NZ704911B2 - Protein material comprising angiogenin and lactoperoxidase - Google Patents

Abstract

The present invention addresses the problem of providing a safe and novel protein material which is useful in the prevention and treatment of various bone disorders such as osteoporosis, bone fractures, rheumatism, and arthritis when taken on a daily basis. The present invention also addresses the problem of providing bone-strengthening food and beverages or feed which is useful in the prevention and treatment of various bone disorders such as osteoporosis, bone fractures, rheumatism, and arthritis when taken orally. A protein material containing 2 to 15 mg/100 mg of angiogenin and/or an angiogenin degradation product, and lactoperoxidase and/or a lactoperoxidase degradation product at a mass ratio of 0.3 to 20 relative to the angiogenin and/or angiogenin degradation product. It is possible to strengthen bones and to prevent and treat various bone disorders such as osteoporosis, bone fractures, rheumatism, and arthritis by taking said protein material. roblem of providing bone-strengthening food and beverages or feed which is useful in the prevention and treatment of various bone disorders such as osteoporosis, bone fractures, rheumatism, and arthritis when taken orally. A protein material containing 2 to 15 mg/100 mg of angiogenin and/or an angiogenin degradation product, and lactoperoxidase and/or a lactoperoxidase degradation product at a mass ratio of 0.3 to 20 relative to the angiogenin and/or angiogenin degradation product. It is possible to strengthen bones and to prevent and treat various bone disorders such as osteoporosis, bone fractures, rheumatism, and arthritis by taking said protein material.

Description

SNOW-192 PROTEIN MATERIAL COMPRISING ANGIOGENIN AND LACTOPEROXIDASE CAL FIELD This invention relates to a novel n material, and a drug, food, drink, or feed that includes the protein material and is useful for prevention and treatment of bone diseases. The protein material has functions of promoting osteoblast proliferation, and suppressing osteoclast differentiation and osteoclastic bone resorption. Therefore, the n material is useful for prevention and treatment of various bone diseases, such as osteoporosis, fracture, rheumatism, and arthritis.

BACKGROUND ART In recent years, various bone diseases, such as osteoporosis, fracture, and backache have increased on a global basis along with aging of society and the like, and have become a serious social m. These diseases are caused by insufficient calcium , sion of calcium absorption ability, hormone imbalance after menopause, and the like. It is considered that increase the body bone mass as much as possible by activating the osteoblast and bone formation from the early stage of life, and increase the maximum bone mass and the bone strength (bone density + bone quality) is ive in preventing various bone diseases, such as osteoporosis, re, and backache. Note that the term “bone quality” refers to the bone microstructure, metabolic turnover, microfracture, and calcification. It is thought that various bone diseases, such as osteoporosis, fracture, and backache may be prevented by suppressing osteoclastic bone tion. Bones are repeatedly resorbed and formed in a balanced manner (remodeling). However, various bone diseases, such as osteoporosis, re, and backache may occur when bone resorption exceeds bone formation due to a change SNOW-192 in hormone balance after menopause, and the like. Therefore, bones can be strengthened by suppressing osteoclastic bone resorption and maintaining the bone strength at a constant level.

In View of the above situation, a drug, food, drink, feed, or the like, in which a calcium salt, such as calcium carbonate, calcium phosphate, or calcium lactate or a natural calcium t, such as Whey calcium, bovine bone powder, or eggshell is added individually, has been administered in order to strengthen bones. A drug, food, drink, feed, or the like that contains such a calcium product together with a substance having a calcium absorption-promoting , such as casein phosphopeptide or oligosaccharide has also been used to strengthen bones. r, the calcium tion rate is 50% or less when a food or drink that contains a calcium salt or a natural calcium product is administered, and the large part of the calcium administered may be discharged from the body without being absorbed. Moreover, even if calcium is absorbed into the body, it does not necessarily exhibit the bone metabolism-improving effect or a bone-strengthening eifect, since the affinity to bones may differ according to its form or the type of nutritional ingredient administered together. An estrogen product, an active n D3 product, a vitamin K2 product, a bisphosphonate product, a onin product, and the like have been known as a drug for ng osteoporosis or thening bones, and new drugs such as an anti—RANKL antibody have been also developed. However, these drugs may have side effects such as buzzing in the ear, a headache, or loss of appetite. Moreover, the above substances are in a situation that they cannot be added to a food or drink at present fiom the viewpoint of safety, cost, and the like. ore, in light of the nature of various bone diseases, such as osteoporosis, fracture, and backache, development of such a bone—strengthening agent, food, drink, or feed that can be administered orally for a long time, increases the bone strength by promoting bone ion and suppressing bone SNOW-192 resorption, and may be expected to have the effect of preventing or treating the various bone diseases has been desired.

There are l food materials that intends to improve the bone strength, for example, it has been reported that a basic protein derived from milk or a peptide fraction of an enzymatically degraded t thereof exhibits osteoblast proliferation activity, osteoclastic bone resorption suppression activity, and thus a bone-strengthening effect (see Patent Document 1). It has also been reported that angiogenin and lactoperoxidase, contained in a basic protein fraction derived from milk, independently have a function to improve the bone metabolism (see Patent Documents 2 to 4).

