TWI574695B

TWI574695B - Bone-fortifying agent

Info

Publication number: TWI574695B
Application number: TW101142659A
Authority: TW
Inventors: 鳥羽保宏; 石田祐子; 大町愛子; 森田如一; 松山博昭; 加藤健
Original assignee: 雪印惠乳業股份有限公司
Priority date: 2012-03-09
Filing date: 2012-11-15
Publication date: 2017-03-21
Also published as: JP6009181B2; TW201336506A; JP2013184959A; WO2013132675A1

Description

骨強化劑 Bone enhancer

本發明係關於一種骨強化劑，其係將鹼性蛋白質級分及/或鹼性蛋白質級分分解物與異黃酮類及/或其代謝物作為有效成分，且使骨密度增加的作用或使骨強化的作用優異，對於骨質疏鬆症或骨折治療、風濕病、關節炎等之種種的骨疾病之預防或治療為有效，而且穩定性及安全性均佳。本發明更關於一種包含該骨強化劑的骨強化用飲食品、骨強化用營養組成物、骨強化用飼料或骨強化用醫藥品。 The present invention relates to a bone strengthening agent which uses an alkaline protein fraction and/or a basic protein fraction decomposition product and an isoflavone and/or a metabolite thereof as an active ingredient, and which increases the bone density or causes The effect of bone strengthening is excellent, and it is effective for prevention or treatment of various bone diseases such as osteoporosis or fracture treatment, rheumatism, arthritis, etc., and is excellent in stability and safety. The present invention further relates to a food or drink for bone strengthening comprising the bone strengthening agent, a nutritional composition for bone strengthening, a feed for bone strengthening, or a medicine for bone strengthening.

近年來全球隨著高齡化，骨質疏鬆症、骨折或腰痛等之種種與骨相關的疾病增加，且成為極大的社會問題。前述被人們認為是因為鈣之攝取不足或鈣吸收能力之下降、停經後的荷爾蒙之失衡等。為了預防骨質疏鬆症或骨折、腰痛等之種種的骨疾病，人們認為從幼年期開始促進利用骨芽細胞的骨形成，儘可能增加體內的骨量，提高最大骨量或骨強度(骨密度+骨質)係為有效。再者，骨質係指骨之細微結構或代謝轉換、微小骨折、石灰化。又，骨不斷地重複取得平衡的吸收與形成(重塑；remodeling)，但因為停經後的荷爾蒙之平衡變化等，所以骨吸收超過骨形成，而前述係成為骨質疏鬆症或骨折、腰痛等之種種的骨疾病之原因。因此，藉由將骨密度或骨強度保持為一定，就結果而言可將骨強化。 In recent years, with the aging of the world, various bone-related diseases such as osteoporosis, fractures, or low back pain have increased, and it has become a great social problem. The foregoing is thought to be due to 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, it is considered that the bone formation of bone bud cells is promoted from the early childhood, and the bone mass in the body is increased as much as possible to increase the maximum bone mass or bone strength (bone density + Bone) is effective. Furthermore, the bone system refers to the fine structure or metabolic conversion, microfracture, and calcification of the bone. Further, the bone continuously repeats the absorption and formation of rebalance (remodeling), but the bone absorption exceeds the bone formation due to the balance of hormones after menopause, and the above-mentioned system becomes osteoporosis, fracture, low back pain, and the like. The cause of various bone diseases. Therefore, by keeping the bone density or bone strength constant, the bone can be strengthened as a result.

根據如前述的現狀，以將骨強化為目的，將碳酸鈣或磷酸鈣、乳酸鈣等之鈣鹽以及乳清鈣或牛骨粉、蛋殼等之天然鈣劑各別單獨添加至醫藥品或飲食品、飼料等而攝取，或者，將該等鈣劑與酪蛋白磷酸胜肽或寡糖等之具有鈣吸收促進效果的物質一起添加至醫藥品或飲食品、飼料等而攝取。然而，有人指出將該等鈣鹽或天然鈣劑添加至飲食品而攝取時，鈣的吸收率為50%以下，一半以上的鈣未被吸收而排出至體外。又，在體內吸收的鈣也因根據其形態或同時攝取之其他的營養成分之種類而對於骨的親和性不同，所以也未必會展現骨代謝之改善或骨強化作用。 According to the current situation as mentioned above, calcium carbonate or calcium phosphate, calcium lactate for the purpose of bone strengthening Calcium salts and natural calcium such as whey calcium or bovine bone powder and eggshell are separately added to pharmaceuticals, foods, foods, feeds, etc., or the calcium and casein phosphate peptides or A substance having a calcium absorption promoting effect such as sugar is added to a pharmaceutical product, a food or beverage, a feed, or the like, and is ingested. However, it has been pointed out that when these calcium salts or natural calcium agents are added to foods and drinks, the absorption rate of calcium is 50% or less, and more than half of the calcium is not absorbed and is discharged to the outside of the body. Further, the calcium absorbed in the body also has different affinity for bone depending on the form or the type of other nutrients taken at the same time, and therefore does not necessarily exhibit an improvement in bone metabolism or a bone strengthening effect.

此外，作為用於骨質疏鬆症治療或骨強化的醫藥，已知有女性荷爾蒙製劑、活性型維他命D3製劑、維他命K2製劑、雙膦酸鹽(bisphosphonate)製劑、降鈣素製劑等，且抗RANKL抗體等之新藥開發進行中。但是，使用該等醫藥品時，有時會伴隨耳鳴、頭痛、食欲不振等之副作用。再者，該等物質從安全性及成本等之觀點，目前有無法添加於飲食品的情況。因此，根據骨質疏鬆症或骨折、腰痛等之種種的骨疾病之疾病性質，吾人希望開發可長期地經口攝取，且提高骨密度或骨強度，並可期待該等之預防或治療效果的骨強化劑或包含該骨強化劑的飲食品、飼料。 Further, as a medicine for osteoporosis treatment or bone strengthening, a female hormone preparation, an active vitamin D3 preparation, a vitamin K2 preparation, a bisphosphonate preparation, a calcitonin preparation, and the like are known, and anti-RANKL is known. The development of new drugs such as antibodies is underway. However, when these medicines are used, side effects such as tinnitus, headache, and loss of appetite may accompany. Furthermore, these substances are currently not available in foods and beverages from the viewpoints of safety and cost. Therefore, according to the nature of the disease of bone diseases such as osteoporosis or fractures and low back pain, it is desirable to develop bones which can be orally ingested for a long period of time and which have increased bone density or bone strength and can be expected to have such prevention or treatment effects. A fortifier or a food or drink or a feed containing the bone fortifier.

作為如前述的骨強化劑，例如，有人報導如專利文獻1中所記載的來自乳汁的鹼性蛋白質級分(以下稱為「來自乳汁的鹼性蛋白質級分」)、或將來自乳汁的鹼性蛋白質級分以蛋白質分解酵素分解而得到的鹼性蛋白質級分分解物(以下稱為「來自乳汁的鹼性蛋白質級分分解物」)。 As a bone reinforcer as described above, for example, a basic protein fraction derived from milk (hereinafter referred to as "alkaline protein fraction derived from milk") as described in Patent Document 1 or a base derived from milk is reported. The basic protein fraction is decomposed by a proteolytic enzyme (hereinafter referred to as "alkaline protein fraction decomposed product from milk").

另一方面，異黃酮類為黃豆等之豆科植物或苜蓿(clover)類所包含的類黃酮之一種，在染料木黃酮(genistein)或黃豆黃酮(daidzein)等之異黃酮類，已知有抗氧化作用或膽固醇下降作用、高血壓改善作用、抗血管新生作用(專利文獻2、非專利文獻1)。又，染料木黃酮或黃豆黃酮、為黃豆黃酮之代謝物的雌馬酚(equol)中，已知有抑制骨量之減少，並預防骨質疏鬆症的效果(非專利文獻1、非專利文獻2)。 On the other hand, the isoflavones are one type of flavonoids contained in legumes or clovers such as soybeans, and isoflavones such as genistein or daidzein are known. Antioxidation, cholesterol lowering action, hypertension improving action, and antiangiogenic effect (Patent Document 2, Non-Patent Document 1). Further, in the equol which is a metabolite of daidzein or soybean flavonoids, it is known that the effect of inhibiting the reduction of bone mass and preventing osteoporosis is achieved (Non-Patent Document 1 and Non-Patent Document 2) ).

