TWI566704B - Bone strengthening agent, products using the bone strengthening agent, and bone strengthening method - Google Patents

Description

Bone strengthening agent, product using the bone strengthening agent, and bone strengthening method

The present invention relates to a bone strengthening agent which is excellent in osteogenic strengthening and which can proliferate osteoblasts, and also has an effect of inhibiting differentiation of osteoclasts or bone resorption by the cells, for osteoporosis or fracture treatment. It is effective in the prevention or treatment of various bone diseases such as rheumatism and arthritis, and has little bitterness, excellent stability and safety. Further, the present invention relates to a food or drink for bone strengthening, a nutritional composition for bone strengthening, a feed for bone strengthening, or a medicine for bone strengthening.

In recent years, with the aging, etc., various bone-related diseases such as osteoporosis or fracture or low back pain have increased to the world scale and become a big social problem. The reason for this is considered to be insufficient calcium intake or a decrease in calcium absorption capacity, and an imbalance of hormones after menopause. In order to prevent bone diseases such as osteoporosis or fractures and low back pain, from the younger age, it promotes the formation of bones from osteoblasts, increases bone mass in the body, and increases maximum bone mass or bone strength (bone density + bone quality). It is considered to be effective. In addition, bone refers to the fine structure or metabolic transformation of bones, micro-fractures, and calcification. In addition, methods for preventing various bone diseases such as osteoporosis or fractures and low back pain can also be considered to inhibit bone resorption caused by osteoclasts. In the bone, the absorption and formation of the balance will continue to be repeated (remodeling), but because of the balance of hormones after menopause, the bone absorption exceeds the bone formation, which can become osteoporosis or fracture, low back pain, etc. The cause of various bone diseases. Therefore, by inhibiting the bone resorption caused by osteoclasts, the bones are made The strength is kept constant and the bones are strengthened as a result.

In view of the current situation, in order to strengthen the bones, calcium salts such as calcium carbonate or calcium phosphate and calcium lactate, and natural calcium agents such as whey calcium or bovine bone powder and eggshells are separately added to pharmaceuticals or foods and drinks. Ingestion of the feed or the like, or the addition of these calcium agents to a drug, food or drink, feed, or the like together with a substance having a calcium absorption-promoting effect such as casein or peptide oligosaccharide. However, when these calcium salts or natural calcium agents are added to foods and drinks, the absorption rate of calcium is 50% or less, and it is considered that more than half of the calcium is not absorbed and excreted. In addition, the affinity of calcium and bone absorbed into the body varies depending on the form or the type of other nutrients taken at the same time, and thus does not necessarily exhibit an improvement in bone metabolism or a bone strengthening effect.

In addition, as a medicine for treating osteoporosis or strengthening bone, a female hormone preparation or an active vitamin D3 preparation or a vitamin K2 preparation, a bisphosphonate preparation, a calcitonin preparation, a new drug such as an anti-RANKL antibody, and the like are known. Developing. However, in the case of using these medicines, there are cases in which side effects such as tinnitus, headache, and loss of appetite are accompanied. Furthermore, in terms of safety and cost, these substances are currently in a state in which they cannot be added to foods and drinks. Therefore, considering the disease properties of various bone diseases such as osteoporosis or fractures and low back pain, it is desired to develop a long-term oral intake to promote bone formation and/or inhibit bone resorption to increase bone strength. A bone strengthening agent for preventing or treating the effect, or a food or drink or a feed containing a bone strengthening agent.

The hydrolyzate of milk protein is used in various commodities in order to prevent food allergy caused by cow's milk or dairy products. In particular, the whey protein of milk is different from the protein of the breast milk, and it is considered to be an allergen. In order to prevent this phenomenon, it is known that the whey protein is hydrolyzed by an enzyme, and Patent Document 1 or Patent Document 2 has been known. 3 was proposed. Further, whey protein is known to promote proliferation of an osteoblast cell line (Non-Patent Document 1) or to increase bone rupture strength of a rat after ovarian ablation (Non-Patent Document 2).

