JP6357266B2 - Protein material for bone disease prevention or treatment - Google Patents

Description

  The present invention relates to a protein material for preventing or treating bone diseases and a method for producing the same. More specifically, the present invention relates to a novel protein material and a pharmaceutical or food / drink product or feed useful for the prevention or treatment of bone diseases containing the protein material. The material promotes the proliferation of osteoblasts and suppresses osteoclast differentiation and bone resorption by osteoclasts, thus preventing and treating various bone diseases such as osteoporosis, fractures, rheumatism and arthritis. Useful.

  In recent years, various bone-related diseases such as osteoporosis, fractures, and back pain are increasing with the aging of the world on a global scale, which is a serious social problem. This is considered to be caused by insufficient intake of calcium, a decrease in calcium absorption capacity, and postmenopausal hormone imbalance. In order to prevent various bone diseases such as osteoporosis, fractures, and back pain, bone formation by osteoblasts is promoted from a young age to increase the amount of bone in the body as much as possible, and maximum bone mass and bone strength (bone density) + Bone quality) is considered effective. Bone quality refers to bone microstructure, turnover, micro fracture, and calcification. In addition, as a method for preventing various bone diseases such as osteoporosis, fractures, and back pain, it is conceivable to suppress bone resorption by osteoclasts. Bone repetitively undergoes balanced resorption and formation (remodeling), but due to changes in hormone balance after menopause, etc., bone resorption exceeds bone formation, which leads to various bone diseases such as osteoporosis, fractures, and back pain Cause. Therefore, it is possible to strengthen the bone as a result by suppressing bone resorption by osteoclasts and keeping the bone strength constant.

Under these circumstances, for the purpose of strengthening bones, calcium salts such as calcium carbonate, calcium phosphate, and calcium lactate, and natural calcium preparations such as whey calcium, beef bone meal, eggshell, etc., alone for pharmaceuticals, foods and drinks, feeds, etc. What is added to is taken. Or these calcium agents are ingested together with substances having a calcium absorption promoting effect such as casein phosphopeptides and oligosaccharides in medicines, foods and drinks, feeds and the like. However, the absorption rate of calcium when ingesting these calcium salts and natural calcium preparations added to foods and drinks is 50% or less, and it is said that a lot of calcium is not absorbed and is discharged outside the body. Yes. In addition, even if calcium is absorbed by the body, the affinity to bone varies depending on its form and the type of other nutritional components that are taken at the same time, so it does not necessarily show an effect of improving bone metabolism or strengthening bone. There is also. In addition, female hormone preparations, active vitamin D ₃ preparations, vitamin K ₂ preparations, bisphosphonate preparations, calcitonin preparations, etc. are known as drugs for osteoporosis treatment and bone strengthening, and new drugs such as anti-RANKL antibodies Development is underway. However, these medicines may have side effects such as tinnitus, headache, and loss of appetite. Furthermore, these substances cannot be added to food and drink at present from the viewpoints of safety and cost. On the other hand, due to the nature of various bone diseases such as osteoporosis, fractures, and back pain, it can be taken orally for a long period of time, acting to promote bone formation and suppress bone resorption to increase bone strength and prevent or Development of bone strengthening agents, foods and drinks, and feeds that can be expected to have therapeutic effects is desired.

  Examples of food materials intended to increase bone strength include, for example, basic protein derived from milk and peptide fractions that are enzymatic degradation products thereof, osteoblast proliferation activity and osteoclast bone resorption inhibition activity, and It has been reported that there is a bone strengthening action through them (Patent Document 1). In addition, it has been reported that angiogenin and lactoperoxidase contained in a milk-derived basic protein fraction each have an effect of improving bone metabolism alone (Patent Documents 2, 3, and 4).

JP-A-8-151331 Japanese Patent Laid-Open No. 10-7585 Japanese Patent Laid-Open No. 2004-238320 JP 2005-60321 A

The present invention is safe and can be ingested daily to promote osteoblast proliferation, suppress osteoclast differentiation and bone resorption by osteoclasts, and strengthen bones It is an object to provide a novel protein material.
Moreover, this invention makes it a subject to provide the bone strengthening pharmaceutical or food-drinks, and feed which are useful for prevention and treatment of various bone diseases, such as osteoporosis, a fracture, rheumatism, and arthritis, by oral ingestion.