[先前技術文獻] [Previous Technical Literature] [專利文獻] [Patent Literature]

專利文獻1：日本特開平8-151331 Patent Document 1: Japanese Patent Laid-Open No. 8-151331

專利文獻2：日本特表2009-533319 Patent Document 2: Japanese Special Table 2009-533319

[非專利文獻] [Non-patent literature]

非專利文獻1：化學與生物2008；46：872-878 Non-Patent Document 1: Chemistry and Biology 2008; 46: 872-878

非專利文獻2：Joumal of Nutrition.2010；140：1373S-1376S Non-Patent Document 2: Joumal of Nutrition. 2010; 140: 1373S-1376S

本發明的課題在於提供一種安全性高，對於骨質疏鬆症或骨折、風濕病、關節炎等之種種的骨疾病之預防或治療為有用的骨強化劑。又，本發明的課題在於提供一種摻合骨強化劑的骨強化用飲食品、骨強化用營養組成物、骨強化用飼料或骨強化用醫藥品。 An object of the present invention is to provide a bone strengthening agent which is useful for the prevention or treatment of bone diseases such as osteoporosis, bone fracture, rheumatism and arthritis. Moreover, an object of the present invention is to provide a food or drink for bone strengthening, a nutritional composition for bone strengthening, a feed for bone strengthening, or a medicine for bone strengthening, which are blended with a bone strengthening agent.

本案發明人等為了解決前述的課題而進行仔細探討時發現：藉由同時攝取來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮及/或其代謝物，相較於各別單獨攝取的情況，更可展現相乘的骨強化作用。 In order to solve the above problems, the inventors of the present invention have found that by simultaneously taking in alkaline protein fractions derived from milk and/or alkaline protein fraction decomposition products derived from milk and isoflavones and/or their metabolites Compared with the individual ingestion alone, it can exhibit multiplication of bone strengthening.

亦即，本發明包含以下態樣。 That is, the present invention encompasses the following aspects.

(1)一種骨強化劑，係將來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物作為有效成分。 (1) A bone strengthening agent which comprises an alkaline protein fraction derived from milk and/or a basic protein fraction decomposed product derived from milk and an isoflavone and/or a metabolite thereof as an active ingredient.

(2)如(1)記載之骨強化劑，其中，該來自乳汁的鹼性蛋白質級分分解物，係利用選自於由胃蛋白酶、胰蛋白酶、胰凝乳蛋白酶及胰酶構成的群組中之至少1種的蛋白質分解酵素將來自乳汁的鹼性蛋白質級分分解而成。 (2) The bone strengthening agent according to (1), wherein the alkaline protein fraction decomposed product derived from milk is selected from the group consisting of pepsin, trypsin, chymotrypsin, and trypsin. At least one of the proteolytic enzymes decomposes the alkaline protein fraction derived from milk.

(3)如(1)或(2)記載之骨強化劑，其中，該來自乳汁的鹼性蛋白質級分，係於其胺基酸組成中含有鹼性胺基酸15重量%以上的級分。 (3) The bone enhancer according to (1) or (2), wherein the alkaline protein fraction derived from milk is a fraction containing 15% by weight or more of the basic amino acid in the amino acid composition. .

(4)如(1)或(2)記載之骨強化劑，其中，該來自乳汁的鹼性蛋白質級分，係使乳汁或來自乳汁的原料與陽離子交換樹脂接觸而使鹼性蛋白質被吸附，並將該樹脂所吸附的級分以鹽濃度0.1M~1.0M的洗提液洗提而得到的級分。 (4) The bone strengthening agent according to (1) or (2), wherein the alkaline protein fraction derived from the milk is such that the milk or the raw material derived from the milk contacts the cation exchange resin to adsorb the basic protein. The fraction obtained by eluting the fraction adsorbed by the resin with an elution solution having a salt concentration of 0.1 M to 1.0 M was obtained.

(5)如(1)記載之骨強化劑，其中，該異黃酮類係選自於染料木黃酮、染料木苷(genistin)、黃豆黃酮、黃豆苷、黃豆黃苷(glycitin)、黃豆黃素(glycitein)、***以及其類似物或糖苷配基(aglycone)中之任1種以上。 (5) The bone enhancer according to (1), wherein the isoflavone is selected from the group consisting of genistein, genistin, soybean flavonoids, daidzin, glycyrin, and glycyrrhizin. Any one or more of (glycitein), estradiol, and the like or aglycone.

(6)如(1)記載之骨強化劑，其中，該異黃酮類係來自黃豆。 (6) The bone strengthening agent according to (1), wherein the isoflavones are derived from soybeans.

(7)如(1)記載之骨強化劑，其中，該異黃酮類的代謝物係為雌馬酚。 (7) The bone strengthening agent according to (1), wherein the metabolite of the isoflavones is equol.

(8)如(1)記載之骨強化劑，其中，該來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與該異黃酮類及/或其代謝物的比例為30：70~70：30。 (8) The bone fortifier according to (1), wherein the ratio of the alkaline protein fraction derived from milk and/or the alkaline protein fraction decomposition product derived from milk to the isoflavones and/or metabolites thereof It is 30:70~70:30.

(9)一種骨強化用飲食品、骨強化用營養組成物、骨強化用飼料或骨強化用醫藥品，其特徵為：包含如(1)至(8)中任一項記載之骨強化劑。 (9) A food or drink for bone reinforcement, a nutrient composition for bone strengthening, a feed for bone strengthening, or a medicine for bone strengthening, comprising the bone strengthening agent according to any one of (1) to (8) .

(10)一種骨強化方法，其係利用同時攝取來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物、與異黃酮類及/或其代謝物。 (10) A bone strengthening method which utilizes simultaneous intake of a basic protein fraction derived from milk and/or a basic protein fraction decomposition product derived from milk, and isoflavones and/or metabolites thereof.

本發明的骨強化劑，係將來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物作為有效成分，且使骨密度增加的作用或骨強化作用顯著，對於骨質疏鬆症或骨折、風濕病、關節炎等之種種的骨疾病之預防或治療為有用。 The bone strengthening agent of the present invention is an alkaline protein fraction derived from milk and/or an alkaline protein fraction decomposed product derived from milk and an isoflavone and/or a metabolite thereof as an active ingredient, and an increase in bone density. The action or bone strengthening effect is remarkable, and is useful for prevention or treatment of bone diseases such as osteoporosis or fracture, rheumatism, and arthritis.

圖1係表示併用乳鹼性蛋白質與異黃酮時之骨吸收抑制活性 Figure 1 shows the bone resorption inhibitory activity when combined with milk basic protein and isoflavones.

本發明之特徵在於將來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物作為有效成分。本發明所使用之來自乳汁的鹼性蛋白質級分，係得自於牛乳、人乳、山羊乳、羊乳等哺乳類的乳汁，而且，本發明所使用之來自乳汁的鹼性蛋白質級分分解物，可使蛋白質分解酵素作用於來自乳汁的鹼性蛋白質級分而得到。 The invention is characterized by alkaline protein fractions from milk and/or alkaline from milk Protein fraction decomposed products and isoflavones and/or their metabolites are used as active ingredients. The alkaline protein fraction derived from milk used in the present invention is derived from milk of mammals such as cow's milk, human milk, goat's milk, goat's milk, and the alkaline protein fraction decomposition product derived from milk used in the present invention. The proteolytic enzyme can be obtained by acting on a basic protein fraction derived from milk.

本發明所使用之來自乳汁的鹼性蛋白質級分，具有以下性質。 The basic protein fraction derived from milk used in the present invention has the following properties.

1)依據十二烷基硫酸鈉-聚丙烯醯胺膠體電泳(SDS-PAGE)，係由分子量3,000~80,000範圍之多種的蛋白質構成。 1) According to sodium dodecyl sulfate-polyacrylamide colloidal electrophoresis (SDS-PAGE), it is composed of a variety of proteins having a molecular weight of 3,000 to 80,000.

2)95重量%以上係蛋白質，且包含其他少量的脂肪、灰分。 2) 95% by weight or more is protein, and contains other small amounts of fat and ash.

