WO2006075558A1

WO2006075558A1 - Egg-derived bone-strengthening composition

Info

Publication number: WO2006075558A1
Application number: PCT/JP2006/300082
Authority: WO
Inventors: Hye-Kyung Kim; Kang-Hyun Leem; Yasuyuki Ooi; Min-Young Ji; Shinsuke Minamoto; Mujo Kim; Sangwoo Cho
Original assignee: Pharma Foods International Co., Ltd.; Doosan Corporation
Priority date: 2005-01-11
Filing date: 2006-01-06
Publication date: 2006-07-20
Also published as: KR20070121639A; JPWO2006075558A1; KR20060082015A; CN101188949A; KR101156703B1; CN101188949B; JP5213332B2

Abstract

A bone-strengthening composition of the invention comprises as an active ingredient an egg-yolk protein hydrolysate in which a proportion of the area of the region of a molecular weight of 500 or more and 20,000 or less to the total proportion of the area of proteins, peptides and amino acids is 50% or higher in a molecular weight distribution analysis by gel filtration chromatography. The composition has an action of osteoblast growth promotion, bone growth and bone strengthening, is effective in the prevention and treatment of a variety of bone diseases such as osteoporosis, and is useful as a safe and inexpensive product that can be widely used for a food and drink, a pharmaceutical or a feed and can be taken daily and continuously. The egg-yolk protein hydrolysate is preferably a hydrolysate obtained by defatting egg yolk with an organic solvent and hydrolyzing the resulting powder with a protein hydrolyzing enzyme.

Description

Egg-derived bone strengthening composition

Technical field

[0001] The present invention relates to a composition containing an egg yolk protein hydrolyzate having bone growth and bone strengthening effects. Moreover, this invention relates to the food / beverage products, the pharmaceutical, or feed which mix | blended this egg yolk protein hydrolyzate composition. The egg yolk protein hydrolyzate of the present invention has the effect of proliferating osteoblasts and promoting the proliferation of chondrocytes at the growth end of bone, so that the prevention and treatment of bone diseases caused by the loss of bone metabolism balance, For young people such as school children

V, useful for strengthening bone during growth.

Background art

[0002] An increase in various bone diseases such as an increase in osteoporosis in recent years and an increase in fracture rate among young people has become a social problem. It is estimated that there are approximately 10 million patients with osteoporosis nationwide, and it is predicted that the number of patients will reach 16 million in 2010 due to the progress of aging. Osteoporosis is the focus of attention on post-menopausal women as a disease in which the loss of female hormones is disrupted by the loss of bone formation and bone destruction. There is a decrease, and there are still about 2 million osteoporosis patients over the age of 50. In addition, the fracture rate of elementary and junior high school students has more than doubled in the last 20 years, and from the standpoint of preventing osteoporosis, bone mineral content is firmly increased in younger years, and higher maximum bone mass is obtained. That is very important. Thus, maintaining bone health is now a social concern regardless of gender or age.

[0003] Conventional methods for preventing or treating bone diseases include dietary calcium supplementation, light exercise, sunbathing, and drug treatment. Calcium supplementation through dietary therapy uses calcium salts such as calcium carbonate and calcium phosphate, and natural calcium preparations such as beef bone meal, eggshell, and fish bone meal. Indispensable in terms of solubility, absorbability, and taste. However, it is not a material suitable for oral intake. Moderate exercise increases bone mass and strengthens the bones, so walking and walking are awkward if the strength of the bones is weak, and light exercise is also troublesome. Almost exercise Kina. Sunbathing is said to be a supplement of active vitamin D3, but this is not enough. Bisphosphonates and calcitonin preparations are used for drug administration and are known to be effective in the treatment of osteoporosis. However, these substances are medicines themselves, and special care is required for their use, and there is a risk of side effects such as tinnitus, headaches, loss of appetite, and they cannot be used as continuous food ingredients. .

