NZ704914B2 - Beverage comprising a specific milk component, and method of producing the same - Google Patents

Abstract

The present invention addresses the problem of providing a safe and novel beverage 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. A beverage containing more than 0.8 mg/100 ml and 150 mg/100 ml or less of angiogenin and/or an angiogenin degradation product, and cystatin and/or a cystatin degradation product at a mass ratio of 0.006 to 1.7 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 beverage. 150 mg/100 ml or less of angiogenin and/or an angiogenin degradation product, and cystatin and/or a cystatin degradation product at a mass ratio of 0.006 to 1.7 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 beverage.

Description

SNOW-193 BEVERAGE COMPRISING A IC MILK COMPONENT, AND METHOD OF PRODUCING THE SAME TECHNICAL FIELD This invention relates to a beverage and a method of producing the same. The drink includes a specific milk component, and may be useful for prevention and treatment of various bone diseases such as orosis, fracture, rheumatism, and arthritis.

BACKGROUND ART In recent years, various bone diseases, such as osteoporosis, fracture, and he have increased on a global basis along with aging of society and the like, and have become a serious social problem. These diseases are caused by insufficient calcium intake, sion of calcium absorption ability, hormone imbalance after use, 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 th (bone density + bone quality) is effective in preventing various bone diseases, such as osteoporosis, fracture, 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 lastic bone resorption. Bones are always repeatedly resorbed and formed in a balanced manner (remodeling). However, various bone diseases, such as osteoporosis, fracture, and backache may occur when bone resorption exceeds bone formation due to a change in e balance after menopause, and the like. Therefore, bones can be strengthened by suppressing osteoclastic bone resorption and maintaining the bone SNOW-193 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 product, such as whey m, bovine bone powder, or eggshell is added individually, has been ingested 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 effect, such as casein phosphopeptide or oligosaccharide has also been used to strengthen bones. However, the calcium absorption rate is 50% or less when a food or drink that contains a calcium salt or a natural calcium product is ingested, and the large part of the calcium ingested may be discharged from the body without being absorbed. Moreover, even if m is absorbed into the body, it does not necessarily exhibit the bone metabolism—improving effect or a bone strengthening effect, since the afiinity to bones may differ according to its form or the type of nutritional ient ingested together. An estrogen product, an active vitamin D3 product, a vitamin K2 t, a bisphosphonate product, a calcitonin product, and the like have been known as a drug for treating orosis or strengthening bones, and new drugs such as an anti-RANKL dy have been also developed. However, these drugs may have side effects such as buzzing in the ear, a headache, or loss of te. Moreover, the above substances are in a situation that they cannot be added to a food or drink at present from the int of safety, cost, and the like. Therefore, in light of the nature of various bone diseases, such as orosis, fracture, and backache, pment of such a food or drink that can be ingested orally for a long time, increases the bone strength by promoting bone formation and suppressing bone resorption, and may be expected to have the effect of preventing or treating the various bone diseases has been desired.

PRIOR—ART DOCUMENT SNOW-193 PATENT NT [Patent Document 1] JP-A-H08-151331 [Patent Document 2] JP-A-H10-7585 [Patent Document 3] JP-A281587 SUMMARY OF THE INVENTION The invention relates to provide a drink that may be useful for prevention and treatment of various bone diseases such as orosis, re, tism, and arthritis.

The present inventors have found that the bone density can be effectively increased by ingesting a drink that includes angiogenin and/or angiogenin hydrolysate, and includes cystatin and/or cystatin hydrolysate in a specific mass ratio with respect to angiogenin and/or enin hydrolysate. This finding has led to the completion of the invention.

