Lactobacillus paracasei L-30 strain and application thereof

Abstract

The present invention relates to a novel Lactobacillus paracasei L-30 and uses thereof, and more particularly, to Lactobacillus paracasei The present invention relates to Lactobacillus paracasei L-30 (KCTC16035BP) and a pharmaceutical composition for treating or preventing bone diseases, and more particularly, to Lactobacillus paracasei L-30 (KCTC16035BP) capable of regenerating bones by having the ability to promote the differentiation or formation of osteoblasts, the ability to inhibit the differentiation or formation of osteoclasts, and the ability to promote immunocompetence, and to a pharmaceutical composition for treating or preventing bone diseases comprising 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 that increases the risk of fractures due to decreased bone density or weakened bone tissue. It is primarily caused by aging, especially in women, with an increased incidence after menopause due to hormonal changes. Throughout life, bones undergo a continuous process of breakdown and regeneration, a process of bone remodeling. In youth, bone regeneration is faster than or roughly equal to the rate of breakdown, but this balance is disrupted with age, leading to a gradual decline in bone density and mass. Contributing factors 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 medication and requires long-term treatment. Therefore, there is an urgent need to develop novel drugs with new mechanisms of action and bone structure, minimal toxicity and side effects, and effective prevention and treatment of osteoporosis. However, bisphosphonates, which currently account for the majority of osteoporosis treatments, have significant side effects due to their inhibition of osteoblasts. Therefore, there is an urgent need to develop natural substances for the prevention and improvement of osteoporosis; these substances should be free of side effects, have low toxicity, and be easily administered.

[0004] The inventors discovered a novel strain of Lactobacillus paracasei with excellent bone regeneration efficacy, making it suitable for use in compositions for the treatment or prevention of bone diseases, thus completing this invention.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: Korean Patent No. 10-2486028 Summary of the Invention

[0008] The purpose of this invention is to provide a novel strain of Lactobacillus paracasei with bone regeneration effects.

[0009] The object of the present invention is to provide a pharmaceutical composition comprising Lactobacillus paracasei L-30 for the treatment or prevention of bone diseases.

[0010] The object of the present invention is to provide a food composition containing Lactobacillus paracasei L-30 for improving bone diseases.

[0011] 1. A strain of Lactobacillus paracasei L-30, which has bone regeneration effects, and its accession number is KCTC16035BP.

[0012] 2. The Lactobacillus paracasei L-30 strain described in 1 above, which has a 16S rRNA sequence with sequence number 1.

[0013] 3. The Lactobacillus paracasei L-30 strain described in 1 above has the effect of promoting osteoblast differentiation or formation.

[0014] 4. The Lactobacillus paracasei L-30 strain described in 1 above has the effect of inhibiting the differentiation or formation of osteoclasts.

[0015] 5. The Lactobacillus paracasei L-30 strain described in section 1 above has the effect of promoting immune activity.

[0016] 6. A pharmaceutical composition for treating or preventing bone diseases, comprising at least one selected from the group consisting of Lactobacillus paracasei L-30 strain as described in any one of 1 to 5 above, lysates of the strain, cultures of the strain, extracts of the strain, and fractions of the extract.

[0017] 7. The pharmaceutical composition for treating or preventing bone diseases as described in 6 above, 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.

[0018] 8. The pharmaceutical composition for treating or preventing bone diseases as described in 6 above, wherein the fraction of the extract has a size of 440 kDa to 600 kDa.

[0019] 9. The pharmaceutical composition for treating or preventing bone diseases as described in section 6 above, wherein the bone disease is selected from the group consisting of osteoporosis, osteoopenia, osteomalacia, rheumatoid arthritis, degenerative arthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc, rickets, fibrous dysplasia, Paget's disease of bone, traumatic fracture, and stress fracture.

[0020] 10. A food composition for improving bone disease, comprising at least one selected from the group consisting of Lactobacillus paracasei L-30 strain as described in any one of 1 to 5 above, lysates of the strain, cultures of the strain, extracts of the strain, and fractions of the extract.

[0021] 11. The food composition for improving bone disease as described in 10 above, 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.

[0022] 12. The food composition for improving bone disease as described in 10 above, wherein the fraction size of the extract is from 440 kDa to 600 kDa.

[0023] 13. The food composition for improving bone diseases as described in 9 above, wherein the bone disease is selected from at least one of the following groups: osteoporosis, osteoopenia, osteomalacia, rheumatoid arthritis, degenerative arthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc, rickets, fibrous dysplasia, Paget's disease of bone, traumatic fracture, and stress fracture.

[0024] The Lactobacillus paracasei L-30 of this invention has bone regeneration effects.

[0025] The Lactobacillus paracasei L-30 of the present invention can promote bone formation by activating the differentiation or formation of osteoblasts.

[0026] The Lactobacillus paracasei L-30 of this invention can promote bone regeneration by inhibiting the differentiation and fusion of osteoclasts.

[0027] The Lactobacillus paracasei L-30 of this invention can promote immune activity. Attached Figure Description

[0028] Figure 1 This study compares the bone differentiation efficacy of various lactic acid bacteria extracts, including strain L-30, from human bone marrow mesenchymal stem cells (hBMSCs).

[0029] Figure 2The results show the size-based separation of L-30 extract by protein liquid chromatography (FPLC).

[0030] Figure 3 The results of comparing the bone differentiation efficacy of different size fractions of L-30 extract from human bone marrow mesenchymal stem cells (hBMSC).

[0031] Figure 4 This study compares the bone differentiation efficacy of L-30 extract and its fractions from human bone marrow mesenchymal stem cells (hBMSCs) with that of extracts from various Lactobacillus paracasei strains.

[0032] Figure 5 This is a confirmation result of cytotoxicity based on the concentration of L-30 extract.

[0033] Figure 6 The results show the comparison of bone differentiation efficacy based on the concentration of L-30 extract and its treatment time.

[0034] Figure 7 shows the results of confirming the expression of bone differentiation-related markers (messenger ribonucleic acid (mRNA) and proteins) based on the treatment with L-30 extract.