3)蛋白質主要由乳鐵蛋白及乳過氧化酶(lactoperoxidase)構成。 3) Protein is mainly composed of lactoferrin and lactoperoxidase.

4)蛋白質之胺基酸組成，含有離胺酸、組胺酸、精胺酸等之鹼性胺基酸15重量%以上。 4) The amino acid composition of the protein, which contains 15% by weight or more of the basic amino acid such as aminic acid, histidine or arginine.

如前述的鹼性蛋白質級分，例如，可藉由以下方法而得到：使脫脂乳或乳清等之乳原料與陽離子交換樹脂接觸而吸附鹼性蛋由質，將吸附於該樹脂的鹼性蛋白質級分以0.1M~1M之鹽濃度的洗提液洗提，並將該洗提級分回收，藉由逆滲透(RO)膜或電透析(ED)法等而脫鹽及濃縮，且視需要而乾燥。又，除前述以外，得到本發明之來自乳汁的鹼性蛋白質級分的方法，也已知有：使乳汁或來自乳汁的原料與陽離子交換體接觸而吸附鹼性蛋白質後，將吸附於該陽離子交換體的鹼性蛋白質級分，以超過pH5且離子強度超過0.5的洗提液洗提而得到的方法(日本特開平5-202098號公報)、使用藻酸膠而得到的方法(日本特開昭61-246198號公報)、使用無機的多孔性粒子，自乳清中得到的方法(日本特開平1-86839號公報)、使用硫酸化酯化合物，自乳汁中得到的方法(日本特開昭63-255300號公報)等，本發明中，可使用藉由如前述的方法而得到的鹼性蛋白質級分。 The basic protein fraction as described above can be obtained, for example, by bringing a milk raw material such as skim milk or whey into contact with a cation exchange resin to adsorb an alkaline egg substance, and adsorbing the alkaline substance adsorbed to the resin. The protein fraction is eluted with an eluent of a salt concentration of 0.1 M to 1 M, and the eluted fraction is recovered, desalted and concentrated by reverse osmosis (RO) membrane or electrodialysis (ED) method, and Dry as needed. Further, in addition to the above, a method for obtaining a basic protein fraction derived from milk of the present invention is also known, in which milk or a raw material derived from milk is brought into contact with a cation exchanger to adsorb a basic protein, and then adsorbed to the cation A method in which an alkaline protein fraction of a exchanger is eluted with an eluent which exceeds pH 5 and has an ionic strength of more than 0.5 (JP-A No. 5-202098) and a method using alginic acid gel (Japanese Patent Laid-Open) JP-A-61-246198, a method obtained from whey using inorganic porous particles (Japanese Unexamined Patent Publication No. Hei No. Hei No. Hei No. 1-86839), and a method of obtaining a sulfating ester compound from milk In the present invention, a basic protein fraction obtained by the method described above can be used.

再者，本發明所使用之來自乳汁的鹼性蛋白質級分分解物，例如，對於藉由前述方法而得到之來自乳汁的鹼性蛋白質級分，使胃蛋白酶、胰蛋白酶、胰凝乳蛋白酶等之蛋白質分解酵素作用，並且可視需要，藉由使胰酶等之蛋白質分解酵素作用，而得到平均分子量4,000以下之胜肽組成物。 Further, the alkaline protein fraction decomposed product derived from milk used in the present invention, for example, pepsin, trypsin, chymotrypsin, etc., is obtained from the alkaline protein fraction derived from milk obtained by the above method. The proteolytic enzyme acts, and if necessary, a propeptide which has an average molecular weight of 4,000 or less is obtained by acting as a proteolytic enzyme such as trypsin.

得到本發明所使用之異黃酮類及/或其代謝物的方法並沒有特別限定，但例如可藉由記載於日本特開平11-9221或WO2005/046702的方法而得到。該等方法中，係於自黃豆萃取黃豆胚芽、或熱萃取該黃豆胚芽後利用液態層析提高異黃酮類的純度。又，異黃酮類的糖苷配基，在精製異黃酮類之際，可藉由利用酸加熱或β-葡萄糖醛酸酶進行水解等而得到。再者，雌馬酚，可使格氏乳球菌(Lactococcus garvieae)等之腸內細菌作用於黃豆黃酮而製備。又，本發明中的異黃酮類，亦可由黃豆等之豆科的植物或紅花苜蓿(Trifolium pratense)等之苜蓿類、或由該等製備的納豆或味噌、醬油、丹貝(tempeh)等之食品形態而製備。 The method for obtaining the isoflavones and/or metabolites thereof used in the present invention is not particularly limited, and can be obtained, for example, by the method described in JP-A-11-221221 or WO2005/046702. In the methods, the purity of the isoflavones is increased by liquid chromatography after extracting the soybean germ from the soybean or thermally extracting the soybean germ. Further, the isoflavone aglycone can be obtained by hydrolysis with acid or β-glucuronidase when the isoflavones are purified. Further, equol can be prepared by allowing intestinal bacteria such as Lactococcus garvieae to act on soybean flavonoids. Further, the isoflavones in the present invention may be those of leguminous plants such as soybeans or terpenes such as Trifolium pratense, or natto or miso, soy sauce, tempeh, etc. prepared therefrom. Prepared by food form.

本發明之來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物，亦可直接作為骨強化劑使用，但視需要也可根據常法，製劑化為粉劑、顆粒劑、錠劑、膠囊劑、飲用劑等而使用。再者，關於利用各式各樣的實驗上以及工業上方法得到的異黃酮類及/或其代謝物與來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物，也可直接作為骨強化劑使用，且也可直接乾燥後使用。又，也可根據常法，予以製劑化而使用 The alkaline protein fraction derived from milk of the present invention and/or the alkaline protein fraction decomposed product and the isoflavones and/or metabolites thereof derived from milk may be directly used as a bone strengthening agent, but may be used as needed In the usual method, the preparation is used as a powder, a granule, a tablet, a capsule, a drink, or the like. Further, regarding isoflavones and/or their metabolites obtained by various experimental and industrial methods, and alkaline protein fractions derived from milk and/or alkaline protein fraction decomposition products derived from milk, It can also be used directly as a bone strengthening agent, and can also be used directly after drying. Also, it can be formulated and used according to the common law.

本發明中，摻合來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物的方法並沒有特別限制，例如，欲在溶液中添加、摻合時，係使來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物懸浮或溶解於去離子水，且在視需要加入乳化劑或穩定劑之後，進行攪拌混合，接著製備為醫藥品、飲食品或飼料的形態而使用。攪拌混合的條件，只要將來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物混合均勻即可，亦可使用超分散器或TK均質機等攪拌混合。又，其溶液為使其易於使用在醫藥品、飲食品或飼料，可視需要以RO膜等濃縮、或冷凍乾燥而使用。本發明中，可實施通常使用於醫藥品、飲食品或飼料之製造的殺菌處理，即使為粉末狀亦可施以乾熱殺菌。因此，可製造本發明之含有來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物之液狀、膠體狀、粉末狀、顆粒狀等各式各樣的形態之醫藥品、飲食品或飼料。再者，在將該等製劑化後，亦可將其摻合於營養劑或優格、乳飲料、威化餅(wafer)等之飲食品、營養組成物、飼料及醫藥品。 In the present invention, a method of blending a basic protein fraction derived from milk and/or a basic protein fraction decomposition product from milk and an isoflavone and/or a metabolite thereof is not particularly limited, for example, in a solution. When added or blended, the alkaline protein fraction derived from milk and/or the alkaline protein fraction decomposition product derived from milk and the isoflavones and/or their metabolites are suspended or dissolved in deionized water, and After the emulsifier or the stabilizer is added, it is stirred and mixed, and then it is used in the form of a pharmaceutical, a food or drink, or a feed. Stir the mixing conditions, as long as the alkaline protein fraction from the milk and/or the alkaline protein fraction decomposed product from the milk is mixed with the isoflavones and/or their metabolites uniformly, or a hyperdisperser or Mix and mix with TK homogenizer. Further, the solution is preferably used in a pharmaceutical product, a food or beverage, or a feed, and may be concentrated by an RO membrane or the like or freeze-dried as needed. In the present invention, sterilization treatment which is generally used for the production of pharmaceuticals, foods, foods or feeds can be carried out, and dry heat sterilization can be carried out even in the form of a powder. Therefore, the present invention can be manufactured Containing alkaline protein fractions from milk and/or alkaline protein fraction decomposed products from milk and isoflavones and/or their metabolites in liquid, colloidal, powdery, granular form, etc. Form of medicine, food or drink or feed. Further, after the preparation is carried out, it may be blended into a nutraceutical, a food or drink such as a yogurt, a milk beverage, a wafer, a nutritional composition, a feed, and a pharmaceutical.