Specifically, the invention provides the following aspects: (1) A drink comprising angiogenin and/or angiogenin hydrolysate in an amount of more than 0.8 mg/100 ml of the drink and not more than 150 mg/100 ml of the drink and cystatin and/or cystatin hydrolysate in the mass ratio to the angiogenin and/or angiogenin hydrolysate of 0.006 to 1.7. (2) A method of ting bone diseases including ingesting the drink SNOW-193 according to (1) in an amount of 200 ml/day or more. (2a) Use of (i) angiogenin and/or angiogenin hydrolysate and (ii) cystatin and/or cystatin hydrolysate in the manufacture of a ment for the prevention of bone disease, fracture, rheumatism and/or arthritis, n the enin and/or enin hydrolysate are present in an amount of more than 0.8 mg per 100 ml of medicament and not more than 150 mg per 100 ml of medicament and wherein the cystatin and/or cystatin hydrolysate are present in a ratio of 0.006 to 1.7 to the mass of angiogenin and/or angiogenin hydrolysate. (3) A method of producing the drink according to (1), comprising mixing angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate with a drink raw material, and sterilizing the obtained mixture. (4) A method of producing the drink according to (1), comprising adding angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate to a sterilized drink raw material.

EFFECTS OF THE INVENTION The drink of the invention exhibits a bone-strengthening effect, and may be useful for prevention and treatment of various bone diseases such as orosis, fracture, rheumatism, and arthritis.

EMBODIMENTS FOR CARRYING OUT THE INVENTION A drink of the invention is characterized in that the drink includes angiogenin and/or angiogenin hydrolysate in a specific amount, and further includes in and/or cystatin hydrolysate in a specific mass ratio with respect to enin and/or angiogenin hydrolysate.

SNOW-193 Cow milk generally contains angiogenin and/or angiogenin hydrolysate in an amount of about 0.2 to 0.8 mg/100 ml, and cystatin and/or cystatin hydrolysate in an amount of about 0.4 to 1 mg/100 ml.

In contrast, the drink of the invention is added with angiogenin and/or angiogenin hydrolysate and cystatin and/or in hydrolysate, and the drink contains angiogenin and/or angiogenin hydrolysate in an amount of more than 0.8 mg/100 ml and not more than 150 mg/100 ml, and cystatin and/or in hydrolysate in a mass SNOW—193 ratio with respect to angiogenin and/or angiogenin hydrolysate of 0.006 to 1.7.

A fraction containing angiogenin and/or angiogenin hydrolysate that is prepared from milk of a mammal, such as human, cow, buffalo, goat, or sheep, a fraction containing cystatin and/or cystatin hydrolysate that is prepared from milk of a , such as human, cow, buffalo, goat, or sheep, a fraction containing angiogenin and/or angiogenin hydrolysate that is produced by genetic engineering, a fraction containing cystatin and/or cystatin hydrolysate that is ed by a genetic ering, angiogenin and/or angiogenin hydrolysate purified from blood or an organ, cystatin and/or in hydrolysate purified from blood or an organ, or the like may be used as the angiogenin and/or angiogenin hydrolysate and the cystatin and/or cystatin hydrolysate included in the drink of the invention. A commercially available purified angiogenin or cystatin reagent may also be used.

The drink of the ion may include angiogenin ysate or cystatin hydrolysate obtained by digesting a fraction containing angiogenin, an angiogenin reagent, a fraction containing cystatin, a cystatin reagent, or the like using one or more proteases.

The drink of the ion may include a protein material prepared by extracting a on containing angiogenin and/or angiogenin ysate and in and/or cystatin ysate directly from milk or a material derived from milk, such as skim milk or whey. Such a protein material may be prepared as follows, for example.

Specifically, milk or a material derived from milk is brought into contact with a cation-exchange resin, and milk—derived proteins adsorbed on the resin is eluted at a salt concentration of 0.1 to 2.0 M, desalted and concentrated using a reverse osmosis membrane, an electrodialysis membrane, an ultrafiltration membrane, a microfiltration membrane, or the like, and ally subjected to proteolysis to a molecular weight of 8000 or less using a protease, such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, or V8 protease. When subjecting to proteolysis using a protease, the lower limit of the molecular weight is ably 500 or more.

The protein material thus obtained may be dried by —drying, spray drying, or the like, and the dried product may be added in the drink.

