Composition for promotng bone formation with fermented oysters and seaweed

Abstract

The present invention relates to a composition for improving bone health, and the composition includes a fermented extract extracted from a fermented material of oysters, which is obtained by fermenting oysters, and the fermented extract may promote bone formation by suppressing the activity of osteoclasts and promoting the activity of osteoblasts. The composition contains large amounts of taurine and vitamins, and natural gamma-aminobutyric acid.

Claims

1. A composition for promoting bone formation, the composition comprising a fermented extract extracted from a fermented material, wherein the fermented material is obtained by fermenting oysters and a seaweed, the fermented material is fermented using a fermentation microbe, and the fermentation microbe is a mixture of Lactobacillus brevis BJ20 (Accession No.: KCTC 11377BP) and Saccharomyces cerevisiae MBP-27, the fermented extract promotes bone formation by suppressing the activity of osteoclasts and promoting the activity of osteoblasts, and the seaweed is algue brune.

2. The composition of claim 1, wherein the fermented extract further comprises taurine and gamma-aminobutyric acid (GABA).

3. The composition of claim 1, wherein the oysters are selected from the group consisting of raw oysters, a hydrolysate of oysters, an extracted concentrate of oysters, and a mixture thereof.

4. A pharmaceutical composition comprising a functional fermented material, comprising the composition for improving bone health according to claim 1.

5. A food composition comprising a functional fermented material, comprising the composition for improving bone health according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A, 1B, 1C and 1D are a set of results of effects of the composition for improving bone health according to an exemplary embodiment of the present invention on the differentiation of osteoclasts.

(2) FIGS. 2A, 2B and 2C are a set of results of effects of the composition for improving bone health according to an exemplary embodiment of the present invention on the expression of proteins associated with the differentiation of osteoclasts.

(3) FIGS. 3A, 3B and 3C are a set of results of effects of the composition for improving bone health according to an exemplary embodiment of the present invention on oxidative stress produced during the differentiation of osteoclasts.

(4) FIGS. 4A, 4B and 4C are a set of results of the change in expression of osteoclast-associated proteins by suppressing the production of ROS by the composition for improving bone health according to an exemplary embodiment of the present invention.

(5) FIG. 5 is a set of results of effects of the composition for improving bone health according to an exemplary embodiment of the present invention on osteoblasts (MTT assay).

(6) FIG. 6 is a set of results of effects of the composition for improving bone health according to an exemplary embodiment of the present invention on the differentiation of essential genes required for the formation of osteoblasts.

(7) FIG. 7 is a set of results of effects of the composition for improving bone health according to an exemplary embodiment of the present invention on the differentiation of essential genes required for the formation of osteoblasts.

(8) FIG. 8 is a set of results of the Alp activation of the composition for improving bone health according to an exemplary embodiment of the present invention.

(9) FIG. 9 is a set of results of the composition for improving bone health according to an exemplary embodiment of the present invention on the promotion of bone formation in the generation step of zebrafish.

(10) FIG. 10 is a set of results of the composition for improving bone health according to an exemplary embodiment of the present invention on the promotion of bone formation in the generation step of zebrafish.

(11) FIG. 11 is a set of results of 3D video images of the composition for improving bone health according to an exemplary embodiment of the present invention obtained by using micro-CT.

(12) FIG. 12 is a set of results of effects of the composition for improving bone health according to an exemplary embodiment of the present invention on bone trait variables of the tibia.

DETAILED DESCRIPTION

(13) Hereinafter, the Examples of the present invention will be described in detail such that a person skilled in the art to which the present invention pertains can easily carry out the present invention. However, the present invention can be implemented in various different forms, and is not limited to the Examples described herein.

Preparation Example 1: Preparation of Composition for Improving Bone Health

(14) 1. Preparation of Composition (FO) for Improving Bone Health Using Raw Oysters and Algue Brune

(15) Impurities and salts were removed by washing and desalting raw oysters and algue brune. Thereafter, a mixed ground material of oysters and algue brune was prepared by drying and grinding raw oysters and algue brune.

(16) After water was mixed with the mixed ground material of oysters and algue brune such that the content of the mixed ground material was 10 to 30% (v/v) based on the total weight, saprophytic bacteria were removed by sterilizing the mixture at 121° C. for 15 minutes. The mixture was cooled to 30 to 37° C. which is a temperature suitable for fermentation.

