Bone-strengthening agent

Abstract

To provide: a bone-strengthening agent which has a rice-derived functional material as an active ingredient, thereby being able to be safely ingested; and a composition for oral ingestion which contains the bone-strengthening agent.SOLUTION: A bone-strengthening agent has a hydrolysate of a rice endosperm protein as an active ingredient. There is also provided the bone-strengthening agent described above, wherein the rice endosperm protein is a rice protein alkali-extracted from rice endosperm. There is also provided the bone-strengthening agent described above, wherein the hydrolysate of the rice endosperm protein is a digest obtained by enzymatic treatment of the rice endosperm protein with a proteolytic enzyme. There is also provided a composition for oral ingestion which contains the bone-strengthening agent in any aspect described above.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a food-derived functional material that has a bone-strengthening effect and is useful for treating or preventing osteoporosis, promoting bone formation during the growth period, etc. [Background technology]

[0002] The main components of proteins contained in rice endosperm are easily digestible glutelin, globulin, and albumin, and indigestible prolamin. Known methods for producing a protein composition extracted from rice endosperm include a method in which heat-resistant amylase is applied to polished rice or rice flour and the remaining proteins are recovered as precipitates (enzyme-treated rice protein), and a method in which an alkaline solution is added to polished rice or rice flour to cause the extracted proteins to form acid precipitates and then recovered (alkali-extracted rice protein). The process of alkaline extraction of proteins from rice is similar to the process of removing proteins when refining starch from rice, so that alkaline-extracted rice protein can be recovered from the wastewater of a starch factory.

[0003] Rice protein has been attracting attention as a functional food ingredient due to its cholesterol-lowering and lipid metabolism-improving effects. For example, it has been reported that rice endosperm protein, which is extracted by soaking rice or rice flour in an alkaline solution and collected as a precipitate by neutralizing the extract, has a liver cholesterol-lowering effect (Patent Document 1). It has also been reported that rice endosperm protein has a serum uric acid-lowering effect (Patent Document 2).

[0004] On the other hand, due to the recent aging of the population, there is a high interest in the treatment and prevention of various bone diseases including osteoporosis. Bones are maintained by bone resorption by osteoclasts and bone formation by osteoblasts, resulting in bone remodeling. In osteoporosis, bone resorption by osteoclasts exceeds bone formation, resulting in weakened bone strength and problems such as fractures. It is expected that bones can be strengthened by suppressing bone resorption or promoting bone formation, but relatively long-term treatment is generally required. For this reason, there is a demand for a highly safe bone strengthening agent that can be taken continuously. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2006-273840 A [Patent Document 2] JP 2016-172693 A Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a bone-strengthening agent which contains a rice-derived functional material as an active ingredient and can be safely ingested, and a composition for oral intake which contains the bone-strengthening agent. [Means for solving the problem]

[0007] As a result of extensive research aimed at solving the above problems, the present inventors discovered that a hydrolysate of rice endosperm protein has a bone-strengthening effect, and thus completed the present invention.

[0008] The bone strengthening agent and the composition for oral ingestion according to the present invention are as follows. [1] A bone strengthening agent whose active ingredient is rice endosperm protein hydrolysate. [2] The bone strengthening agent according to [1], wherein the rice endosperm protein is rice protein extracted from rice endosperm by alkali. [3] The bone strengthening agent according to [1] or [2], wherein the rice endosperm protein hydrolysate is a digest obtained by enzymatically treating rice endosperm protein with a proteolytic enzyme. [4] A composition for oral intake comprising any one of the bone-strengthening agents [1] to [3] above. [5] The oral composition according to [4], which is ingested for the prevention or treatment of a bone disease. [6] The oral composition according to [5], wherein the bone disease is osteoporosis. [7] The oral composition according to [4], which is taken from infancy through the growth period. [8] The composition for oral intake according to any one of [4] to [7] above, which is a food or drink, a medicine, or a feed. Effect of the Invention