  The inventors of the present invention contain angiogenin and / or angiogenin degradation products in a specific range of amounts, and lactoperoxidase and / or lactoperoxidase degradation products with respect to angiogenin and / or angiogenin degradation products. Ingesting a protein material containing a mass ratio within a specific range effectively promotes osteoblast proliferation and suppresses osteoclast differentiation and bone resorption by osteoclasts. The present invention was completed.

That is, this invention consists of the following structures.
(1) It contains 2 to 15 mg / 100 mg of angiogenin and / or angiogenin degradation product, and the mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product A protein material for preventing or treating bone diseases, which is contained in the range of 0.3 to 20.
(2) A food or drink or feed for prevention or treatment of a bone disease containing the protein material according to (1).
(3) A bone strengthening agent comprising the protein material for preventing or treating bone disease according to (1) as an active ingredient.
(4) A bone strengthening method of ingesting 5 mg / day or more of the protein material for prevention or treatment of bone disease according to (1).
(5) The method for producing a protein material for preventing or treating bone disease according to (1), comprising the following steps 1) to 3).
1) A step of preparing angiogenin and / or an angiogenin degradation product.
2) A step of preparing lactoperoxidase and / or a degradation product of lactoperoxidase.
3) The mass ratio of the lactoperoxidase and / or the lactoperoxidase degradation product prepared in the above step 2) to the angiogenin and / or angiogenin degradation product prepared in the above step 1) is 0.3. The process of mix | blending so that it may become ~ 20.
(6) Angiogenin and / or milk so that the mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product is 0.3 to 20 from milk and / or milk raw material. Alternatively, the method for producing a protein material for preventing or treating bone disease according to (1), comprising a step of extracting a fraction containing an angiogenin degradation product and lactoperoxidase and / or a lactoperoxidase degradation product.
(7) The method for producing a protein material for preventing or treating bone disease according to (6), further comprising the step of enzymatically degrading angiogenin and / or lactoperoxidase in the fraction containing angiogenin and lactoperoxidase. .

  The protein material for preventing or treating bone disease of the present invention has a remarkable bone strengthening action through the action of promoting osteoblast proliferation and inhibiting osteoclast differentiation and bone resorption by osteoclasts. In addition, pharmaceuticals, foods and drinks, and feeds that contain the protein material for the prevention or treatment of bone diseases of the present invention are useful for the prevention and treatment of various bone diseases such as osteoporosis, fractures, rheumatism, arthritis, etc. It is.

A feature of the protein material of the present invention is that it contains angiogenin and / or angiogenin degradation products in a specific range of amounts, and lactoperoxidase and / or lactoperoxidase degradation products are angiogenin and / or angiogenin. It exists in containing by mass ratio of a specific range with respect to a decomposition product.
Therefore, in the protein material of the present invention, a fraction containing angiogenin and / or an angiogenin degradation product and a fraction containing lactoperoxidase and / or a lactoperoxidase degradation product are mixed so that the mass ratio is in a specific range. A specific range of mass ratio of angiogenin and / or angiogenin degradation products and lactoperoxidase and / or lactoperoxidase degradation products directly from milk, or milk-derived milk materials such as skim milk and whey And the like prepared by extracting the fraction contained in. In addition, the protein material of the present invention includes those in which an enzyme or the like is allowed to act to decompose angiogenin and / or lactoperoxidase.

When a fraction containing angiogenin and / or an angiogenin degradation product and a fraction containing lactoperoxidase and / or a lactoperoxidase degradation product are mixed to obtain the protein material of the present invention, angiogenin and / or angiogenin degradation As a fraction containing a product and a fraction containing lactoperoxidase and / or a lactoperoxidase degradation product, a fraction prepared from milk of mammals such as humans, cows, buffalos, goats, sheep, and genetic engineering techniques are used. A fraction produced, a fraction purified from blood or an organ, and the like can be used. It is also possible to use purified and commercially available angiogenin and lactoperoxidase reagents, and adjust the mass ratio of angiogenin and / or angiogenin degradation products to lactoperoxidase and / or lactoperoxidase degradation products. Thus, the protein material of the present invention can be used.
In addition, the above-mentioned angiogenin-containing fraction, angiogenin reagent, fraction containing lactoperoxidase, lactoperoxidase reagent, etc., which are decomposed with one or more types of proteolytic enzymes, are obtained as angiogenin degradation products and lactose. It can be used as a peroxidase degradation product.