[Previous Technical Literature] [Patent Literature]

Patent Document 1: Japanese Patent Laid-Open No. 2-002319

Patent Document 2: Japanese Patent Laid-Open No. 2-138991

Patent Document 3: Japanese Special Open 4112753

[Non-patent literature]

Non-Patent Document 1: Biochemical and Biophysical Research Communication. 1996; 223: 445-449

Non-Patent Document 2: Nutrition Research. 1997; 17: 1709-1720

An object of the present invention is to provide a bone strengthening agent which has high safety, promotes proliferation of osteoblasts, inhibits differentiation of osteoclasts and bone resorption by the cells, strengthens bones, and effectively prevents or treats bones. Various bone diseases such as looseness or fracture, rheumatism, and arthritis. In addition, an object of the present invention is to provide a bone-enhanced food or drink for bone strengthening, a nutrient composition for bone strengthening, a bone-enhancing feed or a bone-enhancing medicine, which promotes proliferation and inhibition of osteoblasts. The differentiation of osteoclasts and the bone resorption caused by the cells can strengthen bones and effectively prevent or treat various bone diseases such as osteoporosis or fractures, rheumatism, and arthritis.

In order to solve the above problems, the present inventors have found that the hydrolyzate of whey protein has a bone strengthening effect. That is, the present invention includes the following aspects:

(1) A bone enhancer comprising a whey protein hydrolyzate as an active ingredient.

(2) The bone strengthening agent according to (1), wherein the whey protein hydrolyzate has a decomposition rate of 25% or more.

(3) The bone enhancer according to (1) or (2), wherein the whey protein hydrolyzate has the following characteristics: (A) a molecular weight distribution of 10 kDa or less, a main peak of 200 Da to 3 kDa, and (B) APL (average The peptide chain length is 2~8, (C) The free amino acid content is 20% or less, and (D) the antigenicity is 1/10,000 or less of the antigenicity of β-lactoglobulin.

The bone-enhancing agent of any one of the above-mentioned whey protein hydrolysate which uses heat-resistant protein hydrolysate at pH 6-10, 50-70 degreeC, and milk. The protein is denatured while the enzyme is decomposed and heated to inactivate the enzyme.

(5) The bone-enhancing agent according to any one of (1) to (3), wherein the whey protein hydrolyzate is subjected to a protein hydrolyzing enzyme at pH 6 to 10 and 20 to 55 ° C to carry out whey protein The enzyme is decomposed and heated to 50-70 ° C. The heat-resistant proteolytic enzyme is used at pH 6-10, 50-70 ° C, and the undecomposed whey protein is thermally denatured while the enzyme is decomposed and heated to inactivate the enzyme. Get the winner.

(6) A food or drink for bone reinforcement, a nutrient composition for bone strengthening, a feed for bone strengthening, or a medicine for bone strengthening, which comprises the bone strengthening described in any one of (1) to (5). Agent.

The bone strengthening agent of the present invention has a remarkable bone strengthening effect by promoting the proliferation of osteoblasts and inhibiting the differentiation of osteoclasts and bone resorption by the cells, and the bone strengthening agent can effectively prevent or treat osteoporosis. Various bone diseases such as fractures, rheumatism, and arthritis. Further, since the bone reinforcing agent of the present invention uses whey protein as a raw material, it can be produced simply, economically, and easily.

The whey protein hydrolyzate of the active ingredient in the bone reinforcing agent of the present invention can be obtained, for example, by the method described in Patent Document 3. In this method, by using whey protein at pH 6 to 10 and 50 to 70 ° C, a heat-resistant proteolytic enzyme is added thereto to thermally denature it, and at the same time, the enzyme is decomposed, and the enzyme is heated to inactivate the enzyme to obtain a product. . In addition, if the whey protein is subjected to proteolytic enzymes at pH 6-10, 20-55 °C before the decomposition of the above enzymes, the enzyme is used. The decomposition can be carried out without immediately decomposing the enzyme under the above conditions without cooling it, whereby the yield can be further improved.

Further, the whey protein hydrolyzate prepared as described above may be selected from an ultrafiltration (UF) membrane having a molecular weight cut off of 1 kDa to 20 kDa, preferably 2 kDa to 10 kDa, and/or a molecular weight cutoff of 100 Da to 500 Da, preferably 150 Da to 300 Da. The method of precision filtration (MF) membrane is concentrated. By such a membrane treatment, the average molecular weight of the whey protein hydrolyzate is 300 to 500 Da, which further reduces bitterness and improves transparency.

