NZ704918B2 - Cheese, and method for producing same - Google Patents

Abstract

The present invention addresses the problem of providing a safe and novel cheese product 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 cheese product containing 6.5 to 160 mg/100 g of angiogenin and/or an angiogenin degradation product, and cystatin and/or a cystatin degradation product at a mass ratio of 0.02 to 1.6 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 cheese product. 0 g of angiogenin and/or an angiogenin degradation product, and cystatin and/or a cystatin degradation product at a mass ratio of 0.02 to 1.6 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 cheese product.

Description

SNOW-199 , AND METHOD FOR PRODUCING SAME TECHNICAL FIELD This invention relates to a novel cheese and a method for producing the same.

The cheese includes a ic milk component, and may be useful for prevention and treatment of various bone diseases such as osteoporosis, fracture, tism, 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 problem. These diseases are caused by insufficient calcium intake, depression of calcium absorption ability, hormone imbalance after menopause, and the like. It is considered that se the body bone mass as much as possible and increase the maximum bone mass and the bone strength (bone density + bone quality) by ing osteoblastic bone formation from the early stage of life is effective in preventing s 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 osteoclastic bone resorption. Bones are always repeatedly resorbed and formed in a ed manner (remodeling). However, various bone diseases, such as orosis, fracture, and he may occur when bone resorption exceeds bone formation due to a change in hormone balance after menopause, and the like. Therefore, bones can be strengthened by suppressing osteoclastic bone resorption and maintaining the bone SNOW-199 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 calcium, 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 ns such a calcium product together with a substance having a calcium absorption-promoting effect, such as casein phosphopeptide or accharide 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 rged from the body without being absorbed. Moreover, even if calcium is absorbed into the body, it does not arily exhibit the bone metabolism—improving effect or a bone strengthening effect, since the affinity to bones may differ according to its form or the type of nutritional ingredient ingested together. An estrogen product, an active vitamin D3 product, a vitamin K2 product, a bisphosphonate product, a calcitonin t, and the like have been known as a drug for treating osteoporosis or strengthening bones, and new drugs such as an anti-RANKL antibody have been developed. However, these drugs may bring side s 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 from the viewpoint of safety, cost, and the like. Therefore, in light of the nature of various bone es, such as osteoporosis, fracture, and backache, development of such a food or drink that can be stered 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 PATENT DOCUMENT [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 cheese that may be useful for prevention and treatment of various bone diseases such as osteoporosis, fracture, rheumatism, and arthritis.

The t inventors have found that the bone density can be effectively increased by ingesting a cheese that includes angiogenin and/or angiogenin hydrolysate, and includes cystatin and/or cystatin hydrolysate in a ic mass ratio with respect to angiogenin and/or angiogenin ysate. This g has led to the completion of the invention.

Specifically, the invention provides the following aspects: (1) A cheese comprising angiogenin and/or angiogenin hydrolysate in an amount of 6.5 mg/100 g of the cheese to 160 mg/100 g of the cheese and cystatin and/or in hydrolysate in the mass ratio to the angiogenin and/or angiogenin hydrolysate of 0.02 to 1.6. (2) A method of preventing bone diseases including ingesting the cheese according to (1) in an amount of 20 g/day or more.

SNOW-199 (2a) Use of (i) angiogenin and/or angiogenin hydrolysate and (ii) cystatin and/or cystatin hydrolysate in the cture of a medicament for the prevention of bone disease, fracture, rheumatism and/or arthritis, wherein the angiogenin and/or angiogenin hydrolysate is present in an amount of 6.5 mg/100 g to 160 mg/100 g of the medicament and wherein the cystatin and/or cystatin hydrolysate is t in a ratio of 0.02 to 1.6 to the mass of enin and/or angiogenin hydrolysate. (3) A method of producing the cheese according to (1), comprising mixing angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate with a raw material cheese and/or a cheese curd. (4) A method of producing the cheese according to (1), comprising mixing a raw material cheese with angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate, and emulsifying and cooling the mixture.

S OF THE INVENTION The cheese of the invention exhibits a bone-strengthening effect, and may be useful for prevention and treatment of various bone diseases such as osteoporosis, fracture, rheumatism, and tis.