[0035] Figure 8 The results show the comparison of the expression of bone differentiation-related markers based on the treatment of L-30 extract or fraction.

[0036] Figure 9 To confirm whether treatment with L-30 extract activated the Wnt pathway.

[0037] Figure 10 To confirm whether treatment with p38 or Akt inhibitors promoted bone differentiation, the results were investigated.

[0038] Figure 11 To confirm the results of bone regeneration effects in mouse-derived cells and tissues.

[0039] Figure 12 To compare the results of the osteoclast differentiation inhibition effects of various lactic acid bacteria extracts including strain L-30.

[0040] Figure 13 To compare the results of the osteoclast differentiation inhibition effects of various Lactobacillus paracasei extracts, including strain L-30.

[0041] Figure 14 To analyze the changes in osteoclast differentiation markers after treatment with L-30 extract.

[0042] Figure 15To confirm the results of the osteoclast differentiation inhibition efficacy based on the peak 1 fraction concentration of the L-30 extract.

[0043] Figure 16 shows the results of the analysis of changes in the expression of osteoclast-related genes based on treatment with L-30 extract.

[0044] Figure 17 To confirm the results of treatment with L-30 extract to determine whether osteoclast differentiation receptors were expressed.

[0045] Figure 18 To confirm the results of treatment with L-30 extract to determine whether osteoclast fusion markers were expressed.

[0046] Figure 19 To confirm the results of treatment with L-30 extract to determine whether transcription factors regulating osteoclast differentiation were expressed.

[0047] Figure 20 This is a confirmation result of the toxicity and reactive oxygen species (ROS) expression levels in mouse-derived cells based on treatment with L-30 extract.

[0048] Figure 21 To confirm the results of cytokine production based on treatment with L-30 extract.

[0049] Figure 22 To confirm the results of activation of immune enhancement-related mechanisms following L-30 treatment. Detailed Implementation

[0050] This invention provides a novel strain of Lactobacillus paracasei.

[0051] The novel Lactobacillus paracasei L-30 of this invention was deposited on September 5, 2024, at the Bioresource Center of the Korea Institute of Life Science and Technology (KCTC) (accession number: KCTC 16035BP).

[0052] Lactobacillus paracasei L-30 has a 16S rRNA sequence with sequence number 1.

[0053] Lactobacillus paracasei L-30 has bone regeneration effects.

[0054] "Bone regeneration" refers to all processes that repair or rebuild bone tissue lost due to bone disease or injury. Osteoblasts and osteoclasts play important roles in the natural bone regeneration process.

[0055] The bone regeneration efficacy of the present invention may derive from at least one of the following activities: an activity that promotes the differentiation or formation of osteoblasts; or an activity that inhibits the differentiation or formation of osteoclasts.

[0056] The *Lactobacillus paracasei* L-30 strain of the present invention can promote osteoblast differentiation or formation. This strain can increase the expression of osteoblast marker genes or proteins, thereby facilitating osteoblast differentiation and promoting bone formation.

[0057] In cells treated with the Lactobacillus paracasei L-30 strain of the present invention or its extracts, the expression of markers of bone differentiation promotion may increase. For example, the expression of ALP, RUNX2, or COL1A1, which are genes associated with bone differentiation promotion, may increase, as may the expression of ALP, RUNX2, or COL1A1 proteins.

[0058] In cells treated with the Lactobacillus paracasei L-30 strain of the present invention or its extract, osteoblast differentiation can be promoted, thereby promoting bone formation, and thus it can be used as a composition for the prevention or treatment of bone diseases.

[0059] In one embodiment, treatment with an extract of strain L-30 was used to promote bone differentiation during the induction of bone differentiation from human bone marrow mesenchymal stem cells.

[0060] In one embodiment, bone differentiation was promoted at a significantly higher level in the group treated with extracts of strain L-30 compared with the group treated with extracts of other strains.

[0061] In one embodiment, bone differentiation was induced in mouse-derived cells or tissues, followed by treatment with L-30 extract to promote bone differentiation.

[0062] The *Lactobacillus paracasei* L-30 strain of the present invention can inhibit osteoclast differentiation or formation. This strain can reduce the expression of osteoclast differentiation marker genes or transcription factors, thereby causing osteoclasts to fail to differentiate or form poorly.

[0063] In cells treated with the Lactobacillus paracasei L-30 strain or its extract of the present invention, bone destruction is inhibited and bone density is increased by suppressing osteoclast differentiation. Furthermore, by inhibiting osteoclast fusion, osteoclasts can be prevented from performing their normal bone resorption function.

[0064] In cells treated with the Lactobacillus paracasei L-30 strain of the present invention or its extracts, the expression of osteoclast differentiation regulators may be reduced. For example, the expression of NFATc1, which promotes osteoclast differentiation, may be reduced; the gene expression of C / EBPβ and MafB, transcription factors that have a negative regulatory effect on osteoclast differentiation, may be increased; and the protein expression of p-AKT and p-mTOR may be reduced.

[0065] In one embodiment, when treated with extracts from strain L-30, the expression of TRAP, cathepsin K, and MMP-9, which are representative markers of osteoclast differentiation, was significantly reduced at the mRNA level.

[0066] Unlike other Lactobacillus paracasei strains that only promote osteoblast differentiation, the Lactobacillus paracasei L-30 strain of the present invention not only promotes osteoblast differentiation but also inhibits osteoclast differentiation, thus effectively promoting bone regeneration and treating or preventing bone diseases.

[0067] The Lactobacillus paracasei L-30 of this invention has the effect of promoting immune activity. Cells treated with L-30 extract can increase the production of reactive oxygen species (ROS), thereby promoting the production of cytokines and thus enhancing immune activity.

[0068] This invention provides pharmaceutical compositions for treating or preventing bone diseases.

[0069] The pharmaceutical composition comprises at least one of the following groups: Lactobacillus paracasei L-30 strain, lysate of the strain, culture of the strain, extract of the strain, and fractions of the extract.

