.1g / day).

[0022] Figure 3. Changes in tibia micro-CT images due to orally administered probiotic strains in menopausal animal models. Sham, control; OVX, ovariectomized mice; LR, ovariectomized mice orally administered Limosillactobacillus reuteri MGE 3301 (~10 9 cells / 0.1g / day); WC, ovariectomized mice were orally administered Weissella cibariaMGE 3110 (~10 9 cells / 0.1g / day); M, oral administration of a mixture of two strains to ovariectomized mice (~10 9 cells / 0.1g / day).

[0023] Figure 4. Changes in hematoxylin and eosin (H&E) and Masson's trichrome staining images of the tibia by orally administered lactic acid bacteria in a menopausal animal model. Sham, control; OVX, ovariectomized mice; LR, ovariectomized mice orally administered Limosillactobacillus reuteri MGE 3301 (~10 9 cells / 0.1g / day); WC, ovariectomized mice were orally administered Weissella cibariaMGE 3110 (~10 9 cells / 0.1g / day); M, oral administration of a mixture of two strains to ovariectomized mice (~10 9 cells / 0.1g / day).

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0025] The osteoporosis improvement effect of a composite strain mixed with Limosillactobacillus reuteriMGE 3301 strain isolated from the feces of a healthy infant and Weissella cibariaMGE 3110 strain isolated from kimchi was confirmed.

[0026] Based on this, the present invention provides, from one aspect, a Limosillactobacillus reuteri MGE 3301 strain having the deposit number KCTC 15398BP. The present invention provides a Weissella cibaria MGE 3110 strain having the deposit number KCTC 15399BP. The present invention provides a mixed strain comprising a Limosillactobacillus reuteri MGE 3301 strain having the deposit number KCTC 15398BP and a Weissella cibaria MGE 3110 strain having the deposit number KCTC 15399BP.

[0027] The Limosillactobacillus reuteri according to the present invention is the Limosillactobacillus reuteri MGE 3301 strain deposited with the Korea Research Institute of Bioscience and Biotechnology under the deposit number KCTC 15398BP on April 11, 2023. In addition, the Weissella cibaria according to the present invention is the Weissella cibaria MGE 3110 strain deposited with the Korea Research Institute of Bioscience and Biotechnology under the deposit number KCTC 15399BP on April 11, 2023.

[0028] In this specification, a “mixed strain” is a strain composition that can exhibit a synergistic effect for the desired efficacy without impairing the characteristics of individual strains, and may be in the form of a mixture in which each strain is included at a certain concentration or number.

[0029] In a specific example according to the present invention, the mixed strain according to the present invention may be mixed so that the number of viable cells of the Limosillactobacillus reuteri MGE 3301 strain with the deposit number KCTC 15398BP and the Weissella cibaria MGE 3110 strain with the deposit number KCTC 15399BP are the same, i.e., in a ratio of 1:1.

[0030] The viable cell count of the above strain is, for example, 10 6 10 inland 12 CFU / dose (e.g., 10 8 10 inland 12 In a specific embodiment according to the present invention, the viable cell counts of the Limosillactobacillus reuteri MGE 3301 strain with the deposit number KCTC 15398BP and the Weissella cibaria MGE 3110 strain with the deposit number KCTC 15399BP may be included in the same ratio of 1:1.

[0031] The present invention, from another perspective, relates to a culture of the above mixed strains.

[0032] In this specification, "culture" may refer to a culture of the mixed strains in a culture medium or culture solution. The culture may or may not contain the mixed strains. The culture may be in a liquid or solid form, but is not limited thereto.

[0033] The above culture may be in various forms, for example, a concentrate, dried product or extract of the culture.

[0034] The above extract can be extracted using water, an organic solvent, etc., and for example, can be extracted using water, a lower alcohol having 1 to 4 carbon atoms, hexane, chloroform, ethyl acetate, or a mixed solvent thereof.

[0035] According to the present invention, various forms of the culture may be included, for example, a concentrate, dried product or extract of the culture.

[0036] The strains may be delivered in pharmaceutically acceptable carriers, such as colloidal suspensions, powders, saline solutions, lipids, liposomes, microspheres, or nanospheres. They may be complexed or associated with a carrier, and may be delivered in vivo using carrier systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids.

[0037] Based on this, the present invention relates to a pharmaceutical composition for preventing or treating osteoporosis, comprising the strain, mixed strain or culture thereof from another perspective.

[0038] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and microarchitectural abnormalities, resulting in weakened bones that are more susceptible to fracture. Bone strength is determined by bone quantity and quality. Bone quantity is primarily expressed by bone mineral density (BMD), while bone quality is determined by structure, bone turnover rate, mineralization, and microdamage accumulation. Currently, osteoporosis is diagnosed by measuring bone density.

[0039] The above pharmaceutical composition for preventing or treating osteoporosis can be manufactured in a unit dosage form or by being placed in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the composition can be manufactured. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules or gel (e.g., hydrogel), and may additionally include a dispersing agent or stabilizer.