[0004] Therefore, as a bone strengthening agent using a natural material, a chicken egg (Patent Document 1) reinforced with vitamins and an oyster extract (Patent Document 2) reinforced with zinc are used, a milk-derived basic protein. Products that use quality (Patent Documents 3 and 4) have been developed.

Patent Document 1: Japanese Patent Laid-Open No. 10-56978

Patent Document 2: JP 2002-80380 A

Patent Document 3: Japanese Patent No. 2974604

Patent Document 4: Japanese Patent No. 3112637

Disclosure of the invention

Problems to be solved by the invention

[0005] The present invention provides a novel food material that has a bone strengthening action, is safe and inexpensive, and can be widely used in foods and drinks as a bone strengthening agent that can be ingested on a daily basis. This is an issue.

Means for solving the problem

[0006] Eggs are a biocapsule that creates the next generation, including all the materials that form the living body, as shown by the phenomenon that eggs turn into leopards when heated for 3 weeks at 37 ° C. There is also a lot of physiological activity. Eggs have a long-term dietary experience, their nutritional value is high and safe, and their value as a food ingredient is very high.

[0007] Thus, as a result of searching for components that promote bone growth and bone strengthening in various fractions of eggs, the present inventors have found that egg yolk protein hydrolyzate has osteoblast proliferation and calcification. It was found for the first time that it has a promoting activity, and by ingesting this ingredient orally, it was found that bone growth can be promoted and bone metabolism can be improved, and the present invention has been completed.

[0008] The bone strengthening composition of the present invention is an egg yolk protein hydrolyzate, which is a gel filtration chromatography. In the molecular weight distribution analysis by chromatography, the active ingredient contains a component with an area ratio of 50% or more of the molecular weight of 500 or more and 20,000 or less to the total area ratio of protein, peptide and amino acid. It is characterized by.

[0009] The egg yolk protein hydrolyzate used in the present invention is preferably a product obtained by hydrolyzing a defatted egg yolk with a protein degrading enzyme.

[0010] Degreasing of egg yolk is an organic solvent that can be used for food processing of egg yolk (eg, ethanol).

, Isopropanol, hexane, etc.) is preferred. Proteolytic enzymes include pepsin, trypsin, renin, rennet for cheese containing rennin, carboxypeptidase A, peptase from Bacillus bacteria, and protease from Aspergillus sp. Use a combination.

[0011] Since the strong bone strengthening composition of the present invention is a safe product using natural products, it can be widely used as a bone-strengthening material, food, drink, medicine or feed that can be taken on a daily basis. The

The egg yolk protein hydrolyzate used in the present invention has a molecular weight of 500 or more and 20,000 or less with respect to the total area ratio of protein / peptide / amino acid in molecular weight distribution analysis by gel filtration chromatography. The area ratio of the portion occupies 50% or more, but it is more preferable that the area ratio occupies 55% or more. Particularly, the area ratio occupies 65% or more. Powerful egg yolk protein hydrolyzate: 1) Promote osteoblast proliferation (promote growth of cells that produce bone), 2) Promote osteoblast differentiation (enhance the function of cells that create bone, promote collagen production, lime 3) Inhibition of osteoclast differentiation and proliferation (suppresses the function and proliferation of cells that melt and metabolize bone), 4) Promotion of bone growth! /, It has a depressant effect and also has the activity of promoting the growth of chondrocytes and promoting bone growth.

[0013] Since the bone-strengthening composition mainly composed of strong egg yolk protein hydrolyzate can be taken orally, milk food such as cheese, butter, fermented milk, beverage milk, drink yogurt, coffee beverage Add to beverages such as fruit juice, jelly, pudding, cookies, confectionery such as biscuits and wafers, and other forms of food and drink, such as frozen foods, to make food and drink with bone strengthening action Can do. Furthermore, it can also be used as a supplement in the form of capsules, tablets, etc., and is useful as a material for foods, beverages, medicines or feeds for the purpose of preventing osteoporosis and improving symptoms.