[0070] The extract of Lactobacillus paracasei L-30 strain of the present invention can be a pulverized product of cultured L-30 strain. The extract can be obtained by harvesting and washing the cultured L-30 strain, pulverizing it using ultrasound, removing cell walls and other residues by centrifugation, filtering the supernatant, and lyophilizing it.

[0071] The fraction of the extract can be the fraction of the first peak that appears when separated using Fast Protein Liquid Chromatography (FPLC), and the fraction size can be from 440 kDa to 600 kDa. Compared with the extract of strain L-30 of the present invention or fractions of other peaks of the extract thereof, the fraction has superior bone differentiation efficacy.

[0072] The extract of Lactobacillus paracasei L-30 strain of the present invention, or fractions of the extract of said strain, can promote bone formation and inhibit osteoclast differentiation or formation, thereby exhibiting preventive or therapeutic effects on bone diseases through bone formation and bone regeneration.

[0073] The L-30 strain of the present invention, its extracts or fractions thereof do not exhibit cytotoxicity even when cells are treated at various concentrations.

[0074] The term "bone disease" can be selected from at least one of the following: osteoporosis, osteoopenia, osteomalacia, rheumatoid arthritis, degenerative arthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc, rickets, fibrous dysplasia, Paget's disease of bone, traumatic fracture, and stress fracture.

[0075] As used in this specification, the term "treatment" refers to all actions that improve or change bone disease in a beneficial direction through administration of the pharmaceutical composition of the present invention, and the term "prevention" refers to all actions that inhibit or delay the onset of bone disease.

[0076] The “pharmaceutical composition” can be provided as a pharmaceutical composition containing only the active ingredient or containing at least one pharmaceutically acceptable carrier, excipient or diluent.

[0077] The concentration of the extract of Lactobacillus paracasei L-30 strain included in the pharmaceutical composition of the present invention can be from 0.1 μg / ml to 100 μg / ml, for example, the concentration can be from 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 even at concentrations higher than these values, the pharmaceutical effect may still be achieved.

[0078] The pharmaceutical composition is administered in a pharmaceutically effective amount. "Pharmaceutically effective amount" refers to a dose sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dose level can be determined based on the patient's disease type, severity, drug activity, drug sensitivity, timing of administration, route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and factors known in other medical fields. The compositions of the present invention can be administered as a single therapeutic agent, in combination with other therapeutic agents, sequentially or simultaneously with existing therapeutic agents, or in single or multiple doses. Importantly, the aforementioned factors are considered, and the administered dose achieves maximum efficacy with minimal side effects, which can be easily determined by those skilled in the art. The effective amount of the composition according to the invention can vary depending on the patient's age, sex, and weight.

[0079] The pharmaceutical composition can be administered orally or parenterally. Parenterally administration includes, but is not limited to, topical application or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrapleural injection.

[0080] When formulating the composition, diluents or excipients commonly used in the art can be used, such as fillers, swelling agents, binders, wetting agents, disintegrants, surfactants, etc.

[0081] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, lozenges, etc., while liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, or syrups, etc. In addition to water and liquid paraffin, the liquid formulations may also include various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc.

[0082] Preparations used for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0083] This invention provides a food composition for improving bone diseases.

[0084] The food composition comprises at least one of the following groups: Lactobacillus paracasei L-30 strain, lysates of the strain, cultures of the strain, extracts of the strain, and fractions of the extract.

[0085] The extract of Lactobacillus paracasei L-30 strain or fractions of the extract of said strain in the food composition of the present invention can promote bone formation and inhibit osteoclast differentiation or formation, thereby exhibiting an effect of improving bone diseases through bone formation or bone regeneration.

[0086] The food composition can be formulated into one of the group consisting of tablets, pills, powders, granules, capsules, and liquid preparations, and further includes at least one of a carrier, diluent, excipient, and additive. Various food products, powders, granules, tablets, capsules, syrups, beverages, chewing gum, tea, vitamin complexes, and health functional foods can be added to the food composition.

[0087] As an additive that can be further added to the food composition, at least one component 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.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, antioxidants, glycerin, alcohol, carbonating agents, and fruit pulp may be used.

[0088] When formulating the food composition, diluents or excipients may be used, such as commonly used fillers, leavening agents, binders, wetting agents, disintegrants, surfactants, etc.

[0089] The present invention will be further described in detail below through examples.

[0090] Example

[0091] Example 1. Isolation of a novel Lactobacillus paracasei strain L-30

[0092] Lactobacillus paracasei L-30 strain (KCTC16035BP), purchased from Neorigen Biotech (Gyeonggi Province, South Korea), was pre-cultured in de Man, Rogosa and Sharpe medium (MRS) for 18 hours at 35°C to 37°C. Then, 1% was inoculated into 500 mL of MRS broth and further cultured at 35°C to 37°C for 18 hours. The cultured L-30 was harvested using a centrifuge (10000g for 10 minutes at 4°C) and washed twice with phosphate-buffered saline (PBS). Next, it was washed with distilled water to completely remove the MRS broth and PBS. The L-30, resuspended in 20 mL of distilled water, was sonicated on ice for 30 minutes using an ultrasonic homogenizer. To remove cell wall components and other residues, the supernatant was discarded after centrifugation at 10000g for 20 minutes at 4°C. The supernatant was filtered (0.2 μm) and frozen at -80 °C. It was then lyophilized to obtain the L-30 extract. The obtained L-30 extract was reconstituted with phosphate-buffered saline (PBS) before use. Furthermore, the pH of the L-30 extract was adjusted to 7.0, and the properties of the active molecules in the L-30 extract were confirmed.

