Lactobacillus paracasei L-30 strain and its uses

Abstract

To provide a novel Lactobacillus paracasei strain with bone regeneration effects, and pharmaceutical and food compositions containing the same. [Solution] The present invention relates to a novel Lactobacillus paracasei L-30 and its uses, and more particularly to Lactobacillus paracasei L-30 (KCTC16035BP) which has the ability to promote the differentiation or formation of osteoblasts, the ability to suppress the differentiation or formation of osteoclasts, and the ability to promote immune activity, and which can regenerate bone, and to a pharmaceutical composition for the treatment or prevention of bone diseases containing the same.

Description

[Technical Field]

[0001] This invention relates to a novel Lactobacillus paracasei strain L-30 and its uses. [Background technology]

[0002] Osteoporosis is a disease characterized by decreased bone density and weakening of bone tissue, increasing the risk of fractures. It primarily occurs with age, and in women, the incidence is particularly high due to hormonal changes after menopause. Bones undergo a continuous process of bone remodeling throughout life, but in youth, the rate of bone regeneration is faster than or equal to the rate of bone breakdown. As we age, this balance is disrupted, and bone density and mass gradually decrease. Other contributing factors can include nutritional deficiencies such as calcium, phosphate, and vitamin D deficiencies, endocrine disorders, and lack of exercise.

[0003] Osteoporosis is a disease that is difficult to cure with short-term drug administration alone, and requires long-term drug administration. Therefore, there is a need to develop new drugs that have novel mechanisms of action and skeletal structures, have low toxicity and side effects, and are effective in preventing and treating osteoporosis. However, bisphosphonate drugs, which make up the majority of existing osteoporosis treatments, have been criticized for the side effects that occur when they suppress osteoblasts. Therefore, there is a need to develop natural materials for the prevention and improvement of osteoporosis that have no side effects, low toxicity, and can be easily taken.

[0004] The inventors of this invention have discovered that a novel strain of Lactobacillus paracasei has excellent bone regeneration effects and can be used as a composition for the treatment or prevention of bone diseases, thereby completing the present invention. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Korean Patent No. 10-2486028 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a novel Lactobacillus paracasei strain that exhibits bone regeneration effects.

[0007] The present invention aims to provide a pharmaceutical composition for the treatment or prevention of bone diseases, comprising Lactobacillus paracasei L-30.

[0008] The present invention aims to provide a food composition for improving bone diseases that contains Lactobacillus paracasei L-30. [Means for solving the problem]

[0009] 1. Lactobacillus paracasei L-30 strain, deposit number KCTC16035BP, which has bone regeneration effects.

[0010] 2. The Lactobacillus paracasei L-30 strain having the 16s rRNA sequence of Sequence ID No. 1 as described in item 1 above.

[0011] 3. Lactobacillus paracasei L-30 strain, which has the effect of promoting the differentiation or formation of osteoblasts as described in item 1 above.

[0012] 4. Lactobacillus paracasei L-30 strain, which has the effect of suppressing the differentiation or formation of osteoclasts as described in item 1 above.

[0013] 5. Lactobacillus paracasei L-30 strain, which has an immune-boosting effect as described in item 1 above.

[0014] 6. A pharmaceutical composition for the treatment or prevention of bone disease, comprising one or more selected from the group consisting of Lactobacillus paracasei L-30 strain, crushed strain, culture of strain, extract of strain, and fraction of extract, according to any of items 1 to 5 above.

[0015] 7. In item 6 above, the fraction of the extract is the fraction contained in the peak that first appears when the extract of Lactobacillus paracasei strain L-30 is separated by protein liquid chromatography, and it is a pharmaceutical composition for treating or preventing bone diseases.

[0016] 8. In item 6 above, the size of the fraction of the extract is 440 - 600 kDa, and it is a pharmaceutical composition for treating or preventing bone diseases.

[0017] 9. In item 6 above, the bone disease is any one selected from the group consisting of osteoporosis, osteopenia, osteomalacia, rheumatoid arthritis, osteoarthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc disease, kuru disease, fibrous dysplasia, Paget's disease of bone, traumatic fracture, and stress fracture, and it is a pharmaceutical composition for treating or preventing bone diseases.

[0018] 10. A food composition for improving bone diseases, comprising any one or more selected from the group consisting of Lactobacillus paracasei strain L-30 according to any one of items 1 - 5, the disrupted product of the strain, the extract of the strain, and the fraction of the extract.

[0019] 11. In item 10 above, the fraction of the extract is the fraction contained in the peak that first appears when the extract of Lactobacillus paracasei strain L-30 is separated by protein liquid chromatography, and it is a food composition for improving bone diseases.

[0020] 12. In item 10 above, the size of the fraction of the extract is 440 - 600 kDa, and it is a food composition for improving bone diseases.

[0021] 13. In item 9 above, the bone disease is one or more selected from the group consisting of osteoporosis, osteopenia, osteomalacia, rheumatoid arthritis, osteoarthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc disease, kuru disease, fibrous dysplasia, Paget's disease of bone, traumatic fracture, and stress fracture, and it is a food composition for improving bone diseases.

Advantages of the Invention

[0022] Lactobacillus paracasei L-30 of the present invention has an osteogenic effect.

[0023] Lactobacillus paracasei L-30 of the present invention can activate the differentiation or formation of osteoblasts and promote bone formation.

[0024] Lactobacillus paracasei L-30 of the present invention can suppress the differentiation and fusion of osteoclasts and smoothly regenerate bone.

[0025] Lactobacillus paracasei L-30 of the present invention can promote immunocompetence.