Brief Description of Drawings

FIG. 1 is a view showing the results of molecular weight analysis of gel yolk protein hydrolyzate obtained in Example 1 by gel filtration chromatography. The shaded area indicates the molecular weight of 500 to 20,000.

FIG. 2 shows the analysis results of SDS-polyacrylamide gel electrophoresis of the yolk protein hydrolyzate of Example 1 and the yolk water-soluble fraction obtained from B. The M lane is the molecular weight marker shown on the left in the figure, the 1 lane is the yolk protein-calyzed hydrolyzate, and the 2 lane is the yolk water-soluble fraction obtained in B.

FIG. 3 is a graph comparing the extension of the tibial epiphysis between when the egg yolk protein hydrolyzate of Example 1 was orally administered to rats and when it was not administered.

FIG. 4 is a diagram showing the results of molecular weight analysis of gel yolk protein hydrolyzate obtained in Example 2 by gel filtration chromatography. The shaded area indicates the molecular weight of 500 to 20,000.

FIG. 5 shows the results of measuring the bone growth promoting effect using the egg yolk protein hydrolyzate obtained in Example 2.

FIG. 6 shows the results of measuring the calcification promoting effect using the egg yolk protein hydrolyzate obtained in Example 2.

FIG. 7 is a graph showing the results of measuring osteoblast proliferation promoting activity using egg yolk protein hydrolysates obtained using different enzymes.

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The egg yolk used in the present invention is preferably egg yolk from the viewpoint of power productivity such as egg yolk of chickens, ducks, quail and the like. In addition, when using chicken eggs, egg yolk liquid, egg yolk powder, or defatted egg yolk powder can be used, among which egg yolk powder or defatted egg yolk powder is preferably used.

[0016] The bone strengthening composition of the present invention has an egg yolk powder power and an organic solvent used in food processing. It can be obtained by hydrolyzing a powder obtained by degreasing using a protein hydrolase. As an organic solvent used for degreasing, it is possible to use an organic solvent generally used for food processing such as ethanol, isopropanol, and hexane. However, ethanol is preferably used in terms of simplicity and safety.

[0017] It is also possible to use defatted egg yolk produced as a by-product when producing egg yolk power egg yolk oil or egg yolk lecithin as a raw material for hydrolysis.

[0018] The protein hydrolase used in the present invention includes pepsin (EC. 3. 4. 23. 1), trypsin (EC. 3) which have protease activity or carboxypeptidase activity and can be used for food production. 4. 21. 4), lane (EC. 3. 4. 23. 15), rennet for cheese containing lane, carboxypeptidase A (EC. 3. 4. 17. 1), Protease derived from bacteria belonging to the genus Bacillus (trade name “Alcalase” manufactured by Novozym, product name “Orientase 22BF” manufactured by HIBI Co., Ltd. Manufactured by Enzym Co., Ltd., trade name “Samorose PC10” manufactured by Daiwa Kasei Co., Ltd., etc., Aspergillus genus Protease (trade name “Orientase ONS”) Z 20A ”, manufactured by HBI Corporation, product name“ Protease P “Amano” 3G ”manufactured by Amano Enzym Co., Ltd., product name“ Flavor Zim ”manufactured by Novozym, etc.), or a combination thereof. can do. In the present invention, among the above enzymes, proteases derived from Bacillus bacteria and Z or pepsin are particularly preferably used.

[0019] The concentration of the proteolytic enzyme varies depending on the enzyme used, but the mass ratio of the enzyme to defatted egg yolk is preferably from 1:20 to 1: 1000. In addition, it is preferable to carry out the hydrolysis at a force of 25 to 75 ° C. for 1 to 24 hours, depending on the enzyme reaction temperature and reaction time. If the reaction time is too long or too short, the resulting hydrolyzate cannot provide sufficient osteoblast proliferation promotion, bone growth and bone strengthening action!