[0040] Pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, which are commonly used in the formulation. In addition, in addition to the above ingredients, the formulation may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0041] The above pharmaceutical composition for preventing or treating osteoporosis can be administered orally or parenterally and can be used in the form of a general pharmaceutical preparation. That is, the pharmaceutical composition for preventing or treating osteoporosis of the present invention can be administered in various oral and parenteral dosage forms during actual clinical administration. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include withepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol, and gelatin.

[0042] The concentration of the active ingredient included in the above composition can be determined in consideration of the therapeutic purpose, the patient's condition, the required period, etc., and is not limited to a specific range of concentrations. The pharmaceutical composition for preventing or treating osteoporosis of the present invention is administered in a pharmaceutically effective amount. In the present invention, the 'pharmaceutically effective amount' means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition for preventing or treating osteoporosis of the present invention can be administered as an individual therapeutic agent, or can be administered in combination with other therapeutic agents, and can be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art. The effective dose may vary depending on the patient's age, sex, condition, weight, absorption rate of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitant medication, and may increase or decrease depending on the route of administration, severity of vaginitis, sex, weight, age, etc.

[0043] The above composition may be a quasi-drug composition. "Quasi-drug" refers to products that are used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, and have a milder effect than a pharmaceutical product. For example, according to the Pharmaceutical Affairs Act, a quasi-drug is defined as a product excluding products used for pharmaceutical purposes, and may include products used for the treatment or prevention of diseases in humans or animals, products that have a mild effect on the human body, or products that do not directly affect the human body.

[0044] From another perspective, the present invention relates to a health functional food composition for preventing or improving osteoporosis, comprising the mixed strain or a culture thereof.

[0045] When using the above composition as a food additive, the health functional food composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient can be appropriately used depending on the intended use (prevention or improvement).

[0046] There are no specific restrictions on the types of the above health functional foods. Examples of foods to which the above health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, and vitamin complexes, and can include all health foods in the conventional sense.

[0047] The health functional food composition can be manufactured as a food, particularly a functional food. The functional food of the present invention includes ingredients commonly added during food manufacturing, and may include, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, it may include natural carbohydrates or flavoring agents as additional ingredients in addition to the active ingredient. The natural carbohydrates are preferably monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.), or sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agents may be natural flavoring agents (e.g., thaumatin, stevia extract, etc.) or synthetic flavoring agents (e.g., saccharin, aspartame, etc.).

[0048] In addition to the above health functional food composition, it may further include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, 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.

[0049] Example

[0050] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0051] Example 1: Verification of improvement in osteoporosis in postmenopausal mice by a complex strain of lactic acid bacteria.

[0052] Thirty-five female rats (250-300 g, 12 weeks old) were randomly divided into the control group (Sham), osteoporosis group (OVX), Limosillactobacillus reuteri MGE 3301 strain administration group (LR), Weissella cibaria MGE 3110 strain administration group (WC), and simultaneous administration of both strains group (M).

[0053] OVX, LR, W, and M groups underwent oophorectomy to remove both ovaries and induce osteoporosis. Five weeks after oophorectomy, the probiotic group was administered approximately 10 mg of probiotics orally daily for 16 weeks. 9 After 20 weeks of ovariectomy, bilateral tibiae were extracted and scanned using ex vivo Micro-CT, and pathological evaluation was performed using Hematoxylin & Eosin (H&E) and Masson's trichorme staining.

[0054] The control group (Sham) had higher bone mineral density (BMD, g / cm) compared to the OVX, LR, WC, and M groups. 2), trabecular bone volume (BV / TV, %), trabecular thickness (Tb.Th, mm) and trabecular number (Tb.N, mm -1 ) had the highest value and the value of the fraction separation (Tb.Sp, mm) was the lowest (p<0.05) (Figs. 1, 2).

[0055] In the osteoporosis-induced groups (OVX, LR, WC, and M), bone density, trabecular bone volume, trabecular thickness, trabecular number, and trabecular separation were all improved in the LR, WC, and M groups compared to the OVX group. In particular, the M group showed the best effect (p<0.05), confirming that using a composite strain was more effective than applying individual strains (Figs. 1, 2).

[0056] In addition, ex vivo Micro-CT scan images, H&E, and Massons's trichorme staining results also confirmed that the morphology of the cortical bone and trabecular bone was restored to a thicker thickness in the LR, WC, and M groups compared to the OVX group (Figs. 3, 4).

[0057] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0058]

[0059]

Claims

1. Limosillactobacillus reuteri MGE 3301 strain with accession number KCTC 15398BP.

2. Weissella cibariaMGE 3110 strain with accession number KCTC 15399BP.

3. A mixed strain comprising Limosillactobacillus reuteri MGE 3301 strain with deposit number KCTC 15398BP and Weissella cibaria MGE 3110 strain with deposit number KCTC 15399BP.

4. Culture of mixed strains according to Article 3.

5. A pharmaceutical composition for preventing or treating osteoporosis, comprising the mixed strain of paragraph 3 or the culture of paragraph 4.

6. A health functional food composition for preventing or improving osteoporosis, comprising the mixed strain of paragraph 3 or the culture of paragraph 4.

Images