The drink of the invention is produced by adding enin and/or angiogenin hydrolysate, and cystatin and/or cystatin hydrolysate and a protein material that contains ‘ enin and/or angiogenin hydrolysate and in and/or cystatin hydrolysate, or the like to a drink raw al so that the drink includes angiogenin and/0r angiogenin hydrolysate in an amount of more than 0.8 mg and not more than 150 mg/100 m1, and includes cystatin and/or cystatin ysate in a mass ratio with respect to angiogenin and/or angiogenin hydrolysate of 0.006 to 1.7.

As shown in the test examples described below, when the drink includes angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate as described above, the bone-strengthening effect can be obtained more eifectively than the case of ingesting angiogenin and/or angiogenin hydrolysate or cystatin and/0r cystatin ysate separately. [001 3] The drink of the invention may be produced in the usual manner as long as the drink includes the angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate in specific amounts respectively. For example, the drink of the invention is produced by adding enin and/or angiogenin hydrolysate to a drink raw material, such as a material derived from milk so that the drink includes angiogenin and/or angiogenin hydrolysate in a c amount, and adding cystatin and/or cystatin hydrolysate to the mixture so that the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate is within the specific range. Note that as SNOW-193 the drink raw material, such as a material derived from milk, cow milk, trated skim milk, skim milk powder, Whey, butter, cream, fermented milk, a dairy lactic acid ia beverage, a lactic acid bacteria beverage, or the like can be given, further a milk drink mixed thereof as appropriate, sed milk, composition-modified milk, low-fat milk, fat—flee milk, or the like can be given, for e.

When adding angiogenin and/or angiogenin hydrolysate and cystatin and/0r cystatin hydrolysate to a drink raw material, such as a material derived from milk, angiogenin and/or angiogenin hydrolysate and cystatin and/or in hydrolysate may be added to either unsterilized drink raw material, or a sterilized drink raw material.

When adding to an unsterilized drink raw al, sterilization may be conducted after the addition. In this ce, heat sterilization is preferable. For example, the drink raw material itself, or the drink raw material to which angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate are added, may be heated to 70°C, homogenized using a homogenizer at a pressure of 15 MPa, sterilized at 130°C for 2 seconds, and cooled to 5°C. When sterilizing a mixture prepared by adding angiogenin and/or angiogenin hydrolysate and cystatin and/0r cystatin hydrolysate to a dn'nk raw material, it is preferable to sterilize at 130°C for 2 seconds or less.

It may-be possible that the drink of the invention may be added with a raw material or the like that is ly used for a food or drink, such as a saccharide, a lipid, a protein, a vitamin, a mineral, or a flavor, in addition to angiogenin and/or angiogenin hydrolysate, in and/or cystatin hydrolysate, other than the above drink raw material, and may also be added with another bone-strengthening component such as calcium, vitamin D, vitamin K, or isoflavone.

The drink of the invention can then bones when ingested orally in an amount of 200 ml or more per kg of body weight, as shown in the animal experiments SNOW-193 described below. Since the intake for the experiment animal corresponds to the intake for adults in terms of blood drug concentration (see Mitsuyoshi Nakajima (1993), “Yakkou Hyoka Vol. 8”, wa-Shoten Ltd., pp. 2-18), it is expected that bones can be strengthened, and especially various bone diseases, such as orosis, fracture, rheumatism, and arthritis can be prevented or treated by ingesting the drink of the invention in an amount of 200 ml/day or more per adult.

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 for illustration purposes only, and should not be construed as limiting the invention.

Reference Example 1 Preparation (1) of angiogenin fraction A column filled with 30 kg of cation-exchange resin (Sulfonated Chitopearl; ctured 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 washed the column with deionized water, the ed protein was eluted with a linear gradient of 0.1 to 2.0 M sodium de. The eluted 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 enin-containing fraction was further subjected to gel filtration chromatography (column: Superose 12). The eluate obtained was desalted using a reverse osmosis membrane, and the ed eluate was freeze-dried to obtain 16.5 g of an angiogenin on having an angiogenin purity of 90%. These sive operations were repeated 30 times.