[0020] The egg yolk protein hydrolyzate obtained as described above can be used as a raw material for the bone strengthening composition of the present invention after desalting as appropriate. In addition, ultrafiltration membranes, gel filtration, various column chromatography, membrane filters, methods using isoelectric points, etc. Use it for purification or fractionation.

The egg yolk protein hydrolyzate obtained as described above is subjected to molecular weight distribution analysis by gel filtration chromatography and has a molecular weight of 500 or more to the total area ratio of protein / peptide / amino acid. It is important that the area ratio of the portion of 000 or less occupies 50% or more, and it is more preferable that the area ratio occupies 55% or more. In particular, the area ratio occupies 65% or more. If the molecular weight distribution is out of the above range, the effect will be reduced or lost, and denaturation or aggregation due to heating will easily occur. However, as long as the distribution satisfies the above range, 10 to 20% by mass of undegraded egg yolk protein may remain.

[0022] The egg yolk protein hydrolyzate in the bone-strengthening composition of the present invention preferably has an osteoblast proliferation promoting activity of 1.2 times or more that of a control to which no egg yolk protein hydrolyzate is added. Furthermore, it is preferable that the osteoblast proliferation-promoting activity is maintained at least 1.2 times that of the control even after heat treatment at 65 ° C. to 90 ° C. for 3 minutes to 6 minutes.

[0023] The osteoblast proliferation promoting activity can be measured by a conventional method using MC3T3-T1 cells of mouse osteoblasts or human-derived osteoblast-like cells U20S cells.

[0024] The content of the egg yolk protein hydrolyzate in the bone reinforcing composition of the present invention is not particularly limited, but it is preferable to contain 0.05 to 50% by mass of the egg yolk protein hydrolyzate. 0.1 to 25% by mass is more preferable.

[0025] If the content of the egg yolk protein hydrolyzate is less than 0.05% by mass, the present bone strengthening composition will be added to the food at a high concentration. Since it affects the sex, it is not preferable.

[0026] The bone strengthening composition of the present invention is applied to the prevention or improvement of osteoporosis and various bone diseases.

The effective intake is 50 mg to 5 g in terms of egg yolk protein hydrolyzate per adult day.

It should be noted that the bone strengthening composition of the present invention was not recognized as having acute toxicity by rats.

[0027] The bone reinforcing composition thus prepared is usually used after being powdered. Powdering can be performed by freeze-drying or spray-drying, and is usually administered orally as a food or drink. [0028] In addition to the components described above, the bone strengthening composition of the present invention includes excipients, emulsifiers, stabilizers, sweeteners, PH regulators, thickeners, proteins, peptides, amino acids, lipids, and polysaccharides. It may contain ingredients normally used in food such as oligosaccharides.

[0029] The bone-strengthening composition of the present invention is not only added to food and drink, but also prepared in various forms according to the dosage form, and can also be used as a powder (or granule), a drink, a tablet, a capsule, and the like. . In addition, since eggs are safe ingredients that have been eaten for a long time, they can be used in medicines or feeds as ingredients without any side-effect problems even if they are used continuously.

Example

[0030] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

A.

1 kg of egg yolk powder was charged with 5 L of ethanol, stirred for 30 minutes with a blender, and solids were collected. This operation was repeated three times to degrease the egg yolk force and air-dried to obtain 568 g of defatted egg yolk.

[0031] B. Preparation of old water-soluble fraction of eggs

To 284 g of the defatted egg yolk obtained in A, 3 L of 37 ° C warm water was added and stirred well with a stirrer while maintaining the temperature, followed by centrifugation at 8, OOOrpm for 30 minutes to obtain a supernatant. Then, it was pulverized by freeze-drying to obtain 13.4 g of a water-soluble fraction of egg yolk.

