JP2020176069A - Peptides - Google Patents
Peptides Download PDFInfo
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- JP2020176069A JP2020176069A JP2019077626A JP2019077626A JP2020176069A JP 2020176069 A JP2020176069 A JP 2020176069A JP 2019077626 A JP2019077626 A JP 2019077626A JP 2019077626 A JP2019077626 A JP 2019077626A JP 2020176069 A JP2020176069 A JP 2020176069A
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- bone
- leu
- strengthening
- pro
- lys
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- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
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- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
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- 235000005282 vitamin D3 Nutrition 0.000 description 1
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Landscapes
- Edible Oils And Fats (AREA)
- Bakery Products And Manufacturing Methods Therefor (AREA)
- Non-Alcoholic Beverages (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Jellies, Jams, And Syrups (AREA)
- Peptides Or Proteins (AREA)
- Fodder In General (AREA)
- Dairy Products (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Grain Derivatives (AREA)
Abstract
Description
本発明は、破骨細胞による骨吸収を抑制する作用を有し、骨強化作用に優れ、骨粗鬆症や骨折治療、リウマチ、関節炎などの種々の骨疾患の予防や治療に有効であるペプチドに関する。さらに、本発明は、該ペプチドを含有する、骨強化剤、骨強化用飲食品、骨強化用栄養組成物、骨強化用飼料又は骨強化用医薬品に関する。 The present invention relates to a peptide which has an action of suppressing bone resorption by osteoclasts, has an excellent bone strengthening action, and is effective for prevention and treatment of various bone diseases such as osteoporosis, fracture treatment, rheumatism, and arthritis. Furthermore, the present invention relates to a bone strengthening agent, a food and drink for bone strengthening, a nutritional composition for bone strengthening, a feed for bone strengthening, or a medicine for bone strengthening, which contains the peptide.
近年、世界的規模で、高齢化等に伴い、骨粗鬆症や骨折あるいは腰痛などの種々の骨に関連する疾患が増加しており、大きな社会問題となっている。これは、カルシウムの摂取不足やカルシウム吸収能力の低下、閉経後のホルモンのアンバラスなどが原因であるとされている。骨粗鬆症や骨折、腰痛などの種々の骨疾患を予防するためには、若齢期から骨芽細胞による骨形成を促進して体内の骨量をできるだけ増加させ、最大骨量や骨強度（骨密度＋骨質）を高めることが有効であるとされている。なお、骨質とは、骨の微細構造や代謝回転、微小骨折、石灰化を指すものである。また、骨粗鬆症や骨折、腰痛などの種々の骨疾患を予防する方法としては、破骨細胞による骨吸収を抑制することも考えられる。骨はバランスのとれた吸収と形成を絶えず繰り返している（リモデリング）が、閉経後のホルモンのバランス変化等により、骨吸収が骨形成を上回り、これが骨粗鬆症や骨折、腰痛などの種々の骨疾患の原因となる。したがって、破骨細胞による骨吸収を抑制して骨強度を一定に保つことにより、結果的に骨を強化することが可能である。 In recent years, on a global scale, various bone-related diseases such as osteoporosis, bone fractures, and low back pain have been increasing with the aging of the population, which has become a major social problem. It is said that this is caused by insufficient calcium intake, decreased calcium absorption capacity, and postmenopausal hormone unbalance. In order to prevent various bone diseases such as osteoporosis, fractures, and lower back pain, bone formation by osteoblasts is promoted from an early age to increase bone mass in the body as much as possible, and maximum bone mass and bone strength (bone density). + Bone quality) is said to be effective. Bone quality refers to bone microstructure, metabolic rotation, microfractures, and calcification. In addition, as a method for preventing various bone diseases such as osteoporosis, bone fracture, and low back pain, it is also conceivable to suppress bone resorption by osteoclasts. Bone constantly repeats balanced resorption and formation (remodeling), but bone resorption exceeds bone formation due to changes in the balance of hormones after menopause, which causes various bone diseases such as osteoporosis, fractures, and low back pain. Causes. Therefore, by suppressing bone resorption by osteoclasts and keeping the bone strength constant, it is possible to strengthen the bone as a result.
このような現状から、骨を強化する目的で、炭酸カルシウムやリン酸カルシウム、乳酸カルシウムなどのカルシウム塩ならびに乳清カルシウムや牛骨粉、卵殻などの天然カルシウム剤を、それぞれ単独で医薬品や飲食品、飼料などに添加して摂取する、あるいは、これらのカルシウム剤をカゼインホスホペプチドやオリゴ糖などのカルシウム吸収促進効果を有する物質と共に医薬品や飲食品、飼料などに添加して摂取している。しかしながら、これらのカルシウム塩や天然カルシウム剤を飲食品に添加して摂取した場合、カルシウムの吸収率は５０％以下であり、半分以上のカルシウムが吸収されず体外に排出されてしまうといわれている。また、体内に吸収されたカルシウムも、その形態や同時に摂取される他の栄養成分の種類によって骨への親和性が異なるので、必ずしも骨代謝の改善や骨強化作用を示さないこともある。 Under these circumstances, for the purpose of strengthening bones, calcium salts such as calcium carbonate, calcium phosphate and calcium lactate, and natural calcium agents such as milky calcium, beef bone powder and eggshell are used independently for medicines, foods and drinks, feeds, etc. Or, these calcium agents are added to pharmaceuticals, foods and drinks, feeds, etc. together with substances having a calcium absorption promoting effect such as casein phosphopeptides and oligosaccharides. However, when these calcium salts and natural calcium agents are added to foods and drinks and ingested, the absorption rate of calcium is 50% or less, and it is said that more than half of the calcium is not absorbed and is excreted from the body. .. In addition, calcium absorbed in the body may not necessarily show an improvement in bone metabolism or a bone strengthening effect because its affinity for bone differs depending on its form and the type of other nutritional components ingested at the same time.