SNOW-193 nce Example 2 Preparation (2) of enin fraction A column filled with 10 kg of Heparin Sepharose (manufactured by GE Healthcare) was thoroughly washed with deionized water, and 500 liters of unpasteurized skim milk (pH 6.7) was then applied to the column. After thoroughly washing the column with a 0.5 M sodium de solution, the column was eluted with a 1.5 M sodium chloride solution. The eluate was desalted using a e osmosis membrane, and the desalted eluate was freeze-dried to obtain 18 g of an enin fraction having an angiogenin purity of 5%. The above successive operations were ed 50 times.

Reference Example 3 Preparation of cystatin fraction 100,000 liters of a 5% whey protein solution was heat-treated at 90°C for 10‘ minutes, and a precipitate was removed by centrifugation. A column was filled with a carrier prepared by binding ymethylated papain to ' Tresyl-Toyopearl (manufactured by Tosoh Corporation). After equilibration with a 0.5 M sodium chloride solution, the above whey protein solution was applied to the column. The column was then sequentially washed with a 0.5 M sodium chloride solution and a 0.5 M sodium chloride solution containing Tween 20 (0.1%). After that, a cystatin—containing fraction was eluted with a 20 mM acetic acid-0.5 M sodium de solution. The eluted fraction was immediately neutralized with a 1 M sodium hydroxide solution. The eluate obtained was then desalted using a reverse osmosis membrane, and the desalted eluate was freeze-dried to obtain 9.6 g of a cystatin fraction having a cystatin purity of 90%. The above successive operations were repeated 20 times.

SNOW-193 Measurement of angiogenin and cystatin contained in drink The content of angiogenin, angiogenin hydrolysate, cystatin and cystatin hydrolysate in the drink was measured according to the method described in JP-A164511 with modification. cally, 106 pl of the drink was added to 5 ml of ultrapure water, and a 1/1000—equivalent amount of formic acid was added to the mixture to e a sample solution. Ten microliter (10 pl) of the sample solution was dried up, and dissolved in 20 p1 of 0.1 M ammonium bicarbonate containing 8 M urea and 1 mM tris(carboxyethyl)phosphine (TCEP). The solution was heated at 56°C for 30 minutes. After returning the solution to room temperature, 5 pl of 100 mM iodoacetamide on was added to the solution, and the mixture was reacted for 30 minutes in the dark. After the addition of 54 pl of ultrapure water, 10 p1 of 0.1 pg/ml trypsin and 10 pl of 0.1 pg/ml Lysyl Endopeptidase were added to the e. The mixture was reacted at 37°C for 16 hours. The reaction was then terminated by adding 3 pl of formic acid and used as the sample peptide solution for measurement. The sample on was diluted 6-fold with 10 fmol/pl internal standard e solution containing 0.1% formic acid, 0.02% trifluoroacetic acid (TFA), and 2% acetonitril, and 2.5 p1 of the diluted solution was subjected to LC/MS/MS analysis.

The peptides were ted by gradient elution using an HPLC system. More specifically, the es were ted using a column (MAGIC C18, 0.2 mm (ID) X 50 mm) equipped with a 5 pl-peptide trap on a MAGIC 2002 HPLC system at a flow rate of 2 pl/min. A solution A (2% acetonitrile—0.05% formic acid) and a solution B (90% acetonitrile-0.05% formic acid) were used as eluant for HPLC. Gradient elution was conducted under the elution condition from 2 to 65% using the solution B over 20 minutes.

As object ions for measuring cystatin, parent ion was NHg—QVVSGMNYFLDVELGR—COOH (m/z 914.4), and the MS/MS target ion was NHz—FLDVELGR—COOH (m/z 948.7). As object ions for measuring enin, parent ion was NHz—YIHFLTQHYDAK-COOH (m/z 768.8), and the MS/MS target ion was NHz-FLTQHYDAK-COOH (m/z 1122.8). Regarding the internal standard e parent ion was NHz—ETTVFENLPEK—COOH (wherein, P was labeled with 13C and 15N) (m/z 656.9.), and the MS/MS target ion was NHg—FENLPEK-COOH (wherein, P was labeled with 13‘0 and 15N) (m/z 882.4).