本發明的骨強化用飲食品、骨強化用營養組成物、骨強化用飼料及骨強化用醫藥品，除了僅包含該來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物的情況以外，可含有穩定劑或糖類、脂質、香料、維他命、礦物質、類黃酮、多酚類等通常包含於其他飲食品、飼料及醫藥的原料等。又，除了使用來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物以外，亦可同時使用其他展現骨強化作用的成分，例如，維他命D或維他命K、乳清蛋白質水解物等。 The food for bone for strengthening of the present invention, the nutritional composition for bone strengthening, the feed for bone strengthening, and the medicine for bone strengthening include only the alkaline protein fraction derived from milk and/or the alkaline protein fraction derived from milk. In addition to the decomposition products and isoflavones and/or their metabolites, stabilizers or sugars, lipids, flavors, vitamins, minerals, flavonoids, polyphenols, etc., which are usually included in other foods, foods, feeds, and medicines, may be contained. Raw materials, etc. Further, in addition to the use of alkaline protein fractions derived from milk and/or alkaline protein fraction decomposed products derived from milk and isoflavones and/or their metabolites, other components exhibiting osteoinhibition may be used simultaneously, for example. , vitamin D or vitamin K, whey protein hydrolysate, etc.

又，亦可將如此類的骨強化用飲食品、骨強化用營養組成物、骨強化用飼料或骨強化用醫藥品作為原料，摻合通常包含於其他飲食品等的原料等而製備。 In addition, a food or drink for bone reinforcement, a nutrient composition for bone reinforcement, a bone fortification feed, or a bone fortification pharmaceutical product can be prepared by blending a raw material or the like which is usually contained in other foods and drinks.

骨強化用飲食品、骨強化用營養組成物、骨強化用飼料及骨強化用醫藥品中之來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物的摻合量，並沒有特別限制，但成人每人一天經口攝取來自乳汁的鹼性蛋白質級分或來自乳汁的鹼性蛋白質級分分解物1mg以上、異黃酮類及/或其代謝物2mg以上較為理想，因此，雖然亦根據飲食品、飼料及醫藥的形態而異，但較佳為前述每100g中含有來自乳汁的鹼性蛋白質級分或來自乳汁的鹼性蛋白質級分分解物0.05~200mg，異黃酮類及/或其代謝物相對於總質量一般為0.001~10%(重量/重量)，較佳為0.1~5%(重量/重量)。又，來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物之摻合比例，並沒有特別限定，但以30：70~70：30的比例摻合更為理想。 Basic protein fractions derived from milk and/or alkaline protein fractions derived from milk and isoflavones in bone-enhanced foods and beverages, bone-enhancing nutrient compositions, bone-enhancing feeds, and bone-enhancing pharmaceuticals And/or the amount of the metabolite to be blended is not particularly limited, but an adult per capita oral intake of a basic protein fraction derived from milk or an alkaline protein fraction decomposition product derived from milk of 1 mg or more, isoflavones and It is preferable that it is 2 mg or more of its metabolite. Therefore, although it is also different depending on the form of foods and drinks, feed, and medicine, it is preferred that the above-mentioned basic protein fraction derived from milk or basic protein derived from milk is contained per 100 g. The fraction decomposition product is 0.05 to 200 mg, and the isoflavones and/or their metabolites are generally 0.001 to 10% (weight/weight), preferably 0.1 to 5% (weight/weight) based on the total mass. Further, the ratio of the alkaline protein fraction derived from milk and/or the ratio of the alkaline protein fraction decomposition product derived from milk to the isoflavones and/or metabolites thereof is not particularly limited, but is 30:70 to 70. A ratio of 30: 30 is more desirable.

本發明的骨強化劑，係於前述有效成分中添加適當的助劑而製劑化為任意的形態，且可成為經口投予的骨強化組成物。在製劑化時，可使用通常使用的填充劑、增量劑、黏結劑、崩解劑、界面活性劑、潤滑劑等之稀釋劑或賦形劑。作為賦形劑，例如，可將蔗糖、乳糖、澱粉、結晶性纖維素、甘露醇、輕質無水矽酸、鋁酸鎂、合成矽酸鋁、偏矽酸鋁酸鎂、碳酸鈣、碳酸氫鈉、磷酸氫鈣、羧甲基纖維素鈣等之1種或2種以上組合而添加。 The bone strengthening agent of the present invention is formulated into an arbitrary form by adding an appropriate auxiliary agent to the above-mentioned active ingredient, and can be an orally administered bone strengthening composition. At the time of formulation, 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. As the excipient, for example, sucrose, lactose, starch, crystalline cellulose, mannitol, light anhydrous citric acid, magnesium aluminate, synthetic aluminum citrate, magnesium metasilicate aluminate, calcium carbonate, hydrogencarbonate One or a combination of two or more of sodium, calcium hydrogen phosphate, and calcium carboxymethylcellulose is added.

以下顯示實施例及試驗例，且詳細說明本發明，但該等僅為示例，本發明並非限定於該等。 The embodiments and the test examples are shown below, and the present invention will be described in detail, but these are merely examples, and the present invention is not limited thereto.

實施例1 Example 1

將填充有陽離子交換樹脂之磺化CHITOPEARL(富士紡績股份有限公司製)400g的管柱(直徑5cm×高度30cm)以去離子水充分清洗後，在該管柱中將未殺菌脫脂乳40公升(pH6.7)以流速25ml/min通入。通入後，以去離子水充分清洗該管柱，並以包含0.98M氯化鈉的0.02M碳酸緩衝液(pH7.0)洗提吸附在樹脂上的鹼性蛋白質級分。然後，藉由逆滲透(RO)膜將該洗提液脫鹽並濃縮後，進行冷凍乾燥而得到粉末狀之來自乳汁的鹼性蛋白質級分21g(實施例品1)。關於得到之來自乳汁的鹼性蛋白質級分，利用十二烷基硫酸鈉-聚丙烯醯胺膠體電泳(SDS-PAGE)測定時，分子量分佈於3,000~80,000的範圍，成分組成如表1所示。又，表2顯示以6N鹽酸於110℃水解24小時後，採用胺基酸分析裝置(L-8500型、日立製作所製)分析其胺基酸組成的結果。再者，利用ELISA法分析其蛋白質組成時，如表3所示，係包含40%以上的乳鐵蛋白及乳過氧化酶。 40 g of a sulfonated CHITOPEARL (manufactured by Fujifilm Co., Ltd.) filled with a cation exchange resin was sufficiently washed with deionized water, and then 40 liters of unsterilized skim milk was placed in the column ( pH 6.7) was passed at a flow rate of 25 ml/min. After the introduction, the column was sufficiently washed with deionized water, and the basic protein fraction adsorbed on the resin was eluted with 0.02 M carbonate buffer (pH 7.0) containing 0.98 M of sodium chloride. Then, the eluate was desalted and concentrated by a reverse osmosis (RO) membrane, and then freeze-dried to obtain a powdery alkaline protein fraction 21 g from milk (Example 1). The obtained basic protein fraction derived from milk was measured by sodium dodecyl sulfate-polyacrylamide colloidal electrophoresis (SDS-PAGE), and the molecular weight distribution was in the range of 3,000 to 80,000, and the composition of the components was as shown in Table 1. . Further, Table 2 shows the results of analyzing the amino acid composition of the amino acid analyzer (L-8500 type, manufactured by Hitachi, Ltd.) after hydrolysis at 6 ° C for 24 hours in 6N hydrochloric acid. Further, when the protein composition was analyzed by ELISA, as shown in Table 3, 40% or more of lactoferrin and lactoperoxidase were contained.