Brief Description of the Drawings

[0026] [Figure 1] Figure 1 shows the results of comparing the osteogenic effects of various lactic acid bacteria extracts containing L-30 strain in hBMSC. [Figure 2] Figure 2 shows the results of separating the L-30 extract by size using FPLC. [Figure 3] Figure 3 shows the results of comparing the osteogenic effects of size fractions of the L-30 extract in hBMSC. [Figure 4] Figure 4 shows the results of comparing the osteogenic effects of the L-30 extract and its fractions with extracts of various Lactobacillus paracasei strains in hBMSC. [Figure 5] Figure 5 shows the results of confirming the cytotoxicity of cells by the concentration of the L-30 extract. [Figure 6] Figure 6 shows the results of comparing the osteogenic effects by the concentration and treatment period of the L-30 extract. [Figure 7A] Figure 7A shows the results of confirming the expression of osteogenic-related markers (mRNA, protein) by the treatment with the L-30 extract. [Figure 7B] Figure 7B shows the results of confirming the expression of osteogenic-related markers (mRNA, protein) by the treatment with the L-30 extract. [Figure 7C]Figure 7C shows the results of confirming the expression of osteogenic differentiation-related markers (mRNA, protein) after treatment with L-30 extract. [Figure 8] Figure 8 shows the results of comparing the expression of osteogenic differentiation-related markers after treatment with L-30 extract or fraction. [Figure 9] Figure 9 shows the results of confirming whether or not the Wnt pathway was activated by treatment with L-30 extract. [Figure 10] Figure 10 shows the results of confirming whether or not L-30 extracts were treated with p38 or Akt inhibitors to promote osteogenic differentiation. [Figure 11] Figure 11 shows the results of confirming the bone regeneration effect in mouse-derived cells and tissues. [Figure 12] Figure 12 shows the results of comparing the osteoclast differentiation inhibitory effects of various lactic acid bacteria extracts, including the L-30 strain. [Figure 13] Figure 13 shows the results of comparing the osteoclast differentiation inhibitory effects of various Lactobacillus paracasei extracts, including the L-30 strain. [Figure 14] Figure 14 shows the results of an analysis of changes in osteoclast differentiation markers following treatment with L-30 extract. [Figure 15] Figure 15 shows the results of confirming the inhibitory effect of the peak 1 fraction of the L-30 extract on osteoclast differentiation. [Figure 16A] Figure 16A shows the results of an analysis of changes in the expression of osteoclast-related genes following treatment with L-30 extract. [Figure 16B] Figure 16B shows the results of an analysis of changes in the expression of osteoclast-related genes following treatment with L-30 extract. [Figure 16C] Figure 16C shows the results of an analysis of changes in the expression of osteoclast-related genes following treatment with L-30 extract. [Figure 16D] Figure 16D shows the results of an analysis of changes in the expression of osteoclast-related genes following treatment with L-30 extract. [Figure 16E] Figure 16E shows the results of an analysis of changes in the expression of osteoclast-related genes following treatment with L-30 extract. [Figure 16F] Figure 16F shows the results of an analysis of changes in the expression of osteoclast-related genes following treatment with L-30 extract. [Figure 17] Figure 17 shows the results of confirming the presence or absence of osteoclast differentiation receptor expression after treatment with L-30 extract. [Figure 18] Figure 18 shows the results of confirming the presence or absence of osteoclast fusion marker expression after treatment with L-30 extract. [Figure 19] Figure 19 shows the results of confirming the presence or absence of osteoclast differentiation regulatory transcription factors after treatment with L-30 extract. [Figure 20] Figure 20 shows the results of examining the toxicity and ROS expression levels in mouse-derived cells treated with L-30 extract. [Figure 21] Figure 21 shows the results of confirming cytokine generation by treatment with L-30 extract. [Figure 22] Figure 22 shows the results confirming the activation of immune enhancement-related mechanisms by L-30 treatment. [Modes for carrying out the invention]

[0027] This invention provides a novel Lactobacillus paracasei strain.

[0028] The novel Lactobacillus paracasei L-30 of the present invention was deposited with the Korea Biotechnology Research Institute's Bioresource Center (KCTC) on September 5, 2024 (deposit number: KCTC 16035BP).

[0029] Lactobacillus paracasei L-30 has the 16s rRNA sequence of Sequence ID No. 1.

[0030] Lactobacillus paracasei L-30 has bone regeneration effects.

[0031] "Bone regeneration" refers to all processes of repairing or reconstructing bone tissue lost due to bone disease or injury, and osteoblasts and osteoclasts can play important roles in the natural bone regeneration process.

[0032] The bone regeneration effect of the present invention may be due to one or more activities among those that promote the differentiation or formation of osteoblasts, or activities that suppress the differentiation or formation of osteoclasts.

[0033] The Lactobacillus paracasei L-30 strain of the present invention can promote the differentiation or formation of osteoblasts. The strain can increase the expression of osteoblast marker genes or proteins, thereby enabling smooth differentiation of osteoblasts and promoting bone formation.

[0034] Cells treated with the Lactobacillus paracasei L-30 strain or an extract of the present invention may show increased expression of markers promoting osteogenic differentiation. For example, the expression of ALP, RUNX2, or COL1A1, which are genes associated with promoting osteogenic differentiation, may increase, and the expression of ALP, RUNX2, or COL1A1 proteins may increase.

[0035] Cells treated with the Lactobacillus paracasei L-30 strain or an extract of the present invention show enhanced differentiation of osteoblasts, which allows the composition to be used to promote bone formation and prevent or treat bone diseases.

[0036] In one embodiment, osteogenic differentiation was promoted by treating human adult stem cells with an extract of the L-30 strain during the process of inducing osteogenic differentiation.

[0037] In one embodiment, the group treated with an extract of the L-30 strain showed significantly higher levels of bone differentiation compared to the group treated with extracts of other bacterial species.

[0038] In one embodiment, osteogenic differentiation was promoted by treating mouse-derived cells or tissues with L-30 extract after inducing osteogenic differentiation.

[0039] The Lactobacillus paracasei L-30 strain of the present invention can suppress the differentiation or formation of osteoclasts. The strain can reduce the expression of osteoclast differentiation marker genes or transcription factors, thereby preventing osteoclasts from differentiating or forming properly.

[0040] In cells treated with the Lactobacillus paracasei L-30 strain or its extract according to the present invention, bone destruction may be suppressed and bone density may increase by inhibiting osteoclast differentiation. Furthermore, it may be possible to suppress osteoclast fusion and prevent osteoclasts from performing normal bone resorption functions.

[0041] Cells treated with the Lactobacillus paracasei L-30 strain or its extract may exhibit decreased expression of osteoclast differentiation regulators. For example, the expression of NFATc1, which promotes osteoclast differentiation, may decrease, while the expression of the C / EBPβ and MafB genes, which are negative regulators of this differentiation, may increase, and the expression of the p-AKT and p-mTOR proteins may decrease.