On the other hand, an image containing angiogenin and / or angiogenin degradation products and lactoperoxidase and / or lactoperoxidase degradation products in a specific range of mass ratio directly from milk or milk-derived raw materials such as skim milk and whey. In the case of preparing by extracting a fraction, for example, milk or milk raw material is brought into contact with a cation exchange resin, and then milk-derived protein adsorbed on the resin is eluted at a salt concentration of 0.1 to 2.0 M. After desalting and concentrating with reverse osmosis membrane, electrodialysis membrane, ultrafiltration membrane, microfiltration membrane, etc., trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, V8 as necessary. It can be prepared by limited degradation with a proteolytic enzyme such as protease so that the molecular weight becomes 8,000 or less. In the case of limited degradation with a proteolytic enzyme, the lower limit of the molecular weight is preferably 500 or more. In addition, the protein material thus obtained can be dried by freeze drying, spray drying or the like.
In addition, in order to perform proteome analysis of the protein material of the present invention, after performing limited decomposition with a digestive enzyme under denaturation and reduction according to a conventional method, analysis by LC / MS / MS revealed that the protein material of the present invention was In addition to angiogenin and / or an angiogenin degradation product and lactoperoxidase and / or a lactoperoxidase degradation product, any protein of αs1 casein, αs2 casein, β casein, or κ casein and / or a protein thereof It contained at least one degradation product.

The protein material of the present invention contains 2 to 15 mg / 100 mg of angiogenin and / or angiogenin degradation products, and the mass ratio of lactoperoxidase and / or lactoperoxidase degradation products to angiogenin and / or angiogenin degradation products is It contains in the range of 0.3-20.
As shown in a later test example, the mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product is 0.3 to 20, thereby ingesting each alone. Can also effectively obtain bone strengthening action.

  In addition, although it is reference, the angiogenin and / or angiogenin degradation product in milk is about 0.001%, and the angiogenin and / or angiogenin degradation product of lactoperoxidase and / or lactoperoxidase degradation product The mass ratio is about 20. Further, the whey protein concentrate (WPC) contains about 0.1% of angiogenin and / or angiogenin degradation product, and angiogenin and / or angiogenin of lactoperoxidase and / or lactoperoxidase degradation product. The mass ratio to the decomposition product is about 30.

The protein material of the present invention can be appropriately formulated as an active ingredient and formulated as a bone strengthening agent. It is also possible to use the protein material of the present invention as it is as a bone strengthening agent. In addition, when formulated as a bone strengthening agent, in addition to the protein material of the present invention, which is an active ingredient, other pharmaceuticals, foods and drinks, and feeds such as sugars, lipids, proteins, vitamins, minerals, flavors, etc. It can be mixed with normally used raw materials and the like, and further formulated into powders, granules, tablets, capsules, drinks and the like according to conventional methods. Moreover, it is also possible to use together the protein raw material of this invention and the component which shows other bone reinforcement | strengthening effects, for example, calcium, vitamin D, vitamin K, isoflavone, etc.
The protein material of the present invention can reinforce bone by ingesting 5 mg or more per kg of body weight in a test with an experimental animal described later. The intake in this experimental animal corresponds to the intake per adult in the blood drug concentration (Mitsuyoshi Nakajima (1993) “Volume 8: Efficacy Evaluation”, Yodogawa Shoten, 2-18). By ingesting 5 mg or more of the protein material of the present invention per person per day, bone strengthening, in particular, the effects of prevention and treatment of various bone diseases such as osteoporosis, fractures, rheumatism and arthritis can be expected. Therefore, when blended into a bone strengthening agent or the like, this necessary amount may be ensured.

  The protein material of the present invention can be blended in ordinary foods and drinks such as yogurt, beverages, wafers, desserts and the like. In this case, although depending on the form of the food or drink, it is preferable to blend 0.25 to 1000 mg of the protein material of the present invention per 100 g of the food or drink. It can also be blended with feed such as livestock feed or pet food to make bone strengthening feed. In this case, the protein material of the present invention is preferably blended in an amount of 0.25 to 1000 mg per 100 g of feed.

When the protein material of the present invention is prepared and used in the form of pharmaceuticals, foods and drinks, and feeds, the protein material of the present invention is suspended or dissolved in deionized water and stirred and mixed. As the conditions for stirring and mixing, the protein material of the present invention may be mixed uniformly, and stirring and mixing may be performed using an ultradisperser, a TK homomixer, or the like.
Moreover, the solution of this raw material can be used after desalting, concentrating with a reverse osmosis membrane, etc., or freeze-drying as needed so that it may be easy to use for a pharmaceutical, food-drinks, and feed. In addition, it has been confirmed that the protein material of the present invention maintains the activity related to bone strengthening even when the sterilization treatment that is usually used for the production of pharmaceuticals, foods and drinks, and feeds is performed. When the material is powdered, dry heat sterilization can also be performed. The protein material of the present invention can be used for various forms of pharmaceuticals, foods and drinks, and feeds such as liquid, gel, powder and granules.