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

A cheese generally ns angiogenin and/or angiogenin hydrolysate in an amount of about 1.1 to 6.3 mg/100 g, and cystatin and/or in hydrolysate in an SNOW-199 amount of about 2.1 to 9.3 mg/100 g.

In contrast, the cheese of the ion is added with angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate, and the cheese contains angiogenin and/or angiogenin ysate in an amount of 6.5 mg/100 g to 160 mg/100 g, and cystatin and/or cystatin hydrolysate in a mass ratio with respect to angiogenin and/or angiogenin hydrolysate of 0.02 to 1.6.

A fraction ning angiogenin and/or angiogenin hydrolysate that is prepared from milk of a mammal, such as human, cow, buffalo, goat, or sheep, a fraction ning cystatin and/or cystatin hydrolysate that is prepared from milk of a mammal, 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 produced by a genetic ering, angiogenin and/or angiogenin hydrolysate purified from blood or an organ, cystatin and/or cystatin hydrolysate purified from blood or an organ, or the like may be used as the angiogenin and/or angiogenin hydrolysate and the in and/0r cystatin hydrolysate included in the cheese of the invention. A commercially available purified angiogenin or cystatin reagent may also be used.

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

The cheese of the invention may include a protein material prepared by extracting a fraction containing angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate directly from milk or a material derived from milk, such as skim milk or whey. Such a protein al may be ed as s,.for example.

Specifically, milk or a al 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 odialysis membrane, an ultrafiltration membrane, a microfiltration membrane, or the like, and optionally subjected to lysis to a molecular weight of 8000 or less using a protease, such as trypsin, pancreatin, chymotrypsin, pepsin, papain, SNOW-199 kallikrein, cathepsin, thermolysin, or V8 protease. When subjecting to proteolysis using a protease, the lower limit of the molecular weight is preferably 500 or more.

The protein material thus obtained may be dlied by freeze-drying, spray drying, or the like, and the dried product may be incorporated in the cheese.

The cheese of the invention is produced by mixing the above angiogenin and/or angiogenin ysate, and cystatin and/or cystatin hydrolysate and a protein al that contains angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin ysate, or the like with a raw material and/or a cheese curd, a raw material cheese or the like so that the cheese includes enin and/or enin hydrolysate in an amount of 6.5 mg to 160 mg/100 ml, and includes cystatin and/or cystatin hydrolysate in a mass ratio with respect to angiogenin and/or angiogenin hydrolysate of 0.02 to 1.6.

As shown in the test examples described below, when the cheese includes angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate as bed above, the bone-strengthening effect can be obtained more effectively than the case of ingesting angiogenin and/or angiogenin hydrolysate or in and/or cystatin hydrolysate separately.

The cheese of the ion may be produced in the usual manner as long as the cheese includes the angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate in specific amounts respectively. The term e” used herein includes all types of cheese such as natural cheese, so—called processed cheese preparation which is a food using processed cheese, spreadable processed , processed cheese food specified by the Codex Standard, milk, or the like as a main raw material. For example, natural , such as fresh (unripened) cheese such as cream cheese, mozzarella, ricotta, mascarpone and fromage blanc, white mold cheese such as Camembert and Brie, blue mold cheese such as Gorgonzola, Stilton and Roquefort, SNOW-199 washed rind cheese such as Livarot, semi-hard cheese such as Provolone and Gouda, and hard cheese such as Grana, Emmentaler and Cheddar, processed cheese produced using natural , cheese-like food produced using oils and fats polysaccharides and the like, can be given.

In the case of Gouda cheese, for example, milk that is adjusted in fat content to 2.8% is used as a raw material, and angiogenin and/or angiogenin ysate is added thereto in the specific amount, and cystatin and/or cystatin ysate is further added in the mass ratio to angiogenin and/or enin hydrolysate of the specific range.

The mixture is sterilized at 77°C for 15 seconds, and . A starter, rennet, and the like are added thereto, and stirred. The mixture is then d to stand for about 30 minutes, and the whey is removed to prepare cheese curds. After the cheese curds are optionally added with salt, Gouda cheese can be ed through molding the cheese curds.

