KR20220093719A - Method for preparing soybean extract with bone metabilic function improvement - Google Patents

Abstract

The present invention relates to a method for preparing a soybean extract having an effect of improving bone metabolic function and a food composition for improving bone metabolic function comprising the soybean extract as an active ingredient. The present invention relates to a method for preparing a soybean extract, which comprises the step of sequentially treating a heated and pulverized soybean aqueous solution with a carbohydrate decomposing enzyme and a proteolytic enzyme, followed by fermentation using a microorganism.

Description

Method for producing soybean extract having the effect of improving bone metabolism {METHOD FOR PREPARING SOYBEAN EXTRACT WITH BONE METABILIC FUNCTION IMPROVEMENT}

The present invention relates to a method for producing a soybean extract having an effect of improving bone metabolism, and to a food composition for improving bone metabolism comprising the soybean extract as an active ingredient.

Soybean ( Glycine max ) is an annual grass widely distributed in Asia, Africa, and Australia, and is an edible crop. There are several varieties of soybeans, and depending on the use, they are divided into mature fruiting use, cheongye feed, and green bean. Soybeans have been used as a raw material for traditional foods such as tofu, soy sauce, and soybean paste. Soybeans contain 35-40% protein, 15-20% fat and 20-25% sugar. Soybeans contain essential amino acids and unsaturated fatty acids, and contain physiologically active substances such as dietary fiber, lecithin, sapomin, phytic acid, and isoflavones.

Soy isoflavones exist in the form of glycosides, which are converted into metabolites such as free genistein and daidzein by gastric acid and intestinal microbes and absorbed in the intestine. Therefore, it can be said that eating fermented foods is a more effective intake of isoflavones.

Numerous studies have been published on how to extract non-glycolysate isoflavones from soybeans to facilitate the absorption of isoflavones in the body.

Korean Patent No. 10-1302376 discloses a method for producing soybean powder with an increased content of isoflavones by treating soybeans with far-infrared rays.

Korean Patent No. 10-188152 discloses a method for preparing soybean isoflavone preparations by preparing whole soymilk fortified with non-glycoside isoflavones, high-speed centrifugation, ethanol extraction, filter filtration, concentration, mixing, and freeze-drying steps. start

The present invention increases the total polyphenol content, genistein, daidzein, isoflavone (including aglycone and glycoside) content increases, bone metabolism enhancing effect and An object of the present invention is to provide a method for preparing a soybean extract having an osteoclast activity inhibitory effect. In addition, an object of the present invention is to provide a food composition for improving bone metabolism function, comprising as an active ingredient a soybean extract having an increased content of total polyphenols and active ingredients such as genistein and daidzein.

The above-described task comprises the steps of heating soybeans immersed in purified water for 1 to 2 days at 100° C. for 10 to 30 minutes and then cooling; pulverizing the cooled soybeans and preparing an aqueous solution having a solid content of 5 to 10% by weight; preparing a primary hydrolyzate by adding 1 to 10% by weight of a carbohydrate-degrading enzyme to the aqueous solution based on the solids weight and treating it for 1 to 3 hours; Preparing a secondary hydrolyzate by adding 1 to 10% by weight of a protease to the primary hydrolyzate based on the solids weight and treating for 5 to 7 hours: and Aspergillus oryzae ( Aspergillus oryzae ) or Aspergillus kawachii ( Aspergillus kawachii ) is achieved by a method for producing a soybean extract, comprising the step of inoculation and fermentation.

Preferably, the carbohydrate-degrading enzyme may be at least one selected from the group consisting of AMG, thermamyl, dextrozyme, biscozyme, and cellulose clast.

Also preferably, the protease may be at least one selected from the group consisting of alkalase, flavorzyme and protamex.

Another object of the present invention is to provide a food composition for improving bone metabolism, comprising the soybean extract prepared by the above method as an active ingredient, promoting osteoblast activity of MC3T3-E1 cells, which are osteoblasts, and inhibiting osteoclast activity. is achieved

According to the method for producing a soybean extract according to the present invention, the total polyphenol content and the content of active ingredients such as genistein and daidzein are increased, and a soybean extract having an effect of enhancing bone metabolism can be prepared. can

1 is a graph showing the results of measuring total polyphenol and crude protein content according to complex enzyme treatment of soybeans.
2 is a graph showing the result of measuring the total polyphenol content of fermented soybeans.
3 is a graph showing the result of measuring the content of isoflavones (non-glycosides) in fermented soybeans.
4 is a photograph showing the effect of the soybean extract (SBE) according to the present invention on the actin ring formation of osteoclasts.