[0093] Example 2. Comparison of the bone differentiation efficacy of various lactic acid bacteria extracts from human bone marrow mesenchymal stem cells (hBMSCs)

[0094] In addition to strain L-30 of Example 1, extracts of strains L14 (Lactobacillus plantarum L-14), L15 (Enterococcus faecium L-15), L28 (Lactobacillus Lactis), and MS4 (Lactobacuillus pentosus) were obtained using the same method as in Example 1. To confirm the osteodifferentiation effect of the strains of the present invention, the above-mentioned strains were treated during the induction of osteodifferentiation in human bone marrow mesenchymal stem cells (hBMSCs). The hBMSCs used were cells obtained from human bone marrow, purchased from Promocell GmbH, Germany. The osteodifferentiation effect was visually confirmed using Alizarin Red S (ARS) staining, which visualizes the increased calcium deposits during osteodifferentiation. Differentiated cells were fixed with 4% paraformaldehyde (PFA) and stained with Alizarin red solution (Sigma-Aldrich). Images were taken with a digital camera (Canon, Tokyo, Japan) and observed with an inverted microscope (EVOS™ XL Core Imaging System; Thermo Scientific™, Waltham, MA, USA). The results showed deep and extensive staining in strain L-30, confirming excellent bone differentiation. Figure 1 The novel strain of the present invention was confirmed to promote osteoblast differentiation and bone formation.

[0095] Example 3. Size-based isolation of Lactobacillus paracasei L-30 extract

[0096] To screen for substances effective for bone differentiation from L-30 extract, Fast Protein Liquid Chromatography (FPLC) was used to separate and analyze the components according to size. Specifically, extracellular polysaccharides (EPS, 30 mg / mL) were separated by size exclusion chromatography on a HiLoad 16 / 600 Superdex 200 pg column (GE Healthcare) using phosphate-buffered saline (PBS), and analyzed by AKTA Fast Protein Liquid Chromatography (GE Healthcare). The results showed five fractional peaks, such as... Figure 2 As shown.

[0097] Example 4. Confirmation of the osteodifferentiation efficacy of different size fractions of L-30 extract from human bone marrow mesenchymal stem cells (hBMSCs)

[0098] As described in Example 3, the L-30 extract was separated into 5 fractions according to size and then processed into differentiated human bone marrow mesenchymal stem cells (hBMSCs) to confirm the bone differentiation efficacy of each fraction. Human bone marrow mesenchymal stem cells (hBMSCs) were cultured in osteogenic differentiation medium (10% fetal bovine serum (FBS, Hyclone Laboratories Inc.), α-MEM containing 1% penicillin-streptomycin (Gibco Inc.), 100 nM dexamethasone (Sigma-Aldrich), 10 mM β-glycerol 2-phosphate (Sigma-Aldrich), and 200 μM ascorbic acid (Sigma-Aldrich) at a concentration of 15,000 cells / cm². 2 The culture medium was changed every 2-3 days for a total of 21 days of osteogenic differentiation. Five fractions separated by protein liquid chromatography (FPLC) were treated at concentrations ranging from 1 μL / ml to 4 μL / ml and induced for differentiation for 21 days (including changes in the differentiation medium). The degree of bone differentiation was then observed using ARS staining. The results showed that fraction 1 (peak 1) of the L-30 extract, regardless of its concentration, exhibited excellent differentiation ability, confirming its superior bone differentiation efficacy compared to the L-30 extract (L30EXT). Figure 3 ).

[0099] Example 5. Comparison of the bone differentiation efficacy of various Lactobacillus paracasei extracts from human bone marrow mesenchymal stem cells (hBMSCs).

[0100] The bone differentiation efficacy of the L-30 extract and its first fraction (peak 1) from the above embodiments was compared with that of extracts from other Lactobacillus paracasei strains. Using the same method as in the above embodiments, 1 μg / ml of L-30 extract, 1 μl / 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 into human bone marrow mesenchymal stem cells (hBMSCs), and the degree of bone differentiation was compared. The results showed that bone differentiation efficacy was confirmed in all Lactobacillus paracasei strains containing L-30, particularly the first fraction of the L-30 extract, which exhibited the best bone differentiation efficacy. Figure 4 ).

[0101] Example 6. Cytotoxicity confirmation based on the concentration of L-30 extract

[0102] The cytotoxicity of the L-30 extract at different concentrations described in this embodiment was tested. Human bone marrow mesenchymal stem cells (hBMSCs) were seeded at a density of 1.5 × 10⁴ cells per well in 96-well plates using the EZ-Cytox kit (Daeil LabService, Seoul, Korea) based on the water-soluble tetrazolium salt (WST) method. The hBMSCs were cultured for 3 days in osteogenic differentiation medium containing various concentrations of L-30 extract. WST (water-soluble tetrazolium) solution was added to each well, and the mixture was incubated 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, CA, USA). The results confirmed that there was no cytotoxicity at concentrations within the effective efficacy range of the extract. Figure 5 ).

[0103] Example 7. Comparison of bone differentiation efficacy based on L-30 extract concentration and treatment time

[0104] The L-30 extract from the above-described examples was treated with different concentrations in bone differentiation induction medium. Human bone marrow mesenchymal stem cells (hBMSCs) were cultured in a medium containing 10% fetal bovine serum (FBS, Hyclone Laboratories Inc.), 1% penicillin-streptomycin (Gibco Inc.), α-MEM (Welgene Inc.), 100 nM dexamethasone (Sigma-Aldrich), 10 mM β-glycerol 2-phosphate (Sigma-Aldrich), and 200 μM ascorbic acid (Sigma-Aldrich) at a concentration of 15,000 cells / cm². 2 The culture medium was seeded in 24-well plates. Osteogenic differentiation was induced for a total of 21 days by changing the medium every 2-3 days. ARS staining confirmed that L-30 extract at 0.5 μg / ml and 1 μg / ml effectively induced osteogenic differentiation on day 21 of differentiation. Figure 6 ).

[0105] Example 8. Confirmation of bone differentiation-related mRNA expression based on L-30 extract treatment

[0106] The L-30 extract from the above-described examples was used to treat human bone marrow mesenchymal stem cells (hBMSCs) during bone differentiation induction to confirm that bone differentiation promotes the expression of related markers. Total ribonucleic acid (RNA) was extracted from hBMSCs treated with the L-30 extract using Trizol reagent (Invitrogen), and complementary deoxyribonucleic acid (cDNA) was synthesized using AccuPower RT PreMix & Master Mix (BIONEER Co., KOREA). Real-time quantitative PCR (qRT-PCR) analysis was performed using SYBR Pre-mixEx Taq™ II (Takara, Tokyo, Japan) and a 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). PCR reactions were performed at 95°C for 30 seconds, followed by 40 amplification cycles of 95°C for 5 seconds and 60°C for 34 seconds. Comparative CT methods were used to determine expression levels. Peptidylprolyl isomerase A (PPIA) was used for quantification of the housekeeping gene. The results showed that, compared to the control group, ALP, RUNX2, and COL1A1, representative markers of bone differentiation promotion, were all significantly more expressed at the mRNA level (Figure 7A).

