New types of fermented dairy products and methods for producing the same.

Abstract

New request updated. The invention relates to a powdered milk product that is a combination of... With angiogenin and / or angiogenin hydrolosate in the amount of 0.9 mg per 100 g. 150 mg per 100 g and lactoperoxidase and / or lactoperoxidase hydrolysate. Sets were obtained with a mass-to-angiogenin and / or angiogenin hydrolysate ratio of 0.3 to 23;

Description

[0001] The present invention relates to novel fermented milk products and methods for producing the same. The fermented milk products of the present invention, by containing specific milk components, are useful for the prevention and treatment of various bone diseases such as osteoporosis, fractures, rheumatism, and arthritis.

[0002] In recent years, various bone-related diseases such as osteoporosis, fractures, and lower back pain have been increasing globally due to aging and other factors, becoming a major social problem. This is believed to be caused by insufficient calcium intake, decreased calcium absorption capacity, and hormonal imbalances after menopause. To prevent various bone diseases such as osteoporosis, fractures, and lower back pain, it is considered effective to promote bone formation by osteoblasts from a young age to increase bone mass in the body as much as possible and to increase maximum bone mass and bone strength (bone density + bone quality). Bone quality refers to the microstructure of bone, metabolic turnover, microfractures, and calcification. Another method to prevent various bone diseases such as osteoporosis, fractures, and lower back pain is to suppress bone resorption by osteoclasts. Bones are constantly undergoing a balanced cycle of absorption and formation (remodeling), but changes in hormonal balance after menopause can cause bone resorption to exceed bone formation, which is the cause of various bone diseases such as osteoporosis, fractures, and lower back pain. Therefore, by suppressing bone resorption by osteoclasts and maintaining a constant bone strength, it is possible to ultimately strengthen the bones.

[0003] Given this situation, calcium salts such as calcium carbonate, calcium phosphate, and calcium lactate, as well as natural calcium preparations such as whey calcium, bovine bone meal, and eggshell, are ingested individually as additives to pharmaceuticals, foods, and animal feed for the purpose of strengthening bones. Alternatively, these calcium preparations are ingested together with substances that promote calcium absorption, such as casein phosphopeptides and oligosaccharides, as additives to pharmaceuticals, foods, and animal feed. However, it is said that the absorption rate of calcium when these calcium salts or natural calcium preparations are added to foods is less than 50%, and that much of the calcium is excreted from the body without being absorbed. Furthermore, even if calcium is absorbed into the body, its affinity for bones differs depending on its form and the types of other nutrients ingested at the same time, so it does not necessarily show an effect of improving bone metabolism or strengthening bones. In addition, female hormone preparations and activated vitamin D are used as medicines for the treatment of osteoporosis and strengthening bones. ３ Preparations, Vitamin K ２ Alkaloid preparations, bisphosphonate preparations, and calcitonin preparations are known, and the development of new drugs such as anti-RANKL antibodies is underway. However, these drugs can cause side effects such as tinnitus, headache, and loss of appetite. Furthermore, due to safety and cost considerations, these substances cannot currently be added to food and beverages. On the other hand, given the nature of various bone diseases such as osteoporosis, fractures, and lower back pain, there is a need for the development of food and beverages that can be taken orally over the long term, act to promote bone formation and inhibit bone resorption to increase bone strength, and are expected to have preventive or therapeutic effects.

[0004] Japanese Unexamined Patent Publication No. Hei 8-151331 Japanese Unexamined Patent Publication No. Hei 10-7585 Japanese Unexamined Patent Publication No. 2004-238320 Japanese Unexamined Patent Publication No. 2005-60321

[0005] The object of the present invention is to provide fermented dairy products that are useful for the prevention and treatment of various bone diseases such as osteoporosis, fractures, rheumatism, and arthritis.