The whey protein in the present invention refers to whey prepared from the milk of mammals such as cows, goats, sheep, humans, etc., agglomerates, powders or purified proteins, which are used in an aqueous solution during the enzyme reaction. .

When the whey protein hydrolyzate is prepared by the method described in Patent Document 3, the solution is adjusted to pH 6 to 10, but usually the pH of the whey protein is within this range, so there is no need to specifically adjust the pH, but If necessary, an acid solution such as hydrochloric acid, citric acid or lactic acid or an alkali solution such as sodium hydroxide, calcium hydroxide or sodium phosphate is used to adjust the pH to 6 to 10. The heating is carried out at 50 to 70 ° C. However, from the viewpoint of productivity, it is more suitable to carry out enzyme decomposition after adding the heat-resistant proteolytic enzyme at this temperature.

In addition, the optimum temperature of a general protease is 40 ° C or lower. However, the optimum temperature of the heat-resistant proteolytic enzyme is 45 ° C or higher, and the heat-resistant proteolytic enzyme is a protein hydrolysis which is conventionally known to have heat resistance at such an optimum temperature. The enzyme can be used without particular limitation. Examples of the heat-resistant protein hydrolyzing enzyme include papain, PROTEASE S (trade name), PROLEATHER (trade name), THERMOASE (trade name), ALCALASE (trade name), and PROTEIN A (trade name). The heat-resistant proteolytic enzyme is desirably about 10% or more with a residual activity of heating at 80 ° C for 30 minutes. In addition, the use of multiple enzymes is more effective than using it alone. The reaction is preferably carried out for about 30 minutes to 10 hours.

Finally, the reaction solution is heated to inactivate the enzyme. The inactivation of the enzyme can be achieved by placing the reaction solution at 100 ° C. It is heated by heating for 10 seconds or more.

Next, the reaction solution was centrifuged and the supernatant was recovered, and the supernatant was dried to prepare a powder product. Further, since the precipitate generated at the time of centrifugation has a lower degree of hypoallergenicity than the supernatant, it is preferable to remove it, but it is of course possible to directly dry the reaction solution and use it. The APL (average peptide chain length) of the obtained whey protein hydrolyzate can be measured by a method such as TNBS (2,4,6-trinitrobenzenesulfonic acid). Further, the molecular weight distribution of the whey protein hydrolyzate can be measured by a method such as High Performance size exclusion chromatography (HPSEC), and the free amino acid content can be obtained by extracting a free amino acid with 75% ethanol or the like. Amino acid analyzer or the like is measured. Further, the decomposition rate of the whey protein hydrolyzate can be measured by an o-phthalaldehyde (OPA) method in which a free amine group is modified and measured.

The whey protein hydrolyzate of the present invention can be used as a bone strengthening agent as it is, and if necessary, it can be used in the form of a powder, a granule, a tablet, a capsule, a drink, or the like according to a usual method. Further, the whey protein hydrolyzate obtained by further processing by ultrafiltration (UF) membrane or precision filtration (MF) membrane may be used as a bone strengthening agent as it is, or may be directly dried for use. In addition, it can also be used in accordance with a conventional method. It is also possible to blend the foods, nutrients, feeds, and pharmaceuticals into nutrients or yogurts, milk drinks, wafers, and the like, after the preparation.

The food for bone reinforcement, the nutritional composition for bone strengthening, the feed for bone strengthening, and the medicine for bone strengthening according to the present invention may contain a stabilizer, a saccharide, a lipid, or the like in addition to the whey protein hydrolyzate. Flavoring agents, vitamins, minerals, flavonoids, polyphenols, etc., other foods, feeds, and raw materials usually contained in medicines. Further, in addition to the whey protein hydrolyzate, other components exhibiting osteoinhibition, such as vitamin D or vitamin K, soy isoflavones, milk basic protein (MBP), and the like, may be used at the same time. In addition, it can be prepared by using such a food or drink for bone strengthening, a nutrient composition for bone strengthening, a feed for bone strengthening, or a medicine for bone strengthening as a raw material, and blending a raw material or the like which is usually contained in other foods and drinks.