[0107] Example 9. Confirmation of the expression of bone differentiation-related proteins based on treatment with L-30 extract

[0108] The L-30 extract from the above-described examples was treated with human bone marrow mesenchymal stem cells (hBMSCs) to induce bone differentiation, and proteins were extracted to confirm the expression of bone differentiation-promoting markers by Western blot. Cytoplasmic and nuclear proteins were extracted using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific™), a proteinase inhibitor (MedChemExpress, Monmouth Junction, NJ, USA), and a phosphatase inhibitor (MedChemExpress).The extracted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting was performed using the following antibodies: runx family transcription factor 2 (Runx2; MBL International, USA), ALPI (CUSABIO, USA), COL1A1 (CST (Cell Signaling Technology), AKT (CST (Cell Signaling Technology), USA), p-AKT (CST (Cell Signaling Technology), USA), extracellular signal-regulated kinase (ERK; CST (Cell Signaling Technology), USA), phosphorylated activated ERK (phospho-ERK) (p-ERK; CST (Cell Signaling Technology), USA), c-Jun N-terminal kinase (JNK; CST (Cell Signaling Technology), USA), p-JNK (CST (Cell Signaling Technology), USA). The study included p38 (CST Biotechnology, USA), p-p38 (CST Biotechnology, USA), p-β-catenin (Thermo Fisher, USA), p-GSK3β (CST Biotechnology, USA), GSK3β (CST Biotechnology, USA), Lamin B1 (Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (BioLegend, San Diego, California, USA). GAPDH was used as a housekeeping gene for quantification, and Lamin B1 was used for quantification of β-catenin in the cell nucleus.Compared with the control group, the proteins of human bone marrow mesenchymal stem cells (hBMSCs) treated with L-30 extract were also found to be significantly higher, including ALP, RUNX2 and COL1A1 (Figures 7B and 7C).

[0109] Example 10. Confirmation of bone differentiation-related marker expression based on treatment of L-30 extract or fraction

[0110] The L-30 extract and its first fraction (peak 1) from the above-described embodiments were treated with human bone marrow mesenchymal stem cells (hBMSCs) during osteodifferentiation induction, and the expression of osteodifferentiation-promoting markers was confirmed by fluorescent immunostaining. hBMSCs were fixed with 4% paraformaldehyde and permeabilized for 10 minutes at room temperature using Triton X-100 (dissolved in phosphate-buffered saline (PBS)). After blocking with 3% BSA (dissolved in PBS) for 1 hour, the cells were treated with primary antibody (dissolved in 0.1% BSA) and cultured. The primary antibodies were runx family transcription factor 2 (Runx2; MBL International, USA) and COL1A1 (Cell Signaling Technology, CST). Secondary antibodies used were Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody and Alexa Fluor 594 (Invitrogen). Cell nuclei were stained using ProLong™ Glass Antifade Mountant with NucBlue™ Stain (Invitrogen). Staining was observed using a confocal laser scanning microscope (LSM980 with Airyscan2). Results confirmed that, compared to the control group, both RUNX2 and COL1A1 were significantly expressed when treated with L-30 extract or its peak 1 fraction. In particular, the expression of peak 1 fraction from L-30 extract was significantly higher than that from L-30 extract. Figure 8 ).

[0111] Example 11. Activation analysis of the Wnt pathway based on L-30 extract treatment

[0112] Treatment with si-β-catenin during bone differentiation induction promoted osteoblast differentiation and growth, inducing inhibition of the Wnt pathway, a signaling system crucial for bone tissue maintenance and regeneration. Simultaneous treatment with L-30 extract was performed to verify the counteracting effect of reactivation via alkaline phosphatase staining. β-catenin antisense (Bioneer, Korea) and a negative control were transfected with lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's instructions. si-β-catenin and lipofectamine were cultured separately at room temperature for 5 minutes each before being mixed. This mixture was then cultured at room temperature for 20 minutes and added to 24-well plates of human bone marrow mesenchymal stem cells (hBMSCs). hBMSCs were cultured at 37°C, 5% CO2, and 95% humidity for 24 hours before being used in bone differentiation experiments. ALP staining was performed using the TRACP & ALP double-stain Kit (Takara Bio Inc., JAPAN) according to the manufacturer's instructions. Human bone marrow mesenchymal stem cell (hBMSC) cells were treated with L-30 extract for 7 days. hBMSC cells were fixed with fixation solution for 2 minutes, washed twice with phosphate-buffered saline (PBS), and cultured with ALP staining solution for 30 minutes. After removing the ALP staining solution, the staining was observed using an inverted microscope (EVOS™ XL Core Imaging System; Thermo Scientific™, Waltham, MA, USA).

[0113] After confirming that WNT signaling is reduced due to β-catenin, thereby decreasing the efficiency of bone differentiation, it was confirmed that treatment with L-30 extract could counteract the bone differentiation reduction effect caused by the inhibition of WNT signaling. Based on the above results, it was confirmed that the bone differentiation promoting effect of L-30 extract is related to the activation of the Wnt pathway. Figure 9 ).

[0114] Example 12. Confirmation of bone differentiation induction of L-30 extract treated with p38 or Akt inhibitors

[0115] p38 or AKT was inhibited, and then L-30 extract was treated to confirm whether each pathway was restored. SB203580 (10 μM) was used as a p38 inhibitor, and AKT inhibitor-IV (10 μM) was used as an AKT inhibitor. The inhibitors were treated, and 1 μM of L-30 extract was simultaneously treated. The inhibition and restoration of each signaling pathway were verified by alkaline phosphatase staining. The results confirmed that even when the p38 or AKT pathway was inhibited, treatment with the L-30 extract of this invention could restore and maintain bone differentiation efficacy. Figure 10 ).