その他、骨粗鬆症治療や骨強化のための医薬として、女性ホルモン製剤や活性型ビタミンＤ３製剤 やビタミンＫ２製剤、ビスフォスフォネート製剤、カルシトニン製剤などが知られており、抗ＲＡＮＫＬ抗体などの新薬開発が進められている。しかし、これらの医薬品を用いた場合、耳鳴り、頭痛、食欲不振などの副作用を伴うことがある。また、服用期間に制限があるものもある。さらに、これらの物質は安全性及びコストなどの面から、現在のところ飲食品に添加することができない状況にある。したがって、骨粗鬆症や骨折、腰痛などの種々の骨疾患の疾病の性質から、長期的に副作用が無く、摂取することができ、骨形成促進的及び／または骨吸収抑制的に作用して骨強度を高め、その予防または治療効果が期待できるような骨強化剤や、骨強化剤を含有する飲食品、飼料の開発が望まれている。 In addition, female hormone preparations, active vitamin D3 preparations, vitamin K2 preparations, bisphosphonate preparations, calcitonin preparations, etc. are known as medicines for osteoporosis treatment and bone strengthening, and new drug development such as anti-RANKL antibody is being developed. It is being advanced. However, when these medicines are used, side effects such as tinnitus, headache, and loss of appetite may occur. In addition, there are some that have a limited period of administration. Furthermore, these substances cannot be added to foods and drinks at present in terms of safety and cost. Therefore, due to the nature of various bone diseases such as osteoporosis, bone fracture, and low back pain, it can be taken without side effects in the long term, and it acts to promote bone formation and / or suppress bone resorption to increase bone strength. It is desired to develop bone strengthening agents, foods and drinks containing bone strengthening agents, and feeds that can be enhanced and expected to have preventive or therapeutic effects.
こうしたなかで、最近の研究成果として、例えば、天然植物由来の抽出物が、骨形成を促進することが報告されている（例えば、特許文献１や特許文献２）。また、例えば食品由来成分として、卵黄タンパク質を加水分解処理したペプチドを含む組成物が、骨形成を促進することが報告されている（特許文献３）。しかしながら、これらは植物由来の抽出物あるいは卵黄タンパク質加水分解物を含む、組成物の骨強化作用であり、特定の配列のペプチドに骨吸収抑制作用を通じた骨強化作用があることはこれまで知られていなかった。 Under these circumstances, as a recent research result, it has been reported that, for example, an extract derived from a natural plant promotes bone formation (for example, Patent Document 1 and Patent Document 2). Further, for example, it has been reported that a composition containing a peptide obtained by hydrolyzing egg yolk protein as a food-derived component promotes bone formation (Patent Document 3). However, these are bone-strengthening effects of compositions containing plant-derived extracts or egg yolk protein hydrolysates, and it has been known that peptides of a specific sequence have bone-strengthening effects through bone resorption-suppressing effects. I wasn't.
本発明の課題は、破骨細胞による骨吸収を抑制する作用があり、骨を強化することができ、骨粗鬆症や骨折、リウマチ、関節炎などの種々の骨疾患の予防や治療に有用である骨強化作用を有する素材を提供することである。また、本発明の課題は、破骨細胞による骨吸収を抑制する作用があり、骨を強化することができ、骨粗鬆症や骨折、リウマチ、関節炎などの種々の骨疾患の予防や治療に有用である骨強化作用を有する素材を配合した骨強化剤、骨強化用飲食品、骨強化用栄養組成物、骨強化用飼料又は骨強化用医薬品を提供することである。 The subject of the present invention is bone strengthening which has an action of suppressing bone resorption by osteoclasts, can strengthen bone, and is useful for prevention and treatment of various bone diseases such as osteoporosis, bone fracture, rheumatism, and arthritis. It is to provide a material having an action. Further, the subject of the present invention is that it has an effect of suppressing bone resorption by bone-breaking cells, can strengthen bone, and is useful for prevention and treatment of various bone diseases such as osteoporosis, fracture, rheumatism, and arthritis. It is to provide a bone strengthening agent containing a material having a bone strengthening action, a food and drink for bone strengthening, a nutritional composition for bone strengthening, a feed for bone strengthening or a medicine for bone strengthening.