(17) As a fermentation strain, Lactobacillus brevis BJ20 (Accession No.: KCTC 11377BP) and Saccharomyces cerevisiae MBP-27 were mixed and inoculated, and the inoculated mixture was fermented at a temperature of 30° C. to 37° C. In this case, a fermentation microbe was inoculated so as to have a content of 1 to 5% (v/v) based on the total weight, and the inoculated mixture was fermented for 2 to 7 days, such that the fermentation microbe was inoculated and the fermentation by the fermentation microbe sufficiently occurred. The logarithmically proliferated fermentation microbe during the fermentation period was killed by sterilizing a fermentation solution completely fermented at a high temperature of 121° C. under pressure for 15 to 20 minutes. Only a clear and clean fermentation solution was taken by finely filtering only water-soluble materials of 0.05 to 0.1 um from the sterilized fermentation solution. In order to prepare the finely filtered fermentation solution into a powder, the finely filtered fermentation solution was dried by a method such as spray drying or freeze drying, and then pulverized by grinding.

(18) 2. Preparation of Composition for Improving Bone Health Using Hydrolysate of Oysters and Algue Brune

(19) 2-1. Process of Preparing Hydrolysate of Oysters

(20) The oysters to be used were prepared by washing with water, desalting, and grinding so as to facilitate hydrolysis by enzymes. The pre-treated oysters and water were mixed at 1:1 and hydrolyzed by adding alcalase at 0.2% (v/v) thereto while stirring the mixture at 50 to 70° C. for 20 to 60 minutes. A residue of the hydrolyzed oysters was removed by a vibration sieve of 40 to 200 mesh and a hydrolysate of oysters was collected. Only water-soluble materials were finely filtered from the collected hydrolysate of oysters using an external circulation type vacuum separation membrane having a module with a pore size of 0.05 to 0.1 um.

(21) 2-2. Preparation of Composition for Improving Bone Health

(22) Impurities and salts were removed by washing and desalting algue brune. Thereafter, a ground material of algue brune was prepared by drying and grinding algue brune, and was mixed with the hydrolysate of oysters in 2-1.

(23) After water was mixed with the mixture of the hydrolysate of oysters and algue brune such that the content of the mixture was 10 to 30% (v/v) based on the total weight, saprophytic bacteria were removed by sterilizing the resulting mixture at 121° C. for 15 minutes. The mixture was cooled to 30 to 37° C. which is a temperature suitable for fermentation.

(24) As a fermentation strain, Lactobacillus brevis BJ20 (Accession No.: KCTC 11377BP) and Saccharomyces cerevisiae MBP-27 were mixed and inoculated, and the inoculated mixture was fermented at a temperature of 30° C. to 37° C. In this case, a fermentation microbe was inoculated so as to have a content of 1 to 5% (v/v) based on the total weight, and the inoculated mixture was fermented for 2 to 7 days, such that the fermentation microbe was inoculated and the fermentation by the fermentation microbe sufficiently occurred. The logarithmically proliferated fermentation microbe during the fermentation period was killed by sterilizing a fermentation solution completely fermented at a high temperature of 121° C. under pressure for 15 to 20 minutes. Only a clear and clean fermentation solution was taken by finely filtering only water-soluble materials of 0.05 to 0.1 um from the sterilized fermentation solution. In order to prepare the finely filtered fermentation solution into a powder, the finely filtered fermentation solution was dried by a method such as spray drying or freeze drying, and then pulverized by grinding.

(25) 3. Preparation of Composition for Improving Bone Health Using Extracted Concentrate of Oysters and Algue Brune

(26) 3-1. Process of Preparing Extracted Concentrate of Oysters

(27) The oysters to be used were prepared by washing with water, desalting, and grinding so as to facilitate extraction. The pre-treated oysters were mixed with 10 to 20 times water and extracted while being stirred at 70 to 90° C. for 20 to 60 minutes. A residue of the extracted oysters was removed by a vibration sieve of 40 to 200 mesh and an extract of oysters was collected. Only water-soluble materials were finely filtered from the collected extract of oysters using an external circulation type vacuum separation membrane having a module with a pore size of 0.05 to 0.1 um. The finely filtered extract of oysters was concentrated such that the brix became 20 to 40% by reducing pressure at a temperature of 30 to 50° C.