[0116] Example 13. Confirmation of bone regeneration effects in mouse-derived cells and tissues

[0117] After isolating and culturing cells from mice, bone differentiation was induced using the same methods and differentiation solutions as for human bone marrow mesenchymal stem cells (hBMSCs) described in the example, with L30 extract included during differentiation induction. Primary osteoblast precursors were isolated from mouse calvaria. Animal experiments were approved by the Seoul National University Animal Experimentation Use Committee (IACUC, number SNU-230803-2-2). ICR mouse calvaria from day 4 were cultured in 12-well plates. The calvaria were cultured in BMSC medium with or without 10 μg / mL of L30 extract. The medium was changed every 2 days, and the calvaria were harvested on day 7. The calvaria were fixed in 4% PFA for 24 hours and decalcified in 14% EDTA for 2 days. After decalcification, the calvaria were embedded in paraffin. Tissue blocks were cut to a depth of 800 μm and sagittally sectioned along the midline at a thickness of 10 μm (Leica Microsystems, Wetzlar, Germany). The tissue sections were stained with hematoxylin and eosin (H&E). Isolated skull sections were digested at 37°C for 30 minutes with 0.25% trypsin and 0.2% collagenase. Released cells were grown in 100 mm plates in α-MEM (Welgene Inc.) supplemented with 10% FBS (Hyclone Laboratories Inc.) and then cultured in a 37°C incubator containing 5% CO2. After 3 days, adherent cells were used as osteoblast precursors. The osteoblast precursor is used to treat L-30 extract and peak 1 fraction of the extract for osteogenic differentiation.

[0118] ALP was confirmed on day 14 of differentiation. Figure 11 A), the bone differentiation promoting effect of L-30 was verified by ARS staining on day 21 of differentiation. Figure 11 B). Furthermore, RT-PCR and Western blot confirmed increased expression of differentiation-related markers (B). Figure 11 C and 11D). Furthermore, staining results showed that bone thickness increased after 7 days of treatment with the L-30 extract, confirming that the L-30 extract of this invention promotes bone formation or regeneration (C and 11D). Figure 11 E).

[0119] Example 14. Comparison of the osteoclast differentiation inhibitory effects of various lactic acid bacteria extracts

[0120] Extracts of strains L14, L15, L28, MS4, and L-30 were obtained using the same methods as in Examples 1 and 2 to confirm their inhibitory effect on osteoclast differentiation. Monocytes derived from mouse bone marrow were isolated and cultured to differentiate into osteoclasts, and the lactic acid bacteria extracts were treated with the extracts described above. TRAP staining was used to confirm whether the treatment inhibited osteoclast differentiation. Monocytes were isolated from the femur and tibia tissues of 6-week-old female mice (C57BL6J). After removing all muscle layers around the isolated femur and tibia, the tissues were disinfected with 70% ethanol, and then the joints were cut to expose the cells. Bone marrow cells were collected by washing the interior of the bone tissue with α-MEM media (1% penicillin / streptomycin (P / S)) using a syringe. 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 inactivate the lysis buffer, add twice the volume of α-MEM medium (10% FBS, 1% P / S) and centrifuge at 2000 rpm for 8 minutes. After confirming cell clumps, remove the supernatant, resuspend in fresh α-MEM medium (10% FBS, 1% P / S), remove residues through a 70 μm filter, then treat with M-CSF to a concentration of 5 ng / ml and culture overnight. The next day, collect only floating monocytes that have not attached to the bottom for experiments. Maintain monocytes collected for osteoclast differentiation in α-MEM medium (10% FBS, 1% P / S). To differentiate monocytes into macrophages, treat with M-CSF at 30 ng / ml for 3 days, then, if cell attachment to the bottom is confirmed, treat with 30 ng / ml M-CSF and 100 ng / ml RANKL for 7 days to form osteoclasts in the multinucleated cell form.

[0121] The strain was cultured in MRS medium, and residual medium was removed by washing with phosphate-buffered saline (PBS). The extract was harvested by sonication. Protein concentration was determined by BCA assay, and based on this concentration, osteoclasts were treated with 30 ng / ml M-CSF and 100 ng / ml RANKL. The degree of differentiation of osteoclasts treated with the strain extract was confirmed by TRAP staining. For staining, cells were washed once with phosphate-buffered saline (PBS), fixed with 4% PFA for 5 minutes, and then washed again with PBS to remove residual PFA. Sodium tartrate (10% v / v) was added to the TRAP staining reagent, and the solution was aliquoted into the cells and cultured at 37°C for 45 minutes. The stained cells were washed once with PBS and observed under a microscope. The results confirmed that differentiation was inhibited only in osteoclasts treated with the extract of strain L-30 compared to extracts from other strains. Figure 12 ).

[0122] Furthermore, the inhibitory efficacy of various *Lactobacillus paracasei* extracts on osteoclast differentiation was evaluated using the same method. Extracts were isolated from *Lactobacillus paracasei* strains 13169, 13086, 3169, 3165, and L-30, respectively. After treatment with osteoclasts, the degree of differentiation was observed by TRAP staining. The results showed that only strain L-30 could inhibit osteoclast differentiation. Figure 13 This confirms that this efficacy is unique to the L-30 strain of the present invention among Lactobacillus paracasei strains.

[0123] Example 15. Confirmation of decreased osteoclast differentiation markers after treatment with L-30 extract

[0124] The changes in relevant biomarkers confirmed whether L-30 extract treatment inhibited osteoclast differentiation. Following the same method as in Example 14, after inducing differentiation from monocytes to osteoclasts, the L-30 extract of this invention was treated. The mRNA of osteoclast differentiation-related biomarkers was quantitatively analyzed using the same RT-PCR method as in Example 8. The results confirmed that, compared with the control group, the mRNA levels of TRAP, cathepsin K, and MMP-9, representative biomarkers of osteoclast differentiation, were significantly reduced. Figure 14 ).