In the case of cottage cheese, cream or the like is used as a raw material, angiogenin and/or angiogenin hydrolysate is added thereto in the specific amount, and cystatin and/or cystatin hydrolysate is further added in the mass ratio to angiogenin and/or angiogenin ysate of the specific range. The mixture is uniformly added to cheese curds to be able to produce cottage cheese. Examples of the raw material used for producing the cheese of the invention include milk of a mammal, such as cow, buffalo, goat, or sheep, milk thereof in which the fat content is adjusted, cream prepared from such mammal milk, and the like.

The cheese of the invention may be produced as described below. When producing processed cheese as the cheese of the invention, for example, as an emulsifying salt, sodium citrate, sodium monophosphate, sodium polyphosphate, or the like is added to a raw material cheese in an amount of about 2%. After the addition of water in an amount of about 10%, angiogenin and/or angiogenin hydrolysate is added to SNOW-199 the mixture in the specific amount, and cystatin and/or in hydrolysate is r added to the mixture in the mass ratio to angiogenin and/or angiogenin hydrolysate of the c range. The e is emulsified at 85°C in the usual manner, and the emulsion is placed into a carton, and cooled to 5°C to be able to produce the processed cheese.

As a method of mixing angiogenin and/0r angiogenin hydrolysate in the specific amount and cystatin and/or cystatin hydrolysate in the specific mass ratio to the processed cheese, it may be possible to use a cheese mixture which is previously prepared by added enin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate as a raw material cheese, or to mix appropriate quantities of angiogenin and/or enin hydrolysate and cystatin and/or cystatin hydrolysate with a raw material of the processed cheese.

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

The cheese ofthe invention can strengthen bones when administered orally in an amount of 20 g or more per kg of body weight, as shown in the animal experiments 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”, Hirokawa—Shoten Ltd., pp. 2-18), it is expected that bones can be strengthened, and especially bone diseases, such as osteoporosis, fracture, rheumatism, and arthritis can be prevented or treated by ing the cheese of the invention in an amount of 20 g/day or more per adult, typically.

SNOW—199 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 ng the ion.

Reference Example 1 Preparation (1) of angiogenin fraction A column filled with 30 kg of cation-exchange resin (Sulfonated earl; 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 ed protein was eluted with a linear nt of 0.1 to 2.0 M sodium chloride. The elution on 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 chromatography (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 angiogenin purity of 90%. These successive ions were repeated 30 times.

Reference Example 2 Preparation (2) of angiogenin 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 g the column with a 0.5 M sodium chloride on, the absorbed protein was SNOW-199 eluted with a 1.5 M sodium chloride solution. The eluate was ed using a reverse osmosis membrane, and the desalted eluate was freeze—dried to obtain 18 g of an angiogenin fraction having an angiogenin purity of 5%. The above successive operations were repeated 50 times.

Reference Example 3 ation of cystatin fraction 0 liters of a 5% whey protein solution was heat—treated at 90°C for 10 minutes, and a precipitate was removed by centrifiigation. A column was filled with a carrier prepared by binding carboxymethylated papain to Tresyl-Toyopearl (manufactured by Tosoh Corporation). After equilibration with a 0.5 M sodium chloride solution, the above whey protein on 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 chloride solution. The eluted fraction was immediately neutralized with a 1 M sodium hydroxide solution. The eluate was then desalted using a reverse osmosis membrane, and the ed 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. ement of angiogenin and cystatin contained in cheese The content of angiogenin, angiogenin hydrolysate, cystatin ' and cystatin . hydrolysate in the cheese was measured according to the method described in JP-A—2008-164511 with modification. cally, 190 mg of the cheese was added to 65 ml of ultrapure water, and a 1/1000-equivalent amount of formic acid was added to the mixture to prepare a sample solution. Ten iters (10 ul) of the sample solution was dried up, and dissolved in 20 ul of 0.1 M ammonium bicarbonate SNOW-199 containing 8 M urea and 1 mM tris(carboxyethyl)phosphine (TCEP). The solution was heated at 56°C for 30 minutes. After returning the on to room temperature, 5 p1 of a 100 mM iodoacetamide solution 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 ul of 0.1 ug/ml trypsin and 10 ul of 0.1 ug/ml Lysyl Endopeptidase were added to the mixture. The mixture was reacted at 37°C for 16 hours. The reaction was then terminated by adding 3 ul of formic acid and used as a sample peptide solution for measurement. The sample solution was diluted 6-fold with 10 fmol/ul al standard peptide on containing 0.1% formic acid, 0.02% trifluoroacetic acid (TFA), and 2% itrile, and 2.5 ul of the diluted solution was subjected to LC/MS/MS analysis.