PRIOR-ART NT PATENT DOCUMENT t Document 1] JP-A-H08-151331 [Patent Document 2] JP-A-H10-7585 [Patent Document 3] JP-A238320 t Document 4] JP-A60321 SUMMARY OF THE INVENTION The ion relates to provide a novel protein material that is safe, promotes osteoblast proliferation while suppressing osteoclast differentiation and osteoclastic bone resorption by administering daily, and thus can strengthen bones.

The invention relates to provide a bone-strengthening drug, food, drink, or feed that is useful for prevention and treatment of various bone es, such as osteoporosis, fracture, rheumatism, and tis by administering orally.

SNOW-192 The inventors have found that the effects of effectively promoting osteoblast proliferation, and suppressing osteoclast differentiation and osteoclastic bone resorption can be obtained by administering a protein material that includes enin and/or angiogenin hydrolysate in a specific , and further es lactoperoxidase and/or lactoperoxidase hydrolysate in a specific mass ratio with respect to angiogenin and/or angiogenin hydrolysate. This finding has led to the completion of the invention. ically, the invention es the following aspects: (1) A protein material comprising: angiogenin and/or angiogenin hydrolysate in an amount of 2 to 15 mg/100 mg of the protein material and eroxidase and/or lactoperoxidase hydrolysate in the mass ratio to the angiogenin and/or angiogenin hydrolysate of 0.3 to 20. (2) A food, drink, or feed including the protein material according to (1). (3) A bone-strengthening agent including the protein material according to (1) as an active ingredient. (4) A method of strengthening bones including administering the protein material according to (1) in amount of 5 mg/day or more. (4a) Use of (i) angiogenin and/or angiogenin hydrolysate and (ii) lactoperoxidase and/or lactoperoxidase hydrolysate in the manufacture of a medicament for the strengthening of bones, n the angiogenin and/or angiogenin hydrolysate is in an amount of 2 to 15 mg/100 mg of the medicament and wherein the lactoperoxidase and/or lactoperoxidase hydrolysate is in the mass ratio of 0.3 to 20 to the angiogenin and/or angiogenin hydrolysate. (4b) Use of (i) enin and/or angiogenin hydrolysate and (ii) lactoperoxidase and/or lactoperoxidase hydrolysate in the manufacture of a medicament SNOW-192 for the prevention or treatment of orosis, fracture, rheumatism and/or arthritis, n the angiogenin and/or angiogenin hydrolysate is in an amount of 2 to 15 mg/100 mg of the medicament and wherein the lactoperoxidase and/or lactoperoxidase hydrolysate is in the mass ratio of 0.3 to 20 to the angiogenin and/or angiogenin hydrolysate (5) A method of preparing the protein material ing to (1), including the following steps of 1) to 3): 1) preparing angiogenin and/or angiogenin hydrolysate; 2) preparing lactoperoxidase and/or lactoperoxidase hydrolysate; and 3) mixing the eroxidase and/or lactoperoxidase hydrolysate according to above 2) and the angiogenin and/or angiogenin hydrolysate according to above 1) in the mass ratio to the angiogenin and/or angiogenin hydrolysate of 0.3 to 20. (6) A method of producing the protein al according to (1), comprising a step of extracting a fraction containing angiogenin and/or angiogenin hydrolysate and lactoperoxidase and/or lactoperoxidase hydrolysate from milk and/or a material derived from milk in the mass ratio to the angiogenin and/or the angiogenin hydrolysate of 0.3 to 20. (7) The method according to (6), further comprising another step of enzymatically degrading the angiogenin and/or the lactoperoxidase contained in the fraction.

EFFECTS OF THE INVENTION The protein material of the invention exhibits a remarkable bone-strengthening effect through the function of promoting osteoblast proliferation, and suppressing osteoclast differentiation and osteoclastic bone resorption. The drug, food, drink, or feed of the invention strengthens bones, and is useful for prevention and treatment of various bone diseases, such as osteoporosis, fracture, rheumatism, and arthritis.

EMBODIMENTS FOR CARRYING OUT THE ION A protein material of the invention is characterized in that the n material includes angiogenin and/or angiogenin hydrolysate in a specific amount, and further includes lactoperoxidase and/or lactoperoxidase hydrolysate in a ic mass ratio with respect to angiogenin and/or angiogenin hydrolysate.

The n material of the invention may be a mixture obtained by mixing a on containing angiogenin and/or angiogenin hydrolysate and a fraction containing lactoperoxidase and/or lactoperoxidase hydrolysate in a ic mass ratio, a material prepared by ly extracting a on containing angiogenin and/or angiogenin hydrolysate and lactoperoxidase and/or lactoperoxidase hydrolysate in a specific mass SNOW—1 92 ratio from milk or a material derived from milk, such as skim milk or whey, or the like.

The protein material of the invention may also include a material prepared by enzymatically degrading angiogenin and/or eroxidase.