[0006] The inventors of the present invention have found that by ingesting fermented dairy products containing angiotensin and / or angiotensin degradation products and containing lactoperoxidase and / or lactoperoxidase degradation products in a specific range of mass ratios with respect to angiotensin and / or angiotensin degradation products, an effect of effectively increasing bone density can be obtained, and thus the present invention has been completed.

[0007] That is, the present invention is composed of the following components. (1) Fermented dairy products containing 0.9 mg to 150 mg / 100 g of angiotensin and / or angiotensin degradation products and containing lactoperoxidase and / or lactoperoxidase degradation products in a mass ratio range of 0.3 to 23 with respect to angiotensin and / or angiotensin degradation products. (2) A method for preventing bone diseases by ingesting 100 g / day or more of the fermented dairy products described in (1). (3) A method for producing the fermented dairy products described in (1) above, including a step of adding angiotensin and / or angiotensin degradation products and lactoperoxidase and / or lactoperoxidase degradation products to milk raw materials, sterilizing, and then fermenting. (4) A method for producing the fermented dairy products described in (1) above, including a step of adding angiotensin and / or angiotensin degradation products and lactoperoxidase and / or lactoperoxidase degradation products to sterilized milk raw materials and fermenting.

[0008] The fermented dairy products of the present invention have an effect of strengthening bones and are useful for the prevention and treatment of various bone diseases such as osteoporosis, fractures, rheumatism, and arthritis.

[0009] The characteristics of the fermented dairy products of the present invention are that they contain angiotensin and / or angiotensin degradation products in a specific range of amounts and contain angiotensin and / or angiotensin degradation products and lactoperoxidase and / or lactoperoxidase degradation products in a specific range of mass ratios. Generally, in fermented dairy products, angiotensin and / or angiotensin degradation products are contained at about 0.2 to 0.8 mg / 100 g, and lactoperoxidase and / or lactoperoxidase degradation products are contained at about 1.2 to 6.8 mg / 100 g. In contrast, the fermented milk products of the present invention are prepared by adding angiogenin and / or angiogenin hydrolysate and lactoperoxidase and / or lactoperoxidase hydrolysate, so that they contain 0.9 mg to 150 mg / 100 g of angiogenin and / or angiogenin hydrolysate, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysate to angiogenin and / or angiogenin hydrolysate is in the range of 0.3 to 23.

[0010] As angiogenin and / or angiogenin hydrolysates and lactoperoxidase and / or lactoperoxidase hydrolysates contained in the fermented milk products of the present invention, fractions containing angiogenin and / or angiogenin hydrolysates prepared from the milk of mammals such as humans, cattle, buffaloes, goats, and sheep, fractions containing lactoperoxidase and / or lactoperoxidase hydrolysates, these fractions produced by genetic engineering methods, angiogenin and / or angiogenin hydrolysates, lactoperoxidase and / or lactoperoxidase hydrolysates purified from blood or organs can be used. It is also possible to use purified and commercially available angiogenin and lactoperoxidase reagents. Furthermore, it is also possible to include angiogenin hydrolysates and lactoperoxidase hydrolysates obtained by degrading the aforementioned angiogenin-containing fraction, angiogenin reagent, lactoperoxidase-containing fraction, and lactoperoxidase reagent, respectively, with one or more types of proteolytic enzymes.

[0011] On the other hand, it is also possible to include a protein material prepared by directly extracting a fraction containing angiogenin and / or angiogenin hydrolysate and lactoperoxidase and / or lactoperoxidase hydrolysate from milk or milk-derived raw materials such as skim milk or whey. Such a protein material can be prepared, for example, by contacting milk or milk raw materials with a cation exchange resin, eluting the milk-derived protein adsorbed to the resin with a salt concentration of 0.1 to 2.0 M, desalting and concentrating it using a reverse osmosis membrane, electrodialysis membrane, ultrafiltration membrane, microfiltration membrane, etc., and then, if necessary, degrading it with a proteolytic enzyme such as trypsin, pancreatin, chymotrypsin, pepsin, papain, kallikrein, cathepsin, thermolysin, V8 protease, etc., so that the molecular weight is 8,000 or less. When degrading with a proteolytic enzyme, it is preferable that the lower limit of the molecular weight be 500 or more. Furthermore, the protein material obtained in this way can be dried by freeze-drying or spray-drying, and the dried material may be incorporated into fermented dairy products.