[0125] Example 16. Confirmation of the inhibitory efficacy of L-30 extract fractions on osteoclast differentiation

[0126] The osteoclast differentiation efficacy was confirmed based on the concentration of the first fraction (peak 1) of the L-30 extract. The peak 1 fraction of the L-30 extract was administered to osteoclasts at concentrations of 0.25 μL / ml, 0.5 μL / ml, 1 μL / ml, and 2 μL / ml, respectively. Differentiation was assessed by TRAP staining using the same method as in Example 14, and the results confirmed that osteoclast differentiation was inhibited at all concentrations of the L-30 fraction. Figure 15 ).

[0127] Example 17. Analysis of changes in osteoclast-related gene expression based on treatment with L-30 extract.

[0128] Gene expression changes in osteoclasts were analyzed when treated with L-30 extract. To obtain RNA samples from osteoclasts, cells were processed at 1 x 10⁻⁶ cells / cells. 6Cells were seeded per well in 6-well plates. Osteoclasts were differentiated using the same method as described in the previous example. After treatment with L-30 extract, the cells were washed once with phosphate-buffered saline (PBS), and RNA was isolated using Trizol. Chloroform (20% v / v) was added to the Trizol solution containing lysed cells, vortexed, and then centrifuged at 12,000 rpm for 15 minutes at 4°C, collecting only the RNA fraction. The collected RNA was precipitated with isopropanol to granulate it and then dissolved in RNase-free water. Sample concentration was determined using nanodrop and RNA Screentape. For 1ST cDNA synthesis, the obtained RNA sample was added to oligo dT, dNTP, DTT, and SuperScript III reverse transcriptase, and reacted in a PCR thermocycler at 55°C for 60 minutes and then at 70°C for 15 minutes. 2nd cDNA was synthesized based on the synthesized 1ST cDNA. For synthesis, polymerase buffer, RNase H, and DNA polymerase were used, and the reaction was carried out at 16°C for 2 hours and 30 minutes. The synthesized second-order DNA was purified 2-3 times with magnetic beads and 80% ethanol to obtain the final product, and the DNA concentration was determined using a Quantus fluorometer. For the second-order DNA, a shearing enzyme was added, and fragmentation was performed at 37°C for 7 minutes, followed by fragmentation at 98°C for 10 minutes. Adapters A and B were then added, and ligation was carried out at 37°C for 1 hour. The ligated product was washed 2-3 times with magnetic beads and 80% ethanol using the method described above. The obtained samples were then attached with i5 / i7 index primers and subjected to PCR. Finally, the samples were washed 2-3 times with magnetic beads and 80% ethanol, and the supernatant was transferred to new test tubes.

[0129] The concentration and quality of the obtained RNA library samples were determined using DNA tapestaion. The samples were then mixed with 2 nM using an Illumina P2 300 cycle kit before RNA sequencing was performed. The resulting data were then analyzed.

[0130] First, principal component analysis (PCA) plots were used to appropriately separate the data from each sample (control group, L-30 extract (L30), L-30 fraction (peak), and osteoclast differentiation induction group (Di)), confirming these as suitable samples for analysis (Figure 16A). Volcano plot analysis showed that the control group and the L-30 extract or fraction comparison group (Figure 16B, left) exhibited smaller differences in gene variation compared to the osteoclast differentiation induction group and the L-30 extract or fraction comparison group (Figure 16B, right). Based on this, it was confirmed that L-30 extract or its fractions can inhibit osteoclast differentiation.

[0131] K-means clustering heatmap analysis revealed that the overall gene expression trend in the L-30 extract or fraction treatment groups was closer to that of the control group (CON_1, CON_2). This confirms that L-30 extract or fraction inhibits the differentiation process during osteoclast differentiation induction, thus showing a gene cluster that is closer to that of the control group (Figure 16C).

[0132] Furthermore, gene ontology (GO) analysis confirmed that L-30 is highly correlated with inflammatory responses, immune responses, and metabolic-related mechanisms (Figures 16D and 16E), and is most strongly correlated with osteoclast-related mechanisms, particularly with osteoclast differentiation (Figure 16F).

[0133] Example 18. Confirmation of osteoclast differentiation receptor expression based on L-30 extract treatment

[0134] When osteoclasts were treated with L-30 extract, markers associated with differentiation receptors were confirmed by RT-PCR. Following the induction of differentiation from monocytes to osteoclasts using the same method as in Example 14, L-30 extract was treated. The expression of differentiation receptors in osteoclasts was analyzed using the same RT-PCR method as in Example 8. The results showed that, compared to the control group, both RANK and OSCAR, as differentiation receptors, increased during osteoclast differentiation. Figure 17(Figure 1), but when the L-30 extract was treated, the expression of each receptor was observed to be reduced very effectively. This result indicates that the expression of osteoclast differentiation receptors is regulated at the gene level when the L-30 extract is treated.

[0135] Example 19. Confirmation of osteoclast fusion marker expression based on L-30 extract treatment

[0136] For osteoclasts to perform their normal bone resorption function, the fusion process of multiple cells is essential, and the expression of genes regulating this process, DC-STAMP and ATP6v0d2, has been confirmed. Following differentiation induction from monocytes to osteoclasts using the same method as in Example 14, L-30 extract was applied. Compared to osteoclasts (Di) not treated with L-30 extract, no large multinucleated cells were observed macroscopically. The expression of osteoclast fusion-related genes was analyzed using the same RT-PCR method as in Example 8. The results confirmed that, compared to Di, both DC-STAMP and ATP6v0d2 were significantly reduced in the L-30 extract-treated group. Therefore, it was confirmed that L-30 extract can inhibit osteoclast fusion. Figure 18 ).

[0137] Example 20. Confirmation of the expression of transcription factors regulating osteoclast differentiation based on L-30 extract treatment.