[0042] In one embodiment, when an extract of the L-30 strain was treated, TRAP, Cathepsin K, and MMP-9, which are representative markers of osteoclast differentiation, were all expressed at significantly low mRNA levels.

[0043] Unlike other Lactobacillus paracasei strains that only have an effect of promoting the differentiation of osteoblasts, the Lactobacillus paracasei L-30 strain of the present invention not only promotes the differentiation of osteoblasts but also has an effect of suppressing the differentiation of osteoclasts, thus effectively regenerating bone and treating or preventing bone diseases.

[0044] The Lactobacillus paracasei L-30 of the present invention has an immune activity-enhancing effect. Cells treated with the L-30 extract show an increase in ROS production, which in turn promotes cytokine production and can enhance immune activity.

[0045] This invention provides pharmaceutical compositions for the treatment or prevention of bone diseases.

[0046] The pharmaceutical composition comprises one or more selected from the group consisting of Lactobacillus paracasei L-30 strain, crushed product of the strain, culture of the strain, extract of the strain, and fraction of the extract.

[0047] The extract of the Lactobacillus paracasei L-30 strain of the present invention may also be a pulverized product of the cultured L-30 strain. The extract may be obtained by collecting and washing the cultured L-30 strain, pulverizing it using sonication, removing cell walls and other residues by centrifugation, filtering the supernatant, and freeze-drying it.

[0048] The fraction of the extract may be the fraction of the first peak to appear when separated by fast protein liquid chromatography (FPLC). The size of the fraction may be 440 to 600 kDa. The fraction has a superior osteogenic effect compared to the extract of the L-30 strain of the present invention or fractions of other peaks in that extract.

[0049] The extract of the Lactobacillus paracasei L-30 strain of the present invention, or a fraction of the extract of said strain, can promote bone formation and suppress the differentiation or formation of osteoclasts, thereby exhibiting preventive or therapeutic effects on bone diseases through bone formation and bone regeneration.

[0050] The L-30 strain of the present invention, its extract, or its fractions do not exhibit cytotoxicity when treated with cells at various concentrations.

[0051] The aforementioned "bone disease" may be one or more bone diseases selected from the group consisting of osteoporosis, osteoporosis, osteomalacia, rheumatoid arthritis, osteoarthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc disease, rickets, fibrous dysplasia, Paget's disease of bone, traumatic fractures, and stress fractures.

[0052] In this specification, the term "treatment" means all actions that improve or beneficially alter a bone disease by administering the pharmaceutical compositions of the present invention, and the term "prevention" means all actions that suppress or delay the onset of a bone disease.

[0053] The aforementioned "pharmaceutical composition" may contain an active ingredient alone, or it may contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0054] The extract of Lactobacillus paracasei L-30 strain contained in the pharmaceutical composition of the present invention may be present at a concentration of 0.1 μg / ml to 100 μg / ml, for example, 0.1 μg / ml to 5 μg / ml, 0.1 μg / ml to 10 μg / ml, 0.5 μg / ml to 20 μg / ml, 0.5 μg / ml to 30 μg / ml, 0.5 μg / ml to 40 μg / ml, 0.5 μg / ml to 50 μg / ml, 0.5 μg / ml to 70 μg / ml, 0.5 μg / ml to 100 μg / ml, or 0.1 μg / ml to 100 μg / ml, but the pharmaceutical effect can be observed even at higher concentrations.

[0055] The pharmaceutical composition is administered in a pharmaceutically effective amount. “Pharmaceutically effective amount” means an amount sufficient to treat the disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dose level may be determined by factors including the type and severity of the patient's disease, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration and elimination rate, the duration of treatment, and other factors well known in the medical field, including drugs used concurrently. The composition of the present invention may be administered as a standalone therapeutic agent, in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and in single or multiple doses. Considering all of the above factors, it is important to administer an amount that yields the maximum effect with the minimum amount without side effects, which can be readily determined by those skilled in the art. The effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and weight.

[0056] The aforementioned pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, it may be administered by topical application to the skin, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection, but is not limited to these methods.

[0057] When formulating the aforementioned composition, it may be manufactured using fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, or other diluents or excipients commonly used in the field.

[0058] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and lozenges. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to water and liquid paraffin, the liquid preparations may further contain various excipients, such as humectants, sweeteners, fragrances, and preservatives.

[0059] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, lyophilized preparations, and suppositories.

[0060] This invention provides a food composition for improving bone diseases.

[0061] The food composition comprises one or more selected from the group consisting of Lactobacillus paracasei L-30 strain, crushed product of the strain, culture of the strain, extract of the strain, and fraction of the extract.

[0062] The extract of Lactobacillus paracasei L-30 strain or a fraction of the extract of said strain of the food composition of the present invention can promote bone formation, suppress the differentiation or formation of osteoclasts, and improve bone diseases through bone formation or bone regeneration.

[0063] The food composition may further contain one or more carriers, diluents, excipients, and additives, and be formulated into one of the dosage forms selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquids. Foods to which the food composition can be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

[0064] Additives that may be further included in the food composition include one or more components selected from the group consisting of natural carbohydrates, flavoring agents, nutrients, vitamins, minerals (electrolytes), flavoring agents (synthetic flavoring agents, natural flavoring agents, etc.), coloring agents, fillers (cheese, chocolate, etc.), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohol, carbonation agents, and fruit pulp.

[0065] When formulating the aforementioned food composition, it can be manufactured using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients.

[0066] The present invention will be described in more detail below with reference to examples. [Examples]

[0067] Examples Example 1. Isolation of novel Lactobacillus paracasei strain L-30. Lactobacillus paracasei L-30 strain (KCTC16035BP), obtained from Neoregen Biotech (Gyeonggi-do, South Korea), was pre-cultured in MRS medium at 35-37°C for 18 hours. Next, 1% was inoculated into 500 mL of MRS broth and cultured for another 18 hours at 35-37°C. The cultured L-30 was collected using a centrifuge (10,000 g for 10 minutes at 4°C) and washed twice with PBS. Then, it was washed with distilled water to completely remove MRS broth and PBS. The L-30, resuspended in 20 mL of distilled water, was sonicated on ice for 30 minutes using an ultrasonic shredder. To remove cell wall components and other residues, the pellet was centrifuged at 10,000 g for 20 minutes at 4°C, and then discarded. The supernatant was filtered (0.2 μm) and frozen at -80°C. The L-30 extract was then obtained by lyophilization. The obtained L-30 extract was reconstituted with PBS before use. Furthermore, the L-30 extract was adjusted to pH 7.0, and the properties of the effective molecules within the L-30 extract were identified.