All technical terms used in the present invention, unless otherwise defined, have the following definitions and are consistent with the meanings commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention.

The term "about" means 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length varying by 4, 3, 2 or 1%.

Throughout this specification, unless the context requires otherwise, the terms "comprises" and "comprising" include, but are not limited to, a given step or element, or group of steps or elements, but any other step or element, or It should be understood to imply that a step or group of elements is not excluded.

The present invention relates to a method for producing a soybean extract having an increased content of total flavonoids and isoflavones and an effect of improving bone metabolism. The method comprises the steps of heating and cooling the soybeans immersed in purified water for a certain time (S11); pulverizing the cooled soybeans and preparing an aqueous solution having a solid content of 5 to 10% by weight (12); preparing a primary hydrolyzate by adding 1 to 10% by weight of a carbohydrate-degrading enzyme to the aqueous solution based on the solids weight (S13); Preparing a secondary hydrolyzate by adding 1 to 10% by weight of a protease to the primary hydrolyzate based on the solids weight (S14): and Aspergillus oryzae in the secondary hydrolyzate ) or Aspergillus kawachii ( Aspergillus kawachii ) and inoculated and fermented (S15).

Hereinafter, each step will be described in detail.

First, the soybeans immersed in purified water for a certain time are heated and then cooled (S11). Specifically, the washed soybeans are immersed in purified water for 1 to 2 days, heated at 100° C. for 10 to 30 minutes, and then cooled. In this case, purified water may be used in an amount of 10 to 100 times the weight of soybeans, and more preferably purified water of 100 times the weight of soybeans may be used.

Then, the cooled soybeans are pulverized using a high-speed mixer or the like, and purified water is added so that the solid content is 5 to 10% by weight to prepare an aqueous soybean pulverization solution (S12).

1 to 10% by weight of a carbohydrate-degrading enzyme is added to the aqueous solution based on the weight of the solid content and treated for 1 to 3 hours to prepare a primary hydrolyzate (S13).

The carbohydrate-degrading enzyme may be at least one selected from the group consisting of AMG, thermamyl, dextrozyme, biscozyme, and cellulose clast manufactured by Novozyme. More preferably, it may be AMG. The treatment time of the carbohydrate-degrading enzyme is preferably 1 to 3 hours, and more preferably 2 hours.

Then, 1 to 10% by weight of a protease is added to the primary hydrolyzate based on the solids weight and treated for 5 to 7 hours to prepare a secondary hydrolyzate (S14),

The proteolytic enzyme may be at least one selected from the group consisting of alkalase, flavorzyme, and protamex. More preferably, it may be an alkalase. The treatment time of the proteinase is preferably 5 to 7 hours, more preferably 6 hours. The secondary hydrolyzate can be treated at 100° C. for 20 minutes before the fermentation process to inactivate the enzyme.

Then, the secondary hydrolyzate is inoculated with Aspergillus oryzae or Aspergillus kawachii and fermented (S15). More preferably, Aspergillus oryzae ( Aspergillus oryzae ) may be used, and may be fermented at 25-35° C. for 12 hours to 24 hours. When fermentation is complete, it can be sterilized at 100° C. for 20 minutes, filtered, and subjected to concentration and freeze-drying processes.

In addition, the present invention provides a food composition for improving bone metabolism, comprising the soybean extract prepared by the above method as an active ingredient, promoting osteoblast activity of MC3T3-E1 osteoblasts, and inhibiting osteoclast activity.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited by these Examples.

Example 1 : Preparation of soybean enzyme-treated product and analysis of active ingredients

Soybeans were immersed in purified water in a 1% (w/w) state for 23 hours, and then an aqueous solution of the pulverized solution was heated (sterilized) at 100° C. for 30 minutes, cooled to room temperature, and used as a control and sample. The enzymes used were Nozozyme's AMG, Cellulclast, Dextrozyme, Termamyll, Viscozyme, Alcalase. It is Bromelain. Enzyme treatment conditions are shown in Table 1 below.