(28) 3-2. Preparation of Composition for Improving Bone Health

(29) Impurities and salts were removed by washing and desalting algue brune. Thereafter, a ground material of algue brune was prepared by drying and grinding algue brune, and was mixed with the concentrated extract of oysters in 3-1.

(30) After water was mixed with the mixture of the hydrolysate of oysters and algue brune such that the content of the mixture was 10 to 30% (v/v) based on the total weight, saprophytic bacteria were removed by sterilizing the resulting mixture at 121° C. for 15 minutes. The mixture was cooled to 30 to 37° C. which is a temperature suitable for fermentation.

(31) As a fermentation strain, Lactobacillus brevis BJ20 (Accession No.: KCTC 11377BP) and Saccharomyces cerevisiae MBP-27 were mixed and inoculated, and the inoculated mixture was fermented at a temperature of 30° C. to 37° C. In this case, a fermentation microbe was inoculated so as to have a content of 1 to 5% (v/v) based on the total weight, and the inoculated mixture was fermented for 2 to 7 days, such that the fermentation microbe was inoculated and the fermentation by the fermentation microbe sufficiently occurred. The logarithmically proliferated fermentation microbe during the fermentation period was killed by sterilizing a fermentation solution completely fermented at a high temperature of 121° C. under pressure for 15 to 20 minutes. Only a clear and clean fermentation solution was taken by finely filtering only water-soluble materials of 0.05 to 0.1 um from the sterilized fermentation solution. In order to prepare the finely filtered fermentation solution into a powder, the finely filtered fermentation solution was dried by a method such as spray drying or freeze drying, and then pulverized by grinding.

Experimental Example 1: Suppression of Activity of Osteoclasts

(32) 1. Culture and Differentiation of Osteoclasts

(33) RAW 264.7 cells, which is a murine macrophage cell line used in the present study, were cultured in a CO.sub.2 incubator (37° C., 5% CO.sub.2) using Dulbecco's modified Eagle's media (DMEM; Gibco BRL, Gaithersburg, Md., USA) including 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin for cell culture.

(34) By using RAW 264.7 as an osteoclast progenitor cell, a culture solution in which RANKL as a differentiation factor at 100 ng/mL and the composition for improving bone health (hereinafter, referred to as FO) using raw oysters and algue brune were mixed so as to have a concentration of 0, 200, 400, and 600 μg/mL was aliquoted into the α-MEM media supplemented with 10% FBS, and RAW 264.7 was cultured for 5 days while exchanging the media every two days.

(35) 2. Measurement of Production of Osteoclasts

(36) After RAW 264.7 cells were stabilized in DMEM for 24 hours, RAW 264.7 cells were aliquoted by adding RANKL(100 ng/mL) and FO at each concentration of 0, 200, 400, and 600 μg/mL to the α-MEM. On day 5, TRAP positive cells were acknowledged as osteoclasts by staining with a TRAP solution. The number of cells containing three or more nuclei among the stained osteoclasts was used in the statistics.

(37) 3. Measurement of Actin Ring Formation

(38) After RAW 264.7 cells were differentiated into osteoclasts by treatment with RANKL (100 ng/mL), the osteoclasts were treated with FO at each concentration of 0, 200, 400, and 600 μg/mL while being cultured in α-MEM, matured osteoclasts were stained with Alexa Fluor 488-conjugated phalloidin, and then stained with DAPI for 30 minutes. Actin ring and DAPI staining was observed by a fluorescence microscope.

(39) 4. Western Blot Analysis

(40) In order to observe the signal transduction process by RANKL, RAW 264.7 cells were lysed with a lysis buffer (50 mM tris-Cl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, and protease inhibitors). A supernatant was obtained by centrifuging (14,000 rpm, 30 min) the entire lysate. After the supernatant was weighed, the supernatant was separated by SDS-PAGE and transferred to a PVDF membrane, and then allowed to react using a specific antibody, and the expression level of proteins was confirmed using an image analyzer.