The peptides were separated by gradient elution using an HPLC system. More specifically, the peptides were ted using a column (MAGIC C18, 0.2 mm (ID) X 50 mm) equipped with a 5 til-peptide trap on a MAGIC 2002 HPLC system at a flow rate of 2 ul/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. nt elution was conducted under the elution condition from 2 to 65% the solution B over 20 minutes.

As object ions for measuring cystatin, parent ion was NHz-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 angiogenin, parent ion was NHz—YIHFLTQHYDAK—COOH (m/z 768.8), and the MS/MS target ion was NHz—FLTQHYDAK—COOH (In/z 1122.8). Regarding the al standard peptide parent ion was NHg—ETTVFENLPEK-COOH (wherein, P was labeled with 13C and 15N) (m/z 656.9.), and the MS/MS target ion was NLPEK-COOH (wherein, P was labeled with l3c and 15N) (m/z 882.4).

SNOW-199 A system “LCQ age” was used for MS. The peak area of each protein was calculated from the ing chromatogram, and the concentration was calculated from the ratio with respect to the internal standard peptide.

Example 1 Eight point eight grams (8.8 g) of Gouda cheese and 8.8 g of r cheese were mixed. Next, 0.4 g of sodium citrate as emulsifying salt is added o, and 2 g ofwater, 35 mg of the angiogenin fraction obtained in Reference Example 1 and 0.25 mg of the cystatin fraction obtained in Reference Example 3 were further added to the mixture. The mixture was emulsified at 85°C in the usual manner. After the completion of the emulsification, the emulsion was placed into a carton, and cooled 5°C for two days and nights to obtain a cheese (example product 1). The resulting cheese contained enin and/0r angiogenin hydrolysate in an amount of 160 mg/100 g, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the cheese was 0. 02.

Example 2 Eight point eight grams (8.8 g) of Gouda cheese and 8.8 g of r cheese were mixed. Next, 0.4 g of sodium citrate as emulsifying salt is added thereto, and 2 g of water, 20 mg of the angiogenin on obtained in Reference Example 2 and 1.4 of the cystatin fraction obtained in Reference Example 3 were mixed therewith. The mixture was emulsified at 85°C in the usual manner. After the tion of the emulsification, the emulsion was placed into a carton, and cooled at 5°C for two days and nights to obtain a cheese (example product 2). The resulting cheese contained angiogenin and/or angiogenin hydrolysate in an amount of 6.5 mg/100 g, and the mass ratio of cystatin and/or cystatin hydrolysate t0 angiogenin and/or angiogenin hydrolysate in the cheese was 1.6.

SNOW-199 Example 3 Eight point eight grams (8.8 g) of Gouda cheese and 8.8 g of r cheese were mixed. Next, 0.4 g of sodium citrate as emulsifying salt is added thereto, and 2 g of water, 20 mg of the angiogenin on obtained in Reference Example 1 and 1.4 mg of the cystatin fraction obtained in Reference Example 3 were mixed therewith. The mixture was emulsified at 85°C in the usual manner. After the completion of the emulsification, the emulsion was placed into a , and cooled at 5°C for two days and nights to obtain a cheese (example product 3). The resulting cheese contained angiogenin and/or angiogenin hydrolysate in an amount of 90 mg/100 g, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the cheese was 0.11.

Comparative Example 1 Eight point eight grams (8.8 g) of Gouda cheese and 8.8 g of cheddar cheese were mixed. Next, 0.4 g of sodium citrate as emulsifying salt is added thereto, and 2 g of water, 18mg of the angiogenin fraction obtained in Reference Example 2 and 3.4 of the cystatin fraction ed in Reference Example 3 were mixed therewith. The mixture was emulsified at 85°C in the usual . After the completion of the emulsification, the on was placed into a carton, and cooled at 5°C for two days and nights to obtain a cheese (comparative example product 1). The resulting cheese contained angiogenin and/or angiogenin hydrolysate in an amount of 5.8 mg/100 g, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or enin hydrolysate in the cheese was 3.3.