Sample name enzyme addition amount hour note T1 Thermamyl ▶ Alcalase + Bromelain enzyme star
One% 3 hours ▶ 3 hours : Total 6 hours ▶:
further processing

+:
Complex processing T2 3 hours ▶ 5 hours : Total 8 hours C1 Cellullast ▶
Alcalase + Bromelain 3 hours ▶ 3 hours : Total 6 hours C2 3 hours ▶ 5 hours : Total 8 hours V1 Biscozyme▶
Alcalase + Bromelain 3 hours ▶ 3 hours : Total 6 hours V2 3 hours ▶ 5 hours : Total 8 hours D1 Dextrozyme▶
Alcalase + Bromelain 3 hours ▶ 3 hours : Total 6 hours D2 3 hours ▶ 5 hours : Total 8 hours AMG1 AMG ▶ Alkaline + Bromelain 3 hours ▶ 3 hours : Total 6 hours AMG2 3 hours ▶ 5 hours : Total 8 hours

After the cooled sample was adjusted to the optimum pH of the enzyme, 0.5% by weight of the enzyme was added based on the weight of the raw material, and then hydrolysis was performed in a shaker incubator. Total polyphenol and crude protein content of each treatment group were analyzed.

The total polyphenol content was measured by mixing 20uL of the prepared aqueous solution and 80uL of 7.5% sodium carbonate solution for each enzyme amount and time, 100uL of Folin-ciocalteu's phenol solution was added, reacted for 30 minutes, and absorbance was measured at 725nm. Gallic acid was used as a standard solution, and the result was shown as the gallic acid content in the sample.

To determine the crude protein content, add 2 g of potassium sulfate and 12 mL of concentrated sulfuric acid as a decomposition accelerator to 1 g of the sample solution, carbonize the sample solution in a Kjeldahl decomposition device from light off-white to colorless, and distill, neutralize and It was added dropwise to measure the crude protein content. The analysis results are shown in FIG. 1 .

Referring to Figure 1, after treatment with AMG, the total polyphenol and crude protein content was the highest in the treatment group treated with alkalinease and bromelain. Since isoflavones are a type of protein, the higher the crude protein content, the higher the content of isoflavones. As a result of the total polyphenol content, which can be an indicator of antioxidant substances, AMG was judged to be suitable.

As a result of the repeated experiment, it was finally determined that Alcalase and AMG were the most suitable enzymes in terms of price and efficiency.

Example 2 : Preparation of fermented soybean extract and analysis of active ingredients

It was made into a 10% (W/W) aqueous solution of the enzymatic hydrolyzate prepared by utilizing the two enzymes established in Example 1, sterilized at 100° C. for 20 minutes, cooled, and used as a control and sample. The cooled sample was inoculated with Aspergillus kawachii and Aspergillus oryzae and fermented for 12 hours at an appropriate temperature for each bacteria. The fermented treated group was sterilized at 100° C. for 20 minutes and filtered through filter paper to analyze the content of total polyphenols and isoflavones (non-glycoside) in each treatment group.

The content of isoflavones (non-glycosides) was measured by the following method. First, 70% ethanol was added 20 times to the prepared aqueous solution for each enzyme amount and time compared to the sample solution, followed by reflux extraction and filtration under reduced pressure. The filtered solution was concentrated to evaporate ethanol, and the concentrate was diluted 10-fold, filtered through a 0.45um Syringe Filter, put in a vial, and then the content of isoflavones was quantified using an HPLC instrument and the conditions shown in Table 2 below. The results are shown in FIGS. 2 and 3 .

Instrument HPLC - Agilent A1100 Column YMC triart C18 Detector UV at 254nm mobile phase A: Acetonitril 10: Water 90: Formic acid 0.1
B: Acetonitril 60: Water 40: Formic acid 0.1 flow rate 0.4ml/min Injection volume 2 μl Column temp. 30℃

Referring to FIG. 2 , the total polyphenol content of the soybean fermented product according to the fermentation strain was 340.9 ppm in the control group, 505.7 ppm in the Aspergillus oryzae fermented group, and 506.6 ppm in the kawachii ferment group, confirming the effect of increasing the total polyphenols by fermentation. No difference was found.