(41) 5. Measurement of Production of ROS in Cells

(42) After RAW 264.7 cells were aliquoted at 5×10.sup.4 cells/well into 6-well plates and stabilized for 24 hours, RAW 264.7 cells were induced into differentiation by RANKL (100 ng/mL), treated with FO at each concentration, cultured for 5 days, and then stained at 37° C. for 30 minutes by treatment with 10 μM DCF-DA, and then measured using flow cytometry.

(43) 6. Effects of FO on the Differentiation of Osteoclasts Induced by RANKL (FIG. 1)

(44) RAW 264.7 cells were treated with RANKL to induce differentiation into osteoclasts, and the effects of FO on the differentiation and production of osteoclasts were measured by treatment with FO at various concentrations (0, 200, 400, and 600 μg/ml). As a result of observing TRAP staining of osteoclasts by a microscope, it could be confirmed that as the concentration of FO was increased, the size of TRAP positive cells was decreased (A). And, as a result of measuring the number of TRAP positive cells, it was observed that the number of TRAP positive cells was suppressed in a concentration-dependent manner in the same manner as described above (B, C). Since the actin of osteoclasts is organized as one big ring in order to divide a general extracellular space while osteoclasts perform bone formation during the bone resorption process and are attached to bones, the formation of the actin ring is an important mark for the ability of osteoclasts to resorb bones. As a result of observing the formation of the acting ring by treating differentiated osteoclasts with FO at each concentration, it was confirmed that the formation of the actin ring was suppressed in a concentration-dependent manner (D).

(45) 7. Effects of FO on Expression of Proteins Associated with Differentiation of Osteoclasts (FIG. 2)

(46) After the formation of osteoclasts was induced using RANKL (100 ng/mL) in RAW 264.7 cells, the expressions of proteins associated with differentiation by FO were compared. It could be seen that the expression of TRAF6 and c-Src which are important signal transduction factors for the signal transduction system by RANKL was decreased in a FO concentration-dependent manner, and the phosphorylation of PI3K was also regulated. And, it was confirmed that during the differentiation of osteoclasts, RANKL increased the expression of IκB-α and promoted the nuclear translocation of p65, whereas the expression was suppressed in the FO treatment group. It can be seen that FO suppresses the NF-kB pathway which is a transcription factor essential for the osteoclast differentiation process.

(47) 8. Effects of FO on Oxidative Stress Produced During the Differentiation of Osteoclasts (FIG. 3)

(48) It is known that oxidative stress in cells occurs during the differentiation of osteoclasts induced by RANKL. Therefore, the effects of FO on the production of ROS induced by RANKL was confirmed through DCF-DA staining. ROS increased in the osteoclasts treated with RANKL was remarkably decreased in the FO treatment group. It was confirmed using a microscope that this phenomenon was also similarly observed even in NAC which is an antioxidant.

(49) 9. Change in Expression of Osteoclast-Associated Proteins by Suppressing Production of ROS by FO (FIG. 4)

(50) The NADPH oxidase is one of the main factors of ROS, and among the NOXs constituent members, the expression of NOX1 involved in the production of osteoclasts and a regulatory protein Rac1 was decreased in a FO concentration-dependent manner. In order to confirm the role of NOX1 in effects of FO on the differentiation of osteoclasts, the expression of proteins associated with the differentiation of osteoclasts was analyzed by silencing NOX1. As a result, it was confirmed that TRAF6, c-Src, and p-PI3K signals induced by RANKL were decreased in the FO treatment group, and considerably suppressed in the NOX1 si RNA and FO groups. Therefore, FO may suppress the osteoclast differentiation signal transduction mechanism by inhibiting the NOX1-dependent production of ROS induced by RANKL.

Experimental Example 2: Induction of Activation of Osteoblasts

(51) 1. Culture and Distribution of Osteoblasts

(52) MC3T3-E1 cells used in the present experiment are mouse calvaria-derived osteoblasts and were purchase from the American Type Culture Collection Manassas (ATCC, VA, USA), and the α-minimum essential medium (α-MEM) medium, fetal bovine serum (FBS), penicillin-streptomycin, and the like required for the culture of cells were purchased from GIBCO (Invitrogen, Carlsbad, Calif., USA). The MC3T3-E1 cells were cultured in a CO.sub.2 incubator at 37° C. using the α-MEM medium supplemented with 10% FBS and 1% antibiotic (penicillin-streptomycin). The media were exchanged for the first time after two days, and then exchanged every four days. When the primary culture cells reached a density of 70 to 80% after 2 to 3 days of culture, the primary culture cells were sub-cultured using a 0.025% trypsin solution.