實施例2 Example 2

將填充有陽離子交換樹脂之SP TOYOPEARL(東曹(股)公司製)30kg的管柱(直徑100cm×高度10cm)以去離子水充分清洗後，在該管柱中將於121℃加熱殺菌30秒的乾酪乳清3t(pH6.2)以流速10公升/min通入。通入後，以去離子水充分清洗該管柱，並以包含0.9M氯化鈉的0.1M檸檬酸緩衝液(pH5.7)洗提吸附在樹脂上的鹼性蛋白質級分。然後，藉由電透析(ED)法將該洗提液脫鹽並濃縮後，進行冷凍乾燥而得到粉末狀之來自乳汁的鹼性蛋白質級分183g(實施例品2)。 A 30 kg column (100 cm in diameter × 10 cm in height) filled with SP TOYOPEARL (manufactured by Tosoh Corporation) filled with a cation exchange resin was sufficiently washed with deionized water, and then heat-sterilized at 121 ° C for 30 seconds in the column. The cheese whey 3t (pH 6.2) was passed at a flow rate of 10 liters/min. After the introduction, the column was sufficiently washed with deionized water, and the basic protein fraction adsorbed on the resin was eluted with 0.1 M citrate buffer (pH 5.7) containing 0.9 M sodium chloride. Then, the eluate was desalted and concentrated by an electrodialysis (ED) method, and then freeze-dried to obtain 183 g of a basic protein fraction derived from milk (Example 2).

實施例3 Example 3

將實施例1所得到之來自乳汁的鹼性蛋白質級分50g溶解於蒸餾水10公升後，添加1%胰酶(SIGMA公司製)，在37℃反應2小時。反應後，在 80℃進行加熱處理10分鐘，使酵素失活後，得到來自乳汁的鹼性蛋白質級分分解物48.3g(實施例品3)。 50 g of the alkaline protein fraction derived from the milk obtained in Example 1 was dissolved in 10 liters of distilled water, and then 1% trypsin (manufactured by SIGMA) was added thereto, and the mixture was reacted at 37 ° C for 2 hours. After the reaction, at After heat treatment at 80 ° C for 10 minutes to inactivate the enzyme, 48.3 g of a basic protein fraction decomposition product derived from milk (Example 3) was obtained.

實施例4 Example 4

自黃豆13.5kg熱萃取黃豆胚芽後，利用液態層析進行精製，使異黃酮類的純度成為約92%，得到黃豆異黃酮30g(實施例品4)。該黃豆異黃酮中含有染料木黃酮9.1%、染料木苷46.9%、黃豆黃酮9.8%、黃豆苷22.6%、黃豆黃苷2.5%、黃豆黃素0.7%。 After the soybean extract was heat-extracted from 13.5 kg of soybeans, it was purified by liquid chromatography to obtain a purity of isoflavones of about 92%, and 30 g of isoflavones were obtained (Example 4). The soy isoflavones contain xylenofin 9.1%, genistein 46.9%, soybean flavonoid 9.8%, daidzin 22.6%, xanthoside 2.5%, and soy flavonoid 0.7%.

實施例5 Example 5

使β-葡萄糖醛酸酶作用於實施例4所得到的黃豆異黃酮15g，得到黃豆異黃酮的糖苷配基10g(實施例品5)。 The β-glucuronidase was allowed to act on 15 g of the soybean isoflavone obtained in Example 4 to obtain 10 g of the aglycone of the soy isoflavone (Example 5).

實施例6 Example 6

使格氏乳球菌與實施例4所得到的黃豆異黃酮12g反應96小時，得到雌馬酚含量為6.9%的黃豆異黃酮10g(實施例品6)。 Lactococcus lactis was reacted with 12 g of soy isoflavone obtained in Example 4 for 96 hours to obtain 10 g of soy isoflavone having an equol content of 6.9% (Example 6).

[試驗例1] [Test Example 1] (動物實驗) (Animal experiment)

使用實施例品1、2之來自乳汁的鹼性蛋白質級分與實施例品4之黃豆異黃酮、實施例品6之含有雌馬酚的黃豆異黃酮，對於骨強化作用進行評價。實驗中係使用6週齡的C3H/HeJ小鼠。將小鼠分成投予生理食鹽水的群組(A群組)、小鼠體重每1kg投予實施例品1之來自乳汁的鹼性蛋白質級分1mg的群組(B群組)、小鼠體重每1kg投予實施例品4之黃豆異黃酮2.2mg的群組(C群組)、小鼠體重每1kg投予實施例品1之來自乳汁的鹼性蛋白質級分3mg的群組(D群組)、小鼠體重每1kg投予實施例品4之黃豆異黃酮3.3mg的群組(E群組)、小鼠體重每1kg各別同時投予實施例品1之來自乳汁的鹼性蛋白質級分1mg與實施例品4之黃豆異黃酮2.2mg的群組(F群組)、小鼠體重每1kg各別同時投予實施例品2之來自乳汁的鹼性蛋白質級分1mg與實施例品6之含有雌馬酚的黃豆異黃酮2.2mg的群組(G群組)之7 個試驗群組(各群各10隻)。每天1次各別以胃管經口投予並飼育2週。實施例品1、4及實施例品1與4的混合物、實施例品2與6的混合物，係各別懸浮於生理食鹽水，且各別對B~G群組經口投予。在試驗結束時，使用3D微米級X射線CT(Rigaku(股))測定小鼠之右腳的脛骨之骨密度。其結果示於表4。 The bone strengthening effect was evaluated using the alkaline protein fraction derived from milk of Examples 1 and 2, the soy isoflavones of Example 4, and the equol-containing soy isoflavones of Example 6. Six week old C3H/HeJ mice were used in the experiment. The mice were divided into a group administered with physiological saline (Group A), and a mouse body weight of 1 mg of the alkaline protein fraction derived from the milk of Example 1 (Group B) and mice were administered per kg of the body weight of the mouse. A group (Group C) in which 2.2 mg of soy isoflavones of Example 4 was administered per kg of body weight, and a body weight fraction of 3 mg of milk derived alkaline protein fraction of Example 1 was administered per kg of mouse body weight (D) Group), the body weight of the mouse was administered to the group of the soy isoflavones of Example 4 of 3.3 mg (E group), and the body weight of the mice was administered to the alkaline of the sample 1 at the same time. The protein fraction 1 mg and the soy isoflavone 2.2 mg of Example 4 (Group F), and the mouse body weight of 1 mg of the alkaline protein fraction from the milk of Example 2 were simultaneously administered and administered. Example 6 of group 6 (G group) of equol-containing soy isoflavones 2.2 mg Test groups (10 in each group). Each time, the stomach tube was administered orally once a day for 2 weeks. The mixture of Examples 1 and 4 and Examples 1 and 4 and the mixture of Examples 2 and 6 were each suspended in physiological saline, and each was administered orally to the B-G group. At the end of the experiment, the bone density of the tibia of the right foot of the mouse was measured using 3D micron X-ray CT (Rigaku). The results are shown in Table 4.

其結果：投予2週後的脛骨之骨密度，在小鼠體重每1kg投予實施例品1之來自乳汁的鹼性蛋白質級分1mg或3mg的群組、小鼠體重每1kg投予實施例品4之黃豆異黃酮2.2mg或3.3mg的群組、小鼠體重每1kg各別同時投予實施例品1之來自乳汁的鹼性蛋白質級分1mg與實施例品4之黃豆異黃酮2.2mg的群組、小鼠體重每1kg各別同時投予實施例品2之來自乳汁的鹼性蛋白質級分1mg與實施例品6之含有雌馬酚的黃豆異黃酮2.2mg的群組中，與對照群組相比，骨密度顯著地上升。又，藉由同時投予實施例品1之來自乳汁的鹼性蛋白質級分與實施例品4之黃豆異黃酮、以及同時投予實施例品2之來自乳汁的鹼性蛋白質級分與實施例品6之含有雌馬酚的黃豆異黃酮，相較於各別單獨投予的群組，骨密度更顯著地上升。因此，可知：當同時投予本發明之來自乳汁的鹼性蛋白質級分與異黃酮類時，相較於各別單獨投予的情況，有相乘的提高骨密度之效果。又，可知：該骨強化作用於小鼠體重每1kg同時投予來自乳汁的鹼性蛋白質級分最低 1mg與異黃酮類最低2mg的情況中可確認。 As a result, the bone density of the tibia after 2 weeks of administration was administered to the group of 1 mg or 3 mg of the alkaline protein fraction derived from the milk of Example 1 per kg of the body weight of the mouse, and the body weight of the mouse was administered per kg. In the case of the soy isoflavones of Example 4, 2.2 mg or 3.3 mg of the group, the mouse body weight was administered to the alkaline protein fraction 1 mg of the milk of Example 1 and the soy isoflavones of Example 4 at the same time. The group of mg and the body weight of the mouse were administered to a group of 1 mg of the alkaline protein fraction of the milk of Example 2 and 2.2 mg of the equol-containing soy isoflavones of Example 2, respectively. Bone density increased significantly compared to the control group. Further, the alkaline protein fraction derived from milk of Example 1 and the soy isoflavone of Example 4 and the alkaline protein fraction derived from milk of Example 2 were simultaneously administered and examples. The soy flavonoids containing equol of product 6 showed a more significant increase in bone density compared to the individually administered groups. Therefore, it is understood that when the alkaline protein fraction derived from milk of the present invention and the isoflavones are simultaneously administered, the effect of increasing the bone density is multiplied as compared with the case of separately administered alone. Moreover, it can be seen that the bone strengthening effect is the lowest in the alkaline protein fraction from the milk when the mouse body weight is 1 kg. It can be confirmed in the case where 1 mg and isoflavones are at least 2 mg.