[0138] Differentiation from monocytes to osteoclasts was induced using the same method as in Example 14, followed by treatment of L-30 extract. The gene and protein expression levels of transcription factors regulating osteoclast differentiation were analyzed using the same RT-PCR and Western blot methods as in Examples 8 and 9. NFATc1, which promotes osteoclast differentiation, showed decreased expression at both the gene and protein levels upon L-30 extraction. Conversely, gene levels of C / EBPβ and MafB, transcription factors that negatively regulate NFATc1, increased, while protein expression of p-AKT and p-mTOR decreased. This confirms that L-30 treatment inhibits osteoclast differentiation by suppressing the expression of NFATc1, a major regulator of osteoclast differentiation. Figure 19 ).

[0139] Example 21. Confirmation of cytotoxicity and reactive oxygen species (ROS) expression levels based on treatment with L-30 extract.

[0140] When L-30 cells were treated with mouse-derived cells, the occurrence of cytotoxicity and reactive oxygen species (ROS) expression were confirmed. Mouse-derived RAW 264.7 macrophages were purchased from the American Type Culture Collection (ATCC) and cultured in Durbeco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Cells were cultured at 37°C in a 5% CO2 incubator and passaged at 70-80% concentration.

[0141] Raw 264.7 cells were cultured at 5.0 × 10⁻⁶. 3 Cells / wells were seeded in 96-well plates, then treated with L30 extract, and observed 24 hours later. Cytotoxicity was assessed using the same method as in Example 6, and it was confirmed that no cytotoxicity was observed even after treatment with L-30. Figure 20 A).

[0142] On the other hand, in Raw 264.7 cells, the generation of reactive oxygen species (ROS) was evaluated using a reactive oxygen species (ROS) detection kit (DCFDA, Cayman). The ROS generation was measured at 1.0 × 10⁻⁶ cells. 4 Raw 264.7 cells were aliquoted into 96-well culture plates. The following day, cells were treated with medium containing L30 extract for 24 hours. After 24 hours, reactive oxygen species (ROS) staining buffer (10 μM) was added, and cells were cultured at 37°C for 90 minutes in the dark. Fluorescence was measured using a TECAN microplate reader with an excitation wavelength of 500 nm and an emission wavelength of 550 nm. The results confirmed that treatment with L-30 extract increased the production of reactive oxygen species (ROS), and that ROS generated cytokines and activated the MAPK and NF-κB pathways, thereby contributing to macrophage activation. Therefore, treatment with the L-30 extract of this invention can enhance immune function. Figure 20 B).

[0143] Example 22. Confirmation of the cytokine production effect based on treatment with L-30 extract

[0144] The aim was to confirm the expression of COX-2 and iNOS generated from activated macrophages. Raw 264.7 cells were treated with L-30 extract, and Western blot was performed 48 hours later. iNOS was divided into groups treated for 5 hours and 24 hours, and further observed by RT-PCR. It was confirmed that treatment with L-30 extract increased the secretion of COX-2 and iNOS. Figure 21 ).

[0145] Example 23. Confirmation of activation of immune enhancement-related pathways based on L-30 treatment

[0146] When treating L-30, it was determined whether immune-enhancing mechanisms were activated. Raw 264.7 cells were treated with L-30 extract, and Western blot analysis was performed 24 hours after treatment with markers related to MAPK and NF-κB mechanisms. Activation of the NF-κB and MAPK (p38, ERK, JNK) pathways, which are major pathways involved in the innate immune response, was confirmed, thus demonstrating that L-30 treatment can activate macrophages and thereby enhance immune function. Figure 22 ).

[0147] Registration number

[0148] Name of depositary institution: Korea Center for Biological Resources (KCTC)

[0149] Registration No.: KCTC16035BP

[0150] Registration Date: September 5, 2024

[0151] sequence list

[0152] Attachment: Sequence list electronic file (C:\KipoNet\NKEditor\Data\Hlz\24P08033_sequence) listing.xml).

Claims

1. A strain of Lactobacillus paracasei L-30, characterized in that, It has bone regeneration effects and its accession number is KCTC16035BP.

2. The Lactobacillus paracasei L-30 strain according to claim 1, characterized in that, It has a 16S rRNA sequence with sequence number 1.

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

4. The Lactobacillus paracasei L-30 strain according to claim 1, characterized in that, It has the effect of inhibiting the differentiation or formation of osteoclasts.

5. The Lactobacillus paracasei L-30 strain according to claim 1, characterized in that, It has the effect of promoting immune activity.

6. A pharmaceutical composition for treating or preventing bone diseases, characterized in that, It comprises at least one selected from the group consisting of Lactobacillus paracasei L-30 strain selected from any one of claims 1 to 5, lysates of said strain, cultures of said strain, extracts of said strain, and fractions of said extract.

7. The pharmaceutical composition for treating or preventing bone diseases according to claim 6, characterized in that, 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 using protein liquid chromatography.

8. The pharmaceutical composition for treating or preventing bone diseases according to claim 6, characterized in that, The fractions of the extract range in size from 440 kDa to 600 kDa.

9. The pharmaceutical composition for treating or preventing bone diseases according to claim 6, characterized in that, The bone disease is selected from at least one of the following groups: osteoporosis, osteomalacia, osteomalacia, rheumatoid arthritis, degenerative arthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc herniation, rickets, fibrous dysplasia of bone, osteitis deformans, traumatic fractures, and stress fractures.

10. A food composition for improving bone diseases, characterized in that, It comprises at least one selected from the group consisting of Lactobacillus paracasei L-30 strain selected from any one of claims 1 to 5, lysates of said strain, cultures of said strain, extracts of said strain, and fractions of said extract.

11. The food composition for improving bone disease according to claim 10, characterized in that, 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 using protein liquid chromatography.

12. The food composition for improving bone disease according to claim 10, characterized in that, The fractions of the extract range in size from 440 kDa to 600 kDa.

13. The food composition for improving bone disease according to claim 10, characterized in that, The bone disease is selected from at least one of the following groups: osteoporosis, osteoporosis, osteomalacia, rheumatoid arthritis, degenerative arthritis, osteogenesis imperfecta, osteopenia, bone atrophy, intervertebral disc, rickets, fibrous dysplasia of bone, osteitis deformans, traumatic fracture, and stress fracture.

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