When ing the protein material of the invention by mixing a fraction containing angiogenin and/0r angiogenin hydrolysate and a fraction ning lactoperoxidase and/or lactoperoxidase hydrolysate, a fraction prepared from milk of a mammal, such as human, cow, buffalo, goat, or sheep, a fraction produced by genetic ering, a fraction purified from blood or an internal organ, or the like may be used as the fraction containing angiogenin and/or angiogenin ysate and the on containing lactoperoxidase and/or eroxidase ysate. A commercially available purified angiogenin or lactoperoxidase reagent may also be used. In this case, the protein material of the invention may be prepared by adjusting the mass ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to angiogenin and/or enin hydrolysate.

A product obtained by enzymatically degrading the above fraction containing angiogenin, the angiogenin reagent, the fraction containing lactoperoxidase, the lactoperoxidase t, or the like using one or more proteases may be used as angiogenin hydrolysate or lactoperoxidase hydrolysate.

When preparing the protein material of the invention by directly extracting a fraction which contains angiogenin and/or angiogenin hydrolysate and lactoperoxidase and/0r lactoperoxidase hydrolysate in a specific mass ratio from milk or a material derived from milk, such as skim milk or whey, for example, milk or a material derived from milk may be brought into t with a cation-exchange resin, and then milk-derived proteins adsorbed on the resin may be eluted at a salt concentration of 0.1 to 2.0 M, desalted and concentrated using a reverse osmosis membrane, an SNOW—192 electrodialysis membrane, an ultrafiltration membrane, a microfiltration membrane, or the like, after that optionally subjected to limited degradation to a molecular weight of 8000 or less using a protease, such as trypsin, atin, rypsin, , papain, kallikrein, cathepsin, thennolysin, or V8 protease, When subjecting to limited ation using a protease, it is preferable that the lower limit of the molecular weight is 500 or more. The protein material thus obtained may be dried by freeze-drying, spray drying, or the like.

When subjecting the protein material of the invention to LC/MS/MS analysis, after subjecting to modification and limited degradation under reducing condition using a ive enzyme in the usual manner in order to carry out a proteome analysis of the protein material, it was confirmed that the protein al contained at least one of protein such as asl—casein, usZ—casein, B-casein, or K-casein, and proteolysis product thereof other than angiogenin and/0r angiogenin ysate and lactoperoxidase and/or lactoperoxidase hydrolysate.

The protein al of the invention includes angiogenin and/or angiogenin hydrolysate in an amount of 2 to 15 mg/l 00 mg, and includes lactoperoxidase and/or lactoperoxidase hydrolysate in the mass ratio of 0.3 to 20 to angiogenin and/0r angiogenin hydrolysate.

As shown in the test examples described below, when the mass ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to angiogenin and/or angiogenin hydrolysate is 0.3 to 20, the trengthening effect can be obtained more effectively than the case of administering angiogenin and/0r angiogenin hydrolysate or lactoperoxidase and/01' lactoperoxidase hydrolysate separately.

Note that, for reference only, the content of angiogenin and/or angiogenin hydrolysate in cow milk is about 0.001%, and the mass ratio of lactoperoxidase and/or SNOW-192 lactoperoxidase hydrolysate to angiogenin and/or angiogenin hydrolysate in cow milk is about 20. The t of enin and/or angiogenin hydrolysate in a whey protein concentrate (WPC) is about 0.1%, and the mass ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to angiogenin and/or angiogenin hydrolysate in a Whey protein concentrate is about 30. [001 5] The protein material of the invention may be prepared as a bone—strengthening agent by appropriately adding the protein material as an active ingredient. The protein material of the invention may be used directly as a bone-strengthening agent. When formulating as a bone-strengthening agent, it may be possible to mix a raw material or the like that is y used for drugs, food, drink, and feed, such as a saccharide, a lipid, a protein, a vitamin, a mineral, or a flavor, and it may be also possible to formulate into a powdered drug, es, a tablet, a capsule, a drinkable preparation, or the like in the usual manner. The protein material of the invention may be used together with another ingredient that also ts a bone—strengthening , such as calcium, vitamin D, vitamin K, or isoflavone.

The protein material of the invention can strengthen bones when administered orally in an amount of 5 mg or more per kg of body weight, as shown in the animal experiments described below. Since the intake for this . experimental animal corresponds to the intake for adults in terms of blood drug concentration (see Mitsuyoshi Nakajima (1993), 014 Hyoka Vol. 8”, Hirokawa—Shoten Ltd, pp. 2-18), it is expected that the bone-strengthening effect is obtained, and especially various bone diseases, such as osteoporosis, fracture, tism, and arthritis can be ted or treated by ingesting the protein material of the invention in an amount of 5 mg/day or more per an adult. Therefore, when mixing to a bone-strengthening agent or the like, the protein al may be added thereto so as to ingest the above necessary amount.

SNOW—1 92 The protein material of the invention may be added to a normal food or drink, such as yogurt, beverage, wafer, or dessert). In this case, the protein material of the invention is preferably added in an amount of 0.25 to 1000 mg per 100 g of the food or drink depending on the form of the food or drink. It is expected that the bone-strengthening effect can be obtained by keeping the above mixing amount. The protein material of the invention may also be added to a feed, such as ock feed or pet food to prepare a bone-strengthening feed. In this case, it is able to add the protein material of the invention in an amount of 0.25 to 1000 mg per 100 g ofthe feed.