Referring to FIG. 3 , the total isoflavone content based on the non-glycoside of the soybean fermented product according to the fermentation strain was detected at 28.2 ppm in the control group, 68.7 ppm in the oryzae fermenter, and 11.9 ppm in the kawachii fermenter. Gu was found to be the most effective in increasing isoflavones.

Example 3: Preparation of Soybean Extract

Soybeans were immersed in purified water for one day (soybeans were submerged), and then the soybeans were heated (sterilized) at 100° C. for 20 minutes and cooled to room temperature. The cooled soybeans were pulverized using a high-speed mixer, and purified water was added so that the solid content was 10% by weight. Then, 6% by weight of carbohydrate degrading enzyme (AMG, Novozyme) was added based on the solid weight and hydrolyzed for 2.17 hours. Then, 9% by weight of a proteolytic enzyme (Alcalase, Novozyme) was added based on the solid weight and hydrolyzed for 6 hours. The hydrolyzate was inoculated with Aspergillus oryzae (1 cyrovial (10 ⁸ /g) and fermented at 30° C. for 18 hours. Then, after sterilization at 100° C. for 20 minutes, filtration (Advantec No. 20), concentrated and freeze-dried to prepare soybean extract (SBE).

Experimental Example 1: Evaluation of osteoblast activity

In the bone formation stage, osteoblasts differentiate through the processes of proliferation of bone progenitors, maturation of extracellular matrix, and induction of calcification. there is formation Among them, ALP and collagen are highly expressed at the end of cell proliferation and extracellular matrix formation, so they are used as early differentiation markers of osteoblasts. used

In this experiment, in order to evaluate the bone health improvement effect of the soybean extract prepared in Example 3, the soybean extract was used as an early differentiation marker of ALP activity, collagen formation, and late differentiation marker of osteoblasts (MC3T3-E1 cells). The effect on phosphorus calcified nodule formation was evaluated.

MC3T3-E1 osteoblastic cells derived from mouse bone were purchased from the American Type Culture Collection (ATCC) and used. MC3T3-E1 cells were prepared using a cell culture medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin in α-minimum essential medium (α-MEM, Gibco-Invitrogen) at 37°C. Incubated in a humidified CO ₂ incubator (5% CO ₂ /95% air). When the cells were filled to about 80% of the culture dish, the monolayer of the cells was washed with phosphate-buffered saline (PBS, pH 7.4) and subcultured with 0.25% trypsin-2.65 mM EDTA, and the medium was changed every 2 days.

When MC3T3-E1 cells are about 90% full of the culture dish, 10 mM β-glycerophosphate (Sigma-Aldrich Co.) and 50 μg/mL ascorbic acid (Sigma-Aldrich Co.) were added to α-MEM medium to induce differentiation into osteoblasts. .) was added to the cell culture medium by exchanging the cell culture medium, and the osteoblast differentiation culture medium was exchanged every 3 days.

(1) Effect of soybean extract on cell viability of MC3T3-E1 cells

Since the decrease in cell proliferation due to the toxicity of the soybean extract affects the differentiation and activity of osteoblasts, in order to exclude this, the treatment concentration of the soybean extract that does not affect the proliferation of MC3T3-E1 cells was set. MC3T3-E1 cells were seeded in a 24-well plate at 5 × 10 ⁴ cells/well and cultured for 24 hours at various concentrations (0, 5, 10, 25, 50, 100, 200, 400, 600, 800, 1000 μg). /mL) of soybean extract (Example 3) was treated with a cell culture medium and cultured for 72 hours, followed by MTT assay. The MTT assay is performed by exchanging the cell culture medium with a 1 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Amresco) solution, culturing for 2 hours, and then removing the cells from the living cells. The formed formazan was eluted with isopropanol, and the cell proliferation ability was measured by measuring the absorbance at 570 nm using a SpectraMaxM2 Microplate reader (Molecular Devices). The viability of MC3T3-E1 cells was expressed as a percentage of the control group (0 μg/mL) not treated with the test substance. The results are shown in Table 3. As shown in Table 3, when the soybean extract was treated at a concentration of 800 μg/mL or higher, the viability of MC3T3-E1 cells was significantly reduced. Thereafter, the test was performed by setting the treatment concentration of the test substance to 0, 100, 200, and 400 μg/mL.