[試驗例2] [Test Example 2] (動物實驗) (Animal experiment)

使用實施例品3之來自乳汁的鹼性蛋白質級分分解物與實施例品5之黃豆異黃酮的糖苷配基，對於骨強化作用進行評價。實驗中係使用51週齡的SD系雌性大鼠。使大鼠每6隻為1個群組，分成5個群組，4個群組實施卵巢摘出手術，殘餘的1個群組實施假(pseudo)手術。設定4週的恢復期間，對於實施卵巢摘出手術後的大鼠，使實施例品3以大鼠體重每1kg成為1mg的方式(A群組)、使實施例品5以大鼠體重每1kg成為2.2mg的方式(B群組)、使實施例品3與實施例品5以大鼠體重每1kg同時各別成為1mg與2.2mg的方式(C群組)，1天1次以胃管經口投予，或者，將作為未包含任何實施例品的溶媒即僅是生理食鹽水1天1次以胃管經口投予(對照群組)，並飼養16週。又，在4週的恢復期間之後，對於實施假手術的大鼠，與對照群組同樣地僅將生理食鹽水1天1次以胃管經口投予(假手術群組)。在投予結束後(第16週)，利用骨強度測定裝置(RX-1600、ITECNO)測定大鼠之右大腿骨的骨強度。其結果示於表5。 The bone strengthening effect was evaluated using the alkaline protein fraction decomposed product of the milk of Example 3 and the aglycone of the soy isoflavone of Example 5. In the experiment, 51-week-old SD female rats were used. The rats were divided into 5 groups for 6 groups, and 4 groups were subjected to ovarian extraction surgery, and the remaining group was subjected to pseudo surgery. In the recovery period of 4 weeks, the rats in the ovary-extraction surgery were subjected to the method of the sample product 3 in a case where the rat body weight was 1 mg per 1 kg (group A), and the example product 5 was made in the rat body weight per 1 kg. In the method of 2.2 mg (group B), the product of Example 3 and the product of Example 5 were each 1 kg and 2.2 mg of the rat body weight (C group), and the gastric tube was administered once a day. Oral administration, or as a vehicle not containing any of the examples, that is, only physiological saline was administered orally once a day in a gastric tube (control group), and fed for 16 weeks. Further, after the recovery period of 4 weeks, the rats subjected to the sham operation were orally administered with a gastric tube once a day (sham operation group) 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 using a bone strength measuring device (RX-1600, ITECNO). The results are shown in Table 5.

其結果：在大鼠體重每1kg各別經口投予實施例品3之來自乳汁的鹼性蛋白質級分分解物1mg或實施例品5之黃豆異黃酮的糖苷配基2.2mg的群組、大鼠體重每1kg各別同時經口投予實施例品3之來自乳汁的鹼性蛋白質級分分解物1mg或實施例品5之黃豆異黃酮的糖苷配基2.2mg的群組中，與對照群組相比，骨斷裂強度顯著地上升。又，在大鼠體重每1kg各別同時經口投予實施例品3之來自乳汁的鹼性蛋白質級分分解物與實施例品5之黃豆異黃酮的糖苷配基的群組中，與各別單獨投予的群組相比，骨斷裂強度顯著提高，且其數值與假手術群組為同等級。因此，可知：當同時投予本發明之來自乳汁的鹼性蛋白質級分分解物與異黃酮類時，與各別單獨投予的情況相比，有相乘的提高骨斷裂強度之效果。又，可知：該骨強化作用於大鼠體重每1kg同時投予來自乳汁的鹼性蛋白質級分分解物最低1mg與異黃酮類最低2mg的情況中可確認。 As a result, a group of alkaline protein fraction decomposed product 1 mg of milk of Example 3 or a glycoside of 2.2 mg of soy isoflavone of Example 5 was orally administered per 1 kg of rat body weight, The rat's body weight was administered to the alkaline egg of the milk of Example 3 at the same time every 1 kg. In the group of 2.2 mg of the white matter fraction decomposed product or the aglycone of the soy isoflavone of Example 5, the bone fracture strength was remarkably increased as compared with the control group. Further, in the group of the alkaline protein fraction decomposed product of the milk of Example 3 and the aglycone of the soybean isoflavone of Example 5, each of the rat body weight was administered at a rate of 1 kg each, and each group was administered orally. Compared with the group administered alone, the bone fracture strength was significantly improved, and the value was the same as that of the sham operation group. Therefore, it has been found that when the alkaline protein fraction decomposed product derived from milk of the present invention and the isoflavones are simultaneously administered, the effect of increasing the bone fracture strength is multiplied as compared with the case of separately administered separately. Further, it was found that the bone strengthening effect was confirmed in the case where the body weight of the rat was administered at a minimum of 1 mg of the alkaline protein fraction decomposed product from the milk and the lowest isoflavone content of 2 mg.

[試驗例3] [Test Example 3]

為了調查來自乳汁的鹼性蛋白質級分及異黃酮類之骨吸收抑制效果而實施凹洞分析(pit assay)。將10日齡之兔子的大腿骨摘出，並除去軟組織後，於包含5%FBS(牛胎兒血清)的培養基中機械地切細，使包含破骨細胞的總骨髓細胞成為200,000cells/ml，接種於象牙片上，並添加試驗樣本10%進行2天培養。然後，將破骨細胞完成吸收象牙片的骨吸收窩(凹洞)予以蘇木精染色，並計算其數目(瀬野悍二等人，研究主題別動物培養細胞手冊，pp.199-200,1993)。亦即，凹洞數目少意味著破骨細胞之活性下降且骨吸收被抑制。又，於凹洞分析展現骨吸收抑制效果的物質，在動物實驗也有展現骨吸收之抑制效果(Toba等人、Bone,vol.27,p.403-408,2000)，一般而言，凹洞分析為適合調查骨吸收抑制效果的實驗系。試驗樣本，係使實施例品1之來自乳汁的鹼性蛋白質級分與實施例品4之黃豆異黃酮的重量比成為100：0~0：100的範圍而摻合，而且，使實施例品1之來自乳汁的鹼性蛋白質級分與實施例品4之黃豆異黃酮之合計成為100μg/ml而製備，並將該等添加至培養基。又，也設置作為對照之未包含任何來自乳汁的鹼性蛋白質級分與異黃酮類的對照群組(0：0)。其結果示於圖1。 A pit assay was performed in order to investigate the bone resorption inhibitory effect of the alkaline protein fraction and the isoflavones derived from milk. The thigh bone of a 10-day-old rabbit was removed, and the soft tissue was removed, and then mechanically shredded in a medium containing 5% FBS (bovine fetal serum) to make the total bone marrow cells containing osteoclasts 200,000 cells/ml, inoculated. On the ivory tablets, 10% of the test samples were added for 2 days of culture. Then, the osteoclasts complete the absorption of the osteocytes of the bone-absorbing wells (cavities) for hematoxylin staining, and calculate the number thereof. (瀬野悍二等人, Research Subject Animal Culture Cell Manual, pp. 199-200, 1993 ). That is, a small number of pits means that the activity of osteoclasts is decreased and bone resorption is suppressed. Further, a substance which exhibits a bone resorption inhibiting effect in a pit analysis has an inhibitory effect on bone resorption in animal experiments (Toba et al., Bone, vol. 27, p. 403-408, 2000), in general, a cavity The analysis was an experimental system suitable for investigating the effect of bone resorption inhibition. The test sample was prepared by blending the weight ratio of the alkaline protein fraction derived from milk of Example 1 to the soy isoflavone of Example 4 in the range of 100:0 to 0:100, and further, the example product was used. The total protein fraction derived from milk and the soy isoflavone of Example 4 were prepared to be 100 μg/ml, and these were added to the medium. Further, a control group (0:0) which did not contain any alkaline protein fraction and isoflavones derived from milk was also set as a control. The result is shown in Fig. 1.