When the protein material of the invention is prepared and used in the form of a drug, food, drink, or feed, the protein material of the invention may be used by suspending or dissolving in deionized water, and mixing with stirring. The stirring/mixing conditions are not particularly limited as long as the protein al is uniformly mixed. It is also possible to mix with stirring using an ultra-disperser, a TK-homomixer, or the like.

The solution of the protein material may optionally be desalted or concentrated using a reverse osmosis membrane or the like, or freeze-dried so that the solution can be easily used for a drug, food, drink, or feed.

Notably, it was confirmed that the protein material of the invention ins the bone-strengthening activity even when the protein al is subjected to sterilization treatment that is ly used in the production of a drug, food, drink, or feed. The protein material may be subjected to dry—heat sterilization when the protein material is used as a powder. The protein material of the invention may be used for a drug, food, drink, or feed in various forms, such as , gel, powder, or granular. [001 8] The invention is further described below in more detail by way of reference examples, examples, and test examples. Note that the following examples are intended SNOW—l 92 for illustration es only, and should not be construed as limiting the invention.

Reference Example 1 Preparation (1) of enin fraction A column filled with 30 kg of cation-exchange resin (Sulfonated Chitopearl; manufactured by Fuji Spinning Co., Ltd.) was thoroughly washed with deionized water, and 1000 liters of unpasteurized skim milk (pH 6.7) was then applied to the column.

After thoroughly washing the column with deionized water, the absorbed protein was eluted with a linear gradient of 0.1 to 2.0 M sodium chloride. The elution fraction containing angiogenin was fractionated using an S-Sepharose cation-exchange chromatography (manufactured by Amersham Bioscientific), and the resulted angiogenin—containing fraction was heat-treated at 90°C for 10 minutes, and centrifuged to remove a precipitate. The angiogenin-containing fraction was further subjected to gel filtration tography (column: Superose 12). The eluate obtained was desalted using a reverse osmosis membrane, and the desalted eluate was freeze—dried to obtain 16.5 g of an angiogenin fraction having an enin purity of 90%. These successive operations were repeated 30 times.

Reference Example 2 Preparation (2) of enin fraction A column filled with 10 kg of Heparin Sepharose (manufactured by GE care) was thoroughly washed with deionized water, and 1000 liters of unpasteurized skim milk (pIi 6.7) was then applied to the column.” After thoroughly g the column with a 0.6 M sodium chloride solution, the absorbed protein was eluted with a 1.5 M sodilun chloride solution. The eluate was desalted using a reverse osmosis ne, and the ed eluate was freeze-dried to obtain 32 g of an angiogenin fraction having an angiogenin purity of 2%. The above successive SNOW—1 92 ions were repeated 50 times.

Reference Example 3 Preparation of lactoperoxidase fraction A column (diameter: 5 cm, height: 30 cm) filled with 600 g of cation-exchange resin (Sulfonated Chitopearl; manufactured by Fuji Spinning Co., Ltd.) was thoroughly washed with deionized water, and 360 liters of unpasteurized skim milk (pH 6.7) was applied to the column at a flow rate of 25 ml/min. Afier thoroughly washing the column with deionized water, the absorbed protein was eluted with a 0.02 M carbonate buffer (pH 7.0) containing 2.0 M sodium de. The eluted fraction containing lactoperoxidase was adsorbed on an S-Sepharose FF column (manufactured by Amersham Bioscientific), and the column was ghly washed with zed water.

After equilibration with a 10 mM phosphate buffer (pH 7.0), the adsorbed fraction was eluted with a linear gradient of 0 to 2.0 M sodium chloride to collect a fraction containing lactoperoxidase. The fraction was subjected to gel filtration tography using a HiLoad 16/60 Superdex 75pg actured by Amersham entific). The eluate obtained was desalted using a reverse osmosis membrane, and freeze-dried to obtain 27 g of a lactoperoxidase fraction having a eroxidase purity of 90%. These successive operations were repeated 25 times.

Example 1 Zero point five nine milligrams (0.59 mg) of the angiogenin on obtained in Reference Example 1, 98.58 mg of the angiogenin fraction obtained in Reference Example 2, and 0.83 mg of the lactoperoxidase fraction obtained in Reference Example 3 were mixed to prepare a protein material (example product 1), in which the content of angiogenin and/or angiogenin hydrolysate was 2.5 mg/100 mg, and the mass ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to angiogenin and/or angiogenin SNOW—192 hydrolysate was 0.3.

Example 2 Zero point seven three rams (0.73 mg) of the angiogenin fraction obtained in Reference Example 1, 92.33 mg of the angiogenin fraCtion obtained in nce Example 2, and 6.94 mg of the lactoperoxidase fraction obtained in nce Example 3 were mixed to prepare a protein material (example product 2), in which the content of angiogenin and/or angiogenin hydrolysate was 2.5 mg/100 mg, and the mass ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to enin and/or angiogenin hydrolysate was 2.5.

Example 3 One point eight three milligrams (1.83 mg) of the angiogenin fraction obtained in Reference Example 1, 42.61 mg of the angiogenin fraction ed in Reference Example 2, and 55.56 mg of the lactoperoxidase fraction obtained in Reference Example 3 were mixed to e a protein material (example product 3), in which the content of angiogenin and/or angiogenin hydrolysate was 2.5 mg/l 00 mg, and the mass ratio of lactoperoxidase and/or Iactoperoxidase hydrolysate to angiogenin and/or angiogenin hydrolysate was 20.