  Reference Examples, Examples and Test Examples are shown below, and the present invention will be described in more detail. However, these are merely illustrative, and the present invention is not limited by these.

[Reference Example 1]
(Preparation of angiogenin fraction 1)
A column packed with 30 kg of a cation exchange resin sulfonated chitopearl (Fuji Boseki Co., Ltd.) was thoroughly washed with deionized water, and then 1,000 L (pH 6.7) of non-sterilized skim milk was passed through the column. . The column was then thoroughly washed with deionized water and then eluted with a linear concentration gradient of 0.1-2.0 M sodium chloride. And the elution fraction containing angiogenin is fractionated by S-Sepharose cation exchange chromatography (manufactured by Amersham Biosciences), the obtained angiogenin-containing fraction is heated at 90 ° C. for 10 minutes, and centrifuged. The precipitate was removed by separation. Further, this angiogenin-containing fraction was treated with Superose 12 gel filtration chromatography. This eluate was desalted with a reverse osmosis membrane and then freeze-dried to obtain 16.5 g of an angiogenin fraction having angiogenin purity of 90%. These series of treatments were repeated 30 times.

[Reference Example 2]
(Preparation of angiogenin fraction 2)
A column packed with 10 kg of heparin affinity sepharose (manufactured by GE Healthcare) was thoroughly washed with deionized water, and then 1000 L (pH 6.7) of non-sterilized skim milk was passed through the column. Next, this column was thoroughly washed with 0.6 M sodium chloride solution and then eluted with 1.5 M sodium chloride solution. The eluate was desalted with a reverse osmosis membrane and then freeze-dried to obtain 32 g of an angiogenin fraction with angiogenin purity of 2%. These series of treatments were repeated 50 times.

[Reference Example 3]
(Preparation of lactoperoxidase fraction)
A column (diameter 5 cm × height 30 cm) packed with 600 g of a cation exchange resin sulfonated chitopearl (Fuji Boseki) was thoroughly washed with deionized water, and then 360 L of non-sterilized skim milk (pH 6. 7) was passed at a flow rate of 25 ml / min. After passing, the column was thoroughly washed with deionized water and eluted with 0.02 M carbonate buffer (pH 7.0) containing 2.0 M sodium chloride. Then, the elution fraction containing lactoperoxidase was adsorbed on an S-Sepharose FF column (Amersham Biosciences), washed thoroughly with deionized water, and equilibrated with 10 mM phosphate buffer (pH 7.0). The fraction adsorbed with a linear gradient of 0 to 2.0 M sodium chloride was eluted, and the fraction containing lactoperoxidase was recovered. The fraction was treated by gel filtration chromatography using HiLoad 16/60 Superdex 75 pg (manufactured by Amersham Biosciences). This eluate was desalted with a reverse osmosis membrane and then freeze-dried to obtain 27 g of a lactoperoxidase fraction having a lactoperoxidase purity of 90%. These series of treatments were repeated 25 times.
[Example 1]

The angiogenin fraction of Reference Example 1 0.59 mg, the Angiogenin fraction of Reference Example 2 98.58 mg and the lactoperoxidase fraction of Reference Example 3 0.83 mg were mixed to produce an angiogenin and / or angiogenin degradation product. A protein material (Example product 1) having a content of 2.5 mg / 100 mg and an angiogenin and / or mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin degradation product of 0.3 did.
[Example 2]

An angiogenin fraction of Reference Example 1 0.73 mg, an angiogenin fraction of Reference Example 2 92.33 mg and a lactoperoxidase fraction 6.94 mg of Reference Example 3 were mixed, and angiogenin and / or angiogenin degradation product A protein material (Example product 2) having a content of 2.5 mg / 100 mg and a mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product is 2.5 did.
[Example 3]

  1. Angiogenin fraction of Reference Example 1 1.83 mg, Angiogenin fraction of Reference Example 2 42.61 mg and lactoperoxidase fraction 55.56 mg of Reference Example 3 were mixed together, and angiogenin and / or angiogenin degradation product A protein material (Example product 3) having a content of 2.5 mg / 100 mg and a mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product was 20 was prepared.