Example 1 (Manufacture of egg-old protein hydrolyzate)

Add 3 L of water to 300 g of the defatted egg yolk powder obtained in A, and adjust to pH 3.5 with hydrochloric acid. Thereafter, 3 g of pepsin (manufactured by Sigma Aldrich) was added, and the enzyme reaction was performed at 37 ° C for 3 hours. Then, neutralize to pH7 with sodium hydroxide and add 30g of filter aid (Celite), filter with Buchner funnel, freeze-dry the filtrate and hydrolyze egg yolk protein 44 Obtained 8g.

[0033] The egg yolk protein hydrolyzate obtained in Example 1 was analyzed for molecular weight by gel filtration chromatography under the following conditions.

Column: Diol 60 (6. O X 300mm)

Eluent: 0.2 M potassium phosphate buffer, 0.2 M NaCl (pH 6.9) / acetonitrile (70: 30)

Flow rate: 0.7mlZ min

Detection wavelength: 280nm

The results are shown in Fig. 1. From FIG. 1, the egg yolk protein hydrolyzate of Example 1 has an area ratio (shaded area) of a molecular weight of 500 to 20,000 with respect to the total area showing the total of protein, peptide, and amino acid, approximately 57%. It became clear to occupy.

Next, SDS-polyacrylamide gel electrophoresis was carried out in a conventional manner using the egg yolk protein hydrolyzate of Example 1 and the egg yolk water-soluble fraction obtained in B. The result is shown in figure 2. As is clear from Fig. 2, many bands with a molecular weight of 20,000 or less were smaller than those of the yolk water-soluble fraction.

Test 1 (Measurement of osteoblast proliferation activity)

The osteoblast proliferation activity of the egg yolk protein hydrolyzate obtained in Example 1 was measured as follows. In other words, MC3T3-E1 cells of mouse osteoblasts were confluent at 37 ° C, 5% CO-95% air using α-MEM culture medium containing 10% FBS.

2

The cells were collected by trypsin treatment. The collected cells were suspended in the α-MEM culture solution to prepare a cell suspension (1 × 10 ⁴ ZmL). Each 0.5 mL of this cell suspension was seeded on a 24-well plate and cultured at 37 ° C under 5% CO-95% air. The next day, eggs

2

Dissolve the yellow protein hydrolyzate in 50 (v / v)% DMSO and add the egg yolk protein hydrolyzate to the cells to 100, 25, 12.5 _i ug / mL (DMSO l (v / v)%) and α-glyceport phosphate (2 mmol ZL), which is a calcification accelerator, was added. The culture medium is changed every other day, and after 20 days, the number of osteoblasts is measured using MTT (3- (4,5-Dimethyto 2-thiazolyl) -2,5-diphenyltetrazolium Bromide) according to a conventional method. (MTT method).

The osteoblast proliferation promoting activity of egg yolk protein hydrolyzate (100 μg / mL) was 150 ± 11 when expressed as a relative value when the growth number of the control was 100. On the other hand, the osteoblast proliferation promoting activity of the yolk water-soluble fraction obtained in B above was 128 ± 18 when expressed in the same relative value.

The egg yolk protein hydrolyzate obtained in Example 1 was heat-treated at 80 ° C for 5 minutes. Similarly, when the proliferation promoting activity of osteoblasts was measured, it was 130 ± 10 as a relative value when the proliferation number of the control group was 100. On the other hand, when the same heat treatment was performed on the egg yolk water-soluble fraction obtained in B above, the osteoblast proliferation promoting activity was expressed as 105 ± 11 in the same relative value. That is, although the activity of egg yolk protein hydrolyzate was slightly decreased with respect to heat treatment, it maintained a sufficiently high activity of promoting osteoblast proliferation and exhibited thermal stability.

[0035] Test 2 (Bone growth promoting effect)

The bone growth promoting effect of the egg yolk protein hydrolyzate obtained in Example 1 was evaluated using Sprague-Dawley rats. Specifically, the growth of the tibia is measured by administering a fluorescent substance, tetracycline, and observing the fluorescence band formed in the bone by utilizing the phenomenon that tetracycline chelates calcium and deposits on the bone. did.