[0009] Since the bone-strengthening agent of the present invention is derived from rice, it can be taken relatively safely over a long period of time and is useful for treating and preventing osteoporosis, which requires continuous intake, and for assisting healthy bone formation during the growth period. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1(A) shows the results of measuring the bone breaking force of the femur (FIG. 1(A)) and the bone breaking energy (FIG. 1(B)) for the postmenopausal osteoporosis model in Test Example 1, which includes a control group, a group fed with feed containing 0.01% rice protein digest A (A0.01% group), a group fed with feed containing 0.1% rice protein digest A (A0.1% group), a group fed with feed containing 0.01% rice protein digest B (B0.01% group), and a group fed with feed containing 0.1% rice protein digest B (B0.1% group). [Diagram 2]FIG. 2(A) shows the results of measuring the bone breaking force of the femur (FIG. 2(A)) and the bone breaking energy (FIG. 2(B)) for the postmenopausal bone loss model in Test Example 2, which includes a control group, a group fed with a feed containing 0.01% rice protein digest A (A0.01% group), a group fed with a feed containing 0.1% rice protein digest A (A0.1% group), a group fed with a feed containing 0.01% rice protein digest B (B0.01% group), and a group fed with a feed containing 0.1% rice protein digest B (B0.1% group). [Diagram 3] FIG. 3(A) shows the results of measuring the bone breaking force of the femur (FIG. 3(A)) and the bone breaking energy (FIG. 3(B)) for the control group, the group fed with feed containing 0.1% rice protein digest B (B 0.1% group), and the group fed with feed containing 0.3% undigested rice protein (R 0.3% group) of growing rats in Test Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The bone strengthening agent according to the present invention contains a hydrolysate of rice endosperm protein as an active ingredient. Because rice is widely consumed, the hydrolysate of rice endosperm protein is expected to be highly safe and suitable for long-term continuous intake.

[0012] The rice endosperm protein, which is the raw material of the active ingredient of the bone strengthening agent according to the present invention, can be recovered by extracting it from the rice endosperm with alkali and then precipitating it with acid. That is, the "rice endosperm protein" in the present invention and this specification refers to an alkali-soluble protein in the endosperm.

[0013] The rice endosperm protein used in the present invention is extracted by soaking rice or rice flour, which contains rice endosperm, in an alkaline solution. When extracting from rice, if rice soaked in an alkaline solution is pulverized by wet grinding or the like, the protein contained in the rice tissue is also extracted, and the recovery rate can be increased. When using rice flour, rice endosperm protein can be extracted with high efficiency by soaking it in an alkaline solution for one hour to one day and night.

[0014] The rice and rice flour used as raw materials are not particularly limited as long as they contain rice endosperm, and may be any of brown rice, germ rice, polished rice, and germinated brown rice. In the case of polished rice, the polishing degree is not particularly limited, and may be about 50% or 90% or more. The variety of rice used as raw materials is not particularly limited, and may be japonica (Oryza sativa subsp. japonica), indica (Oryza sativa subsp. Indica), or javanica (Oryza sativa subsp. javanica). Among japonica varieties, various varieties of rice such as Nipponbare, Koshihikari, and Sasanishiki, and their rice flours can be used.

[0015] The alkali used for extraction is not particularly limited, but from the viewpoint of safety when it is introduced into the final product, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate, etc. The concentration of the alkali used for extraction is preferably 0.1 to 2 mass%, more preferably 0.15 to 0.25 mass%.

[0016] The protein solution obtained by the alkaline extraction contains a large amount of alkali-soluble fiber in addition to the target rice endosperm protein, and therefore it is preferable to remove this using a fine mesh sieve, filter cloth, or the like. The protein solution after the removal process is adjusted to pH 5 to 6 by adding an acid, and the aggregated protein can be recovered as a precipitate. From the viewpoints of neutralizing power and safety, the acid is preferably hydrochloric acid, sulfuric acid, or the like. The precipitation can be performed by a known solid-liquid separation process such as centrifugation or filtration. The recovered protein aggregates are appropriately washed with water and dried using airflow drying, fluidized bed drying, freeze drying, or the like, to obtain rice endosperm protein.

[0017] When a protein solution is heated to about 50°C beforehand and then neutralized, protein aggregates are obtained even at pH 7. When this is heated to 80°C or higher, the aggregates grow even larger. Therefore, protein aggregates can be efficiently recovered by filtration using a sieve or filter press without the need for centrifugation.