[0012] In the present invention, the above-mentioned angiogenin and / or angiogenin hydrolysate, lactoperoxidase and / or lactoperoxidase hydrolysate, protein material containing these, etc., are added to the milk raw material to contain angiogenin and / or angiogenin hydrolysate at a concentration of 0.9 mg to 150 mg / 100 g, and lactoperoxidase and / or lactoperoxidase hydrolysate are contained in a mass ratio of 0.3 to 23 to angiogenin and / or angiogenin hydrolysate. As will be shown in later test examples, by including angiogenin and / or angiogenin hydrolysates, lactoperoxidase and / or lactoperoxidase hydrolysates, as described above, bone strengthening effects can be obtained more effectively than by ingesting each component individually.

[0013] The fermented milk products of the present invention are not particularly limited except that they contain angiogenin and / or angiogenin hydrolysate and lactoperoxidase and / or lactoperoxidase hydrolysate in specific amounts, and may be manufactured according to general methods. The fermented milk products manufactured by the present invention include all fermented milk products such as fermented milk, dairy lactic acid bacteria beverages, and lactic acid bacteria beverages. For example, the milk raw materials are mixed as needed, angiogenin and / or angiogenin hydrolysate are added to this mixture in a specific range of amounts, and lactoperoxidase and / or lactoperoxidase hydrolysate are added in a specific mass ratio to angiogenin and / or angiogenin hydrolysate to prepare the product. Here, the milk raw materials include milk, skimmed condensed milk, skimmed milk powder, whey, butter, cream, etc., as well as milk beverages, processed milk, adjusted milk, low-fat milk, non-fat milk, etc., which are mixtures of these as needed. Subsequently, lactic acid bacteria such as Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus kefiri, Kluyveromyces marxianus, and Saccharomyces unisporus were also found. The fermented milk products of the present invention can be prepared by inoculating a starter culture, prepared by fermenting yeasts such as *Unisporus*, in an appropriate amount, and then fermenting it according to a conventional method. Furthermore, the fermented products can be sterilized afterward.

[0014] When added to milk raw materials, angiogenin and / or angiogenin hydrolysates and lactoperoxidase and / or lactoperoxidase hydrolysates may be added to unpasteurized milk raw materials or to pasteurized milk raw materials. When added to unpasteurized milk raw materials, pasteurization should be performed after addition. Pasteurization is preferably performed by heat sterilization. However, when pasteurization is performed after adding angiogenin and / or angiogenin hydrolysates and / or lactoperoxidase hydrolysates to milk raw materials, it is preferable to heat at 130°C for 2 seconds or less. In addition to angiogenin and / or angiogenin hydrolysates and lactoperoxidase and / or lactoperoxidase hydrolysates, the fermented milk products of the present invention may also contain the above-mentioned milk ingredients, as well as sugars, lipids, proteins, vitamins, minerals, flavors, and other ingredients commonly used in food and beverages. Other components that exhibit bone-strengthening effects, such as calcium, vitamin D, vitamin K, and isoflavones, may also be included.

[0015] In tests on experimental animals described later, the fermented milk products of the present invention can strengthen bones when administered orally at a dose of 100 g or more per kg of body weight. Since the intake amount in these experimental animals corresponds to the intake amount per adult in terms of blood drug concentration (Nakajima, Mitsuyoshi (1993) "Vol. 8 Pharmacological Evaluation" Hirokawa Shoten, pp. 2-18), it is generally possible for an adult to strengthen bones by consuming 100 g or more of the fermented milk products of the present invention per day, and particular effects can be expected in preventing and treating various bone diseases such as osteoporosis, fractures, rheumatism, and arthritis.