Comparative Example 2 Eight point eight (8.8 g) of Gouda cheese and 8.8 g of cheddar cheese were SNOW—199 mixed. Next, 0.4 g of sodium citrate as emulsifying salt is added thereto, and 2 g of water, 35.2 mg of the angiogenin fraction obtained in Reference Example 1 and 0.05 mg of the cystatin fraction obtained in Reference Example 3 were mixed therewith. The mixture was emulsified at 85°C in the usual manner. After the completion of the emulsification, the emulsion was placed into a carton, and cooled at 5°C for two days and nights to obtain a cheese (comparative example product 2). The resulting cheese contained angiogenin and/or angiogenin hydrolysate in an amount of 161 mg/100 g, and the mass ratio of in and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the cheese was 0.14.

Test Example 1 The bone-strengthening s of the e 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. Each cheese of the example products 1 to 3 and the comparative e products 1 and 2 was added to hot water (60°C) so that the content of the cheese was 20%, and the mixture was homogenously stirred. After 1 week acclimation, the mice were divided into six groups (10 roup). The mice were orally administered each of the example products 1 to 3 and the comparative example ts 1 and 2 in an amount of g (as cheese)/day per 1 kg of mouse weight daily in two divided dose using a tube.

The control group was not administrated any example products 1 to 3 and the comparative example ts 1 and 2. After completion of administration (second week), the bone density of the right tibia of each mouse was measured using a micro-CT actured by Rigaku Corporation). The results are shown in Table I. As shown in Table 1, the groups that were orally administered the example products 1 to 3 showed a significant increase in bone density as compared with the control group and the comparative example groups that were orally administered the ative example SNOW—199 product 1 or 2.

TABLE 1 Reference Example 4 A column (diameter: 4 cm, height: 30 cm) filled with 400 g of -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 . After thoroughly washing the column with deionized water, proteins adsorbed on the resin were eluted using a 0.02 M carbonate buffer (pH 7.0) containing 0.78 M sodium de. The eluate was desalted using a reverse osmosis membrane, and the desalted eluate was freeze-dried to obtain 18 g of a powdery protein material (reference example product 4).

Reference Example 5 Four grams (4 g). of protein material of the reference example product 4 was dissolved in 800 m1 of water. After the addition of n (manufactured by Sigma), which is a se, 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 protease SNOW-199 through heat—treatment at 90°C for 5 minutes, the mixture was freeze-dried to obtain 3.0 g of a y protein material ence example product 5).

Example 4 [003 1] Forty milligrams (40 mg) of the reference example product 4 was mixed with 3 g of 30% cream. The e was homogenously added to 17 g of cottage cheese curds to obtain a cheese (example product 4). The resulting cheese contained angiogenin and/or angiogenin hydrolysate in an amount of 11 mg/l 00 ml, and the mass ratio of cystatin and/or in hydrolysate to angiogenin and/or angiogenin hydrolysate in the cheese was 0.35.

Example 5 Forty milligrams (40 mg) of the reference example product 5 was mixed with 3 g of 30% cream. The mixture was homogenously added to 17 g of cottage cheese curds to obtain a cheese (example product 5). The resulting cheese contained angiogenin and/or angiogenin hydrolysate in an amount of 11 mg/100 g, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin ysate in the cheese was 0.36.

Forty milligrams (40 mg) of the reference example product 4 was added to 100 ml of milk that was adjusted in fat content to 2.8%, and the mixture was sterilized at 77°C for 15 seconds. After g, starter, rennet, and the like were added thereto, and the mixture was allowed to stand for 30 minutes. After that, the whey was removed to prepare cheese curds. The cheese curds were salted, and the salted cheese curds were placed in a mold to obtain a cheese (example product 6). The ing cheese contained angiogenin and/or angiogenin hydrolysate in an amount of 16 mg/100 SNOW-199 g, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin hydrolysate in the cheese was 0.6 Comparative Example 3 Thirty milligrams (30 mg) of the reference example product 4 and 10 mg of the cystatin fraction obtained in Reference Example 3 were mixed with 3 g of 30% cream.