[Comparative Example 1]
The angiogenin fraction of Reference Example 1 0.57 mg, the Angiogenin fraction 99.15 mg of Reference Example 2 and the lactoperoxidase fraction 0.28 mg of Reference Example 3 were mixed to produce angiogenin content and / or angiogenin degradation. A protein material (Comparative Example Product 1) having a mass of 2.5 mg / 100 mg and a mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product was 0.1 .

[Comparative Example 2]
Angiogenin fraction of Reference Example 1, 2.08 mg, Angiogenin fraction of Reference Example 3 31.25 mg, and Lactoperoxidase fraction 66.67 mg of Reference Example 3 were mixed to produce angiogenin and / or angiogenin degradation product. A protein material (Comparative Example Product 2) having a content of 2.5 mg / 100 mg and a mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product was 24 was prepared.

[Test Example 1]
With respect to Examples 1 to 3 and Comparative Examples 1 and 2, the osteoblast proliferation effect, the effect of suppressing bone resorption by osteoclasts, and the effect of suppressing osteoclast differentiation were investigated.
The osteoblast proliferation effect was examined as follows. The established osteoblast (MC3T3-E1) was seeded in a 96-well plate cell culture plate so as to be 2 × 10 ³ cells / well, and cultured in α-MEM medium containing 10% fetal bovine serum for 24 hours. After removing all of the medium, 90 μl of α-MEM medium not containing fetal calf serum was added, 10 μl of each of the solutions in which Examples 1 to 3 and Comparative Examples 1 and 2 were dissolved was further added for 24 hours. The culture was continued. Bromodeoxyuridine (BrdU) attached to Cell Proliferation kit (manufactured by GE Healthcare) was added and incubated for 2 hours, followed by reaction with peroxidase-labeled anti-BrdU antibody, and the substrate 3,3 ′, 5,5′-tetra By adding methylbenzidine and measuring the absorbance at 450 nm, the amount of BrdU incorporated into the cells was measured to determine the osteoblast proliferation activity. When the absorbance of the added group was significantly higher than the absorbance at 450 nm of the group (control) in which Examples 1 to 3 and Comparative Examples 1 and 2 were not added to the medium, osteoblast proliferation activity Positive.

The effect of inhibiting bone resorption by osteoclasts was examined as follows. After removing tibia and femur from 5-day-old rabbits and removing soft tissue, 1 × 10 ⁶ total bone marrow cells including osteoclasts mechanically minced in α-MEM medium containing 5% FBS were obtained. The cells were plated on the wells of a crystalline calcium phosphate plate (Cornig) so as to be cells / well and cultured. After 2 hours of culturing, all the medium was removed, and α-MEM medium containing 5% FBS was added by 180 μl each, and 20 μl each of the solutions in which Examples 1 to 3 and Comparative Examples 1 and 2 were dissolved were added. Cultured for 72 hours. After removing cells by adding 5% sodium hypochlorite solution, the bone resorption pits (pits) formed on the wells of the calcium phosphate plate were photographed under a stereomicroscope, and the area was measured by image analysis. Thus, the effect of suppressing bone resorption by osteoclasts was examined (Seno Junji et al., Animal Culture Cell Manual by Research Theme, pp. 199-200, 1993). When the pit area of the added group was significantly lower than the pit area of the group to which Examples 1 to 3 and Comparative Examples 1 and 2 were not added to the medium (control), osteoclast bone resorption Suppressive activity was positive.

The effect of inhibiting osteoclast differentiation was examined as follows. Bone marrow cells collected from the femurs of ddy mice (7 or 8 weeks old, male) were seeded in 96-well plates to 4 × 10 ⁴ cells / well, and 10% FBS containing 25 ng / ml M-CSF. The culture was performed in 200 μl of a containing α-MEM medium (GIBCO) at 37 ° C. and 5% CO ₂ . After 2 days of culture, the culture medium was removed, and α-MEM medium containing 10% FBS containing 5 ng / ml RANKL and 25 ng / ml M-CSF was added at 180 μl / well, and Examples 1 to 3 and Comparative Examples 1 and 2 were added. After adding 20 μl of the solution in which the solution was dissolved, the cells were cultured under the conditions of 37 ° C. and 5% CO ₂ for 2 days, and then the medium was changed and further cultured for 1 day. After completion of the culture, the culture solution was removed, washed with PBS, fixed with an acetone-ethanol (1: 1) solution for 1 minute, and then 1.5 mg / ml disodium p-nitrophenyl phosphate- 100 μl / well of 20 mM sodium tartrate-50 mM citrate buffer (pH 4.5) was added and reacted at room temperature for 30 minutes. After stopping the reaction by adding 50 μl / well of 1M sodium hydroxide solution, the absorbance at 405 nm was measured and used as an index of osteoclast differentiation and maturation. Inhibition of osteoclast differentiation when the absorbance of the added group was significantly lower than the absorbance at 405 nm of the group (control) to which Examples 1 to 3 and Comparative Examples 1 and 2 were not added to the medium. The activity was positive.
These results are shown in Table 1.