[0016] The present invention will be described in more detail below with reference examples, examples and test examples, but these are for illustrative purposes only and the present invention is not limited in any way by them.

[0017] [Reference example 1] (Preparation of the angiogenin fraction 1) A column packed with 30 kg of sulfonated chitopearl (manufactured by Fuji Spinning Co., Ltd.), a cation exchange resin, was thoroughly washed with deionized water, and then 1,000 L of unpasteurized skim milk (pH 6.7) was passed through the column. Next, the column was thoroughly washed with deionized water, and then eluted using a linear concentration gradient of sodium chloride from 0.1 to 2.0 M. The eluted fraction containing angiogenin was then fractionated using S-Sepharose cation exchange chromatography (manufactured by Amersham Biosciences), and the resulting angiogenin-containing fraction was heated at 90°C for 10 minutes, followed by centrifugation to remove the precipitate. Furthermore, this angiogenin-containing fraction was treated with Superose 12 gel filtration chromatography. After desalting the eluate using a reverse osmosis membrane, it was freeze-dried to obtain 16.5 g of an angiogenin fraction with an angiogenin purity of 90%. This series of processes was repeated 30 times.

[0018] [Reference example 2] (Preparation of the angiogenin fraction 2) A column packed with 10 kg of heparin affinity Sepharose (GE Healthcare) was thoroughly washed with deionized water, and then 500 L of unpasteurized skim milk (pH 6.7) was passed through the column. Next, the column was thoroughly washed with a 0.5 M sodium chloride solution, and then eluted with a 1.5 M sodium chloride solution. The eluate was then desalted using a reverse osmosis membrane and freeze-dried to obtain 18 g of an angiogenin fraction with an angiogenin purity of 5%. This series of processes was repeated 50 times.

[0019] [Reference example 3] (Preparation of lactoperoxidase fraction) A column (5 cm in diameter x 30 cm in height) packed with 600 g of sulfonated chitopearl (manufactured by Fuji Spinning Co., Ltd.), a cation exchange resin, was thoroughly washed with deionized water. Then, 360 L of unpasteurized skim milk (pH 6.7) was passed through the column at a flow rate of 25 ml / min. After passing the milk through, the column was thoroughly washed with deionized water and eluted with 0.02 M carbonate buffer (pH 7.0) containing 2.0 M sodium chloride. The eluted fraction containing lactoperoxidase was then adsorbed onto an S-SepharoseFF column (manufactured by Amersham Biosciences), thoroughly washed with deionized water, and equilibrated with 10 mM phosphate buffer (pH 7.0). Finally, the adsorbed fraction was eluted with a linear gradient of 0-2.0 M sodium chloride, and the lactoperoxidase-containing fraction was recovered. The fraction was then treated by gel filtration chromatography using HiLoad 16 / 60 Superdex 75 pg (Amersham Biosciences). After desalting the eluate using reverse osmosis, it was freeze-dried to obtain 27 g of lactoperoxidase fraction with a lactoperoxidase purity of 90%. This series of processes was repeated 25 times.

[0020] (Measurement of angiogenin and lactoperoxidase contained in fermented milk products) The measurement of angiogenin, angiogenin hydrolysates, lactoperoxidase, and lactoperoxidase hydrolysates contained in fermented milk products was carried out by modifying the method described in Japanese Patent Publication No. 2008-164511. Specifically, 86 μl of fermented milk products were added to 5 ml of ultrapure water, and 1 / 1,000 of the volume of formic acid was added to prepare the sample solution. After drying 10 μl of this solution, it was dissolved in 20 μl of 0.1 M ammonium bicarbonate containing 8 M urea and 1 mM tris(carboxyethyl)phosphine (TCEP), and heated at 56°C for 30 minutes. After returning to room temperature, 5 μl of 100 mM iodoacetamide solution was added, and the mixture was reacted for 30 minutes under light protection. To this, 54 μl of ultrapure water was added, followed by 10 μl of 0.1 μg / ml trypsin and 10 μl of 0.1 μg / ml lysyl endopeptidase, and the mixture was reacted at 37°C for 16 hours. Subsequently, 3 μl of formic acid was added to stop the reaction and prepare the peptide solution for measurement. Each sample solution was diluted six-fold with 10 fmol / μl of an internal standard peptide solution (containing 0.1% formic acid, 0.02% trifluoroacetic acid (TFA), and 2% acetonitrile), and 2.5 μl of the diluted solution was analyzed by LC / MS / MS.