Soybean Extract
(Example 3) Cell viability after 72 hours treatment
(% of control) 0 μg/mL 100 ± 2.1 5 μg/mL 98.2 ± 2.1 10 μg/mL 97.9 ± 2.1 25 μg/mL 98.2 ± 0.9 50 μg/mL 98.2 ± 2.7 100 μg/mL 97.7 ± 0.2 200 μg/mL 95.2 ± 2.4 400 μg/mL 95.3 ± 1.6 600 μg/mL 95.3 ± 1.6 800 μg/mL 94.3 ± 0.6 ^* 1,000 μg/mL 92.7 ± 0.5 ^*

Values are expressed as mean ± SEM.

* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of 0 μg/mL group.

(2) Measurement of the effect of soybean extract on ALP (Alkaline phosphatase) activity

To evaluate ALP activity, MC3T3-E1 cells were aliquoted in a 96-well plate to 1 × 10 ⁴ cells/well, and stabilized for 24 hours. The cell culture medium was exchanged with an osteoblast differentiation culture medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 5 days. ALP activity of cells induced to differentiate for 5 days was measured using the TRACP & ALP assay kit (Takara Bio Inc.) according to the method suggested by the manufacturer. The results are shown in Table 4.

As shown in Table 4 When differentiation of MC3T3-E1 cells was induced, ALP activity was significantly increased at 21.3 ± 0.6 U/L, compared to 0.8 ± 0.1 U/L when undifferentiated. When the soybean extract was treated at concentrations of 100, 200, and 400 μg/mL, the ALP activity was 22.1 ± 0.8 U/L, 27.4 ± 2.6 U/L, and 31.0 ± 2.0 U/L, respectively, compared to the osteoblastic differentiation control group (G1). Thus, from the concentration of 200 μg/mL, the ALP activity significantly increased as the concentration increased. When estradiol, a positive control, was treated with 100 nM, ALP activity was 2.53 ± 0.3 U/L, which significantly increased ALP activity compared to the osteoblast differentiation control group (G1).

Differentiation Soybean Extract ALP activity (U/L) G0 - 0 μg/mL 0.8 ± 0.1 G1 + 0 μg/mL 21.3 ± 0.6 ^*** G2 + 100 μg/mL 22.1 ± 0.8 G3 + 200 μg/mL 27.4 ± 2.6 ^† G4 + 400 μg/mL 31.0 ± 2.0 ^†† G5 + Estradiol (100 nM) 25.3 ± 0.3 ^††

Values are expressed as mean ± SEM.

* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.

(3) Collagen synthesis measurement (Sirius red staining)

Collagen is an important protein distributed in each connective tissue of the human body. Type 1 collagen is abundantly present in the skin, bone, and ligaments, is synthesized mainly by osteoblasts, and accounts for 85-90% of the total bone protein. The degree of collagen synthesis was measured by staining the cells using Sirius Red, an anionic dye that strongly binds to collagen molecules. MC3T3-E1 cells were aliquoted in a 24-well plate so as to become 5 × 10 ⁴ cells/well, and then stabilized for 24 hours. The cell culture medium was exchanged with an osteoblast differentiation culture medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 8 days. Collagen synthesis of cells induced for differentiation for 8 days was measured using a Sirius red collagen detection kit (Chondrex) according to the method suggested by the manufacturer. Table 5 shows the effect of soybean extract on collagen synthesis in osteoblasts.

As shown in Table 5, when differentiation of MC3T3-E1 cells was induced (G1), the synthesized collagen was increased 5.63 times compared to 82.8 ± 8.0 μg/mL of (G0) when undifferentiated at 466.5 ± 20.6 μg/mL (G0). . As for the collagen synthesis increased by differentiation induction, from the concentration of 200 μg/mL by the treatment of soybean extract, the collagen synthesis increased significantly as the treatment concentration increased, and when treated with 400 μg/mL (G4) 546.4 ± 16.6 At μg/mL, collagen synthesis was increased by 17.1% compared to the osteoblast differentiation control group (G1). When estradiol, a positive control, was treated with 100 nM (G5), it was increased by 12.9% to 526.9 ± 5.1 μg/mL.