A system “LCQ Advantage” was used for MS. The peak area of each protein was calculated from the resulting chromatogram, and the concentration was calculated from the ratio with t to the internal standard peptide.

Example 1 Three hundred and thirty milligrams (330 mg) of the angiogenin fi'action obtained in nce Example 1 and 1 mg of the cystatin fraction obtained in Reference Example 3 were mixed with 200 ml of cow milk. The e was sterilized at 130°C for 2 seconds, and cooled to 10°C to obtain a drink (example product 1). The resulting drink ned angiogenin and/or angiogenin hydrolysate in an amount of 150 mg/100 ml, and the mass ratio of cystatin and/or cystatin hydrolysate to enin and/or angiogenin hydrolysate in the drink was 0.006.

Example 2 Twenty four milligrams (24 mg) of the angiogenin fiaction obtained in Reference Example 2 and 2 mg of the cystatin fraction obtained in Reference Example 3 were mixed with 200 ml of cow milk. The mixture was sterilized at 130°C for 2 seconds, and cooled to 10°C to obtain a drink (example product 2). The resulting drink contained angiogenin and/0r angiogenin hydrolysate in an amount of 0.81 mg/100 ml, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the drink was 1.7.

SNOW-193 Example 3 Twenty four milligrams (24 mg) of the angiogenin fraction obtained in nce Example 1 and 2 mg of the cystatin fraction obtained in Reference Example 3 were mixed with 200 m1 of cow milk. The mixture was sterilized at 130°C for 2 seconds, and cooled to 10°C to obtain a drink (example product 3). The resulting drink contained angiogenin and/or angiogenin hydrolysate in an amount of 11 mg/100 m1, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the drink was 0.12.

Comparative e 1 Twenty two milligrams (22 mg) of the angiogenin fraction obtained in Reference Example 2 and 4 mg of the cystatin fraction obtained in Reference Example 3 were mixed with 200 ml of cow milk. The mixture was sterilized at 130°C for 2 seconds, and cooled to 10°C to obtain a drink (comparative example product 1). The resulting drink ned angiogenin and/or angiogenin ysate in an amount of 0.75 mg/ l 00 ml, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the drink was 3.0.

Comparative Example 2 Three hundred thirty milligrams (330 mg) of the enin on obtained in Reference Example ’1 and 0.25 mg of the cystatin on ed in Reference Example 3 were mixed with 200 m1 of cow milk. The mixture was sterilized at 130°C for 2 seconds, and cooled to 10°C to obtain a drink (comparative example t 2).

The resulting drink contained angiogenin and/or angiogenin hydrolysate in an amount of 150 mg/100 ml, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the drink was 0.004.

SNOW-193 Test Example 1 The bone-strengthening effects of the example products 1 to 3 and the comparative example products 1 and 2 were determined by animal experiments.

C3H/HeJ mice (5 weeks old, male) were used for the animal experiments. After 1 week acclimation, the mice were divided into six groups (10 mice/group). The mice were orally stered each product of the example products 1 to 3 and the comparative example products 1 and 2 in an amount of 200 ml per 1 kg of mouse weight once a day for 2 weeks using a tube. The control group was not administrated any example products 1 to 3 and the comparative example products 1 and 2 were not stered. After completion of administration (second week), the bone density of the right tibia of each mouse was measured using a CT (manufactured by Rigaku Corporation). The results are shown in Table 1. As shown in Table 1, the groups that were orally administered the e products 1 to 3 showed a significant increase in bone y compared with the control group and the comparative example groups that were orally administered the comparative example product 1 0r 2.