[0018] The active ingredient of the bone strengthening agent according to the present invention may be a hydrolysate obtained by hydrolyzing rice endosperm protein by any method. The hydrolysate of rice endosperm protein used as the active ingredient may be a digest obtained by digesting rice endosperm protein with a protease, a digest obtained by acid hydrolysis of rice endosperm protein, or a digest obtained by alkaline hydrolysis of rice endosperm protein. The acid hydrolysis treatment and the alkaline hydrolysis treatment may be carried out by a conventional method.

[0019] The active ingredient of the bone strengthening agent according to the present invention is preferably a digest obtained by digesting rice endosperm protein with a protease. The protease is not particularly limited, and may be an endoprotease (proteinase) or a peptidase. Examples of the protease include proteinase K; serine proteases such as trypsin, chymotrypsin, and subtilisin; cysteine ​​proteases such as papain and calpain; aspartic acid proteases such as pepsin and rennin; and zinc proteases such as thermolysin and carboxypeptidase A. The active ingredient of the bone strengthening agent according to the present invention is preferably a digest obtained by enzymatically treating rice endosperm protein with one or a combination of two or more of these proteases.

[0020] The enzyme reaction temperature, reaction time, and composition of the reaction solution in the digestion reaction of rice endosperm protein with a protease are appropriately adjusted depending on the enzyme used. For example, an enzymatic digest of rice endosperm protein can be obtained by adding a protease to a solution in which rice endosperm protein is dissolved in water or a buffer of an appropriate composition, and incubating the solution at pH 6.0 to 10.0 and at 20 to 55°C for 10 minutes to 6 hours. After completion of the enzyme reaction, it is preferable to inactivate the enzyme by heat treatment.

[0021] The active ingredient of the bone strengthening agent according to the present invention is preferably a product in which at least a portion of rice endosperm protein has been digested to peptides having a length of 30 or less amino acid residues. In particular, the active ingredient of the bone strengthening agent according to the present invention is preferably a product in which the proportion of peptides having a length of 30 or less amino acid residues in the total peptides in the hydrolysate is 20% by mass or more, more preferably 25% by mass or more, and even more preferably 30% by mass or more. The upper limit of the proportion of peptides having a length of 30 or less amino acid residues in the total peptides in the hydrolysate is not particularly limited, and may be 100% by mass, 90% by mass or less, 75% by mass or less, or 60% by mass or less.

[0022] The digested product after hydrolysis can be used as it is as an active ingredient of the bone strengthening agent according to the present invention, but it is preferable to use a purified product obtained by separating and purifying the hydrolyzate and undecomposed matter as the active ingredient. For example, the digested product is centrifuged with a centrifugal force such that the target hydrolyzate is present in the supernatant fraction and the undifferentiated matter is precipitated. By this, the supernatant fraction can be collected to obtain the hydrolyzate separated from the undifferentiated matter. Other methods for the purification include various methods for fractionating proteins and peptides by molecular size, such as molecular size chromatography, ultrafiltration membrane processing, and microfiltration membrane processing.

[0023] The digested matter or its purified product after hydrolysis may be used as an active ingredient of the bone strengthening agent according to the present invention in the form of a solution, or may be dried and powdered. The drying method for powdering is not particularly limited, and various drying methods such as freeze drying, spray drying, and hot air drying can be used.

[0024] The bone strengthening agent according to the present invention may consist of only the rice endosperm protein hydrolysate as the active ingredient, or may be formulated with other ingredients. The other ingredients may be any ingredients that do not impair the bone strengthening effect of the rice endosperm protein hydrolysate, and may contain, for example, various substances used as excipients, binders, flow improvers (anti-caking agents), stabilizers, preservatives, pH adjusters, solubilizers, suspending agents, emulsifiers, thickeners, flavoring agents, sweeteners, acidulants, flavoring agents, coloring agents, etc., as appropriate depending on the desired product quality. These additives can be appropriately selected from those used as additives for medicines and foods and beverages.

[0025] The dosage form of the bone strengthening agent according to the present invention is not particularly limited, and various dosage forms can be applied. As the dosage form, for example, tablets, capsules, granules, powders, syrups, sprays, and the like suitable for oral administration are preferable.