本発明者らは、上記課題を解決するため鋭意検討を進めた結果、多数存在するジペプチドまたはトリペプチドの中から特定配列のジペプチドあるいはトリペプチドに骨強化作用があることを見出したことにもとづく発明であり、以下の構成が含まれる。
（１）Ｉｌｅ−Ａｒｇ、Ｉｌｅ−Ｇｌｎ、Ｌｅｕ−Ｌｙｓ、Ｓｅｒ−Ｐｒｏ、Ｌｙｓ−Ｇｌｙ、Ｔｈｒ−Ｐｒｏ、Ｖａｌ−Ｌｅｕ、Ｌｅｕ−Ｇｌｕ、Ａｓｎ−Ｐｒｏ−Ｔｈｒからなる群から選択される１つ以上のペプチドを有効成分として含む骨強化用組成物。
（２）前記（１）に記載の骨強化用組成物を含むことを特徴とする骨強化用飲食品、骨強化用栄養組成物、骨強化用飼料又は骨強化用医薬品。
As a result of diligent studies to solve the above problems, the present inventors have found that a dipeptide or tripeptide having a specific sequence has a bone-strengthening effect among a large number of dipeptides or tripeptides. And includes the following configurations:
(1) One or more selected from the group consisting of Ile-Arg, Ile-Gln, Leu-Lys, Ser-Pro, Lys-Gly, Thr-Pro, Val-Leu, Leu-Glu, Asn-Pro-Thr. Bone strengthening composition containing the peptide of.
(2) A bone-strengthening food or drink, a bone-strengthening nutritional composition, a bone-strengthening feed, or a bone-strengthening drug, which comprises the bone-strengthening composition according to (1) above.
本発明の骨強化用組成物は、安全性が高く、破骨細胞の分化ならびに該細胞による骨吸収を抑制する作用を介した骨強化作用が顕著であり、該骨強化用組成物は、骨粗鬆症や骨折、リウマチ、関節炎などの種々の骨疾患の予防や治療に有用である。また、本発明は、破骨細胞の分化ならびに該細胞による骨吸収を抑制する作用があり、骨を強化することができ、骨粗鬆症や骨折、リウマチ、関節炎などの種々の骨疾患の予防や治療に有用である骨強化用組成物を配合した骨強化用飲食品、骨強化用栄養組成物、骨強化用飼料又は骨強化用医薬品を提供するものである。 The bone-strengthening composition of the present invention is highly safe and has a remarkable bone-strengthening action through the action of suppressing the differentiation of bone fracture cells and bone resorption by the cells, and the bone-strengthening composition is osteoporosis. It is useful for the prevention and treatment of various bone diseases such as bone fractures, rheumatism, and arthritis. In addition, the present invention has an effect of suppressing the differentiation of bone-breaking cells and bone resorption by the cells, can strengthen bone, and is used for prevention and treatment of various bone diseases such as osteoporosis, fracture, rheumatism, and arthritis. It provides a bone-strengthening food and drink, a bone-strengthening nutritional composition, a bone-strengthening feed, or a bone-strengthening drug containing a useful bone-strengthening composition.
本発明のＩｌｅ−Ａｒｇ、Ｉｌｅ−Ｇｌｎ、Ｌｅｕ−Ｌｙｓ、Ｓｅｒ−Ｐｒｏ、Ｌｙｓ−Ｇｌｙ、Ｔｈｒ−Ｐｒｏ、Ｖａｌ−Ｌｅｕ、Ｌｅｕ−Ｇｌｕ、及び／またはＡｓｎ−Ｐｒｏ−Ｔｈｒの配列からなるペプチドについて、以下に詳細に説明する。 Regarding the peptide consisting of the sequences of Ile-Arg, Ile-Gln, Leu-Lys, Ser-Pro, Lys-Gly, Thr-Pro, Val-Leu, Leu-Glu, and / or Asn-Pro-Thr of the present invention. This will be described in detail below.
（本発明のペプチドとその製造方法）
本発明のペプチドは、Ｉｌｅ−Ａｒｇ、Ｉｌｅ−Ｇｌｎ、Ｌｅｕ−Ｌｙｓ、Ｓｅｒ−Ｐｒｏ、Ｌｙｓ−Ｇｌｙ、Ｔｈｒ−Ｐｒｏ、Ｖａｌ−Ｌｅｕ、Ｌｅｕ−Ｇｌｕ、及びＡｓｎ−Ｐｒｏ−Ｔｈｒからなる群からから選択される１つ以上を用いることができる。
これらのペプチドは、上記した配列を有するものであればどのように調製されたものでもよく、調製方法としてペプチド合成装置を用いた固相法、液相法などの化学合成法、遺伝子工学的手法、酵素工学的手法、微生物による産生等を例示できる。また、精製され、市販されているペプチドを使用することも可能である。さらに、該アミノ酸配列を有する各種のタンパク質やペプチドを原料に、タンパク質加水分解酵素を用いて酵素分解して得ることもできる。原料とするタンパク質に制限はないが、動物性タンパク質や植物性タンパク質を用いることが可能であり、例えば、牛乳、卵、大豆、小麦、とうもろこし、牛肉、鶏肉、豚肉などが挙げられる。
(Peptide of the present invention and its production method)
The peptides of the present invention are selected from the group consisting of Ile-Arg, Ile-Gln, Leu-Lys, Ser-Pro, Lys-Gly, Thr-Pro, Val-Leu, Leu-Glu, and Asn-Pro-Thr. One or more of the above can be used.