根據圖1，因為與未包含任何來自乳汁的鹼性蛋白質級分與異黃酮類的對照群組相比較，其他全部的群組織凹洞數目顯著較少，所以可知其具有骨吸收抑制活性。又，與來自乳汁的鹼性蛋白質級分與黃豆異黃酮之重量比為100：0~80：20以及20：80~0：100的情況相比，來自乳汁的鹼性蛋白質級分與黃豆異黃酮之重量比為70：30~30：70的範圍之情況的凹洞數目顯著較少，可確認其具有各別單獨添加來自乳汁的鹼性蛋白質或黃豆異黃酮以上的骨吸收抑制活性。再者，本試驗中，除對照群組以外，使各群組之來自乳汁的鹼性蛋白質級分與黃豆異黃酮之合計重量為相同而製備，且藉由改變該等之比進行實驗。因此，本試驗例中，不是單純的相加效果，而來自乳汁的鹼性蛋白質級分與異黃酮類會相乘地進行作用，且明確地展現抑制骨吸收。 According to Fig. 1, since the number of all other group tissue pits was significantly smaller than that of the control group containing no alkaline protein fraction derived from milk and isoflavones, it was found to have bone resorption inhibiting activity. Also, with the weight of the alkaline protein fraction from the milk and the weight of the soy isoflavones Compared with the case of 100:0 to 80:20 and 20:80 to 0:100, the weight ratio of the alkaline protein fraction from the milk to the isoflavone of the soybean is in the range of 70:30 to 30:70. The number of pits was remarkably small, and it was confirmed that the bone resorption inhibitory activity of the basic protein or the soy isoflavones derived from the milk alone was added. Further, in this test, except that the control group was prepared by making the total weight of the alkaline protein fraction derived from the milk and the isoflavone of the respective groups the same, and experimenting was carried out by changing the ratios. Therefore, in this test example, it is not a simple addition effect, and the alkaline protein fraction derived from milk acts synergistically with the isoflavones, and clearly exhibits inhibition of bone resorption.

實施例7 Example 7 (骨強化用錠劑之製備) (Preparation of bone reinforcement tablets)

以表6所示之摻合混合原料後，採常法成型、打錠為1g，製造本發明的骨強化用錠劑。再者，該錠劑1g中含有實施例品1之來自乳汁的鹼性蛋白質級分25mg、實施例品4的黃豆異黃酮50mg。 After the mixed raw materials shown in Table 6 were blended, the conventional method was used to form and ingot 1 g, and the bone reinforcing tablet of the present invention was produced. Further, 1 g of the tablet contained 25 mg of a basic protein fraction derived from milk of Example 1 and 50 mg of soy isoflavones of Example 4.

實施例8 Example 8

(骨強化液狀營養組成物之製備) (Preparation of bone-enhanced liquid nutritional composition)

將實施例品3之來自乳汁的鹼性蛋白質級分分解物25g與實施例品5之黃豆異黃酮的糖苷配基50g溶解於4,925g的去離子水，加熱至50℃後，採用TK均質機(TKROBO MICS；特殊機化工業社製)，以6,000rpm攪拌混合30分鐘而得到含有實施例品3之來自乳汁的鹼性蛋白質級分分解物25g/5kg與實施例品5之黃豆異黃酮的糖苷配基50g/5kg的溶液。在該溶液 5.0kg中，摻合酪蛋白(casein)5.0kg、黃豆蛋白質5.0kg、魚油1.0kg、紫蘇油3.0kg、糊精17.0kg、礦物質混合物6.0kg、維他命混合物1.95kg、乳化劑2.0kg、穩定劑4.0kg、香料0.05kg，並填充至200ml的殺菌袋，以加壓殺菌機(第1種壓力容器、TYPE：RCS-4CRTGN、日阪製作所製)於121℃殺菌20分鐘，製造本發明的骨強化用液狀營養組成物50kg，。再者，在該骨強化用液狀營養組成物中，每100g含有實施例品3之來自乳汁的鹼性蛋白質級分分解物50mg、實施例品5之黃豆異黃酮的糖苷配基100mg。 25 g of the alkaline protein fraction decomposed product from the milk of Example 3 and 50 g of the aglycone of the soybean isoflavone of Example 5 were dissolved in 4,925 g of deionized water, and heated to 50 ° C, and then a TK homogenizer was used. (TKROBO MICS; manufactured by Kekyo Kogyo Co., Ltd.), which was stirred and mixed at 6,000 rpm for 30 minutes to obtain 25 g/5 kg of the alkaline protein fraction decomposition product derived from the milk of Example 3 and the isoflavone of the product of Example 5. A solution of aglycone 50g/5kg. In the solution In 5.0kg, 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, 2.0kg of emulsifier, stable The present invention was prepared by sterilizing at a temperature of 121 ° C for 20 minutes in a pressure sterilization machine (first type pressure vessel, TYPE: RCS-4 CRTGN, manufactured by HISAKA Co., Ltd.) in a sterilization bag (manufactured by a pressure vessel). The liquid nutrient composition for bone strengthening is 50 kg. In addition, in the liquid nutritional composition for bone strengthening, 50 mg of the basic protein fraction decomposition product derived from the milk of Example 3 and 100 mg of the aglucone isoflavone of Example 5 were contained per 100 g.

實施例9 Example 9 (骨強化用飲料之製備) (Preparation of Bone Fortified Beverage)

將脫脂奶粉300g溶解於408.5g的去離子水後，溶解實施例品2之來自乳汁的鹼性蛋白質級分0.5g與實施例品5之黃豆異黃酮的糖苷配基1g，加熱至50℃後，採用超分散器(ULTRA-TURRAX T-25；IKAJapan公司製)，以9,500rpm攪拌混合30分鐘。添加麥芽糖醇100g、酸味料2g、還原麥芽糖20g、香料2g、去離子水166g後，填充至100ml的玻璃瓶，於95℃殺菌15秒後栓緊，製備本發明的骨強化用飲料10瓶(容量100ml)。再者，在該骨強化用飲料中，每100ml含有實施例品2之來自乳汁的鹼性蛋白質級分50mg與實施例品5之黃豆異黃酮的糖苷配基100mg。 After dissolving 300 g of skim milk powder in 408.5 g of deionized water, 1 g of the alkaline protein fraction derived from milk of Example 2 and 1 g of the aglucone isoflavone of Example 5 were dissolved and heated to 50 ° C. A hyperdisperser (ULTRA-TURRAX T-25; manufactured by IKA Japan) was used, and the mixture was stirred and mixed at 9,500 rpm for 30 minutes. After adding 100 g of maltitol, 2 g of sourdil, 20 g of reduced maltose, 2 g of perfume, 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 tightened to prepare 10 bottles of the bone reinforcing beverage of the present invention ( Capacity 100ml). Further, in the bone fortification drink, 50 mg of the alkaline protein fraction derived from the milk of Example 2 and 100 mg of the aglycone of the soy isoflavone of Example 5 were contained per 100 ml.