Differentiation Soybean Extract Collagen (μg/mL) G0 - 0 μg/mL 82.8 ± 8.0 G1 + 0 μg/mL 466.5 ± 20.6 ^*** G2 + 100 μg/mL 517.2 ± 3.0 G3 + 200 μg/mL 542.2 ± 3.4 ^† G4 + 400 μg/mL 546.4 ± 16.6 ^† G5 + Estradiol (100 nM) 526.9 ± 5.1 ^†

Values are expressed as mean ± SEM.

* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.

(4) Mineralization measurement (Alizarin red staining)

The degree of mineralization of osteoblasts was investigated by measuring the calcium content deposited in osteoblasts using Alizarin Red S, an anthraquinonoid derivative. Alizarin stains the mineralized cell matrix, so the amount of calcification and the degree of staining are proportional to each other. Therefore, Alizarin red staining was used to investigate mineral deposition following treatment with soybean extract. MC3T3-E1 cells were aliquoted in a 24-well plate so as to become 5 × 10 ⁴ cells/well, and then stabilized for 24 hours. The cell culture medium was exchanged with an osteoblast differentiation medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 18 days. Cell mineral deposition was measured using the Osteogenesis assay kit (Millipore) according to the method suggested by the manufacturer. The results are shown in Table 6. As shown in Table 6, mineral deposition increased by induction of osteoblastic differentiation was 39.79 ± 1.45 µM, 42.50 ± 2.45 µM, and 65.64 ± 5.97 µM at the treatment concentrations of SEB 100, 200, and 400 µg/mL, respectively, in the osteoblastic differentiation control group (G1, 37.64 ± 1.78 μM), but only significantly increased mineralization at a treatment concentration of 400 μg/mL.

Differentiation Soybean Extract Mineralization (μM) G0 - 0 μg/mL 4.61 ± 1.63 G1 + 0 μg/mL 37.64 ± 1.78 ^*** G2 + 100 μg/mL 39.79 ± 1.45 G3 + 200 μg/mL 42.50 ± 2.45 G4 + 400 μg/mL 65.64 ± 5.97 ^†† G5 + Estradiol (100 nM) 56.07 ± 2.01 ^†††

Values are expressed as mean ± SEM.

* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.

(5) Osteocalcin (OCN) content measurement

OCN is mainly produced by osteoblasts and is a matrix protein known as a specific marker of active osteoblasts. OCN is synthesized in osteoblasts as a biochemical marker of bone formation. MC3T3-E1 cells were aliquoted in a 24-well plate so as to become 5 × 10 ⁴ cells/well, and then stabilized for 24 hours. The cell culture medium was exchanged with an osteoblast differentiation medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 18 days. Cell cultures were collected in which cells were conditioned for 24 hours. The level of osteoblast-secreted osteocalcin (OCN) in the cell culture medium conditioned by the cells was measured using the OCN ELISA Kit (Takara Bio Inc.) according to the manufacturer's method. The results are shown in Table 7. Referring to Table 7, the OCN content of the osteoblastic differentiation control group (G1) was 0.804 ± 0.044 ng/mL, and when SBE was treated at a concentration of 100, 200, and 400 μg/mL, 1.218 ± 0.068 ng/mL and 1.361 ± 0.092 ng, respectively. OCN production was significantly increased from a concentration of 100 μg/mL to /mL, 1.830 ± 0.158 ng/mL.

Differentiation Soybean Extract Osteocalcin (ng/mL) G0 - 0 μg/mL 0.321 ± 0.016 G1 + 0 μg/mL 0.804 ± 0.044 ^*** G2 + 100 μg/mL 1.218 ± 0.068 ^†† G3 + 200 μg/mL 1.361 ± 0.092 ^†† G4 + 400 μg/mL 1.830 ± 0.158 ^††† G5 + Estradiol
(100 nM) 1.438 ± 0.036 ^†

Values are expressed as mean ± SEM.

* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.

Through this experiment, it was confirmed that the soybean extract according to the present invention significantly increased ALP activity, collagen synthesis, mineralization deposition, and osteocalcin production, which are indicators of osteoblast differentiation, at a concentration that was not toxic to MC3T3-E1 cell proliferation. .

Experimental Example 2: Evaluation of osteoclast activity

In order to evaluate the bone health improvement effect of the soybean extract prepared in Example 3, the effect of the soybean extract on osteoclast activity was measured.

RAW 264.7 cells, which are macrophages derived from mice, were purchased from the American Type Culture Collection (ATCC) and used. RAW 264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Welgene) in a cell culture medium supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin in a 37°C humidified CO ₂ incubator (5% CO ₂ / 95% air). When the cells were about 80% full of the culture dish, the cell monolayer was washed with phosphate buffer saline (PBS, pH 7.4), the cell culture solution was added, the cells were removed and subcultured, and the medium was exchanged every 2 days.

In order to induce the differentiation of RAW 264.7 cells into osteoclasts, the cell culture medium was exchanged with the osteoclast differentiation medium containing 50 ng/mL RANKL (Sigma-Aldrich Co.) and 10 μM PD98059 (Santa Cruz) added to α-MEM. was cultured, and the osteoclast differentiation culture medium was exchanged every 3 days. The osteoclasts thus obtained were used in the experiments below.

(1) Effect of soybean extract on cell viability of RAW 264.7 cells

The cell viability of RAW 264.7 cells was measured by the MTT assay method.

RAW 264.7 cells were seeded at 5 × 10 ⁴ cells/well in a 24-well plate and cultured for 24 hours at various concentrations (0, 5, 10, 25, 50, 100, 200, 400, 600, 800, 1,000 μg/ mL) of soybean extract (Example 3) was treated with a cell culture medium and cultured for 72 hours, followed by MTT assay. MTT assay is performed by exchanging the cell culture medium with 1 mg/mL MTT (Amresco) solution and eluting formazan formed from living cells with isopropanol after culturing the cells for 2 hours at 570 nm using a SpectraMaxM2 Microplate reader (Molecular Devices). By measuring the absorbance, the proliferative capacity of the cells was measured. The viability of RAW 264.7 cells was expressed as a percentage relative to the control (0 μg/mL) not treated with the test substance. The results are shown in Table 8. As shown in Table 8, the treatment with the test substance did not show a significant difference in the viability of RAW264.7 cells, indicating that there is no cytotoxicity. Thereafter, the test was performed by setting the treatment concentration of the test substance to 0, 100, 200, and 400 μg/mL.

Soybean Extract Cell viability after 72 hours treatment
(% of control) 0 μg/mL 100.0 ± 6.2 5 μg/mL 99.8 ± 4.7 10 μg/mL 98.7 ± 4.9 25 μg/mL 99.1 ± 3.5 50 μg/mL 98.0 ± 3.9 100 μg/mL 96.6 ± 3.7 200 μg/mL 94.5 ± 4.7 400 μg/mL 90.8 ± 6.2 600 μg/mL 90.9 ± 4.3 800 μg/mL 85.2 ± 3.7 1,000 μg/mL 85.6 ± 3.8

Values are expressed as mean ± SEM.

* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.

(2) Effect of soybean extract (SBE) on TRAP activity

Since TRAP is a specific marker of osteoclasts and its secretion increases during bone resorption, the degree of osteoclast differentiation is confirmed by measuring the activity of TRAP. To evaluate TRAP activity, RAW 264.7 cells were aliquoted in a 96-well plate to 5 × 10 ³ cells/well, and stabilized for 24 hours. The cell culture medium was exchanged with the osteoclast differentiation medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 5 days. After inducing differentiation for 5 days, TRAP activity of cells was measured using the TRACP & ALP assay kit (Takara Bio Inc.) according to the method suggested by the manufacturer. The results are shown in Table 9. As shown in Table 9 When RAW 264.7 cells were induced to differentiate into osteoclasts, TRAP activity was significantly increased at 6.71 ± 0.10 U/L, compared with 2.47 ± 0.02 U/L when undifferentiated. The increased TRAP activity was 5.69 ± 0.06 U/L, 5.64 ± 0.13 U/L, and 4.36 ± 0.04 U/L when SBE was treated at a concentration of 100, 200, and 400 μg/mL, respectively, in the osteoclast differentiation control group (G1, 0 μg/mL), the TRAP activity was significantly decreased as the concentration increased from the concentration of 100 μg/mL.