These peptides may be prepared in any way as long as they have the above-mentioned sequences, and as the preparation method, a solid phase method using a peptide synthesizer, a chemical synthesis method such as a liquid phase method, or a genetic engineering method , Enzyme engineering methods, production by microorganisms, etc. can be exemplified. It is also possible to use purified and commercially available peptides. Further, it can also be obtained by enzymatically decomposing various proteins and peptides having the amino acid sequence with a protein hydrolase. The protein used as a raw material is not limited, but animal protein or vegetable protein can be used, and examples thereof include milk, eggs, soybeans, wheat, corn, beef, chicken, and pork.
（ペプチドの摂取量）
本発明のペプチドの摂取量は、破骨細胞の分化ならびに該細胞による骨吸収を抑制する作用を介した骨強化作用を示す量であれば特に制限はないが、Ｉｌｅ−Ａｒｇ、Ｉｌｅ−Ｇｌｎ、Ｌｅｕ−Ｌｙｓ、Ｓｅｒ−Ｐｒｏ、Ｌｙｓ−Ｇｌｙ、Ｔｈｒ−Ｐｒｏ、Ｖａｌ−Ｌｅｕ、Ｌｅｕ−Ｇｌｕ、及び／またはＡｓｎ−Ｐｒｏ−Ｔｈｒからなる群から選択されるペプチドを成人１日あたり０．１ｍｇ以上摂取すればよく、１ｍｇ以上が好ましく、１０ｍｇ以上がさらに好ましく、５０ｍｇ以上が最も好ましい。前記群から複数種のペプチドを摂取する場合は、その合計量が上記量以上になればよい。本発明のペプチドは、そのまま経口摂取してもよいが、飲食品、栄養組成物、飼料および経口用医薬等への配合する場合は、上記した摂取量となるように配合量を調整すればよい。なお、非経口医薬の場合は、経口投与した場合の上記投与量と同等の効果を示す有効量を投与すればよい。
(Peptide intake)
The intake of the peptide of the present invention is not particularly limited as long as it exhibits a bone strengthening effect through the action of suppressing the differentiation of osteoclasts and bone resorption by the cells, but Ile-Arg, Ile-Gln, and the like. Adults ingest 0.1 mg or more of peptide selected from the group consisting of Leu-Lys, Ser-Pro, Lys-Gly, Thr-Pro, Val-Leu, Leu-Glu, and / or Asn-Pro-Thr. 1 mg or more is preferable, 10 mg or more is more preferable, and 50 mg or more is most preferable. When a plurality of kinds of peptides are ingested from the above group, the total amount may be equal to or more than the above amount. The peptide of the present invention may be orally ingested as it is, but when it is added to foods and drinks, nutritional compositions, feeds, oral medicines, etc., the amount may be adjusted so as to have the above-mentioned intake amount. .. In the case of parenteral medicine, an effective amount showing the same effect as the above-mentioned dose when orally administered may be administered.
（本発明のペプチドの飲食品及び医薬品への使用方法について）
本発明のペプチドは、それぞれを単独もしくは、任意の組み合わせでそのまま骨強化用組成物として使用してもよいが、破骨細胞の分化ならびに該細胞による骨吸収を抑制する作用を介した骨強化作用を失わない範囲で必要に応じて常法に従い、粉末剤、顆粒剤、錠剤、カプセル剤、ドリンク剤等に製剤化して用いることもできる。また、そのまま又は製剤化した後に、栄養剤や粉乳、乳飲料、乳酸菌飲料、発酵乳、清涼飲料水、チーズ、マーガリン、クリーム、プリン、ゼリー、ウエハース等の飲食品、栄養組成物、飼料及び医薬品に配合することも可能である。
本発明のペプチドは、他の飲食品、飼料及び医薬に通常含まれる安定剤や糖類、脂質、フレーバー、ビタミン、ミネラル、フラボノイド、ポリフェノール等の原材料とともに使用することができる。
(About the method of using the peptide of the present invention in foods and drinks and pharmaceuticals)
The peptides of the present invention may be used alone or in any combination as they are as a bone strengthening composition, but they have a bone strengthening action through an action of suppressing osteoclast differentiation and bone resorption by the cells. It is also possible to formulate and use powders, granules, tablets, capsules, drinks and the like according to a conventional method as long as the above is not lost. In addition, foods and drinks such as nutritional supplements, milk powder, milk drinks, lactic acid bacteria drinks, fermented milk, soft drinks, cheese, margarine, cream, pudding, jelly, and wafers, nutritional compositions, feeds, and pharmaceuticals as they are or after formulation. It is also possible to blend in.
The peptides of the present invention can be used together with stabilizers and raw materials such as sugars, lipids, flavors, vitamins, minerals, flavonoids and polyphenols usually contained in other foods and drinks, feeds and pharmaceuticals.