Comparative Example 1 Zero point five seven (0.57 mg) of the angiogenin fraction ed in Reference Example 1, 99.15 mg of the angiogenin fraction obtained in Reference Example 2, and 0.28 mg of the lactoperoxidase fraction obtained in Reference Example 3 were mixed to prepare a protein material (comparative example product 1), in which the t of angiogenin and/or angiogenin hydrolysate was 2.5 mg/100 mg, and the mass ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to angiogenin and/or angiogenin SNOW-192 hydrolysate was 0.1.

Comparative Example 2 Two point zero eight milligrams (2.08 mg) of the angiogenin fraction obtained in nce Example 1, 31.25 mg of the angiogenin on obtained in Reference Example 2, and 66.67 mg of the lactoperoxidase fraction obtained in Reference Example 3 were mixed to e a protein material (comparative example product 2), in which the content of angiogenin and/or angiogenin hydrolysate was 2.5 mg/100 and the mass ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to angiogenin and/or enin hydrolysate was 24.

Test Example 1 The osteoblast proliferation effect, the suppressive effect on osteoclastic bone resorption, and the suppressive effect on osteoclast differentiation of the example products 1 to 3 and the comparative example products 1 and 2 were ined.

The osteoblast proliferation effect was determined as described below. An osteoblastic cell line (MC3T3—E1) was seeded on a l cell culture plate at a density of 2X103 cells/well, and cultured for 24 hours using an a—MEM medium (manufactured by GIBCO) supplemented with 10% fetal bovine serum (PBS). After the medium was completely removed, 90 ul of a PBS free a—MEM medium , and 10 pl of a solution containing any of the example products 1 to 3 and the ative example products 1 and 2 is added to each well. The cells were further cultured for 24 hours. After the addition of bromodeoxyuridine (BrdU)which was included in the Cell eration Kit (manufactured by GE Healthcare), the cells were cultured for 2 hours, and reacted with a peroxidase-labelled anti—BrdU antibody. After the addition of 3,3’,5,5’-tetramethylbenzidine (substrate), the osteoblast proliferation activity was determined by ing the amount of BrdU introduced into the cells through SNOW-192 measuring the absorbance at 450 nm. The osteoblast proliferation activity was determined to be positive when the absorbance at 450 nm was significantly higher than that of a group (control), in which none of the example products 1 to 3 and the comparative example products 1 and 2 were added to the medium.

The suppressive effect on osteoclastic bone resorption was determined as described below. The tibia and the one were taken out from a rabbit (5 days old).

After removing the soft tissue, these bones were mechanically chopped and the total bone marrow cells containing the osteoclasts were dispersed in an a-MEM medium mented with 5% FBS, and then seeded on the wells of a crystalline calcium phosphate plate (manufactured by Coming) at a density of 1X106 cells/well. The medium was completely removed at 2 hours after ng the culture, and 180 pl of an a—MEM medium supplemented with 5% FBS, and 20 pl of a solution containing any of the example products 1 to 3 and the ative example products 1 and 2 was added to each well. The cells were cultured for 72 hours. After removing the cells by addition of a 5% sodium hypochlorite solution, tion pits formed on the wells of the calcium phosphate plate were photographed using a stereoscopic microscope, and the area thereof was measured by image analysis to determine the suppressive effect on osteoclastic bone resorption (Takeshi Seno et al., “Manual of selected cultured cell lines for bioscience hnology”, pp. 199-200, 1993). The suppressive activity against osteoclastic bone resorption was determined to be positive when the pit area was significantly smaller than that of a group (control), in which any of the e ts 1 to 3 and the comparative example products 1 and 2 was not added to the medium.

The suppressive effect on osteoclast ditferentiation was ined as described below. The bone marrow cells collected from the thighbone of a ddy mouse (7 or 8 SNOW~192 weeks old, male) were seeded on a 96—well plate at a density of 4X104 well, and cultured in 200 pl of a a—MEM medium supplemented with 10% FBS and M—CSF (25 ng/ml) at 37°C and 5% C02. After the medium was completely removed on 2 days after starting the culture 180 pl of a OL-MEM medium supplemented with 10% FBS, RANKL (5 ng/ml) and M-CSF (25 ng/ml), and 20 pl of a solution containing any of the example products 1 to 3 and the comparative example products 1 and 2 was added to each well, and the cells were cultured under the condition of 37°C and 5% C02 for 2 days. After changing the medium, the cells were further cultured for 1 day. At the completion 'of the culture, the culture solution was d, washed with PBS, and treated with an e—ethanol (1:1) on for 1 minute to fix the cells. After that, 1.5 mg/ml of a disodium p—nitrophenylphosphate—ZO mM sodium tartrate-SO mM citrate buffer (pH 4.5) was added (100 til/well), and reacted at room temperature for 30 minutes, and then, a 1 M sodium hydroxide solution (50 til/well) was added to terminate the reaction. The absorbance at 405 nm was measured, and taken as an index of osteoclast differentiation/mutation. The suppressive activity against osteoclast difierentiation was determined to be positive when the absorbance at 405 nm of group adding example products 1 to 3 or the comparative example products 1 0r 2 was cantly lower than that of a group (control), in which any product of example products 1 to 3 and the comparative example products 1 and 2 was not added to the medium.