From the results of Table 1, Examples 1 to 3 which are protein materials of the present invention were positive in all cell assay activities. In contrast, in Comparative Examples 1 and 2, the activity of some cell assays was positive, but there were also negative cell assays.
[Example 4]

A column (diameter 5 cm × height 30 cm) packed with 600 g of a cation exchange resin sulfonated chitopearl (Fuji Boseki Co., Ltd.) was thoroughly washed with deionized water, and 40 L of unsterilized skim milk (pH 6.7) was added to this column. At a flow rate of 25 ml / min. After passing through the column, the column was thoroughly washed with deionized water, and the protein adsorbed on the resin was eluted with a 0.02 M carbonate buffer solution (pH 7.0) containing 0.78 M sodium chloride. The eluate was desalted with a reverse osmosis membrane and then freeze-dried to obtain 18 g of a powdered protein material (Example Product 4). This protein material has an angiogenin and / or angiogenin degradation product content of 2 mg / 100 mg, and a mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product is 18. It can be used as it is as a bone reinforcing agent or as an active ingredient of a bone reinforcing agent. As a result of proteome analysis, this protein material contained degradation products of β-casein and κ-casein.
[Example 5]

A column packed with 30 kg of cation exchange resin SP Toyopearl (Tosoh Corporation) (diameter 20 cm × height 100 cm) was thoroughly washed with deionized water, and then whey sterilized by heating at 75 ° C. for 15 seconds. 3t (pH 6.2) was passed at a flow rate of 10 L / min. After passing through the column, the column was thoroughly washed with deionized water, and the protein adsorbed on the resin was eluted with 0.1 M citrate buffer (pH 5.7) containing 0.68 M sodium chloride. The eluate was desalted with an electrodialysis membrane and then freeze-dried. This series of operations was repeated 20 times to obtain 3.3 kg of a powdery protein material (Example Product 5). This protein material has an angiogenin and / or angiogenin degradation product content of 15 mg / 100 mg, and a mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product is 0.8. And can be used as it is as a bone strengthening agent or as an active ingredient of a bone strengthening agent. As a result of proteome analysis, this protein material contained degradation products of αs1 casein and κ casein.
[Example 6]

4 g of the protein material of Example product 4 was dissolved in 800 ml of water, pancreatin (manufactured by Sigma) as a proteolytic enzyme was added to a final concentration of 0.02% by weight, and the enzyme treatment was performed at 37 ° C. for 8 hours. And after heat-processing at 90 degreeC for 5 minute (s) and deactivating an enzyme, it lyophilized | freeze-dried and 3.2 g of protein raw materials were obtained (Example product 6). The protein material thus obtained has an angiogenin degradation product content of 2.0 mg / 100 mg, a mass ratio of the lactoperoxidase degradation product to the angiogenin degradation product of 16, and a molecular weight of 8,000 or less. Therefore, it can be used as it is as a bone reinforcing agent or an active ingredient of a bone reinforcing agent. As a result of proteome analysis, this protein material contained degradation products of β-casein and κ-casein.
[Example 7]

  4 g of the protein material of Example product 5 was dissolved in 800 ml of water, and trypsin (manufactured by Sigma) as a proteolytic enzyme was added to a final concentration of 0.03% by weight, followed by enzyme treatment at 37 ° C. for 8 hours. And after heat-processing at 90 degreeC for 5 minute (s) and deactivating an enzyme, it lyophilized | freeze-dried and obtained 3.0 g of protein raw materials (Example goods 7). The protein material thus obtained has an angiogenin degradation product content of 14 mg / 100 mg, a mass ratio of lactoperoxidase degradation product to an angiogenin degradation product of 0.7, and a molecular weight of 8,000 or less. Therefore, it can be used as it is as a bone reinforcing agent or an active ingredient of a bone reinforcing agent. As a result of proteome analysis, this protein material contained degradation products of αs1 casein and κ casein.