製剤化に際しては、通常使用される充填剤、増量剤、結合剤、崩壊剤、界面活性剤、滑沢剤等の希釈剤又は賦形剤を用いることができる。賦形剤としては、例えばショ糖、乳糖、デンプン、結晶性セルロース、マンニット、軽質無水珪酸、アルミン酸マグネシウム、合成珪酸アルミニウム、メタ珪酸アルミン酸マグネシウム、炭酸カルシウム、炭酸水素ナトリウム、リン酸水素カルシウム、カルボキシルメチルセルロースカルシウム等の１種又は２種以上を組み合わせて加えることができる。 Diluents or excipients such as commonly used fillers, bulking agents, binders, disintegrants, surfactants, and lubricants can be used for formulation. Excipients include, for example, sucrose, lactose, starch, crystalline cellulose, mannit, light anhydrous silicic acid, magnesium aluminate, synthetic aluminum silicate, magnesium aluminometasilicate, calcium carbonate, sodium hydrogen carbonate, calcium hydrogen phosphate. , Calcium carboxylmethylcellulose, etc., or a combination of two or more.
以下に実施例、試験例を示し、本発明について詳細に説明するが、本発明はこれらによって限定されるものではない。 Examples and test examples are shown below, and the present invention will be described in detail, but the present invention is not limited thereto.
（実施例１）
カゼインタンパク質１０％水溶液５Ｌに、パパイン５０Ｕ／ｇ・カゼインタンパク質及びプロレザー(天野エンザイム社製)１５０Ｕ／ｇ・カゼインタンパク質を加え、ｐＨ８に調整し、５５℃において６時間カゼインタンパク質を変性させながら酵素分解を行った。反応液を１００℃で１５秒間以上加熱して酵素を失活させ、遠心分離して上清を回収して、カゼインタンパク質加水分解物を得た。このカゼインタンパク質加水分解物をＳｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅ（ＧＥヘルスケア社製）を充填したカラムに通液し、分子サイズで分画し、平均分子量２００〜３５０Ｄａの画分を得た。この画分を、Ｐｏｒｏｓｈｅｌｌ １２０ ＳＢ−Ｃ１８（アジレント・テクノロジー社製）を充填した逆相カラムに通液して、Ｉｌｅ−Ｇｌｎ、Ｓｅｒ−Ｐｒｏ、Ｔｈｒ−Ｐｒｏを含む画分をそれぞれ回収した。これらのフラクションを凍結乾燥して、Ｉｌｅ−Ｇｌｎ、Ｓｅｒ−Ｐｒｏ、Ｔｈｒ−Ｐｒｏ（実施例品１）を得た。これらＩｌｅ−Ｇｌｎ、Ｓｅｒ−Ｐｒｏ、Ｔｈｒ−ＰｒｏをＨＰＬＣによって分析したところ、純度はそれぞれ９０％以上であった。このようにして得られたＩｌｅ−Ｇｌｎ、Ｓｅｒ−Ｐｒｏ、Ｔｈｒ−Ｐｒｏは、そのまま本発明の骨強化作用を有するペプチドとして使用できる。
(Example 1)
50 U / g of papain, 150 U / g of casein protein and Proleather (manufactured by Amano Enzyme) 150 U / g of casein protein are added to 5 L of a 10% aqueous solution of casein protein, adjusted to pH 8, and the enzyme is denatured at 55 ° C. for 6 hours. It was disassembled. The reaction solution was heated at 100 ° C. for 15 seconds or longer to inactivate the enzyme, and the supernatant was collected by centrifugation to obtain a casein protein hydrolyzate. This casein protein hydrolyzate was passed through a column packed with Superdex Peptide (manufactured by GE Healthcare Japan) and fractionated by molecular size to obtain a fraction having an average molecular weight of 200 to 350 Da. This fraction was passed through a reverse phase column packed with Poroshell 120 SB-C18 (manufactured by Agilent Technologies), and the fractions containing Ile-Gln, Ser-Pro, and Thr-Pro were collected. These fractions were lyophilized to give Ile-Gln, Ser-Pro and Thr-Pro (Example Product 1). When these Ile-Gln, Ser-Pro, and Thr-Pro were analyzed by HPLC, the purity was 90% or more, respectively. The Ile-Gln, Ser-Pro, and Thr-Pro thus obtained can be used as they are as the peptide having the bone strengthening action of the present invention.