The mixture was homogenously added to 17 g of cottage cheese curds to obtain a cheese (comparative example product 3). The obtained cheese contained angiogenin and/or angiogenin hydrolysate in an amount of 8.8 mg/100 g, and the mass ratio of cystatin and/or cystatin hydrolysate to angiogenin and/or angiogenin ysate in the cheese was 5.6.

Test Example 2 The bone—strengthening effects of the example products 4 to 6 and the comparative example product 3 were determined by animal experiments. Forty eight SD female rats (51 weeks old) were used for the animal experiments. Each of the e products 4 to 6 and the comparative example product 3 was added to hot water (60°C) so that the content of the cheese was 20%, and the mixture was homogenously mixed and stirred. The rats were divided into six groups (8 rats/group). Five groups underwent ovariectomy and the ing one group sham surgery. After a 4-week recovery period, the ovareactomized rats were orally administered the e products 4 to 6 or the comparative example product 3 in an amount of 20 g (as cheese) per 1kg of rat weight daily in six divided dose using a tube. The control group was not administrated any e products 4 to 5 and the comparative example product 3.

After a 4-week recovery period, the rats underwent sham y were fed for 16 weeks in the same manner as the control group. After completion of administration enth week), the bone density of the right tibia of each rat was measured using a SNOW—199 micro-CT actured by Rigaku ation).

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 Example 7 Fifty milligrams (50 mg) of the reference e product 4 was added to 100 ml of milk that was adjusted in fat content to 3.6%, and the mixture was sterilized at 77°C for 15 seconds. The mixture was then . A starter, rennet, and the like were added thereto, and the mixture was allowed to stand for 40 minutes. A tarter, rennet, and the like, were added thereto and stirred, after that the mixture was allowed to stand for 40 minutes. The whey was then removed to prepare cheese curds. After the addition of 0.05% of blue mold (P. roqueforti) was added to the cheese curds at 0.05% with t to the cards, the cheese curds were placed in a cheese hoop, and allowed to stand at 20°C for 20 hours. The cheese curds were taken out from the hoop, and the SNOW-199 surface of the cheese was rubbed with a salt for 3 days. After the completion of the salting, needling was conducted at the upper and lower sides of the cheese curds.

After needling, the surface of the cheese was wrapped with a film, and the cheese was then matured at 8oC for 60 days. The ed cheese contained angiogenin and/or angiogenin hydrolysate in an amount of 19 mg/100 g, and the mass ratio of cystatin and/or in hydrolysate to angiogenin and/or angiogenin hydrolysate in the cheese was 0.5.

SNOW-199

Claims (7)

1. A cheese comprising enin and/or angiogenin hydrolysate in an amount of 6.5 mg/100 g of the cheese to 160 mg/100 g of the cheese and cystatin and/or in hydrolysate in the mass ratio to the angiogenin and/or angiogenin hydrolysate of 0.02 to 1.6.

2. Use of (i) angiogenin and/or angiogenin hydrolysate and (ii) cystatin and/or cystatin ysate in the manufacture of a medicament for the prevention of bone disease, fracture, rheumatism and/or tis, wherein the angiogenin and/or angiogenin hydrolysate is present in an amount of 6.5 mg/100 g to 160 mg/100 g of the medicament and wherein the cystatin and/or cystatin hydrolysate is present in a ratio of 0.02 to 1.6 to the mass of angiogenin and/or angiogenin hydrolysate.

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

4. Use according to claim 2 or 3 wherein the medicament is for administration at an amount of 20 g/day or more.

5. Use according to any one of claims 2 to 4 wherein the ment is a cheese.

6. A method of producing the cheese according to claim 1, comprising mixing angiogenin and/or angiogenin hydrolysate and cystatin and/or cystatin hydrolysate with a raw material cheese and/or a cheese curd.

7. A method of producing the cheese ing to claim 1, comprising mixing a raw material cheese with angiogenin and/or angiogenin hydrolysate and in and/or cystatin hydrolysate, and emulsifying and cooling the mixture.