[0068] Example 2. Comparison of the bone differentiation effects of various lactic acid bacteria extracts in hBMSC. In addition to the L-30 strain of Example 1, extracts of the L14 strain (Lactobacillus plantarum L-14), L15 strain (Enterococcus faecium L-15), L28 (Lactobacillus Lactis), and MS4 (Lactobacillus pentosus) strain were obtained in the same manner as in Example 1. To confirm the osteogenic differentiation effect of the strains according to the present invention, the aforementioned strains were used to treat human adult bone marrow stem cells (hBMSCs) during osteogenic differentiation induction. As hBMSCs, cells obtained from human bone marrow were purchased from promocell GmbH (Germany), and the osteogenic differentiation effect was confirmed macroscopically using the ARS staining method, which can visualize calcium deposits that increase during osteogenic differentiation. Differentiated cells were fixed with 4% paraformaldehyde (PFA) and stained with Alizarin red solution (Sigma-Aldrich). Taken with a digital camera (Canon, Tokyo, Japan) and an inverted microscope (EVOS) TM XL Core Imaging System;Thermo Scientific TM Observations were made in Waltham, Massachusetts, USA. As a result, the L-30 strain showed strong and widespread staining, demonstrating excellent bone differentiation effects (Figure 1). This confirmed that the novel strain of the present invention can promote the differentiation of osteoblasts and promote bone formation.

[0069] Example 3. Separation of Lactobacillus paracasei L-30 extract by size. To select substances effective for bone differentiation from the L-30 extract, the sample was fractionated by size using Fast Protein Liquid Chromatography (FPLC) and analyzed. Specifically, EPS (30 mg / mL) was separated by size exclusion chromatography using PBS on a HiLoad® 16 / 600 Superdex 200 pg column (GE Healthcare) and analyzed by AKTA High-Performance Protein Liquid Chromatography (GE Healthcare). As a result, the peaks of the five fractions were separated as shown in Figure 2.

[0070] Example 4. Confirmation of the osteogenic differentiation effect of size-specific fractions of L-30 extract in hBMSC. After separating the L-30 extract into five fractions according to size as in Example 3, the osteogenic differentiation effect of each fraction was confirmed by treating differentiating hBMSCs with each fraction. The hBMSCs were then fed into a 24-well plate at a rate of 15,000 cells / cm² in an osteogenic differentiation medium consisting of 10% FBS (Hyclone Laboratories Inc.), 1% penicillin-streptomycin (Gibco Inc.) in α-MEM (Welgene Inc.), 100 nM dexamethasone (Sigma-Aldrich), 10 mM β-glycerol 2-phosphate (Sigma-Aldrich), and 200 μM ascorbic acid (Sigma-Aldrich). 2 Seeds were seeded. The osteogenic differentiation medium was changed every 2-3 days, and osteogenic differentiation was carried out for a total of 21 days. Five peaks of the fractions separated by FPLC were added to the differentiation medium at concentrations of 1 ul / ml to 4 ul / ml each, and differentiation was induced for 21 days. After that, the degree of osteogenic differentiation was observed by ARS staining. As a result, fraction 1 of the L-30 extract (peak 1) showed superior differentiation ability regardless of the concentration of all fractions, and it was confirmed that it had a superior osteogenic differentiation effect compared to the L-30 extract (L30 EXT) (Figure 3).

[0071] Example 5. Comparison of the bone differentiation effects of various Lactobacillus paracasei extracts in hBMSC. The osteogenic differentiation effect of the L-30 extract from the above example and the first fraction (peak 1) of that extract was compared with extracts of other Lactobacillus paracasei strains. 1 μg / ml of L-30 extract, 1 μg / ml of the peak 1 fraction of L-30 extract, 1 μg / ml of Lactobacillus paracasei 13169, 2 μg / ml of Lactobacillus paracasei 13169, 1 μg / ml of Lactobacillus paracasei 3165, and 2 μg / ml of Lactobacillus paracasei 3165 were treated with hBMSC in the same manner as in the above example, and the degree of osteogenic differentiation was compared. As a result, osteogenic differentiation effects were confirmed in all Lactobacillus paracasei strains, including L-30. In particular, the osteogenic differentiation effect of the first fraction of the L-30 extract was the best (Figure 4).

[0072] Example 6. Confirmation of cytotoxicity based on the concentration of L-30 extract. Cell viability tests were performed on the L-30 extracts from the above-mentioned examples at different concentrations. The EZ-Cytox kit (Daeil Lab Service, Seoul, South Korea) based on the water-soluble tetrazolium salt (WST) method was used. hBMSC cells were 1.5 × 10⁶ per well. 4 The cells were seeded in 96-well plates at a given cell density. hBMSC cells were cultured for 3 days in osteogenic differentiation medium with various concentrations of L-30 extract. WST solution was added to each well, and the mixture was cultured at 37°C for 30 minutes. The absorbance of each well was measured at 450 nm using an Emax Plus microplate reader (Molecular Devices, Sunnyvale, California, USA). The results confirmed that the extract was non-cytotoxic at concentrations within the effective range (Figure 5).

[0073] Example 7. Comparison of bone differentiation effects depending on the concentration and treatment period of L-30 extract. The L-30 extract from the above example was treated in osteogenic medium at varying concentrations. hBMSCs were fed into a 24-well plate at a rate of 15,000 cells / cm² in a medium consisting of 10% FBS (Hyclone Laboratories Inc.), 1% penicillin-streptomycin (Gibco Inc.) in α-MEM (Welgene Inc.), 100 nM dexamethasone (Sigma-Aldrich), 10 mM β-glycerol 2-phosphate (Sigma-Aldrich), and 200 μM ascorbic acid (Sigma-Aldrich). 2 Seeds were sowed. The culture medium was changed every 2-3 days, and bone differentiation was carried out for a total of 21 days. Observation using ARS staining confirmed that both treatment with L-30 extract at 0.5 μg / ml and 1 μg / ml effectively induced bone differentiation on day 21 (Figure 6).