（実施例２）
卵白タンパク質１０％水溶液５Ｌに、パパイン５０Ｕ／ｇ・卵白タンパク質及びプロレザー(天野エンザイム社製)１５０Ｕ／ｇ・卵白タンパク質を加え、ｐＨ８に調整し、５５℃において６時間卵白タンパク質を変性させながら酵素分解を行った。反応液を１００℃で１５秒間以上加熱して酵素を失活させ、遠心分離して上清を回収して、卵白タンパク質加水分解物を得た。この卵白タンパク質加水分解物をＳｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅ（ＧＥヘルスケア社製）を充填したカラムに通液し、分子サイズで分画し、平均分子量２００〜３５０Ｄａの画分を得た。この画分を、Ｐｏｒｏｓｈｅｌｌ １２０ ＳＢ−Ｃ１８（アジレント・テクノロジー社製）を充填した逆相カラムに通液して、Ｉｌｅ−Ａｒｇ、Ｖａｌ−Ｌｅｕ、Ｌｅｕ−Ｇｌｕを含む画分をそれぞれ回収した。これらのフラクションを凍結乾燥して、Ｉｌｅ−Ａｒｇ、Ｖａｌ−Ｌｅｕ、Ｌｅｕ−Ｇｌｕ（実施例品２）を得た。これらＩｌｅ−Ａｒｇ、Ｖａｌ−Ｌｅｕ、Ｌｅｕ−ＧｌｕをＨＰＬＣによって分析したところ、純度はそれぞれ９０％以上であった。このようにして得られたＩｌｅ−Ａｒｇ、Ｖａｌ−Ｌｅｕ、Ｌｅｕ−Ｇｌｕは、そのまま本発明の骨強化作用を有するペプチドとして使用できる。
(Example 2)
50 U / g of papine, egg white protein and 150 U / g of proleather (manufactured by Amano Enzyme), egg white protein are added to 5 L of a 10% aqueous solution of egg white protein, adjusted to pH 8, and the enzyme is denatured at 55 ° C. for 6 hours. It was disassembled. The reaction solution was heated at 100 ° C. for 15 seconds or longer to inactivate the enzyme, and the supernatant was collected by centrifugation to obtain an egg white protein hydrolyzate. This egg white protein hydrolyzate was passed through a column packed with Superdex Peptide (manufactured by GE Healthcare Japan) and fractionated by molecular size to obtain a fraction having an average molecular weight of 200 to 350 Da. This fraction was passed through a reverse phase column packed with Poroshell 120 SB-C18 (manufactured by Agilent Technologies), and the fractions containing Ile-Arg, Val-Leu, and Leu-Glu were collected. These fractions were lyophilized to give Ile-Arg, Val-Leu, and Leu-Glu (Example 2). When these Ile-Arg, Val-Leu, and Leu-Glu were analyzed by HPLC, the purity was 90% or more, respectively. The Ile-Arg, Val-Leu, and Leu-Glu thus obtained can be used as they are as the peptide having the bone strengthening action of the present invention.
（実施例３）
卵黄タンパク質１０％水溶液５Ｌに、パパイン５０Ｕ／ｇ・卵黄タンパク質及びプロレザー(天野エンザイム社製)１５０Ｕ／ｇ・卵黄タンパク質を加え、ｐＨ８に調整し、５５℃において６時間卵黄タンパク質を変性させながら酵素分解を行った。反応液を１００℃で１５秒間以上加熱して酵素を失活させ、遠心分離して上清を回収して、卵黄タンパク質加水分解物を得た。この卵黄タンパク質加水分解物をＳｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅ（ＧＥヘルスケア社製）を充填したカラムに通液し、分子サイズで分画し、平均分子量２００〜３５０Ｄａの画分を得た。この画分を、Ｐｏｒｏｓｈｅｌｌ １２０ ＳＢ−Ｃ１８（アジレント・テクノロジー社製）を充填した逆相カラムに通液して、Ｉｌｅ−Ｇｌｎ、Ｌｙｓ−Ｇｌｙ、Ａｓｎ−Ｐｒｏ−Ｔｈｒを含む画分をそれぞれ回収した。これらのフラクションを凍結乾燥して、Ｉｌｅ−Ｇｌｎ、Ｌｙｓ−Ｇｌｙ、Ａｓｎ−Ｐｒｏ−Ｔｈｒ（実施例品３）を得た。これらＩｌｅ−Ｇｌｎ、Ｌｙｓ−Ｇｌｙ、Ａｓｎ−Ｐｒｏ−ＴｈｒをＨＰＬＣによって分析したところ、純度はそれぞれ９０％以上であった。このようにして得られたＩｌｅ−Ｇｌｎ、Ｌｙｓ−Ｇｌｙ、Ａｓｎ−Ｐｒｏ−Ｔｈｒは、そのまま本発明の骨強化作用を有するペプチドとして使用できる。
(Example 3)
Add 50 U / g of papine, egg yolk protein and 150 U / g of proleather (manufactured by Amano Enzyme), egg yolk protein to 5 L of 10% aqueous egg yolk protein, adjust to pH 8, and denature the egg yolk protein at 55 ° C for 6 hours. Disassembly was performed. The reaction solution was heated at 100 ° C. for 15 seconds or longer to inactivate the enzyme, centrifuged, and the supernatant was collected to obtain an egg yolk protein hydrolyzate. This egg yolk protein hydrolyzate was passed through a column packed with Superdex Peptide (manufactured by GE Healthcare Japan) and fractionated by molecular size to obtain a fraction having an average molecular weight of 200 to 350 Da. This fraction was passed through a reverse phase column packed with Poroshell 120 SB-C18 (manufactured by Agilent Technologies), and fractions containing Ile-Gln, Lys-Gly, and Asn-Pro-Thr were collected. .. These fractions were lyophilized to give Ile-Gln, Lys-Gly, Asn-Pro-Thr (Example 3). When these Ile-Gln, Lys-Gly, and Asn-Pro-Thr were analyzed by HPLC, the purity was 90% or more, respectively. The Ile-Gln, Lys-Gly, and Asn-Pro-Thr thus obtained can be used as they are as the peptide having the bone strengthening action of the present invention.