That is, during the test period (1 week), a sample dissolved in purified water at a concentration of 20 mgZml was orally administered to a rat with a stomach tube (lOOmgZkg body weight Z1 day). Tetracycline was administered by injection on days 3 and 5 during the study period. During the test period, drinking water and feed for rats (trade name “MF”, manufactured by Oriental Yeast Co., Ltd.) were freely consumed. On the 7th day, the tibia was removed, and the interval between each fluorescent band formed in the lower part of the tibia growth plate (new bone formation) by administration of tetracycline The elongation of the tibial epiphysis was measured by observing under a fluorescence microscope.

Figure 3 shows the bone elongation rate (%) obtained when egg yolk protein hydrolyzate was administered with the yolk protein hydrolyzate administered as a force (control). Shown in From FIG. 3, it was confirmed that when egg yolk protein hydrolyzate was administered, there was a bone growth promoting effect in which the interval between the two fluorescent bands was 31% wider than that of the control.

Example 2 (Production of egg hydrolyzate)

To 500 kg of defatted egg yolk powder obtained in the same way as A, add 2.5 tons of water and 25 kg of Alkalase (trade name, protease derived from Bacillus licheniformis) of Novozymes. Enzymatic reaction at pH 7 and 55 ° C for 3 hours After that, the enzyme is inactivated by heat treatment at 80 ° C for 15 minutes, centrifuged at 3000g for 20 minutes to remove insoluble matter, filtered, and the filtrate is spray-dried to about 140kg. Egg yolk protein hydrolyzate was obtained. [0037] The molecular weight analysis of the egg yolk protein hydrolyzate obtained in Example 2 was performed in the same manner as in Example 1. The results are shown in Fig. 4. From Fig. 4, the obtained egg yolk protein hydrolyzate has an area ratio (shaded area) of about 85% to a molecular weight of 500 to 200,000 with respect to the total area showing the total of protein, peptide and amino acid. It became clear that it occupied.

[0038] Next, the following test was performed on the egg yolk protein hydrolyzate of Example 2. The control in each test is the one without egg yolk protein hydrolyzate.

Test 1 (Bone growth promoting effect)

The bone growth promoting effect of the egg yolk protein hydrolyzate obtained in Example 2 was examined in the same manner as in Test 2 of Example 1. That is, a sample (concentration 20 mgZml) dissolved in purified water was orally administered to a Sprague-Dawley rat using a gastric sonde (10 mg, 20 mg or 50 mg per kg body weight was administered daily). Then, a fluorescent substance (tetracycline lOmgZkg) was administered by injection on the 3rd and 5th day of the study period, and on the 7th day, the tibia was removed and observed under a fluorescence microscope. The fluorescence band interval (the force on the third day when tetracycline was administered and the extension of the tibial epiphysis during the two days on the fifth day) was measured. The results are shown in Fig. 5. From FIG. 5, it can be seen that a bone growth promoting effect is obtained in a dose-dependent manner.

[0039] Test 2 (calcification promoting effect)

3. Seed 6 × 10 ⁵ MC3T3-E1 cells in a ⁵ cm petri dish-MEM medium, 10% F CS, 2 mmol / L glycephosphate), and then add the egg yolk protein hydrolyzate obtained in Example 2 to 10 0 Add at a concentration of gZml, change with medium every 2 days, continue culture for 4 weeks, rinse cells with PBS (phosphate buffer), add 5 (v / v)% perchloric acid, Calcium deposited on the cells was eluted by sonication, and the amount of calcium eluted was measured using “Calcium E Test Sakai Koichi” (trade name, manufactured by Wako Pure Chemical Industries, Ltd.) to obtain calcification activity. . The results are shown in Fig. 6. From FIG. 6, it can be seen that the addition of egg yolk protein hydrolyzate significantly increases the mineralization activity (p 0.001).