In the food for bone fortification, the nutrient composition for bone strengthening, the feed for bone strengthening, and the medicine for bone strengthening, the blending amount of the whey protein hydrolyzate is not particularly limited, so that each adult is required to Ingestion of whey protein hydrolyzate of 2 mg or more in the celestial cavity is preferably 0.001 to 10% (w/w) based on the form of food, food, feed, and medicine, but it is generally 0.001 to 10% (w/w) based on the total mass. It should be 0.1~5% (w/w).

The bone strengthening agent of the present invention can be formulated into any form by adding an appropriate auxiliary agent to the above-mentioned active ingredient to prepare a bone-strengthening composition which can be administered orally. A diluent or an excipient which is usually used as a filler, a bulking agent, a binder, a disintegrating agent, a surfactant, a lubricant or the like can be used in the formulation. The excipient may be added, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous citric acid, magnesium aluminate, synthetic aluminum citrate, magnesium metasilicate aluminate, calcium carbonate, sodium hydrogencarbonate One or a combination of two or more of calcium hydrogen phosphate and calcium carboxymethyl cellulose.

The invention is described in detail below with reference to the embodiments, the comparative examples and the test examples, which are merely illustrative, and the invention is not limited by the scope.

[Example 1]

Add papaya enzyme 50U/g to 1L of whey protein 10% aqueous solution. Whey protein and PROLEATHER (made by Amano Enzyme Co., Ltd.) 150U/g. The whey protein was adjusted to pH 8, and the whey protein was denatured at 55 ° C for 6 hours while the enzyme was decomposed. The reaction solution was heated at 100 ° C for 15 seconds or more to inactivate the enzyme, centrifuged, and the supernatant was collected and dried to obtain a whey protein hydrolyzate (Example 1). The obtained whey protein hydrolyzate had a molecular weight distribution of 10 kDa or less, a main peak of 1.3 kDa, an APL of 7.2, and a free amino acid content of 18.9% with respect to all constituent components. The antigenicity of β-lactoglobulin was measured by the Inhibition ELISA method, and the result was 1/10,000 or less, and the decomposition rate was 28%, and the yield (the enzyme reaction solution was centrifuged, and the supernatant dry weight was relative to The ratio (%) of the dry weight of the charged amount was 80.3%, and the bitterness was 2. The whey protein hydrolyzate obtained in this manner can be directly used as the bone strengthening agent of the present invention.

[Embodiment 2]

In the whey protein 10% aqueous solution 1L, add papaya enzyme 50U / g. Whey protein and PROLEATHER (made by Amano Enzyme Co., Ltd.) 150U/g. Whey protein, fermented at pH 8, 50 ° C The protein is decomposed for 3 hours. The temperature was raised to 55 ° C, and the protein was denatured at this temperature for 3 hours, and the enzyme decomposition of the protein was carried out, and the mixture was heated at 100 ° C for 15 seconds or more to inactivate the enzyme. The reaction liquid was treated with a UF membrane (manufactured by STC Corporation) having a molecular weight cut-off of 10 kDa and an MF membrane (manufactured by STC Corporation) having a molecular weight cut off of 300 Da, and the fraction of the concentrate was collected and dried to obtain a whey protein hydrolyzate (Example) Product 2). The molecular weight distribution of the obtained whey protein hydrolyzate was 10 kDa or less, the main peak was 500 Da, the APL was 3.0, and the content of the free amino acid with respect to all the constituent components was 15.2%. The antigenicity of β-lactoglobulin was measured by the Inhibition ELISA method, and as a result, it was 1/10,000 or less, and the decomposition rate was 32%, the yield was 65.4%, and the bitterness was 2. The whey protein hydrolyzate obtained in this manner can be directly used as the bone strengthening agent of the present invention.