Differentiation Soybean Extract TRAP activity (U/L) G0 - 0 μg/mL 2.47 ± 0.02 G1 + 0 μg/mL 6.71 ± 0.10 ^*** G2 + 100 μg/mL 5.69 ± 0.06 ^††† G3 + 200 μg/mL 5.64 ± 0.13 ^††† G4 + 400 μg/mL 4.36 ± 0.04 ^††† G5 + Estradiol (100 nM) 5.18 ± 0.1 ^†††

Values are expressed as mean ± SEM.

* p < 0.05, ** p < 0.01, *** p < 0.001 significantly different from that of G0; † p < 0.05, †† p < 0.01, ††† p < 0.001 significantly different from that of G1.

(3) Effect of soybean extract (SBE) on actin ring formation of osteoclasts

The actin ring structure on the bone cell surface is essential, and through this, the bone matrix is absorbed. When osteoclasts differentiate, their size becomes huge and the surface forms an actin ring, which is involved in the bone resorption process. Therefore, the actin ring formation of osteoclasts is an important indicator of the cell's ability to resorb bone. RAW 264.7 cells were aliquoted in a 24-well plate with a cover glass so that the concentration was 1 × 10 ⁴ cells/well, and the cells were stabilized for 24 hours. The cell culture medium was exchanged with the osteoclast differentiation medium containing the soybean extract of Example 3 at various concentrations (0, 100, 200, 400 μg/mL) and cultured for 5 days. After removing the medium and washing with PBS, the cells were fixed by treatment with 4% paraformaldehyde and 0.1% Triton X-100. The fixed cells were stained with Alexa Fluor 594 Phalloidin and DAPI to stain actin and nuclei, respectively. The stained cells were observed for morphological changes in the actin ring of differentiated osteoclasts using an optical microscope (Carl Zeiss). The results are shown in FIG. 4 . In the case of the osteoclast differentiation control group (G1), the actin ring was the clearest and the cells were dense, but the actin ring was significantly reduced as the concentration of soybean extract (SBE) increased. This indicates that the treatment of soybean extract inhibited differentiation to a stage capable of bone resorption.

Through this experiment, it was confirmed that the soybean extract of the present invention reduced TRAP activity, an osteoclast differentiation index, and inhibited actin ring formation at a concentration that was not toxic to the cell proliferation of RAW 264.7 cells.

From the above experimental results, it can be confirmed that the soybean extract according to the present invention improves bone metabolism by enhancing osteoblastic activity and suppressing osteoclast activity.

Claims (4)

Heating soybeans immersed in purified water for 1 to 2 days at 100° C. for 10 to 30 minutes and then cooling;
pulverizing the cooled soybeans and preparing an aqueous solution having a solid content of 5 to 10% by weight;
preparing a primary hydrolyzate by adding 1 to 10% by weight of a carbohydrate-degrading enzyme to the aqueous solution based on the solids weight and treating it for 1 to 3 hours;
Preparing a secondary hydrolyzate by adding 1 to 10% by weight of a protease to the primary hydrolyzate based on the solids weight and treating it for 5 to 7 hours: And
A method for producing a soybean extract, comprising inoculating the secondary hydrolyzate with Aspergillus oryzae or Aspergillus kawachii and fermenting the same. The method according to claim 1, wherein the carbohydrate-degrading enzyme is at least one selected from the group consisting of AMG, thermamyl, dextrozyme, biscozyme and cellulclast. The method according to claim 1, wherein the proteolytic enzyme is at least one selected from the group consisting of alkalase, flavorzyme and protamex. The improvement of bone metabolism function comprising the soybean extract prepared by the method according to any one of claims 1 to 3 as an active ingredient, and promoting osteoblastic activity of MC3T3-E1 cells, which are osteoblasts, and inhibiting osteoclast activity food composition for use.

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