The results are shown in Table 1.

TABLE 1 osteoblast suppressive activity suppressive activity proliferation against osteoclastic against osteoclast ty bone resorption differentiation SNOW-192 Emple3 Comparative ositive ositive ne ative example 1 p p g Comparative positive ne example 2 gative positive As shown in Table l, the example ts 1 to 3 which correspond to the protein material of the invention exhibited positive activity in all cell assays. The comparative example products 1 and 2 also ted positive ty in the some cell assays, but there were one cell assay that exhibited negative activity.

Example 4 A column (diameter: 5 cm, height: 30 cm) filled with 600 g of cation—exchange resin (Sulfonated Chitopearl; manufactured by Fuji Spinning Co., Ltd.) was thoroughly washed with deionized water, and 40 liters of unpasteurized skim milk (pH 6.7) was applied to the column at a flow rate of 25 ml/min. After thoroughly washing the column with deionized water, proteins ed on the resin were eluted using a 0.02 M carbonate buffer (pH 7.0) containing 0.78 M sodium chloride. The eluate was desalted using a reverse osmosis membrane, and the desalted eluate was fieeze-dried to obtain 18 g of a y protein materiai (example product 4). The protein al contained angiogenin and/0r angiogenin hydrolysate in an amount of 2 mg/100 mg, and the mass ratio of eroxidase and/or lactoperoxidase ysate to angiogenin and/or enin hydrolysate was 18. The protein material may be used directly as a bone-strengthening agent or an active ingredient of a bone-strengthening agent. As a result of proteome analysis, it was found that the protein material contained degraded product of B-casein and degraded product of K—casein.

Example 5 SNOW-192 A column (diameter: 20 cm, height: 100 cm) filled with 30 kg of cation-exchange resin (SP Toyopearl; manufactured by Tosoh ation) was thoroughly washed with deionized water, and 3 t of whey (pH 6.2) which was heat—sterilized at 75°C for 15 s was applied to the column at a flow rate of 10 l/min. After ghly washing the column with deionized water, proteins adsorbed on the resin were eluted using a 0.1 M citrate buffer (pH 5.7) containing 0.68 M sodium chloride. The eluate was desalted using an electrodialysis membrane, and the desalted eluate was freeze-dried. The above successive operations were ed 20 times to obtain 3.3 kg of a powdery protein material le product 5). The protein material contained angiogenin and/or angiogenin hydrolysate in an amount of 15 mg/100 and the mass ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to angiogenin and/or angiogenin hydrolysate was 0.8. The protein material may be used directly as a bone-strengthening agent or an active ingredient of a bone—strengthening agent. As a result of me is, it was found that the protein material contained degraded products of asl-casein and K—casein.

Example 6 Four grams (4 g) of protein material of the example product 4 was dissolved in 800 ml of water. After the addition ofpancreatin (manufactured by Sigma), which was a protease, at the final concentration of 0.02 wt%, and then the mixture was subjected to tic treatment at 37°C for 8 hours. After inactivating the protease through reatment at 90°C for 5 minutes, the mixture was freeze-dried to obtain 3.2 g of a protein al (example product 6). The protein material thus obtained contained angiogenin hydrolysate in an amount of 2.0 mg/100 mg, and the mass ratio of lactoperoxidase hydrolysate to angiogenin hydrolysate was 16, and the molecular weight of the protein material was 8000 or less. Therefore, the protein material may SNOW—1 92 be used directly as a bone-strengthening agent or an active ient of a bone-strengthening agent. As a result of proteome analysis, it was found that the protein material contained ed products of B—casein and K—casein.

Example 7 Four grams (4 g) of protein material of the example product 5 was dissolved in 800 m1 of water. After the addition of n (manufactured by Sigma), which was a protease, so as to obtain at the final concentration of 0.03 wt%, the mixture was subjected to enzymatic treatment at 37°C for 8 hours. After inactivating the protease through heat-treatment at 90°C for 5 minutes, the mixture was freeze-dried to obtain 3.0 g of a protein material (example product 7). The protein material thus obtained contained angiogenin hydrolysate in an amount of 14 mg/100 mg, and the mass ratio of lactoperoxidase hydrolysate to angiogenin hydrolysate in the protein material was 0.7, and the molecular weight of the protein material was 8000 or less. Therefore, the protein material may be used directly as a bonerstrengthening agent or an active ingredient of a bone-strengthening agent. As a result of proteome analysis, it was found that the n material contained degraded ts of (131 —casein and Ic-casein.

Comparative Example 3 Ten milligrams (10 mg)of the lactoperoxidase fraction obtained in Reference Example 3 and 100 mg of the n al of the example product 4 were mixed to e a protein material (comparative example product 3), in which the content of angiogenin and/0r angiogenin hydrolysate was 1.8 mg/lOO mg, and the mass ratio of lactoperoxidase and/or lactoperoxidase hydrolysate to enin and/or angiogenin hydrolysate was 22.5.