[Example 3]

A hydrolyzate of whey protein is prepared by the method reported in Japanese Laid-Open Patent Publication No. Hei-4-69315. 120 g of whey protein was dissolved in 1800 ml of pure water, and the pH was adjusted to 7.0 with a 1 M sodium hydroxide solution. Subsequently, the mixture was sterilized by heating at 60 ° C for 10 minutes, and kept at 45 ° C and 20 g of AMINO A (manufactured by Amano Enzyme Co., Ltd.) was added thereto to carry out a reaction for 2 hours. The enzyme was inactivated by heating at 80 ° C for 10 minutes, and lyophilized to obtain a whey protein hydrolyzate (Example 3). The molecular weight distribution of the obtained whey protein hydrolyzate was 14 kDa or less, the main peak was 3.1 kDa, the APL was 17.2, and the content of the free amino acid was 13.2% with respect to all the constituent components. The antigenicity of β-lactoglobulin was measured by the Inhibition ELISA method, and as a result, it was 1/5,000 or less, and the decomposition rate was 18%, the yield was 80.6%, and the bitterness was 2. The whey protein hydrolyzate obtained in this manner can be directly used as the bone strengthening agent of the present invention.

[Example 4]

A hydrolyzate of whey protein is prepared by the method reported in Japanese Laid-Open Patent Publication No. Hei-4-69315. 120 g of whey protein was dissolved in 1800 ml of pure water, and the pH was adjusted to 7.0 with a 1 M sodium hydroxide solution. Subsequently, the mixture was sterilized by heating at 60 ° C for 10 minutes, and kept at 45 ° C and 20 g of AMINO A (manufactured by Amano Enzyme Co., Ltd.) was added thereto to carry out a reaction for 8 hours. The enzyme was inactivated by heating at 80 ° C for 10 minutes, and lyophilized to obtain a whey protein hydrolyzate (Example 4). The obtained whey protein hydrolyzate has a molecular weight distribution of 10 kDa or less, a main peak of 1.8 kDa, and an APL of 10.0. The content of the free amino acid relative to all the constituent components was 19.3%. The antigenicity of β-lactoglobulin was measured by the Inhibition ELISA method, and as a result, it was 1/10,000 or less, and the decomposition rate was 25%, the yield was 80.6%, and the bitterness was 2. The whey protein hydrolyzate obtained in this manner can be directly used as the bone strengthening agent of the present invention.

[Comparative Example 1]

A hydrolyzate of the following casein reported in Japanese Laid-Open Patent Publication No. Hei-4-69315 was prepared as a comparative sample. 200 g of casein was suspended in 2000 ml of pure water, and the pH was adjusted to 8.0 with a 1 M sodium hydroxide solution to completely dissolve it. Then, it was sterilized by heating at 80 ° C for 10 minutes, and kept at 50 ° C, and 20 g of Pancreatin F (manufactured by Amano Enzyme Co., Ltd.) and 20 g of AMANO A (manufactured by Amano Enzyme Co., Ltd.) were added and reacted for 10 hours. The enzyme was inactivated by heating at 80 ° C for 10 minutes, and lyophilized to obtain a casein hydrolyzate (Comparative Example 1). The molecular weight distribution of the obtained casein hydrolyzate was 10 kDa or less, the main peak was 1.4 kDa, the APL was 7.8, and the content of the free amino acid with respect to all the constituent components was 19.8%. The antigenicity of β-lactoglobulin was measured by the Inhibition ELISA method, and as a result, it was 1/10,000 or less, and the decomposition rate was 27%, the yield was 77.8%, and the bitterness was 2.

[Comparative Example 2]

A hydrolyzate of the following casein reported in Japanese Laid-Open Patent Publication No. Hei-4-69315 was prepared as a comparative sample. 200 g of casein was suspended in 2000 ml of pure water, and the pH was adjusted to 8.0 with a 1 M sodium hydroxide solution to completely dissolve it. Subsequently, the mixture was sterilized by heating at 80 ° C for 10 minutes, and kept at 40 ° C, and 15 g of Pancreatin F (manufactured by Amano Enzyme Co., Ltd.) was added and allowed to react for 5 hours. The enzyme was inactivated by heating at 80 ° C for 10 minutes, and lyophilized to obtain a casein hydrolyzate (Comparative Example 2). The molecular weight distribution of the obtained casein hydrolyzate was 12 kDa or less, the main peak was 2.5 kDa, the APL was 13.8, and the content of the free amino acid relative to all the constituent components was 15.1%. The antigenicity of β-lactoglobulin was measured by the Inhibition ELISA method, and as a result, it was 1/5,000 or less, and the decomposition rate was 20%, the yield was 79.1%, and the bitterness was 2.