（実施例４）
小麦タンパク質１０％水溶液５Ｌに、パパイン５０Ｕ／ｇ・小麦タンパク質及びプロレザー(天野エンザイム社製)１５０Ｕ／ｇ・小麦タンパク質を加え、ｐＨ８に調整し、５５℃において６時間小麦タンパク質を変性させながら酵素分解を行った。反応液を１００℃で１５秒間以上加熱して酵素を失活させ、遠心分離して上清を回収して、小麦タンパク質加水分解物を得た。この小麦タンパク質加水分解物をＳｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅ（ＧＥヘルスケア社製）を充填したカラムに通液し、分子サイズで分画し、平均分子量２００〜３５０Ｄａの画分を得た。この画分を、フェニルイソチオシアネートにより誘導化し、Ｓｕｐｅｒｓｐｈｅｒｅ ＲＰ−１８（アジレント・テクノロジー社製）を充填した逆相カラムに通液して、Ｓｅｒ−Ｐｒｏ、Ｔｈｒ−Ｐｒｏを含む画分をそれぞれ回収した。これらのフラクションを凍結乾燥して、Ｓｅｒ−Ｐｒｏ、Ｔｈｒ−Ｐｒｏ（実施例品４）を得た。これらＳｅｒ−Ｐｒｏ、Ｔｈｒ−ＰｒｏをＨＰＬＣによって分析したところ、純度はそれぞれ９０％以上であった。このようにして得られたＳｅｒ−Ｐｒｏ、Ｔｈｒ−Ｐｒｏは、そのまま本発明の骨強化作用を有するペプチドとして使用できる。
(Example 4)
Papain 50 U / g, wheat protein and Proleather (manufactured by Amano Enzyme) 150 U / g, wheat protein are added to 5 L of a 10% wheat protein aqueous solution, adjusted to pH 8, and the enzyme is denatured at 55 ° C. for 6 hours. It was disassembled. The reaction solution was heated at 100 ° C. for 15 seconds or longer to inactivate the enzyme, and the supernatant was collected by centrifugation to obtain a wheat protein hydrolyzate. This wheat protein hydrolyzate was passed through a column packed with Superdex Peptide (manufactured by GE Healthcare Japan) and fractionated by molecular size to obtain a fraction having an average molecular weight of 200 to 350 Da. This fraction was derivatized with phenyl isothiocyanate and passed through a reverse phase column packed with Supersphere RP-18 (manufactured by Agilent Technologies) to recover the fractions containing Ser-Pro and Thr-Pro, respectively. .. These fractions were freeze-dried to obtain Ser-Pro and Thr-Pro (Example product 4). When these Ser-Pro and Thr-Pro were analyzed by HPLC, the purity was 90% or more, respectively. The Ser-Pro and Thr-Pro thus obtained can be used as they are as the peptide having the bone strengthening action of the present invention.
（実施例５）
大豆タンパク質１０％水溶液５Ｌに、パパイン５０Ｕ／ｇ・大豆タンパク質及びプロレザー(天野エンザイム社製)１５０Ｕ／ｇ・大豆タンパク質を加え、ｐＨ８に調整し、５５℃において６時間大豆タンパク質を変性させながら酵素分解を行った。反応液を１００℃で１５秒間以上加熱して酵素を失活させ、遠心分離して上清を回収して、大豆タンパク質加水分解物を得た。この大豆タンパク質加水分解物をＳｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅ（ＧＥヘルスケア社製）を充填したカラムに通液し、分子サイズで分画し、平均分子量２００〜３５０Ｄａの画分を得た。この画分を、フェニルイソチオシアネートにより誘導化し、Ｓｕｐｅｒｓｐｈｅｒｅ ＲＰ−１８（アジレント・テクノロジー社製）を充填した逆相カラムに通液して、Ｌｅｕ−Ｌｙｓ、Ｌｙｓ−Ｇｌｙ、Ｌｅｕ−Ｇｌｕを含む画分をそれぞれ回収した。これらのフラクションを凍結乾燥して、Ｌｅｕ−Ｌｙｓ、Ｌｙｓ−Ｇｌｙ、Ｌｅｕ−Ｇｌｕ（実施例品５）を得た。これらＬｅｕ−Ｌｙｓ、Ｌｙｓ−Ｇｌｙ、Ｌｅｕ−ＧｌｕをＨＰＬＣによって分析したところ、純度はそれぞれ９０％以上であった。このようにして得られたＬｅｕ−Ｌｙｓ、Ｌｙｓ−Ｇｌｙ、Ｌｅｕ−Ｇｌｕは、そのまま本発明の骨強化作用を有するペプチドとして使用できる。
(Example 5)
Papain 50 U / g, soy protein and Proleather (manufactured by Amano Enzyme) 150 U / g, soy protein are added to 5 L of a 10% soy protein aqueous solution, adjusted to pH 8, and the enzyme is denatured at 55 ° C. for 6 hours. It was disassembled. The reaction solution was heated at 100 ° C. for 15 seconds or longer to inactivate the enzyme, and the supernatant was collected by centrifugation to obtain a soybean protein hydrolyzate. This soy protein hydrolyzate was passed through a column packed with Superdex Peptide (manufactured by GE Healthcare Japan) and fractionated by molecular size to obtain a fraction having an average molecular weight of 200 to 350 Da. This fraction is derivatized with phenyl isothiocyanate and passed through a reverse phase column packed with Supersphere RP-18 (manufactured by Agilent Technologies) to contain a fraction containing Leu-Lys, Lys-Gly and Leu-Glu. Was collected respectively. These fractions were lyophilized to give Leu-Lys, Lys-Gly, and Leu-Glu (Example product 5). When these Leu-Lys, Lys-Gly, and Leu-Glu were analyzed by HPLC, the purity was 90% or more, respectively. The Leu-Lys, Lys-Gly, and Leu-Glu thus obtained can be used as they are as the peptide having the bone strengthening action of the present invention.