實施例10 Example 10 (犬用骨強化飼料之製備) (Preparation of canine bone fortified feed)

將實施例品1之來自乳汁的鹼性蛋白質級分1kg與實施例品6之含有雌馬酚的黃豆異黃酮2kg溶解於97kg的去離子水，加熱至50℃後，採用TK均質機(MARKII 160型；特殊機化工業社製)，以3,600rpm攪拌混合40分鐘，得到含有實施例品1之來自乳汁的鹼性蛋白質級分1g/100g與實施例品6之含有雌馬酚的黃豆異黃酮2g/100g的溶液。在該溶液10kg中摻合黃豆粕12kg、脫脂奶粉14kg、黃豆油4kg、玉米油2kg、棕櫚油23.2kg、玉米澱粉14kg、麵粉9kg、糠麩2kg、維他命混合物5kg、纖維素2.8kg、礦物質混合物2kg，於120℃殺菌4分鐘，製造本發明的犬用骨強化飼料100kg。再者，在該犬用骨強化飼料中，每100g含有實施例品1之來自乳汁的鹼性蛋白質級分100mg與實施例品6之含有雌馬酚的黃豆異黃酮200mg。 1 kg of the alkaline protein fraction from the milk of Example 1 and 2 kg of the equol-containing soy isoflavones of Example 6 were dissolved in 97 kg of deionized water, and heated to 50 ° C, and then a TK homogenizer was used (MARKII Model 160 (manufactured by Special Kogyo Co., Ltd.), and stirred and mixed at 3,600 rpm for 40 minutes to obtain a basic protein fraction 1 g/100 g of milk containing the product of Example 1 and an equol-containing soy bean of Example 6. A solution of flavonoids 2g/100g. 10kg of soybean meal, 14kg of skim milk powder, 4kg of soybean oil, 2kg of corn oil, 2kg of palm oil, 14kg of corn starch, 9kg of corn starch, 2kg of bran, 2kg of bran, 5kg of vitamin mixture, 2.8kg of cellulose, minerals in 10kg of this solution 2 kg of the mixture was sterilized at 120 ° C for 4 minutes to prepare 100 kg of the dog bone fortified feed of the present invention. Furthermore, in the bone fortified feed for dogs, the milk derived from Example 1 is contained per 100 g. The alkaline protein fraction of the juice was 100 mg and the equol-containing soy isoflavone 200 mg of the product of Example 6.

實施例11 Example 11

將實施例品1之來自乳汁的鹼性蛋白質級分與Fujiflavone P40(Fujicco股份有限公司、異黃酮類含量40%)以重量比2：5之比例混合(來自乳汁的鹼性蛋白質級分：異黃酮類=1：1)，得到本發明的骨強化劑。 The alkaline protein fraction derived from milk of Example 1 was mixed with Fujiflavone P40 (Fujicco Co., Ltd., isoflavone content 40%) at a weight ratio of 2:5 (basic protein fraction from milk: different Flavonoids = 1:1), the bone enhancer of the present invention is obtained.

實施例12 Example 12 將實施例品2之來自乳汁的鹼性蛋白質級分與SoyAct(Kikkoman股份有限公司、異黃酮類含量30%)以重量比1：2之比例混合(來自乳汁的鹼性蛋白質級分：異黃酮類=5：3)，得到本發明的骨強化劑。 The alkaline protein fraction from the milk of Example 2 was mixed with SoyAct (Kikkoman Co., Ltd., isoflavone content 30%) in a weight ratio of 1:2 (alkaline protein fraction from milk: isoflavones) Class = 5:3), the bone strengthening agent of the present invention is obtained. 實施例13 Example 13

將實施例品1之來自乳汁的鹼性蛋白質級分300mg混合於無調整豆乳(Kibun Food Chemifa股份有限公司、異黃酮類含量210mg/L)1L(來自乳汁的鹼性蛋白質級分：異黃酮類=10：7)，攪拌後，於120℃進行4秒之加熱殺菌，得到本發明的骨強化劑。 The alkaline protein fraction from the milk of Example 1 was mixed with unadjusted soymilk (Kibun Food Chemifa Co., Ltd., isoflavone content 210 mg/L) 1 L (basic protein fraction from milk: isoflavones) =10:7), after stirring, heat sterilization at 120 ° C for 4 seconds to obtain a bone strengthening agent of the present invention.

Claims

一種骨強化劑，係將來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物、與黃豆異黃酮類及/或其代謝物作為有效成分，且該來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與該黃豆異黃酮類及/或其代謝物的比例為30：70~70：30。 A bone fortifier which comprises an alkaline protein fraction derived from milk and/or an alkaline protein fraction decomposed product derived from milk, and soy isoflavones and/or metabolites thereof as an active ingredient, and the base derived from milk The ratio of the protein fraction and/or the alkaline protein fraction decomposed from the milk to the soy isoflavones and/or its metabolites is from 30:70 to 70:30.

如申請專利範圍第1項之骨強化劑，其中，該來自乳汁的鹼性蛋白質級分分解物，係利用選自於由胃蛋白酶、胰凝乳蛋白酶及胰酶構成的群組中之至少1種的蛋白質分解酵素將來自乳汁的鹼性蛋白質級分分解而成。 The bone strengthening agent according to claim 1, wherein the alkaline protein fraction decomposed product derived from milk is at least 1 selected from the group consisting of pepsin, chymotrypsin and trypsin. A proteolytic enzyme that breaks down the alkaline protein fraction from milk.

如申請專利範圍第1或2項之骨強化劑，其中，該來自乳汁的鹼性蛋白質級分，係於其胺基酸組成中含有鹼性胺基酸15重量%以上的級分。 The bone reinforcing agent according to claim 1 or 2, wherein the alkaline protein fraction derived from milk is a fraction containing 15% by weight or more of a basic amino acid in the amino acid composition.

如申請專利範圍第1或2項之骨強化劑，其中，該來自乳汁的鹼性蛋白質級分，係使乳汁或來自乳汁的原料與陽離子交換樹脂接觸，使鹼性蛋白質被吸附，並將該樹脂所吸附的級分以鹽濃度0.1M~1.0M的洗提液洗提而得到的級分。 The bone fortifier according to claim 1 or 2, wherein the alkaline protein fraction derived from milk is such that the milk or the raw material derived from the milk is contacted with the cation exchange resin to cause the alkaline protein to be adsorbed, and the The fraction obtained by eluting the fraction adsorbed by the resin with an elution solution having a salt concentration of 0.1 M to 1.0 M was obtained.

如申請專利範圍第1項之骨強化劑，其中，該黃豆異黃酮類係選自於染料木黃酮、染料木苷、黃豆黃酮、黃豆苷、黃豆黃苷、黃豆黃素、***以及其類似物或糖苷配基中之任1種以上。 The bone enhancer according to claim 1, wherein the soybean isoflavone is selected from the group consisting of genistein, genistein, soybean flavonoid, daidzin, daidzin, glycyrrhizin, estradiol, and the like Any one or more of an analog or a aglycone.

如申請專利範圍第1項之骨強化劑，其中，該黃豆異黃酮類的代謝物係為雌馬酚。 The bone enhancer according to claim 1, wherein the metabolite of the soybean isoflavone is equol.

一種骨強化用飲食品，其特徵為：包含如申請專利範圍第1至6項中任一項之骨強化劑。 A food or drink for bone reinforcement, which comprises the bone strengthening agent according to any one of claims 1 to 6.

一種骨強化用營養組成物，其特徵為：包含如申請專利範圍第1至6項中任一項之骨強化劑。 A nutrient composition for bone strengthening, which comprises the bone strengthening agent according to any one of claims 1 to 6.

一種骨強化用飼料，其特徵為：包含如申請專利範圍第1至6項中任一項之骨強化劑。 A bone fortification feed comprising the bone strengthening agent according to any one of claims 1 to 6.

一種骨強化用醫藥品，其特徵為：包含如申請專利範圍第1至6項中任一項之骨強化劑。 A medicinal product for bone strengthening, which comprises the bone strengthening agent according to any one of claims 1 to 6.

一種來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物與異黃酮類及/或其代謝物之用途，用於製造骨強化之藥劑，係利用同時攝取來自乳汁的鹼性蛋白質級分及/或來自乳汁的鹼性蛋白質級分分解物、與異黃酮類及/或其代謝物。 An alkaline protein fraction derived from milk and/or an alkaline protein fraction decomposed product derived from milk and an isoflavone and/or a metabolite thereof, used for the manufacture of a bone strengthening agent, which utilizes simultaneous intake of milk from milk Alkaline protein fraction and/or alkaline protein fraction decomposition from milk And isoflavones and/or their metabolites.