[Comparative Example 3]
10 mg of the lactoperoxidase fraction of Reference Example 3 and 100 mg of the protein material of Example Product 4 are mixed, the content of angiogenin and / or angiogenin degradation products is 1.8 mg / 100 mg, and angiogenin and / or angiogenin A protein material (comparative product 3) having a mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to degradation product of 22.5 was prepared.

[Comparative Example 4]
Trypsin (produced by Sigma), which is a proteolytic enzyme, is prepared by mixing 1 g of the angiogenin fraction of Reference Example 1 and 2 g of the protein material of Example Product 5 and dissolving in 800 ml of water to a final concentration of 0.02% by weight. ) And enzyme-treated at 37 ° C. for 12 hours. And after heat-processing at 90 degreeC for 5 minute (s) and deactivating an enzyme, it lyophilized | freeze-dried and obtained 2.8g of protein raw materials (comparative example goods 4). The protein material thus obtained had an angiogenin degradation product content of 39 mg / 100 mg, and the mass ratio of the lactoperoxidase degradation product to the angiogenin degradation product was 0.2.

[Comparative Example 5]
A column (diameter 5 cm × height 5 cm) packed with 100 g of a cation exchange resin CM Sepharose FF (GE Healthcare) was thoroughly washed with deionized water, and then this column was subjected to non-sterilized skim milk (pH 6.7). 40 L was passed at a flow rate of 40 ml / min. After passing through the column, the column was thoroughly washed with deionized water, and the protein adsorbed on the resin was eluted with 0.02 M carbonate buffer (pH 6.8) containing 0.98 M sodium chloride. The eluate was desalted with a reverse osmosis membrane and then freeze-dried to obtain 20 g of a powdered protein material (Comparative Product 5). This protein material has an angiogenin and / or angiogenin degradation product content of 1.5 mg / 100 mg, and a mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product is 30. Met.

[Test Example 2]
The bone strengthening effects of Examples 4 and 5 and Comparative Examples 3 and 5 were examined by animal experiments. For the experiment, C3H / HeJ male mice aged 5 weeks were used. After one week of pre-breeding, the mice were divided into 5 groups of 6 mice, and Examples 4 and 5 and Comparative Examples 3 and 5 were orally administered once a day with a sonde to give 5 mg per kg of mouse body weight. And raised for 4 weeks. In addition, those in which Example products 4 and 5 and Comparative product 3 and 5 were not administered were used as a control group. After completion of the administration (week 4), the bone density of the right tibia of the mouse was measured by micro CT (manufactured by Rigaku Corporation). The results are shown in Table 2.

  As shown in Table 2, the bone mineral density was significantly higher in the group in which the examples 4 and 5 of the protein material of the present invention were orally administered for 4 weeks compared to the group to which the control group or the comparative examples 3 and 5 were administered. Rose.

[Test Example 3]
The bone strengthening effect of Examples 6 and 7 and Comparative Examples 4 and 5 was examined by animal experiments. In the experiment, 48 51-week-old SD male rats were used. Rats were divided into 6 groups of 8 rats, 5 groups were subjected to ovariectomy and the remaining 1 group was subjected to sham surgery. A 4-week recovery period was provided, and rats 6 and 7 and comparative products 4 and 5 were orally administered once a day with a sonde to give 5 mg per 1 kg of the body weight of rats. Raised for 16 weeks. The control group was one that did not receive Example Products 6 and 7 and Comparative Example Products 4 and 5. In addition, after the recovery period of 4 weeks, rats subjected to sham operation were bred for 16 weeks as in the control group. After the end of administration (16 weeks), the bone density of the right femur of the rat was measured by micro CT (manufactured by Rigaku Corporation). The results are shown in Table 3.

As shown in Table 3, the bone mineral density was significantly higher in the group in which the examples 6 and 7 of the protein material of the present invention were orally administered for 16 weeks compared to the group in which the control group or the comparative examples 4 and 5 were administered. The value rose to a level close to that of the sham operation group.
[Example 8]

(Preparation of liquid nutrition composition for bone strengthening)
5 g of the protein material of Example Product 4 was dissolved in 4995 g of deionized water, and the mixture was stirred and mixed at 6000 rpm for 30 minutes with a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). A solution containing 100 g was obtained. 5.0 kg of this solution, 4.0 kg of casein, 5.0 kg of soy protein, 1.0 kg of fish oil, 3.0 kg of perilla oil, 18.0 kg of dextrin, 6.0 kg of mineral mixture, 1.95 kg of vitamin mixture, 2.0 kg of emulsifier 4.0 kg of stabilizer and 0.05 kg of fragrance are blended and filled into a 200 ml retort pouch, and the retort sterilizer (type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Nisaka Seisakusho) at 121 ° C., 20 Sterilized for a minute to produce 50 kg of a liquid nutritional composition for bone strengthening. In the thus obtained liquid nutrition composition for strengthening bone, no precipitation or the like was observed, and no abnormal flavor was felt.
[Example 9]