[0026] The bone strengthening agent according to the present invention is also suitable as an active ingredient of compositions for oral ingestion such as food and drink, medicines, and feed. By containing the bone strengthening agent according to the present invention as an active ingredient, a composition for oral ingestion to be orally ingested for bone strengthening can be produced. Food and drink, medicines, feed, etc. containing the bone strengthening agent according to the present invention can be produced by a conventional method, except that the bone strengthening agent according to the present invention is used as a raw material.

[0027] The bone strengthening agent according to the present invention is particularly useful as an active ingredient of medicines, functional foods, feeds, etc., ingested for the treatment or prevention of bone diseases in which bone strengthening is expected to improve the pathology. Examples of such bone diseases include osteoporosis, bone fractures, arthritis, rheumatism, and bone development disorders. The bone strengthening agent according to the present invention is also effective for strengthening bones in healthy individuals and can be ingested relatively safely over a long period of time, so that the bone strengthening agent according to the present invention is also suitable as an active ingredient of functional foods and feeds ingested for the purpose of maintaining normal bone development from infancy to the growth period and improving bone strength in adults.

[0028] The dosage of the bone strengthening agent according to the present invention or the composition for oral intake containing the same may be sufficient to exert the bone strengthening effect of the hydrolysate of rice endosperm protein, and varies depending on the species, sex, age, body weight, diet, form of administration, state of bone metabolism, etc. of the subject of administration. For example, the daily dosage of the active ingredient for an adult (body weight 60 kg) is preferably 0.1 to 20 g, more preferably 0.1 to 10 g, and even more preferably 0.1 to 1 g, of the hydrolysate of rice endosperm protein. Such a dosage can be administered once or in divided doses.

[0029] The bone strengthening agent of the present invention is preferably one that is orally ingested by mammals, more preferably one that is ingested by humans or livestock or laboratory animals such as mice, rats, rabbits, guinea pigs, hamsters, monkeys, sheep, horses, cows, pigs, donkeys, dogs, and cats, and even more preferably one that is ingested by humans. EXAMPLES

[0030] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0031] In the following description, unless otherwise specified, "%" means "% by mass."

[0032] <Rat> In the following experiments, 5-week-old SD female rats (obtained from CLEA Japan) were used. They were kept in an environment with a room temperature of 25±1°C, humidity of 50±5%, and a 12-hour light / dark cycle, and were allowed to consume food and deionized distilled water ad libitum.

[0033] [Example 1] A digest of rice endosperm protein was prepared by enzymatic treatment. The purified rice endosperm protein used as the raw material was prepared from rice flour by the method described in Patent Document 2.

[0034] The purified rice endosperm protein and a proteolytic enzyme (Zn protease or serine protease) were dissolved in water to prepare a reaction solution (pH 7.0) with a final rice endosperm protein concentration of 50 g / L. The reaction solution was incubated at 37°C for 4 hours to carry out the enzyme reaction. After completion of the enzyme reaction, the reaction solution (digestion solution) was boiled (100°C, 10 minutes) to terminate the enzyme reaction, and then centrifuged (5000 x g, 10 minutes) to recover the supernatant. The recovered supernatant was dried using a freeze dryer to obtain a rice endosperm protein digest (rice protein digest A or B).

[0035] [Test Example 1] The rice protein digest A or B prepared in Example 1 was administered to a postmenopausal osteoporosis model to examine the in vivo effects. Specifically, the effect of administration of rice endosperm protein digest B on bone strength in rats was examined.

[0036] Five-week-old rats were ovariectomized (OVX) and then fed a low calcium (Ca) diet (0.01%) for four weeks to create a postmenopausal osteoporosis model.

[0037] The postmenopausal osteoporosis model was divided into five groups, namely, a group that received the feed as is (control group), a group that received feed supplemented with 0.01% rice protein digest A (A0.01% group), a group that received feed supplemented with 0.1% rice protein digest A (A0.1% group), a group that received feed supplemented with 0.01% rice protein digest B (B0.01% group), and a group that received feed supplemented with 0.1% rice protein digest B (B0.1% group), and was bred for four weeks. The calcium content of the feed during the test diet feeding period was 0.3%. After four weeks, the animals were dissected and the femurs were collected.