[0074] Example 8. Confirmation of osteogenic differentiation-related mRNA expression by treatment with L-30 extract. The L-30 extract from the above example was used to treat hBMSCs during osteogenic differentiation induction, and the expression of osteogenic differentiation-related markers was investigated. Total RNA was extracted from hBMSCs treated with the L-30 extract using TRIzol® reagent (Invitrogen), and cDNA was synthesized using AccuPower® RT PreMix & Master Mix (BIONEER Co., Korea). SYBR Pre-mix Ex Taq TMqRT-PCR analysis was performed using the II (Takara, Tokyo, Japan) and 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, California, USA). The PCR reaction was performed for 30 seconds at 95°C, followed by 40 amplification cycles of 5 seconds at 95°C and 34 seconds at 60°C. Comparative CT was used to measure expression levels, and peptidylprolyl isomerase A (PPIA) was used as the housekeeping gene for quantification. As a result, it was observed that ALP, RUNX2, and COL1A1, representative markers of osteogenic differentiation, were all expressed at significantly higher mRNA levels compared to the control group (Figure 7A).

[0075] Example 9. Confirmation of the expression of bone differentiation-related proteins by treatment with L-30 extract. After treating hBMSCs with the L-30 extract from the above example during osteogenic differentiation induction, proteins were extracted, and the expression of osteogenic differentiation-related markers was confirmed by Western blot. Cytoplasmic and nuclear proteins were analyzed using NE-PER. TM Nuclear and Cytoplasmic Extraction Reagents(Thermo Scientific TMExtraction was performed using a proteinase inhibitor (MedChemExpress, Monmouth Junction, New Jersey, USA) and a phosphatase inhibitor (MedChemExpress). The extracted proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting was performed with the following antibodies: runx family transcription factor 2 (Runx2; MBL International, USA), ALPI (CUSABIO, USA), COL1A1 (Cell Signaling Technology), AKT (Cell Signaling Technology), p-AKT (Cell Signaling Technology), extracellular signal-regulated kinase (ERK; Cell Signaling Technology), phospho-ERK (p-ERK; Cell Signaling Technology), c-Jun N-terminal kinase (JNK; Cell Signaling Technology), p-JNK (Cell Signaling Technology), p38 (Cell Signaling Technology), p-p38 (Cell Signaling Technology), p-β-catenin (Thermo Fisher, USA), β-catenin (Cell Signaling Technology), p-GSK3β (Cell Signaling Technology), GSK3β (Cell Signaling Technology) Lamin B1 (Santa Cruz Biotechnology, Dallas, Texas, USA), GAPDH (BioLegend, San Diego, California, USA). GAPDH was used as a housekeeping gene for quantification, and Lamin B1 was used to quantify nuclear β-catenin.In the proteins of hBMSCs treated with the L-30 extract, it was also confirmed that ALP, RUNX2, and COL1A1 were all significantly highly expressed compared to the control group (Figures 7B and 7C).

[0076] Example 10. Confirmation of the expression of bone differentiation-related markers by treatment with the L-30 extract or fraction After treating the hBMSCs with the L-30 extract and its first fraction (peak 1) of the above example during osteogenic induction, an attempt was made to confirm the expression of osteogenic promotion-related markers by fluorescence immunostaining. After fixing the hBMSC cells with 4% paraformaldehyde, permeabilization was performed at room temperature for 10 minutes using Triton X-100 (in PBS). After blocking with 3% BSA (in PBS) for 1 hour, the cells were cultured after treatment with the primary antibodies (in 0.1% BSA). The primary antibodies were runx family transcription factor2 (Runx2; MBL International, USA) and COL1A1 (Cell Signaling Technology), and the secondary antibody was Goat anti-Mouse IgG(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Invitrogen). For nuclear staining, ProLong TM Glass Antifade Mountant with NucBlue TM Stain (Invitrogen) was used. The staining was observed using a confocal laser scanning microscope (LSM980 with Airyscan2). As a result, it was confirmed that when the L-30 extract or its peak 1 fraction was treated, both RUNX2 and COL1A1 were significantly expressed compared to the control group. In particular, the peak 1 fraction of the L-30 extract was significantly more highly expressed than when the L-30 extract was treated (Figure 8).

[0077] Example 11. Analysis of Wnt pathway activation by treatment with the L-30 extract We attempted to induce suppression of the WNT pathway, a signaling pathway crucial for bone tissue maintenance and regeneration, by treating cells with si-β-catenin during osteogenic differentiation induction to promote osteocyte differentiation and growth. Simultaneously, we investigated the counteracting effect of reactivation by treating cells with L-30 extract and verifying it by ALP staining. β-catenin antisense (Bioneer, Korea) and negative control were pheno-infected with lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's protocol. After culturing si-β-catenin and lipofectamine separately at room temperature for 5 minutes, they were mixed. This mixture was cultured at room temperature for 20 minutes, and the pheno-infected mixture was added to hBMSC cells in a 24-well plate. The hBMSC cells were cultured at 37°C, 5% CO2, and 95% humidity for 24 hours before being used in the osteogenic differentiation experiment. ALP staining was performed using the TRACP & ALP double-stain Kit (Takara Bio Inc., JAPAN) according to the manufacturer's instructions. hBMSC cells were treated with L-30 extract for 7 days. hBMSC cells were fixed with fixative solution for 2 minutes, washed twice with PBS, and cultured with ALP staining solution for 30 minutes. The ALP staining solution was removed and the cells were cultured under an inverted microscope (EVOS). TM XL Core Imaging System;Thermo Scientific TM Staining was observed in Waltham, Massachusetts, USA.

[0078] After confirming that si-β-catenin reduces WNT signaling and decreases the efficiency of osteogenic differentiation, we treated the samples with L-30 extract and confirmed that the effect of suppressing WNT signaling on reducing osteogenic differentiation was offset. From these results, it was confirmed that the osteogenic differentiation-promoting effect of L-30 extract is related to the activation of the WNT pathway (Figure 9).