[0021] Peptides were separated by gradient elution using an HPLC system. Specifically, a MAGIC C18 0.2 mm ID × 50 mm column with a 5 μl peptide trap was used, and the flow rate was 2 μl / min using a MAGIC2002 HPLC system. The solvents used for HPLC were Solution A: 2% acetonitrile - 0.05% formic acid and Solution B: 90% acetonitrile - 0.05% formic acid. The elution conditions involved gradient elution of Solution B from 2% to 65% over 20 minutes. The target ions for lactoperoxidase were measured with a parent ion of NH2-IHGFDLAAINLQR-COOH at m / z 734.4 and an MS / MS target ion of NH2-IHGFDLA-COOH at m / z 754.4. The target ions for angiogenin were measured with a parent ion of NH2-YIHFLTQHYDAK-COOH at m / z 768.8 and an MS / MS target ion of NH2-FLTQHYDAK-COOH at m / z 1122.8. The internal standard peptide was prepared with a parent ion of NH2-ETTVFENLPEK-COOH (where the carbon atom of P is labeled with C13 and the nitrogen atom with N15) and a m / z of 656.9. The target ion for MS / MS was NH2-FENLPEK-COOH (where the carbon atom of P is labeled with C13 and the nitrogen atom with N15) and a m / z of 882.4. LCQ Advantage was used for MS. The peak area of ​​each protein was determined from the obtained chromatogram, and the concentration was calculated from the ratio to the internal standard peptide.

[0022] 100 g of 10% reduced skim milk powder, which had been heat-sterilized at 100°C for 10 minutes, was mixed with 166 mg of the angiogenin fraction from Reference Example 1 and 45 mg of the lactoperoxidase fraction from Reference Example 3, and fermented according to a conventional method to obtain fermented milk products (Example 1). The obtained fermented milk products contained 150 mg / 100 g of angiogenin and / or angiogenin hydrolysates, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysates to angiogenin and / or angiogenin hydrolysates was 0.3.

[0023] 100 g of 10% reduced skim milk powder, which had been heat-sterilized at 100°C for 10 minutes, was mixed with 12 mg of the angiogenin fraction from Reference Example 2 and 18 mg of the lactoperoxidase fraction from Reference Example 3, and fermented according to a conventional method to obtain fermented milk products (Example 2). The obtained fermented milk products contained 0.9 mg / 100 g of angiogenin and / or angiogenin hydrolysates, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysates to angiogenin and / or angiogenin hydrolysates was 23.

[0024] 100 g of 10% reduced skim milk powder, which had been heat-sterilized at 100°C for 10 minutes, was mixed with 12 mg of the angiogenin fraction from Reference Example 1 and 18 mg of the lactoperoxidase fraction from Reference Example 3, and fermented according to a conventional method to obtain fermented milk products (Example 3). The obtained fermented milk products contained 11 mg / 100 g of angiogenin and / or angiogenin hydrolysates, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysates to angiogenin and / or angiogenin hydrolysates was 1.9.

[0025] [Comparative Example 1] 100 g of 10% reduced skim milk powder, heat-sterilized at 100°C for 10 minutes, with starter culture added, was mixed with 8 mg of the angiogenin fraction from Reference Example 2 and 22 mg of the lactoperoxidase fraction from Reference Example 3, and fermented according to a conventional method to obtain fermented milk products (Comparative Example 1). The obtained fermented milk products contained 0.9 mg / 100 g of angiogenin and / or angiogenin hydrolysates, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysates to angiogenin and / or angiogenin hydrolysates was 27.