(53) 2. MTT Assay

(54) The proliferation degree of osteoblasts was measured by MTT assay, which obtains the survival rate of living cells. After 100 μl of MC3T3-E1 cells were each aliquoted at a concentration of 1×10.sup.4 cells/well into 96 well plates, and then cultured in a CO.sub.2 incubator at 37° C. for 24 hours, the medium was removed, and a new culture solution prepared by distributing FO at various conditions (0, 50, 100, and 200 μg/ml) was aliquoted into a medium which was not supplemented with FBS. After 48 hours of culture, the medium was removed, an MTT reagent (10 μl/well) at a concentration of 0.05 mg/ml was aliquoted, and then after the cells were further cultured for 4 hours, the culture solution was removed and the precipitate was dissolved in dimethyl sulfoxide (DMSO) to measure the absorbance at 570 nm using a microplate reader (Bio-rad, Benchmark, Hercules, Calif., USA). A control was cultured by putting a medium which was not supplemented with a sample, and the survival rate of cells was calculated based on the absorbance of the control.

(55) 3. Measurement of Activity of Alkaline Phosphatase of Osteoblasts

(56) MC3T3-E1 cells were inoculated at 1×10.sup.4 cells per well into 96-well culture plates and cultured for 24 hours, and cultured for 48 hours by exchanging the medium with a medium containing FO at various concentrations (0, 50, 100, and 200 μg/ml). The culture solution was removed, and the cells were lysed with 1% Triton X-100 and sonicated. After 50 μl/well of a buffer solution containing 0.4 mM tris-HCl, 2 mM MgCl.sub.2, and 4 mM p-nitrophenol phosphate (PNPP) was added thereto, the cells were allowed to react for 30 minutes, the reaction was terminated by adding 150 μl of 1 N NaOH thereto, and then the absorbance of decomposed p-nitrophenol (PNP) was measured at 405 nm using a microplate reader (Bio-rad, Benchmark, Hercules, Calif., USA). The amount of protein was measured using a bovine serum albumin protein assay reagent, and the enzyme activity was expressed as a percentage relative to the control.

(57) 4. Measurement of Promotion of Bone Formation in Zebrafish Model

(58) In order to confirm an effect of FO in vivo on the promotion of bone formation, the zebrafish fed with FO at various concentrations (0, 50, and 100 μg/ml) for 35 days were exposed to calcein and stained.

(59) Specifically, the zebrafish fed with FO at various concentrations (0, 50, and 100 μg/ml) and the zebrafish fed with no FO were exposed to calcein and stained, when the zebrafish were appropriately stained, the solution was discarded, and the zebrafish were washed with PBS, and then dried. Subsequently, images were obtained using a Nikon Eclipse Ti microscope, and the effect of FO in vivo on the promotion of bone formation was evaluated by measuring the staining intensity among the images.

(60) 5. Effects of FO on Osteoblasts (FIG. 5)

(61) In the FO treatment group, it could be confirmed that a clump was formed between cells from day 3, and MTT activation was rapidly increased from day 3. As a result of confirming the effects on the change in the actual number of total cells and the survival of cells, the survival rate of total cells was shown to be the same as that of the group which was not treated with the drug during the experimental time, but the number of total cells was noticeably reduced from day 5 (the number of total cells was reduced due to the differentiation into osteoblasts).

(62) 6. Effects of FO on Expression of Genes Essential for Formation of Osteoblasts (FIGS. 6 and 7)

(63) After a treatment in order to understand the effect of FO (100 ug/ml) on the expression of genes essential for the formation of osteoblasts, as a result of confirming the expression of genes on days 1, 3, 5, and 7 after treatment for 7 days, the expression of Alp, RUNX2, Col1a1, OSX, Bglap, BMP.sub.2, and BMP was strongly increased.