TABLE 1 SNOW-193 Reference Example 4 A column (diameter: 4 cm, : 30 cm) filled with 400 g of cation-exchange resin (Sulfonated Chitopearl; manufactured by Fuji Spinning Co., Ltd.) was thoroughly washed with zed 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 zed water, proteins adsorbed 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 freeze-dried to obtain 18 g of a powdery protein al (reference example product 4). nce Example 5 Four grams (4 g) of protein material of the nce example product 4 was dissolved in 800 ml of water. After the addition of trypsin (manufactured by , which is a protease, so as to obtain the final concentration of 0.03 wt%, the mixture was subjected to enzymatic treatment at 37°C for 8 hours. After inactivating the se through heat-treatment at 90°C for 5 minutes, the mixture was freeze-dried to obtain 3.0 g of a powdery protein material (reference example product 5).

Example 4 [003 l] Forty milligrams (40 mg) of the reference example product 4 was mixed with 200 ml of sterilized cow milk to obtain a drink (example product 4). The resulting drink contained angiogenin and/or angiogenin hydrolysate in an amount of 1.2 mg/100 ml, and the mass ratio of cystatin and/0r cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the drink was 0.39.

Example 5 Forty rams (40 mg) of the reference example product 5 was mixed with SNOW—193 200 m1 of ized cow milk to obtain a drink (example product 5). The resulting drink contained angiogenin and/or angiogenin hydrolysate in an amount of 1.15 mg/100 ml, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or enin hydrolysate in the drink was 0.4.

Comparative Example 3 Thirty rams (30 mg) of the nce example product 4 and 10 mg of the cystatin fraction obtained in Reference Example 3 were mixed with 200 ml of sterilized cow milk to obtain a drink (comparative example product 3). The resulting drink contained angiogenin and/or enin hydrolysate in an amount of 0.95 mg/100 m1, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the drink was 5.2 Test Example 2 The bone-strengthening efi‘ects of the example products 4 and 5 and the comparative example t 3 was determined by animal experiments. Forty SD female rats (51 weeks old) were used for the animal experiments. The rats were divided into five groups (8 rats/group). Four groups underwent ovariectomy, and the remaining one group sham surgery. After a 4-week recovery , the ovariectomized rats were orally administered the example products 4 or 5 or the comparative example product 3 in an amount of 200 ml per 1kg of rat weight daily in six divided dose for 16 weeks using a tube. The control group was not administrated any example products 4 and 5 and the comparative example product 3. 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 administration (sixteenth week), the bone density of the right tibia of each rat was measured using a micro—CT actured by Rigaku Corporation).

SNOW-193 The results are shown in Table 2. As shown in Table 2, the groups that were orally administered the example products 4 and 5 showed a significant increase in bone density as compared with the control group and the group that was orally administered the comparative example product 3. Moreover, the bone density approached that of the sham surgery group.

TABLE 2 Bone density (mg/cm3) Control group 552 ± 10 Sham surgery group 601 ± 8 Example product 4 596 ± 10 Example product 5 595 ± 9 ative example product 3 555 ± 12 SNOW-193

Claims (7)

1. A drink comprising angiogenin and/or angiogenin hydrolysate in an amount of more than 0.8 mg/100 ml of the drink and not more than 150 mg/100 ml of the drink and cystatin and/or cystatin hydrolysate in the mass ratio to the angiogenin and/or angiogenin hydrolysate of 0.006 to 1.7.

2. Use of (i) angiogenin and/or angiogenin hydrolysate and (ii) cystatin and/or cystatin hydrolysate in the manufacture of a medicament for the prevention of bone disease, fracture, rheumatism and/or arthritis, wherein the angiogenin and/or angiogenin hydrolysate are t in an amount of more than 0.8 mg per 100 ml of medicament and not more than 150 mg per 100 ml of medicament and wherein the cystatin and/or cystatin hydrolysate are present in a ratio of 0.006 to 1.7 to the mass of angiogenin and/or angiogenin hydrolysate.

3. Use according to claim 2 n the bone disease is osteoporosis.

4. Use according to claim 2 or 3 wherein the medicament is for stration at an amount of 200 mg/day or more.

5. Use according to any one of claims 2 to 4 wherein the medicament is a drink.

6. A method of ing the drink according to claim 1, comprising mixing angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate with a drink raw material, and izing the obtained mixture.

7. A method of producing the drink according to claim 1, sing adding enin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate to a sterilized drink raw material.