[0026] [Comparative Example 2] 100 g of 10% reduced skim milk powder, heat-sterilized at 100°C for 10 minutes, with starter culture added, was mixed with 175 mg of the angiogenin fraction from Reference Example 1 and 30 mg of the lactoperoxidase fraction from Reference Example 3, and fermented according to a conventional method to obtain fermented milk products (Comparative Example 2). The obtained fermented milk products contained 158 mg / 100 g of angiogenin and / or angiogenin hydrolysates, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysates to angiogenin and / or angiogenin hydrolysates was 0.2.

[0027] [Test Example 1] The bone-strengthening effects of Examples 1-3 and Comparative Examples 1 and 2 were investigated through animal experiments. Five-week-old C3H / HeJ male mice were used in the experiments. After a one-week preliminary rearing period, the mice were divided into six groups of 10 each. Examples 1-3 and Comparative Examples 1 and 2 were administered orally twice daily via a tube at a dose of 100 g per kg of mouse body weight for two weeks. A control group was also provided with no administration of Examples 1-3 or Comparative Examples 1 and 2. After the completion of administration (week 2), the bone density of the right tibia of the mice was measured using micro-CT (manufactured by Rigaku Corporation). The results are shown in Table 1. As shown in Table 1, the groups that received Examples 1-3 orally for two weeks showed a significant increase in bone density compared to the control group or the groups that received Comparative Examples 1 and 2.

[0028]

[0029] [Reference example 4] A column (4 cm in diameter x 30 cm in height) packed with 400 g of sulfonated chitopearl cation exchange resin (manufactured by Fuji Spinning Co., Ltd.) was thoroughly washed with deionized water. Then, 40 L of unpasteurized skim milk (pH 6.7) was passed through the column at a flow rate of 25 ml / min. After the flow, the column was thoroughly washed with deionized water, and the proteins adsorbed to the resin were eluted with 0.02 M carbonate buffer (pH 7.0) containing 0.78 M sodium chloride. The eluate was then desalted using a reverse osmosis membrane, and freeze-dried to obtain 18 g of powdered protein material (Reference Example 4).

[0030] [Reference example 5] Four grams of protein material from Reference Example 4 were dissolved in 800 ml of water, and trypsin (manufactured by Sigma-A) was added to achieve a final concentration of 0.03% by weight. The enzyme was then treated at 37°C for 8 hours. After inactivating the enzyme by heat treatment at 90°C for 5 minutes, the material was freeze-dried to obtain 3.0 g of powdered protein material (Reference Example 5).

[0031] 97 g of 10% reduced skim milk powder was mixed with 40 mg of Reference Example 4, heat-sterilized at 93°C for 6 minutes, then 3 g of starter culture was added and fermented according to a conventional method to obtain fermented milk products (Example 4). The obtained fermented milk products contained 2.4 mg / 100 g of angiogenin and / or angiogenin hydrolysates, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysates to angiogenin and / or angiogenin hydrolysates was 5.4.

[0032] 97 g of 10% reduced skim milk powder was mixed with 40 mg of Reference Example 5, heat-sterilized at 93°C for 6 minutes, then 3 g of starter culture was added and fermented according to a conventional method to obtain fermented milk products (Example 5). The obtained fermented milk products contained 2.3 mg / 100 g of angiogenin and / or angiogenin hydrolysates, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysates to angiogenin and / or angiogenin hydrolysates was 5.3.

[0033] [Comparative Example 3] 97 g of 10% reduced skim milk powder was mixed with 15 mg of Reference Example 4 and 25 mg of the lactoperoxidase fraction of Reference Example 3, and heat-sterilized at 93°C for 6 minutes. Then, 3 g of starter culture was added, and fermentation was carried out according to a conventional method to obtain fermented milk products (Comparative Example 3). The obtained fermented milk products contained 1.2 mg / 100 g of angiogenin and / or angiogenin hydrolysate, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysate to angiogenin and / or angiogenin hydrolysate was 27.