[0079] Example 12. Confirmation of osteogenic differentiation induction of L-30 extract by treatment with p38 or Akt inhibitor. After inhibiting p38 or AKT, we examined whether each pathway was restored by treating it with L-30 extract. SB203580 (10 μM) was used as the p38 inhibitor, and AKT inhibitor-IV (10 μM) was used as the AKT inhibitor. While treating with the inhibitors, 1 μM of L-30 extract was simultaneously applied, and the results of inhibition and restoration of each signaling pathway were verified by ALP staining. The results confirmed that even if the p38 or AKT pathway was inhibited, it could be restored by treatment with the L-30 extract of the present invention, thus maintaining the osteogenic differentiation effect (Figure 10).

[0080] Example 13. Confirmation of bone regeneration effect in mouse-derived cells and tissues. After isolating and culturing cells from mice, osteogenic differentiation was induced using the same method and differentiation solution as in the hBMSC example described above. L30 extract was included in the differentiation induction process. Primary osteoblast precursor cells were isolated from the mouse calvaria. The animal experiments were approved by the Seoul National University Animal Experiment Utilization Committee (IACUC, number SNU-230803-2-2). On postnatal day 4, the calvaria of ICR mice were cultured in 12-well culture plates. The calvaria were cultured in BMSC culture medium in or without the presence of 10 μg / mL of L30 extract. The medium was changed every 2 days, and the calvaria were collected on day 7. The calvaria were fixed with 4% PFA for 24 hours, and calcification was removed with 14% EDTA for 2 days. After calcification, the calvaria were embedded in paraffin wax. The block was cut to a depth of 800 μm and sagittally sectioned at a thickness of 10 μm (Leica Microsystems, Wetzlar, Germany) from the midline. The sectioned tissue was stained with H&E (hematoxylin and eosin). The separated cranial vault sections were digested with 0.25% trypsin and 0.2% collagenase at 37°C for 30 minutes. The released cells were grown in α-MEM (Welgene Inc.) supplemented with 10% FBS (Hyclone Laboratories Inc.) on a 100 mm plate, and then cultured in a 37°C incubator containing 5% CO2. After 3 days, the attached cells were used as osteoblast precursors. These osteoblast precursors were treated with L-30 extract and the peak 1 fraction of the extract and used during bone formation and differentiation.

[0081] ALP was confirmed on day 14 of differentiation (Figure 11A), and the bone differentiation-promoting effect of L-30 was verified by ARS staining on day 21 of differentiation (Figure 11B). Furthermore, RT-PCR and Western blot confirmed an increase in the expression of differentiation-related markers (Figures 11C and 11D). In addition, staining results showed that bone thickness increased when treated with L-30 extract for 7 days, confirming that the L-30 extract of the present invention promotes bone formation or regeneration (Figure 11E).

[0082] Example 14. Comparison of the inhibitory effects of various lactic acid bacteria extracts on osteoclast differentiation. Extracts of L14, L15, L28, MS4, and L-30 strains were obtained using the same method as in Examples 1 and 2, and their effect on inhibiting osteoclast differentiation was investigated. Mononuclear cells (monocytes) derived from mouse bone marrow were isolated and cultured to differentiate into osteoclasts. Simultaneously, the lactic acid bacteria extracts were treated with these extracts, and it was confirmed whether osteoclast differentiation could be inhibited by TRAP staining. Mononuclear cells were isolated from the femur and tibia tissue of 6-week-old female mice from C57BL6J. After removing all the muscle layers around the isolated femur and tibia, the tissue was disinfected with 70% ethanol, and the joint area was cut to expose the cells. Using a syringe, the inside of the bone tissue was washed with α-MEM medium (media) (1% P / S), and bone marrow cells were collected. The collected cells were centrifuged at 2000 rpm for 8 minutes, and then erythrocyte lysis buffer was added to lyse the erythrocytes for 10 minutes. To deactivate the lysis buffer, twice the volume of α-MEM medium (10% FBS, 1% P / S) was added, and the mixture was centrifuged at 2000 rpm for 8 minutes. Once a cell pellet was confirmed, the supernatant was removed, the cells were resuspended in fresh α-MEM medium (10% FBS, 1% P / S), and the residue was removed using a 70 μm filter. Subsequently, the cells were treated with M-CSF at a concentration of 5 ng / ml and cultured overnight. The following day, only mononuclear cells that were floating and not adhering to the bottom were collected and used for the experiment. To differentiate the mononuclear cells into osteoclasts, the collected mononuclear cells were maintained in α-MEM (10% FBS, 1% P / S) medium. To differentiate the mononuclear cells into macrophages, they were treated with M-CSF at 30 ng / ml for 3 days. Once it was confirmed that the cells adhered to the bottom, they were further treated with M-CSF at 30 ng / ml and RANKL at 100 ng / ml for 7 days to induce the formation of multinucleated osteoclasts.

[0083] The bacterial strains were cultured in MRS medium, then residual medium was removed by PBS washing, and extracts of the strains were obtained by sonication. Protein concentrations were measured by BCA assay, and osteoclasts were treated with 30 ng / ml of M-CSF and 100 ng / ml of RANKL, using these concentrations as a baseline. The degree of differentiation of osteoclasts treated with each of the strain extracts was confirmed by TRAP staining. After washing the cells once with PBS for staining, they were fixed with 4% PFA for 5 minutes, and residual PFA was removed again by washing with PBS. Sodium tartrate (10% v / v) was added to the TRAP staining reagent and mixed, and the solution was dispensed into the cells and cultured at 37°C for 45 minutes. The stained cells were washed once with PBS and observed under a microscope. As a result, it was confirmed that differentiation was suppressed only in osteoclasts treated with the L-30 strain extract, in contrast to the extracts of other strains (Figure 12).

[0084] Furthermore, the inhibitory effect of various Lactobacillus paracasei extracts on osteoclast differentiation was further evaluated using a similar method. Extracts were isolated from Lactobacillus paracasei 13169, Lactobacillus paracasei 13086, Lactobacillus paracasei 3169, Lactobacillus paracasei 3165, and the L-30 strain, treated with them, and the degree of differentiation was observed by TRAP staining. As a result, only the L-30 strain was able to suppress osteoclast differentiation (Figure 13). This confirmed that this effect is possessed only by the L-30 strain of the present invention among the Lactobacillus paracasei species.