[0040] Test 3 (Osteoclast differentiation and proliferation inhibitory effect)

Bone marrow cells were isolated from mouse bone marrow and measured for osteoclast differentiation and proliferation. First, bone marrow cells from the femur of mice are differentiated with the differentiation-inducing factor RANKL (receptor activate The cells were cultured in a-MEM medium containing r of NF-KB Ligand: NF-κB activating receptor ligand) for 3 days, and after the sample treatment, the cells were cultured again for 4 days. Then, after fixing the cells with a fixative, TRAP staining was performed using a TRAP staining kit (trade name “387-A Acid Phosphatase, Leukocyte” manufactured by Sigma Aldrich Japan). The number was observed with a microscope. The frequency of osteoclasts was measured for the egg yolk protein hydrolyzate from Example 2 added with 50 gZml, lOO ^ g / ml added and 200 g / ml added, and without addition. The results are shown in the following table.

[0041]. Egg yolk protein hydrolyzate Osteoclasts (red stained cells)

(a _S / mi) Cattle. (%)

. 0 22. 5

.50 16

.100 2.5

. 200 0

From the above table, it can be seen that the frequency of osteoclast generation is suppressed depending on the concentration of egg yolk protein hydrolyzate.

[0042] ¾ Example 3 (egg old protein-added 7k degradation product)

Add 3 L of water to 300 g of the defatted egg yolk powder obtained in A, perform the enzyme reaction under the enzyme conditions shown in Table 1, and then inactivate the enzyme by heating for 10 minutes after reaching 80 ° C in a boiling water bath and filtering. 30 g of auxiliary agent (Celite) was added and filtered through a Buchner funnel, and the obtained filtrate was freeze-dried to obtain 7 kinds of egg yolk protein hydrolysates.

[0043] [Table 1]

Reaction conditions

Hydrolyzate

I) Molecule a Use ffl Fermentation type ll Time Temperature

No mm (hr) (in)

1 8 δ% Nova "S 'Rei's" Alcala-Ze "7.0 3 55

(Bacillus 1 icheniforniisrti come)

2 74% "Chibiai's" Oriental "10.5 3 65

(Derived from Bacillus subtilis)

3 73% HI: Kureshin 7.2 3 55

(Bacil lus subtil is t † l come)

4 67% “Protea-ze s Γama /” の of Amano Nozim 7.0 3 70

(Bacillus stearothr 卿 hi lusrtl came)

o 57% Sigma Alto 'Rich's "Pepsin" 3.5 3 37

6 56% “Samoa-Se 'PC 10” by Daiwa Kasei Co., Ltd. 7.0 3 65

(Bacillus thermoproteolyt icus cause)

7 52% Amano Enza "Akatsuki's" P D Thea-Ze P "Ama G j 8.0 3 45

(Aspergillus mel leus purse)

"Lambda" 'Λ' ratio of molecule a is the gel filtration chromatographic analysis of egg ¾ protein hydrolyzate.

In the molecular a distribution analysis by GRAPHI, protein 'peptide' amino

The surface of the part of the molecule as 00 to 20,000

Set the fi¾ ratio. Test 1 (osteoblast proliferation promoting activity)

Mouse osteoblast MC3T3-E1 cells are cultured in α-MEM culture medium containing 10% FBS at 37 ° C under 5% CO -95% air until confluent, then trypsin

2

Cells were collected by treatment. The collected cells were suspended in the above α-MEM culture solution to prepare a cell suspension (2 × 10 ⁴ ZmL). This cell suspension is seeded at 0.1 mL each in a 96-well plate, and a mineralization accelerator a-glyceport phosphate (2 mmol / L) is added to the mixture at 37 ° C, 5% CO —. Cultured under 95% air. After 24 hours, each sample obtained in Example 3 was

2

It was dissolved in 0 (v / v)% DMSO and added to the cells so that the sample was 100 μg ZmL (DMSO was added so as to be 1 (v / v)% of the culture solution). Then, after 24 hours of culture, cell proliferation activity was measured by the MTT method in the same manner as in Test 1 of Example 1.