[Test Example 1] (Animal experiment)

The bone strengthening effect was evaluated using the whey protein hydrolyzate of Examples 1, 3, and 4 and the casein hydrolyzate of Comparative Examples 1 and 2. The experiment used 6-week-old C3H/HeJ mice. The mice were divided into a population (Group A) to which physiological saline was administered, and 2 mg of the whey protein hydrolyzate of Example 1 (Group B) was administered per 1 kg of mouse body weight, and 5 mg per 1 kg of mouse body weight was administered. The population of the whey protein hydrolyzate of Example 1 (Group C), the body weight of 10 mg of the whey protein hydrolyzate of Example 1 (Group D) per 1 kg of mouse body weight, and the administration of 10 mg per 1 kg of mouse body weight The population of the whey protein hydrolyzate of Example 3 (Group E), the body of 10 mg of the whey protein hydrolyzate of Example 4 (F group), and the body weight of each 1 kg of mice were administered 10 mg per 1 kg of mouse body weight. The test group (G group) of casein hydrolysate of Example 1 and the test group (H group) of 10 mg of the casein hydrolysate of Comparative Example 2 were administered to the body weight of 1 kg of mice (10 per group). . Each group was orally administered by intubation once a day for 2 weeks. The product samples 1, 3, and 4 and the comparative samples 1 and 2 were suspended in physiological saline, respectively, and orally administered to the B-H group. The bone density of the right ankle bone of the mouse was measured at the end of the test using 3D micro X-ray CT (Rigaku Co., Ltd.). The results are disclosed in Table 1.

Values represent mean ± standard deviation (n = 10). * indicates a significant difference (p < 0.05) from the group A as a control group.

As a result, regarding the bone density of the tibia after 2 weeks of administration, the population of 2 mg or more of the whey protein hydrolyzate of Example 1, 3, and 4 was administered orally to the body weight of 1 kg of the mouse, respectively, compared with the control group. The density has increased significantly. On the other hand, a population of 10 mg of the casein hydrolyzate of Comparative Examples 1 and 2 was orally administered to exhibit the same bone density as the control group. From this result, the whey protein of the present invention is known. The hydrolysate has a bone strengthening effect. It was also found that this osteoinhibition can be observed in the case where a minimum of 2 mg of whey protein hydrolyzate is administered per kg of mouse body weight.

[Test Example 2] (Animal experiment)

The bone strengthening effect was evaluated using the whey protein hydrolyzate of Example 2 and the casein hydrolyzate of Comparative Example 1. The SD female rats of 51 weeks old were used in the experiment. The rats were divided into 6 groups for every 6 groups, 5 groups were subjected to ovarian ablation, and the remaining 1 group was subjected to a simulated operation. During the recovery period of 4 weeks, the rats subjected to ovarian ablation surgery were orally administered with 2 mg or 10 mg once a day for 1 kg of the rat body, and the test article 2 and the comparative sample 1 were orally administered once or once a day. Physiological saline (control group; 0 mg) containing no solvent of any of the examples or test examples was orally administered to the cannula for 16 weeks. In addition, after the recovery period of 4 weeks, the rats subjected to the simulated surgery were orally administered with physiological saline (simulated surgery group; 0 mg) once a day in the same manner as the control group. After the end of the administration (week 16), the bone strength of the right thigh bone of the rat was measured by a bone strength measuring device (RX-1600, ITECNO). The results are disclosed in Table 2.

Values represent mean ± standard deviation (n = 6). * indicates a significant difference (p < 0.05) compared with the control group.

As a result, the population of 2 mg or 10 mg of the Example 2 was orally administered per 1 kg of the body weight of the rats over a period of 16 weeks, and the bone rupture strength was remarkably increased as compared with the control group, and this value was the same as that of the simulated surgery group. On the other hand, oral administration of 2 mg or 10 mg per 1 kg of rat body weight over 16 weeks The population of Comparative Example 1 exhibited the same skeletal fracture strength as the control group. From this result, it is understood that the whey protein hydrolyzate of the present invention has a bone strengthening effect. Further, it was also found that this osteoinhibition can be observed in the case where a minimum of 2 mg of whey protein hydrolyzate per 1 kg of rat body weight is administered.