Comparative Example 4 SNOW-192 One gram (1 g) of the enin fraction obtained in Reference Example 1 and 2 g ofthe protein material of the example product 5 were mixed and dissolved in 800 ml of water. After the addition of trypsin (manufactured by Sigma), which is a protease, at the final concentration of 0.02 wt%, the mixture was subjected to enzymatic treatment at 37°C for 12 hours. After inactivating the protease h heat-treatment at 90°C for 5 minutes, the mixture was freeze-dried to obtain 2.8 g of a protein material (comparative example product 4). The protein material thus obtained contained angiogenin hydrolysate in an amount of 39 mg/100 mg, and the mass ratio of eroxidase hydrolysate to angiogenin hydrolysate was 0.2. [003 8] Comparative Example 5 A column (diameter: 5 cm, height: 5 cm) filled with 100 g of cation-exchange resin (CM Sepharose FF; manufactured by GE Healthcare) was thoroughly washed with deionized water, and 40 liters of unpasteurized skim milk (pH 6.7) was applied to the column ata flow rate of 40 ml/min. After thoroughly washing the column with deionized water, proteins adsorbed on the resin were eluted using a 0.02 M carbonate buffer (pH 6.8) containing 0.98 M sodium chloride. The eluate was ed using a e osmosis membrane, and the desalted eluate was freeze-dried to obtain 20 g of a powdery protein al (comparative example product 5). The n material contained angiogenin and/or angiogenin hydrolysate in an amount of 1.5 mg/100 mg, and the mass ratio of eroxidase and/or lactoperoxidase hydrolysate to angiogenin and/or angiogenin hydrolysate was 30.

Test Example 2 Each trengthening effect of the example products 4 and 5 and the comparative example products 3 and 5 were determined by animal experiments.

C3H/HeJ mice (5 weeks old, male) were used for the animal experiments. After 1 SNOW-192 - week acclimation, the mice were divided into five groups (6 mice/group). The mice were orally stered the example products 4 or 5 or the ative example products 3 or 5 in an amount of 5 mg per 1 kg of body weight once a day for 4 weeks using a tube. The control group was not administrated any example products 4 and 5 and the comparative example ts 3 and 5 were not administered. After completion of administration (fourth week), the bone density of the right tibia of each mouse was measured using a micro-CT (manufactured by Rigaku Corporation). The results are shown in Table 2.

TABLE 2 As shown in Table 2, the groups that were orally stered the example product 4 or 5 that were the protein material of the invention showed a significant increase in bone density as compared with the control group and the groups that were orally administered the comparative example t 3 or 5.

Test Example 3 Each bone—strengthening effect of the example products 6 and 7 and the comparative example products 4 and 5 was determined by animal experiments.

Forty—eight SD rats (51 weeks old, female) were used for the animal experiments.

SNOW-192 The rats were divided into six groups (8 rats/group). Five groups underwent ovariectomy, and the remaining one group was subjected to sham surgery. After a 4~week recovery period, the rats underwent ovariectomy were orally administered the example ts 6 or 7 or the comparative example products 4 or 5) in an amount of 5 mg per 1 kg of rat weight once a day for 16 weeks using a tube. The control group was not strated any example products 6 and 7 and the comparative example products 4 and 5. After a 4-week recovery period, the rats underwent sham surgery were fed for 16 weeks in the same manner as the control group. After completion of stration (sixteenth week), the bone density of the right tibia of each rat was measured using a micro-CT (manufactured by Rigaku Corporation). The results are shown in Table 3.

TABLE 3 As shown in Table 3, the groups that were orally administered the example product 6 or 7 that was the n material of the invention showed a significant increase in bone density as ed with the control group and the groups that were orally administered the comparative example product 4 or 5. Moreover, the bone density approached that of the sham surgery group.

SNOW-1 92 Example 8 ation of bone—strengthening liquid nutritional supplement Five grams (5 g) of the protein material of the example product 4 was dissolved in 4995 g of deionized water. The on was stirred at 6000 rpm for 30 minutes using a TK-homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo co., ltd.) to obtain a solution containing the example product 4 in an amount of 100 mg/1 00 g. Then, 4.0 kg of casein, 5.0 kg of a soybean protein, 1.0 kg of fish oil, 3.0 kg of perilla oil, 18.0 kg of dextrin, 6.0 kg of a l mixture, 1.95 kg of a vitamin mixture, 2.0 kg of an emulsifying agent, 4.0 kg of a stabilizer, and 0.05 kg of essence were added to 5.0 kg of the solution. The mixture was charged in a retort pouch (200 ml) and sterilized at 121°C for 20 minutes using a retort sterilizer (class-1 pressure vessel, RCS-4CRTGN; ctured by Hisaka Works, Ltd.) to produce 50 kg of a bone-strengthening liquid nutrient composition. Any precipitation was ed, and no abnormal flavor was felt in the trengthening liquid nutrient composition thus obtained.