The results of expressing the osteoblast proliferation promoting activity of each egg yolk protein hydrolyzate (100 μg / mL) as a relative value when the number of growth of the control (no egg yolk protein hydrolyzate added) is 100. Figure 7 shows. Fig. 7 shows that egg yolk obtained by hydrolysis using proteases derived from Bacillus bacteria, pepsin, and Aspergillus spp. Protein hydrolyzate has a high osteoblast proliferation promoting activity (it is an egg yolk protein hydrolyzate obtained by hydrolysis using protease and pepsin derived from Bacillus sp. Are significantly different from the control (p 0.005).

In the following examples, “%” represents “mass%” unless otherwise specified.

Example 4 (soft drink)

A soft drink containing the egg yolk protein hydrolyzate obtained in Example 1 was prepared.

. That is, the composition is mixed isomer sucrose 15.0%, fruit juice 10%, egg yolk protein hydrolyzate

2. 0%, fragrance 0.1%, calcium 0.1%, water 72.8% are mixed and sterilized using a plate sterilizer at 90 ° C for 15 seconds to have bone strengthening effect A soft drink was produced.

[0046] Decorative 15 (yogurt)

A yogurt containing the egg yolk protein hydrolyzate obtained in Example 1 was prepared. That is, yogurt having a bone strengthening action is manufactured by mixing raw materials having a composition of egg yolk protein hydrolyzate 3.0%, sucrose 7%, flavoring 0.1%, yogurt 89.9% and filling the container. did.

[0047] Example 6 (cheese)

Processed cheese containing the egg yolk protein hydrolyzate obtained in Example 1 was prepared. That is, after mixing each raw material so that it contains 35% Gouda cheese, 35% Cheddar cheese, 20% Parmesan cheese, 2.0% egg yolk protein hydrolyzate, 1.0% calcium phosphate, 7.0% water, emulsification temperature Emulsified at 85 ° C to produce processed cheese with bone strengthening action.

[0048] Example 7 (capsule)

60% egg yolk protein hydrolyzate obtained in Example 1, 30% corn starch, lactose 1

Each ingredient is mixed so that it contains 0% and filled into gelatin capsules (20 per capsule)

Omg) to produce a capsule having a bone strengthening action.

[0049] Decoration 18 (tablet)

Each ingredient was blended to contain 60% egg yolk protein hydrolyzate obtained in Example 1, 18% reduced maltose, 18% crystalline cellulose, and 4% sucrose ester, and then compressed into tablets (300 mg per tablet). Thus, a tablet having a bone strengthening action was produced. Industrial applicability

The bone-strengthening composition of the present invention and food and drink containing the same are made from defatted egg yolk, which is a by-product of producing safe and inexpensive egg yolk and oil of yolk, as a raw material, promoting osteoblast proliferation, bone growth and bone strengthening action Therefore, it can be used as a health food for preventing osteoporosis, promoting bone growth and strengthening bone.

Claims

The scope of the claims

[1] An egg yolk protein hydrolyzate that has a molecular weight distribution analysis by gel filtration chromatography, the area ratio of the portion with a molecular weight of 500 to 20,000 is 50 to the total area ratio of protein / peptide 'amino acid. A bone-strengthening composition comprising an active ingredient that occupies% or more.

[2] The egg yolk protein hydrolyzate is obtained by defatting egg yolk with at least one organic solvent used for food processing and then hydrolyzing with a protein hydrolase. Bone strengthening composition.

[3] The proteolytic enzyme is selected from the group power of pepsin, trypsin, renin, rennet for cheese containing rennin, carboxypeptidase A, protease derived from Bacillus bacteria and protease derived from Aspergillus spp. The bone strengthening composition according to claim 1 or 2, wherein the composition is one kind.

[4] A food, beverage, medicine or feed comprising the bone reinforcing composition according to any one of claims 1 to 3.