[0085] Example 15. Confirmation of the decrease in osteoclast differentiation markers by treatment with L-30 extract. We confirmed whether treatment with L-30 extract suppressed osteoclast differentiation by observing changes in related markers. After completing the induction of differentiation from mononuclear cells to osteoclasts in the same manner as in Example 14, we treated them with the L-30 extract of the present invention. Using the same RT-PCR method as in Example 8, we quantitatively analyzed the mRNA levels of markers related to osteoclast differentiation. As a result, it was confirmed that TRAP, cathepsin k, and MMP-9, which are representative markers of osteoclast differentiation, were all expressed at significantly lower mRNA levels compared to the control group (Figure 14).

[0086] Example 16. Confirmation of the inhibitory effect of L-30 extract fractions on osteoclast differentiation. The effect of the concentration of the first fraction (peak 1) of the L-30 extract on osteoclast differentiation was investigated. Osteoclasts were treated with the peak 1 fraction of the L-30 extract at concentrations of 0.25 ul / ml, 0.5 ul / ml, 1 ul / ml, and 2 ul / ml, respectively. The degree of differentiation was confirmed by TRAP staining using the same method as in Example 14, and it was confirmed that osteoclast differentiation was suppressed at all concentrations of the L-30 fraction (Figure 15).

[0087] Example 17. Analysis of changes in osteoclast-related gene expression after treatment with L-30 extract. We analyzed changes in gene expression when osteoclasts were treated with L-30 extract. To obtain RNA samples from osteoclasts, cells were placed in a 6-well plate at a rate of 1 × 10⁶ 6Cells were seeded at a given number per well. Osteoclasts were differentiated using the same method as in the previous example, treated with L-30 extract, washed once with PBS, and RNA was separated using Trizol. Chloroform (20% v / v) was added to the Trizol solution containing the lysed cells, vortexed, and then centrifuged at 12000 rpm for 15 minutes at 4°C to collect only the RNA portion. The collected RNA was precipitated with isopropanol and pelletized, then dissolved in RNase-free water, and the sample concentration was measured using nanodrop and RNA Screentape. For 1ST cDNA synthesis, oligo dT, dNTPs, DTT, and SuperScript III Reverse Transcriptase were added to the obtained RNA samples, and the mixture was reacted in a PCR thermocycler at 55°C for 60 minutes and at 70°C for 15 minutes, respectively. Based on the synthesized 1ST cDNA, 2nd cDNA was synthesized. For synthesis, polymerase buffer, RNase H, and DNA polymerase were used and reacted at 16°C for 2 hours and 30 minutes. The synthesized 2nd DNA was purified 2-3 times with magnetic beads and 80% ethanol to obtain the final product, and the DNA concentration was measured using a Quantus fluorometer. The 2nd DNA underwent fragmentation with shearing enzyme at 37°C for 7 minutes and then at 98°C for 10 minutes. Adapters A and B were added, and the ligation process was carried out at 37°C for 1 hour. The ligated product was washed 2-3 times with magnetic beads and 80% ethanol as described above. The obtained samples were attached with i5 / i7 index primers and subjected to PCR, and finally washed 2-3 times with magnetic beads and 80% ethanol, after which the supernatant was transferred to a new tube.

[0088] The obtained RNA library samples were checked for concentration and sample quality using a DNA tape station. After mixing each sample at a concentration of 2 nM using an Illumina P2 300-cycle cartridge, the instrument was activated to perform RNA sequencing, and the resulting data was analyzed.

[0089] First, PCA plot analysis confirmed that the data for each sample (control group (CON), L-30 extract (L30), L-30 fraction (peak), and osteoclast differentiation induction group (Di)) were properly separated and suitable for analysis (Figure 16A). Volcano plot analysis revealed that the control group and the L-30 extract or fraction comparison group (Figure 16B, left) showed less difference in genetic changes compared to the osteoclast differentiation induction group and the L-30 extract or fraction comparison group (Figure 16B, right). This confirmed that L-30 extract or its fraction can suppress the osteoclast differentiation process.

[0090] K-means clustering heatmap analysis revealed that the expression trends of all genes were closer to those of the control groups (CON_1, CON_2) in the L-30 extract or fractional treatment groups. During osteoclast differentiation induction, the L-30 extract or fractional treatment suppressed the differentiation process and showed gene clusters even closer to those of the control group (Figure 16C).

[0091] Furthermore, GO analysis revealed that L-30 is highly associated with inflammatory responses, immune responses, and metabolic mechanisms (Figures 16D and 16E), and was most strongly associated with osteoclast differentiation among osteoclast-related mechanisms (Figure 16F).

[0092] Example 18. Confirmation of osteoclast differentiation receptor expression by treatment with L-30 extract. When osteoclasts were treated with L-30 extract, markers related to differentiation receptors were identified by RT-PCR. After completing the differentiation induction from mononuclear cells to osteoclasts in the same manner as in Example 14, the cells were treated with L-30 extract. The expression of differentiation receptors in osteoclasts was analyzed using the same RT-PCR method as in Example 8. As a result, both the differentiation receptors RANK and OSCAR increased during osteoclast differentiation compared to the control group (Figure 17, Di graph), but it was observed that the expression of each receptor decreased very effectively when treated with L-30 extract. This result indicates that the expression of differentiation receptors in osteoclasts is regulated at the gene level by treatment with L-30 extract.

[0093] Example 19. Confirmation of osteoclast fusion marker expression by treatment with L-30 extract. For osteoclasts to perform bone resorption normally, the fusion process in which multiple cells combine is essential. Therefore, we checked the expression of DC-STAMP and ATP6v0d2, genes that regulate this process. After completing the differentiation induction from mononuclear cells to osteoclasts in the same manner as in Example 14, the cells were treated with L-30 extract. Compared to osteoclasts that were not treated with L-30 extract (Di), no macroscopically large multinucleated cells were observed. Using the same RT-PCR method as in Example 8, we analyzed the expression of fusion-related genes in osteoclasts. The results showed that both DC-STAMP and ATP6v0d2 were significantly reduced in the L-30 extract-treated group compared to Di. This confirmed that L-30 extract can suppress osteoclast fusion (Figure 18).