[0034] [Correction based on Rule 91, 12.07.2013] [Test Example 2] The bone-strengthening effects of Examples 4 and 5 and Comparative Example 3 were investigated through animal experiments. Forty 51-week-old female SD rats were used in the experiment. The rats were divided into five groups of eight rats each. Four groups underwent ovariectomy, and the remaining group underwent a simulated surgery. After a four-week recovery period, the rats that underwent ovariectomy were orally administered Examples 4 and 5 and Comparative Example 3 six times a day via a tube at a dose of 100 g each per kg of rat body weight, and were reared for 16 weeks. Rats that did not receive Examples 4 and 5 or Comparative Example 3 were used as the control group. After the four-week recovery period, the rats that underwent the simulated surgery were also reared for 16 weeks in the same manner as the control group. After the completion of administration (16 weeks), the bone density of the right femur of the rats was measured using micro-CT (manufactured by Rigaku Corporation). The results are shown in Table 2. As shown in Table 2, in the group that received oral administration of Example 4 and 5 for 16 weeks, bone density was significantly higher compared to the control group or the group that received Comparative Example 3, and the values ​​were close to those of the group that underwent quasi-surgery.

[0035]

[0036] 98 g of 2.5% reduced skim milk powder, which had been heat-sterilized at 100°C for 10 minutes, was mixed with 50 mg of Reference Example 4, 2 g of starter culture was added, and fermentation was carried out according to the conventional method. After heat-sterilization at 130°C for 2 seconds, it was cooled to 10°C to obtain fermented milk products (Example 6). The obtained fermented milk products contained 2.9 mg / 100 g of angiogenin and / or angiogenin hydrolysates, and the mass ratio of lactoperoxidase and / or lactoperoxidase hydrolysates to angiogenin and / or angiogenin hydrolysates was 5.2.

Claims

Revised 5 / 02 / 2016 1. Fermented milk products containing angiogenin and / or angiogenin hydrolysate in amounts of 0.9 mg / 100 g to 150 mg / 100 g, lactoperoxidase and / or lactoperoxidase hydrolysate in a mass-to-angiogenin and / or angiogenin hydrolysate ratio of 0.3 to 2.

3.

2. The use of angiogenin and / or angiogenin hydrolysate in amounts greater than 0.9 mg / 100 ml to 150 mg / 100 ml...

3. The method of fermenting the dairy product according to claim 1 involves mixing angiogenin and / or angiogenin hydrolysate and lactoperoxidase and / or lactoperoxidase hydrolysate with dairy raw materials and sterilizing the resulting mixture, which is then fermented. 4.Method of fermenting milk products according to claim 1, which involves the addition of angiogenin and / or angiogenin hydrolysate and lactoperoxidase and / or lactoperoxidase hydrolysate to milk product raw materials and sterilization. ---------------------------------------------------------------New Application Amendment 1. Fermented milk product containing angiogenin and / or angiogenin hydrolysate in an amount of 0.9 mg / 100 g to 150 mg / 100 g and lactoperoxidase and / or lactoperoxidase hydrolysate.

1. Lactoperoxidase hydrolysate in a mass-to-angiogenin and / or angiogenin hydrolysate ratio of 0.3 to 232.

2. A method of preventing bone disease comprising the administration of fermented dairy products according to claim 1 in amounts of 100 g / day or more.

3. A method of preparing fermented dairy products according to claim 1 comprising the mixing of angiogenin and / or angiogenin hydrolysate and lactoperoxidase and / or lactoperoxidase hydrolysate with dairy raw materials and sterilizing the resulting mixture and subsequently fermenting it. 4.The method of producing fermented dairy products under claim 1 involves the addition of angiogenin and / or angiogenin hydrolysate and lactoperoxidase and / or lactoperoxidase hydrolysate to dairy raw materials and sterilization;