Example 9 Preparation of bone-strengthening gel—like food Two grams (2 g) of the protein material of the example product 5 was dissolved in 708 g of deionized water. The solution was d and mixed using an disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan) at 9500 rpm for minutes. 40 g sorbitol, 2 g of a sour agent, 2 g of essence, 5 g of pectin, 5 g of a whey protein concentrate, 1 g of calcium lactate, and 235 g of deionized water were added to the solution. After stirring and mixing, the mixture was charged into a 200 m1 cheer pack, and sterilized at 85°C for 20 minutes, and the pack was sealed to obtain five packs (200 g) of a bone-strengthening gel-like food. Any precipitation was SNOW-l 92 ed, and no abnormal flavor was felt in the bone-strengthening gel—like food thus obtained.

Example 10 Preparation ofbone-strengthening drink Two grams (2 g) of an acidifier was dissolved in 706 g of deionized water, and 4 g of the protein material of the example product 6 was dissolved in the solution. The solution was stirred and mixed using an ultra-disperser (ULTRA-TURRAX T—25; manufactured by IKA Japan) at 9500 rpm for 30 minutes. After the addition of 100 g of maltitol, 20 g of reduced starch syrup, 2 g of essence, and 166 g of deionized water, the mixture was charged into a 100 ml glass bottle. After sterilized at 95°C for 15 seconds, the bottle was closely sealed to obtain ten bottles (100 ml) of a bone-strengthening drink. Any precipitation was observed, and no al flavor was felt in the bone—strengthening drink thus obtained Example 11 Preparation of bone-strengthening feed Two kilograms (2 kg) of the protein material of the example product 7 was dissolved in 95 kg of deionized water. The solution was stirred and mixed using a TK-homomixer (MARK II 160; manufactured by PRIMIX ation) at 3600 rpm for 40 minutes to obtain a solution ning the example product 7 in an amount of 2 g/100 g. Then, 12 kg of soybean meal, 14 kg of powdered skim milk, 4 kg of soybean oil, 2 kg of corn oil, 23.2 kg of palm oil, 14 kg of corn starch, 9 kg of flour, 2 kg of branu kg of a Vitamin mixture, 2.8 kg of cellulose, and 2 kg of a l mixture were added to 10 kg of the on. The mixture was sterilized at 120°C for 4 s to obtain 100 kg of a bone-strengthening dog food.

Example 12 SNOW-192 Preparation of bone-strengthening agent (tablet) The raw materials were mixed in the ratio shown in Table 4. Then, 1 g of the mixture was formed and tableted in the usual manner to prepare a bone-strengthening agent.

TABLE 4 Hydrous crystalline glucose 92.5 % (wt%) n material (example product 1) 1.0 % Mineral mixture 5.0 % Sugar ester 1.0 % Essence 0.5 % SNOW-192

Claims (10)

1. A protein material comprising angiogenin and/or angiogenin hydrolysate in an amount of 2 to 15 mg/ 100 mg of the protein material and lactoperoxidase and/or lactoperoxidase ysate in the mass ratio to the angiogenin and/or enin hydrolysate of 0.3 to 20.

2. A food, drink, or feed comprising the protein al according to claim 1.

3. A bone-strengthening agent comprising the protein material according to claim 1 as an active ingredient.

4. Use of (i) angiogenin and/or angiogenin hydrolysate and (ii) lactoperoxidase and/or lactoperoxidase hydrolysate in the manufacture of a medicament for thening bones, wherein the angiogenin and/or angiogenin hydrolysate is in an amount of 2 to 15 mg/100 mg of the medicament and wherein the lactoperoxidase and/or lactoperoxidase hydrolysate is in the mass ratio of 0.3 to 20 to the angiogenin and/or angiogenin hydrolysate.

5. Use of (i) angiogenin and/or angiogenin hydrolysate and (ii) lactoperoxidase and/or lactoperoxidase ysate in the manufacture of a medicament for the prevention or treatment of orosis, fracture, rheumatism and/or arthritis, wherein the angiogenin and/or angiogenin hydrolysate is in an amount of 2 to 15 mg/100 mg of the ment and wherein the lactoperoxidase and/or lactoperoxidase hydrolysate is in the mass ratio of 0.3 to 20 to the angiogenin and/or angiogenin hydrolysate.

6. Use according to claim 4 or 5 wherein the medicament is for administration at an amount of 5 mg/day or more.

7. Use according to any one of claims 4 to 6 wherein the ment is a protein material .

8. A method of preparing the protein material according to claim 1, comprising following steps 1) to 3): SNOW-192 1) preparing angiogenin and/or angiogenin ysate; 2) preparing lactoperoxidase and/or lactoperoxidase hydrolysate; and 3) mixing the lactoperoxidase and/or lactoperoxidase hydrolysate according to above 2) and the angiogenin and/or angiogenin hydrolysate according to above 1) in the mass ratio to the enin and/or angiogenin hydrolysate of 0.3 to 20.

9. A method of preparing the protein material according to claim 1, comprising a step of extracting a on containing angiogenin and/or angiogenin hydrolysate and lactoperoxidase and/or lactoperoxidase hydrolysate from milk and/or a material d from milk in the mass ratio to the angiogenin and/or the angiogenin hydrolysate of 0.3 to 20.

10. The method according to claim 9, further comprising another step of enzymatically degrading the angiogenin and/or the lactoperoxidase contained in the fraction.