[0094] Example 20. Confirmation of the expression of osteoclast differentiation regulatory transcription factors by treatment with L-30 extract. After inducing differentiation from mononuclear cells to osteoclasts in the same manner as in Example 14, the cells were treated with L-30 extract. The expression levels of genes and proteins of transcription factors that regulate osteoclast differentiation were analyzed using the same RT-PCR method and Western blot method as in Examples 8 and 9. NFATc1, which promotes osteoclast differentiation, showed decreased expression at both the gene and protein levels when L-30 was extracted. On the other hand, the gene levels of C / EBPβ and MafB, transcription factors that negatively regulate NFATc1, increased, while the protein expression of p-AKT and p-mTOR decreased. This confirmed that L-30 treatment can suppress osteoclast differentiation by inhibiting the expression of NFATc1, a major regulator of osteoclasts (Figure 19).

[0095] Example 21. Confirmation of cytotoxicity and ROS expression levels after treatment with L-30 extract. We investigated the occurrence of cytotoxicity and ROS expression levels during L-30 treatment using mouse-derived cells. Mouse-derived RAW 264.7 macrophages were purchased from the American Type Culture Collection (ATCC), and the cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The cells were cultured in an incubator at 37°C and 5% CO2, and subcultured at a concentration of 70-80%.

[0096] Raw 264.7 cells were placed in a 96-well plate in 5.0 × 10⁶ units. 3 After seeding in cell / wells, the cells were treated with L30 extract. After 24 hours, cytotoxicity was observed using the same method as in Example 6, and it was confirmed that there was no cytotoxicity even after treatment with L-30 (Figure 20A).

[0097] On the other hand, the generation of reactive oxygen species (ROS) in Raw 264.7 cells was evaluated using a ROS detection cell-based assay kit (DCFDA, Cayman). Raw 264.7 cells were cultured in 96-well plates in a 1.0 × 10⁶ container. 4The cells were dispensed at a density of cell number / well. The following day, the cells were treated with a medium containing the L30 extract for 24 hours. After 24 hours, ROS staining buffer (10 μM) was added, and the cells were cultured at 37°C for 90 minutes under light shielding. Fluorescence was measured using a fluorescence plate reader (TECAN) with an excitation wavelength of 500 nm and an emission wavelength of 550 nm. As a result, it was confirmed that treatment with the L-30 extract increased the amount of ROS produced. Since ROS can generate cytokines and activate the MAPK and NF-κB pathways, thereby contributing to macrophage activation, it was confirmed that treatment with the L-30 extract of the present invention can enhance immune function (Figure 20B).

[0098] Example 22. Confirmation of cytokine generation effect by treatment with L-30 extract. We aimed to confirm the expression of COX-2 and iNOS produced by activated macrophages. Western blot was performed 48 hours after treatment of Raw 264.7 cells with L-30 extract. iNOS was further observed by RT-PCR in groups 5 and 24 hours after treatment. It was confirmed that L-30 extract treatment increased the secretion of COX-2 and iNOS (Figure 21).

[0099] Example 23. Confirmation of activation of immune enhancement-related pathways by L-30 treatment. We investigated whether L-30 treatment activates mechanisms related to immune enhancement. Raw 264.7 cells were treated with L-30 extract, and markers related to MAPK and NF-κB mechanisms were observed by Western blot 24 hours after treatment. The NF-κB and MAPK (p38, ERK, JNK) pathways, which are major pathways involved in the innate immune response, were activated. This confirmed that L-30 treatment can enhance immune function by activating macrophages (Figure 22). [Accession Number]

[0100] Depository name: Korea Institute of Biotechnology, Bioresource Center (KCTC) Accession number: KCTC16035BP Entrustment date: September 5, 2024 JPEG2026073990000002.jpg197137

Claims

1. Lactobacillus paracasei L-30 strain, deposit number KCTC16035BP, which has bone regeneration effects.

2. The Lactobacillus paracasei L-30 strain according to claim 1, having the 16s rRNA sequence of Sequence ID No.

1.

3. The Lactobacillus paracasei L-30 strain according to claim 1, which has the effect of promoting the differentiation or formation of osteoblasts.

4. The Lactobacillus paracasei L-30 strain according to claim 1, which has the effect of suppressing the differentiation or formation of osteoclasts.

5. The Lactobacillus paracasei L-30 strain according to claim 1, which has an immune-activating effect.

6. A pharmaceutical composition for the treatment or prevention of bone disease, comprising one or more selected from the group consisting of Lactobacillus paracasei L-30 strain according to any one of claims 1 to 5, a crushed product of the strain, a culture of the strain, an extract of the strain, and a fraction of the extract.

7. The pharmaceutical composition for the treatment or prevention of bone disease according to claim 6, wherein the fraction of the extract is the fraction contained in the first peak that appears when the extract of Lactobacillus paracasei L-30 strain is separated by protein liquid chromatography.

8. The pharmaceutical composition for the treatment or prevention of bone disease according to claim 6, wherein the size of the fraction of the extract is 440 to 600 kDa.

9. The pharmaceutically active composition for the treatment or prevention of a bone disease according to claim 6, wherein the bone disease is one or more selected from the group consisting of osteoporosis, osteoporosis, osteomalacia, rheumatoid arthritis, osteoarthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc disease, rickets, fibrous dysplasia, Paget's disease of bone, traumatic fracture, and stress fracture.

10. A food composition for improving bone disease, comprising one or more selected from the group consisting of Lactobacillus paracasei L-30 strain according to any one of claims 1 to 5, crushed product of the strain, extract of the strain, and fraction of the extract.

11. The food composition for improving bone disease according to claim 10, wherein the fraction of the extract is the fraction contained in the first peak that appears when the extract of Lactobacillus paracasei L-30 strain is separated by protein liquid chromatography.

12. The food composition for improving bone disease according to claim 10, wherein the size of the fraction of the extract is 440 to 600 kDa.

13. The bone disease is one or more selected from the group consisting of osteoporosis, osteoporosis, osteomalacia, rheumatoid arthritis, osteoarthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc disease, rickets, fibrous dysplasia, Paget's disease of bone, traumatic fracture, and stress fracture, as per claim 10.

Images