(64) 7. Alp Activation which is Core Factor for Bone Formation (FIG. 8)

(65) The synthesis of ALP, which increases the concentration of local phosphoric acid ions, was measured at a site where bone reformation and regeneration occurred by administering FO at 50 μg/ml and 100 μg/ml to MC3T3-E1 cells. On day 3, there was no significant difference between the experimental group and the control, but the ALP activity was significantly increased in the two FO treatment group from day 5. Dex was used as a positive control.

(66) 8. Promotion of Bone Formation in Generation Step of Zebrafish (FIGS. 9 and 10)

(67) After treatment with FO (50 ug/ml and 100 ug/ml) from day 3 to day 9, bone formation was confirmed in the zebrafish. About 9 days later, it was confirmed that the formation of the vertebrae was promoted by treatment with FO. It was confirmed that the area and fluorescence intensity of the vertebrae entirely produced were also increased.

Experimental Example 3: Therapeutic Effect of Osteoporosis in Ovariectomy Model

(68) 1. Manufacture of Osteoporosis Animal Model

(69) After 8-week-old female ICR mice were purchased and acclimatized for one week, the mice were divided into 5 groups of 8 animals each for experiments.

(70) The respective groups were classified into 1) a non-ovariectomy normal control (Sham) in which the skin and the muscle layer were sutured without taking out the ovaries, 2) a control (OVX) in which osteoporosis was caused by excising the ovaries, 3) an experimental group (OVX+FO 100 mg/kg) in which FO was orally administered at 100 mg/kg each daily after the excision of the ovaries, 4) an experimental group (OVX+FO 200 mg/kg) in which FO was orally administered at 200 mg/kg each daily after the excision of the ovaries, and 5) a positive control (OVX+E2) in which 17-beta-estradial (E2) was intraperitoneally administered daily after the excision of the ovaries.

(71) After the ICR mice were anaesthetized with Avertin, the hairs on the backs of the mice were removed, the skin and the muscle layer were excised with a pair of fine scissors, and then the ovaries on both sides were completely excised and sutured with sutures.

(72) 2. Micro-CT Analysis

(73) After the experimental animals were sacrificed and the tibias were taken out and fixed 4 weeks after the ovariectomy, 3-D video images were reconstructed using micro-CT (Skyscan 1272, Bruker, Kontich, Belgium), and various bone indices (BMD, BV/TV, Tb.N, Tb.Th, and Tb.Sp) were analyzed.

(74) 3. Therapeutic Effect of Osteoporosis (FIGS. 11 and 12)

(75) Through the 3-D video images obtained by Micro-CT, the bone loss was more severe in the control in which osteoporosis was caused than in the non-ovariectomy normal control, and the bone loss was suppressed in all the groups to which FO was orally administered at 100 mg/kg and 200 mg/kg.

(76) It was shown that the administration of FO had a significant improvement effect in bone density (BMD) and bone volume (BV/TV (%)). Further, it was confirmed that the trabecular number, trabecular separation, and trabecular thickness were also significantly recovered.

(77) Therefore, it is determined that FO exhibits effective effects on the improvement of osteoporosis.

Experimental Example 4: Comparison of Effects of Composition for Improving Bone Health on Improvement of Bone Health

(78) 1. Preparation of Composition (FO′) for Improving Bone Health Using Raw Oysters and Algue Brune

(79) Impurities and salts were removed by washing and desalting raw oysters and algue brune. Thereafter, a mixed ground material of oysters and algue brune was prepared by drying and grinding raw oysters and algue brune. Thereafter, a composition for improving bone health was prepared in the same manner as in Preparation Example 1, except that as fermentation strains, Lactobacillus fermentum JS and Aspergillus Usamii were used.

(80) 2. Preparation of Composition (FM1) for Improving Bone Health

(81) A composition (FM1) for improving bone health was prepared by mixing FO and FO′ at a weight ratio of 1:0.5.

(82) 3. Preparation of Composition (FM2) for Improving Bone Health

(83) A composition (FM2) for improving bone health was prepared by mixing FO and FO′ at a weight ratio of 1:1.

(84) 4. Preparation of Composition (FM3) for Improving Bone Health

(85) A composition (FM3) for improving bone health was prepared by mixing FO and FO′ at a weight ratio of 1:2.