[Test Example 3]

The whey protein hydrolyzate of Examples 1 to 4 and the casein hydrolyzate of Comparative Examples 1 and 2 were examined for osteoblast proliferation effect. The osteoblast cell line (MC3T3-E1) was inoculated into a 96-well plate culture plate and cultured in α-MEM medium containing 10% fetal calf serum for 24 hours. After all the culture medium was removed, 90 μl of each of α-MEM medium containing no fetal bovine serum was added, and 10 μl of each of Examples 1 to 4 and Comparative Examples 1 and 2 was added, and the cultivation was further continued for 24 hours. Brodated deoxyuridine (BrdU) attached to a Cell Proliferation kit (manufactured by GE Healthcare) was added, and after incubation for 2 hours, it was reacted with a peroxidase-labeled anti-BrdU antibody to add 3,3' as a matrix. 5,5'-tetramethylbenzidine was measured for absorbance at 450 nm, and the amount of BrdU absorbed into the cells was measured, thereby obtaining osteoblast proliferation activity. The results are disclosed in Table 3.

Values represent mean ± standard deviation (n = 5). * indicates a significant difference (p < 0.05) compared with physiological saline.

As a result, when the whey protein hydrolyzate of the example products 1 to 4 was added to the culture medium, the proliferation of the osteoblasts was remarkably promoted as compared with the case where the physiological saline was added to the culture medium. On the other hand, in the case where the casein hydrolyzate of Comparative Examples 1 and 2 was added to the medium, no proliferation of osteoblasts was observed. From this result, it is understood that the whey protein hydrolyzate of the present invention has an effect of promoting proliferation of osteoblasts.

[Test Example 4]

The whey protein hydrolyzate of Examples 1 to 4 and the casein hydrolyzate of Comparative Examples 1 and 2 were examined for the effect of inhibiting bone resorption by osteoclasts.

The tibia and thigh bone of the 5-day-old rabbit were removed, and after the soft tissue was removed, the mechanically shredded osteoclasts were contained at 1000,000 cells/well in DMEM/F12 medium containing 5% FBS. The bone marrow cells were cultured by culturing the wells of a crystalline calcium phosphate plate (manufactured by Cornig Co., Ltd.). After the culture for 2 hours, the medium was replaced with a new medium, and then the solutions in which Examples 1 to 4 and Comparative Examples 1 and 2 were dissolved were added to a concentration of 10%, and cultured for 72 hours. Next, after the cells were removed by adding a 5% sodium hypochlorite solution, the bone resorption pits appearing on the calcium phosphate disc holes were photographed under a stereoscopic microscope, and the area was measured by image analysis. This investigation inhibits the effect of bone resorption by osteoclasts (Handbook of Animal Culture Cells, classified by the research topic, pp. 199-200, 1993). The results are disclosed in Table 4.

Values represent mean ± standard deviation (n = 5). * indicates a significant difference (p < 0.05) compared with physiological saline.

As a result, when the whey protein hydrolyzate of the example products 1 to 4 was added to the medium, the area of the dimple was significantly reduced as compared with the case where the physiological saline was added to the medium. On the other hand, when the casein hydrolyzate of Comparative Examples 1 and 2 was added to the medium, the area of the dimple did not decrease. From this result, it is understood that the whey protein hydrolyzate of the present invention has an effect of inhibiting bone resorption by osteoclasts.

[Example 5] (Preparation of lozenge for bone strengthening)

After the raw materials were mixed in the formulation disclosed in Table 5, and molded and tableted in an amount of 1 g by a usual method, the bone reinforcing tablet of the present invention was produced. Further, 50 g of the whey protein hydrolyzate of Example 1 was contained in 1 g of the tablet.