(Preparation of gel food for bone strengthening)
2 g of the protein material of Example product 5 was dissolved in 708 g of deionized water, and the mixture was stirred and mixed at 9500 rpm for 30 minutes with an ultradisperser (ULTRA-TURRAX T-25; manufactured by IKA Japan). To this solution, 40 g of sorbitol, 2 g of acidulant, 2 g of fragrance, 5 g of pectin, 5 g of whey protein concentrate, 1 g of calcium lactate and 235 g of deionized water were added and mixed with stirring. After sterilizing at 20 ° C. for 20 minutes, it was tightly sealed to prepare 5 bags (200 g) of gel food for bone strengthening. In the thus obtained bone-strengthening gel food, no precipitation was observed, and no abnormal flavor was felt.
[Example 10]

(Preparation of bone strengthening beverage)
After 2 g of the sour agent was dissolved in 706 g of deionized water, 4 g of the protein material of Example product 6 was dissolved and stirred for 30 minutes at 9500 rpm with an ultradisperser (ULTRA-TURRAX T-25; manufactured by IKA Japan). Mixed. After adding 100 g maltitol, 20 g reduced starch syrup, 2 g fragrance, and 166 g deionized water, it is filled into a 100 ml glass bottle, sterilized at 95 ° C. for 15 seconds, sealed, and prepared 10 bone-strengthening beverages (100 ml). did. In the bone-strengthening beverage thus obtained, no precipitation or the like was observed, and no abnormal flavor was felt.
[Example 11]

(Preparation of bone strengthening feed)
2 kg of the protein material of Example Product 7 was dissolved in 98 kg of deionized water, and the mixture was stirred and mixed at 3600 rpm for 40 minutes with a TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.). Of 2 g / 100 g was obtained. 10 kg of this solution is mixed with 12 kg of soybean meal, 14 kg of skim milk powder, 4 kg of soybean oil, 2 kg of corn oil, 23.2 kg of palm oil, 14 kg of corn starch, 9 kg of wheat flour, 2 kg of bran, 5 kg of vitamin mixture, 2.8 kg of cellulose, and 2 kg of mineral mixture. Then, it was sterilized at 120 ° C. for 4 minutes to produce 100 kg of dog-raising feed for bone reinforcement.
[Example 12]

(Preparation of bone strengthening agent (tablet))
After mixing the raw materials with the formulation shown in Table 4, it was molded into 1 g and tableted according to a conventional method to produce a bone strengthening agent.

Claims (6)

  2 to 15 mg / 100 mg of angiogenin and / or angiogenin degradation product, and the mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product is 0.3 to 0.3 A protein material for preventing or treating bone diseases, which is contained in 20 ranges.   A food or drink or feed for the prevention or treatment of a bone disease comprising the protein material according to claim 1.   A bone strengthening agent for preventing or treating a bone disease comprising the protein material according to claim 1 as an active ingredient. The method for producing a protein material for prevention or treatment of bone disease according to claim 1, comprising the following steps 1) to 3).
1) A step of preparing angiogenin and / or an angiogenin degradation product.
2) A step of preparing lactoperoxidase and / or a degradation product of lactoperoxidase.
3) The mass ratio of the lactoperoxidase and / or the lactoperoxidase degradation product prepared in the above step 2) to the angiogenin and / or angiogenin degradation product prepared in the above step 1) is 0.3. The process of mix | blending so that it may become ~ 20.   Angiogenin and / or angiogenin so that the mass ratio of lactoperoxidase and / or lactoperoxidase degradation product to angiogenin and / or angiogenin degradation product is 0.3 to 20 from milk and / or milk raw material. The method for producing a protein material for preventing or treating bone disease according to claim 1, comprising a step of extracting a fraction containing a degradation product and lactoperoxidase and / or a lactoperoxidase degradation product. The method for producing a protein material for preventing or treating bone disease according to claim 5 , further comprising the step of enzymatically degrading angiogenin and / or lactoperoxidase in the fraction containing angiogenin and lactoperoxidase.