(86) 2. Comparison of Effects of Suppressing Formation of Osteoclasts

(87) The effects of FO, FO′, FM1, FM2, and FM3 on the suppression of formation of osteoclasts were compared and evaluated. The effect of FO on the suppression of formation of osteoclasts was set to an index of 5, and degrees to which FO′, FM1, FM2, and FM3 suppressed the formation of osteoclasts were evaluated and expressed as indices.

(88) A higher index means that the effect is excellent.

(89) TABLE-US-00001 TABLE 1 Change in expression of Expression of proteins proteins associated with Suppression of associated with osteoclasts osteoclast differentiation Oxidative by suppression differentiation of osteoclasts stress of ROS production FO 5 5 5 5 FO′ 6 7 7 7 FM1 3 4 3 3 FM2 8 8 8 7 FM3 5 5 4 5

(90) (Unit index) According to Table 1, it was confirmed that according to the difference in fermentation strains, the effect of FO′ on the suppression of the formation of osteoclasts was better than that of FO on the suppression of the formation of osteoclasts.

(91) More specifically, after the treatment with FO and FO′ in the same concentration range, the formation of an actin ring was observed, but it was confirmed that the formation of the actin ring was suppressed in FO′ as compared to in FO, so that it was confirmed based on the result that the differentiation of osteoclasts was suppressed.

(92) The expression of TRAF6 and c-Src as signal transduction factors was decreased in FO′ as compared to in FO, and the phosphorylation of PI3K was regulated. Further, it was confirmed that the effect of FO′ on the suppression of the NF-kB pathway was better than that of FO on the suppression of the NF-kB pathway.

(93) It was confirmed that ROS increased in the RANKL-treated osteoclasts was remarkably decreased in FO′ as compared to in FO and the TRAF6, c-Src, and p-PI3K signals induced by RANKL were further decreased in the FO′ treatment group, so that in summary, the effect of FO′ on the suppression of formation of osteoclasts is better than that of FO.

(94) It was confirmed that FM 1 to FM 3 in which FO and FO′ were mixed exhibited the difference in effect of suppressing the formation of osteoclasts according to the mixing ratio of FO and FO′, and among them, FM2 exhibited the best effect.

(95) 3. Comparison of Effects of Inducing Activation of Osteoblasts

(96) Effects of FO and FO′ on the induction of activation of osteoblasts were compared and evaluated. The effect of FO on the induction of activation of osteoblasts was set to an index of 5, and degrees to which FO′, FM1, FM2, and FM3 induced the activation of osteoblasts were evaluated and expressed as indices.

(97) A higher index means that the effect is excellent.

(98) TABLE-US-00002 TABLE 2 Expression of essential genes (expression of Alp, Promotion of RUNX2, Col1a1, bone formation Number of OSX, Bglap, BMP2, Alp (generation step total cells and BMP) activation of zebrafish) FO 5 5 5 5 FO′ 7 7 7 7 FM1 4 4 4 3 FM2 8 8 8 7 FM3 5 5 5 5

(99) (Unit index) According to Table 2, it was confirmed that according to the difference in fermentation strains, the effect of FO′ on the induction of the activation of osteoblasts was better than that of FO on the induction of the activation of osteoblasts.

(100) More specifically, it was confirmed through experiments that due to the differentiation of osteoblasts, a decrease in number of total cells was marked in FO′ as compared to in FO, and the expression of essential genes was also increased in FO′ as compared to in FO.

(101) It was confirmed that the Alp activity, which is a core factor for bone formation, was also increased in FO′ as compared to in FO, and the area and fluorescence intensity of the vertebrae in the generation step of zebrafish were also increased considerably.

(102) It was confirmed that FM 1 to FM 3 in which FO and FO′ were mixed exhibited the difference in effect of inducing the activation of osteoblasts according to the mixing ratio of FO and FO′, and among them, FM2 exhibited the best effect.

(103) Although preferred Examples of the present invention have been described in detail hereinabove, the right scope of the present invention is not limited thereto, and it should be understood that many variations and modifications of those skilled in the art using the basic concept of the present invention, which is defined in the following claims, will also fall within the right scope of the present invention.