[Embodiment 6] (Preparation of bone-enhanced liquid nutrient composition)

50 g of the whey protein hydrolyzate of Example 2 was dissolved in 4,950 g of deionized water, and the mixture was heated to 50 ° C, and then stirred and mixed at 6,000 rpm for 30 minutes using a TK homomixer (TK ROBO MI CS; manufactured by Kosei Kogyo Co., Ltd.). A whey protein hydrolyzate solution of Example 2 whey protein hydrolysate content of 50 g/5 kg was obtained. In this 5.0kg of whey protein hydrolysate solution, 5.0kg of casein, 5.0kg of soybean protein, 1.0kg of fish oil, 3.0kg of perilla oil, 17.0kg of dextrin, 6.0kg of mineral mixture, 1.95kg of vitamin mixture, emulsifier 2.0 kg, stabilizer 4.0 kg, and perfume 0.05 kg, and filled into a 200 ml sterilization bag, and sterilized at 121 ° C for 20 minutes with a high-temperature autoclave (first type pressure vessel, TYPE: RCS-4CRTGN, manufactured by HISAKA). 50 kg of the liquid nutritional composition for bone strengthening of the present invention was produced. In addition, the liquid nutritional composition for bone strengthening contains 100 mg of the whey protein hydrolyzate of Example 2 per 100 g.

[Embodiment 7] (Preparation of Bone Fortified Beverage)

After dissolving 300 g of skim milk powder in 409 g of deionized water, 1 g of the whey protein hydrolyzate of Example 1 was dissolved, and the mixture was heated to 50 ° C, and then a super disperser (ULTRA-TURRAXT-25; manufactured by IKA Japan Co., Ltd.) was used. Mix and mix for 30 minutes at 9,500 rpm. Add 100g of maltitol, 2g of sour sauce, After reducing 20 g of leeches, 2 g of scent, and 166 g of deionized water, the mixture was filled in a 100 ml glass bottle, sterilized at 95 ° C for 15 seconds, and then capped to prepare 10 cans (100 ml) of the bone reinforcing beverage of the present invention. Further, 100 mg of the whey protein hydrolyzate of Example 1 was contained in this 100 ml of the bone-enhancing beverage.

[Embodiment 8] (Preparation of canine bone fortified feed)

2 kg of the whey protein hydrolyzate of Example 2 was dissolved in 98 kg of deionized water, and the mixture was heated to 50 ° C, and then stirred and mixed at 3,600 rpm for 40 minutes using a TK homomixer (MARK II 160 type; manufactured by Kosei Kogyo Co., Ltd.). A whey protein hydrolyzate solution of Example 2 whey protein hydrolyzate content 2 g/100 g was obtained. In this 10kg of whey protein hydrolyzate solution, 12kg of soybean residue, 14kg of skim milk powder, 4kg of soybean oil, 2kg of corn oil, 23.2kg of palm oil, 14kg of corn starch, 9kg of flour, 2kg of bran, 5kg of vitamin mixture, 5kg of vitamin mixture, cellulose 2.8 kg and 2 kg of a mineral mixture were sterilized at 120 ° C for 4 minutes to produce 100 kg of a bone fortified feed for dogs of the present invention. Further, 200 mg of the whey protein hydrolyzate of Example 2 was contained per 100 g of the bone fortified feed for dogs.

Claims (6)

A bone strengthening agent characterized in that a whey protein hydrolyzate is used as an active ingredient, and an average molecular weight of the whey protein hydrolyzate is 300 to 500 Da. The bone strengthening agent according to claim 1, wherein the whey protein hydrolyzate has a decomposition rate of 25% or more. The bone fortifier according to claim 1 or 2, wherein the whey protein hydrolyzate uses heat-resistant proteolytic enzyme at pH 6-10, 50-70 ° C to thermally denature whey protein while enzymatic decomposition And heat to get the enzyme inactivated. The bone fortifier according to claim 1 or 2, wherein the whey protein hydrolyzate is decomposed with a protein hydrolyzate at a pH of 6 to 10 and 20 to 55 ° C to heat the whey protein to 50 °. At 70 ° C, a thermostable proteolytic enzyme is used at pH 6 to 10 and 50 to 70 ° C to thermally decompose the undecomposed whey protein while decomposing the enzyme and heating to deactivate the enzyme. A food or drink for bone reinforcement, a nutrient composition for bone strengthening, a feed for bone strengthening, or a medicine for bone strengthening, which comprises the bone strengthening agent according to any one of claims 1 to 4. A bone strengthening method characterized by ingesting whey protein hydrolysate.