[0038] To examine the bone fracture properties of the retrieved femurs, the bone fracture force (×10 6 dyn) and bone fracture energy (×10 5 erg) was measured using a stress-strain measuring device. The measurement results of bone fracture force and bone fracture energy are shown in Figure 1(A) and Figure 1(B), respectively. As shown in Figure 1(A), the bone fracture force of the femur in the A0.1% group and the B0.1% group was significantly higher than that of the control group (A0.1% group: p=0.001, B0.1% group: p=0.007). As shown in Figure 1(B), the bone fracture energy of the femur in the A0.1% group and the B0.1% group was significantly higher than that of the control group (A0.1% group: p=0.001, B0.1% group: p=0.061). These results clearly show that rice endosperm protein digest has a bone strengthening effect, suggesting that it is effective in treating osteoporosis.

[0039] [Test Example 2] The rice protein digest A or B prepared in Example 1 was administered to a postmenopausal bone loss model to examine its effect on bone strength.

[0040] Five-week-old rats were ovariectomized (OVX) to create a postmenopausal osteopenia model.

[0041] The postmenopausal bone loss models were divided into five groups, namely, a group that received the feed as is (control group), a group that received feed containing 0.01% rice protein digest A (A0.01% group), a group that received feed containing 0.1% rice protein digest A (A0.1% group), a group that received feed containing 0.01% rice protein digest B (B0.01% group), and a group that received feed containing 0.1% rice protein digest B (B0.1% group), and were bred for 8 weeks. The calcium content of the feed during the test diet feeding period was 0.3%. After 8 weeks, the animals were dissected, and the femurs were collected and assayed for bone breaking strength (×10 6 dyn) and bone fracture energy (×10 5 erg) was measured.

[0042] The results of the bone breaking force and bone breaking energy measurements of the rats in each group are shown in Figure 2(A) and Figure 2(B), respectively. As shown in Figure 2(A), the femoral bone breaking force of the A0.01% group, the B0.1% group, and the B0.1% group was significantly higher than that of the control group. As shown in Figure 2(B), the femoral bone breaking energy of the A0.01% group was significantly higher than that of the control group. Although no significant difference was observed between the B0.1% group and the B0.1% group, it was higher than that of the control group. From these results, it is clear that the rice endosperm protein digest has a bone strengthening effect, and it is also suggested that it is effective in preventing osteoporosis.

[0043] [Test Example 3] The rice protein digest B prepared in Example 1 was administered to 5-week-old healthy rats to examine its effect on bone formation during the developmental period.

[0044] After a one-week acclimation period, 5-week-old SD female rats were divided into three groups: a group that received the feed as is (control group), a group that received feed supplemented with 0.1% rice protein digest B (B 0.1% group), and a group that received feed supplemented with 0.3% rice protein (undigested) (R 0.3% group), and bred for 60 days. The calcium content of the feed during the test diet feeding period was 0.3%. After 60 days, the rats were dissected, and the femurs were collected and assayed for bone breaking strength (×10 5 dyn) and bone fracture energy (×10 6 erg) was measured.

[0045] The results of measuring the bone breaking force of the rats in each group are shown in Figure 3 (A), and the results of measuring the bone breaking energy are shown in Figure 3 (B). As shown in Figures 3 (A) and (B), both the femoral bone breaking force and bone breaking energy were significantly higher in the B 0.1% group compared to the control group. These results clearly show that the bone strengthening effect of rice endosperm protein digest is also effective in bone formation during development. On the other hand, since the R 0.3% group was at the same level as the control group (Figures 3 (A) and (B)), it is thought that undigested rice endosperm protein does not have a bone strengthening effect.

Claims

1. A bone strengthening agent whose active ingredient is rice endosperm protein hydrolysate.

2. 2. The bone strengthening agent according to claim 1, wherein the rice endosperm protein is rice protein extracted from rice endosperm with an alkali.

3. 2. The bone strengthening agent according to claim 1, wherein the rice endosperm protein hydrolysate is a digest obtained by enzymatically treating rice endosperm protein with a protease.

4. An oral composition comprising the bone strengthening agent according to any one of claims 1 to 3.

5. The oral composition according to claim 4 , which is ingested for the prevention or treatment of bone diseases.

6. The oral composition according to claim 5 , wherein the bone disease is osteoporosis.

7. The orally ingestible composition according to claim 4, which is ingested from infancy through the growth period.

8. The composition for oral ingestion according to claim 4 , which is a food or drink, a medicine, or a feed.

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