（実施例６）
トリ由来コラーゲンタンパク質１０％水溶液５Ｌに、パパイン５０Ｕ／ｇ・トリ由来コラーゲンタンパク質及びプロレザー(天野エンザイム社製)１５０Ｕ／ｇ・トリ由来コラーゲンタンパク質を加え、ｐＨ８に調整し、５５℃において６時間トリ由来コラーゲンタンパク質を変性させながら酵素分解を行った。反応液を１００℃で１５秒間以上加熱して酵素を失活させ、遠心分離して上清を回収して、トリ由来コラーゲンタンパク質加水分解物を得た。このトリ由来コラーゲンタンパク質加水分解物をＳｕｐｅｒｄｅｘ Ｐｅｐｔｉｄｅ（ＧＥヘルスケア社製）を充填したカラムに通液し、分子サイズで分画し、平均分子量２００〜３５０Ｄａの画分を得た。この画分を、フェニルイソチオシアネートにより誘導化し、Ｓｕｐｅｒｓｐｈｅｒｅ ＲＰ−１８（アジレント・テクノロジー社製）を充填した逆相カラムに通液して、Ｉｌｅ−Ａｒｇ、Ｌｅｕ−Ｌｙｓ、Ｖａｌ−Ｌｅｕを含む画分をそれぞれ回収した。これらのフラクションを凍結乾燥して、Ｉｌｅ−Ａｒｇ、Ｌｅｕ−Ｌｙｓ、Ｖａｌ−Ｌｅｕ（実施例品６）を得た。これらＩｌｅ−Ａｒｇ、Ｌｅｕ−Ｌｙｓ、Ｖａｌ−ＬｅｕをＨＰＬＣによって分析したところ、純度はそれぞれ９０％以上であった。このようにして得られたＩｌｅ−Ａｒｇ、Ｌｅｕ−Ｌｙｓ、Ｖａｌ−Ｌｅｕは、そのまま本発明の骨強化作用を有するペプチドとして使用できる。
(Example 6)
To 5 L of a 10% aqueous solution of collagen protein derived from birds, 50 U / g of papain, collagen protein derived from birds and 150 U / g of proleather (manufactured by Amano Enzyme), collagen protein derived from birds was added to adjust the pH to 8, and the chicken was adjusted to pH 8 for 6 hours. Enzymatic degradation was performed while denaturing the derived collagen protein. The reaction solution was heated at 100 ° C. for 15 seconds or longer to inactivate the enzyme, centrifuged, and the supernatant was collected to obtain a bird-derived collagen protein hydrolyzate. This chicken-derived collagen protein hydrolyzate was passed through a column packed with Superdex Peptide (manufactured by GE Healthcare Japan) and fractionated by molecular size to obtain a fraction having an average molecular weight of 200 to 350 Da. This fraction is derivatized with phenyl isothiocyanate and passed through a reverse phase column packed with Supersphere RP-18 (manufactured by Agilent Technologies) to contain a fraction containing Ile-Arg, Leu-Lys and Val-Leu. Was collected respectively. These fractions were lyophilized to give Ile-Arg, Leu-Lys, and Val-Leu (Example 6). When these Ile-Arg, Leu-Lys, and Val-Leu were analyzed by HPLC, the purity was 90% or more, respectively. The Ile-Arg, Leu-Lys, and Val-Leu thus obtained can be used as they are as the peptide having the bone strengthening action of the present invention.
（試験例１）
ウサギの全骨髄細胞を用いて、実施例品２のＩｌｅ−Ａｒｇ、実施例品１のＩｌｅ−Ｇｌｎ、及び実施例品５のＬｅｕ−Ｌｙｓの配列からなるペプチドの、骨代謝改善効果を調べた。５日齢のウサギの脛骨及び大腿骨を摘出し、軟組織を除去した後、５％ＦＢＳを含むα‐ＭＥＭ培地中で機械的に細切した破骨細胞を含む全骨髄細胞を５００，０００ｃｅｌｌｓ／ｗｅｌｌになるように結晶性リン酸カルシウムプレート（Ｃｏｒｎｉｎｇ社製）のウェル上に撒き込み、培養した。培養２時間後に、新しい培地へと交換した後、本発明のペプチドＩｌｅ−Ａｒｇを２ｍＭ、Ｉｌｅ−ＧｌｎとＬｅｕ−Ｌｙｓを４ｍＭの濃度となるように添加して７２時間培養した。そして、５％次亜塩素酸ナトリウム溶液を添加することで細胞を取り除いた後、リン酸カルシウムプレートのウェル上にできた骨吸収窩（ピット）を実体顕微鏡下で撮影し、画像解析によってその面積を測定することにより破骨細胞による骨吸収を抑制する効果を調べた（瀬野悍二ら，研究テーマ別動物培養細胞マニュアル，ｐ．１９９−２００，１９９３）。その結果を表１に示す。
(Test Example 1)
Using rabbit whole bone marrow cells, the effect of improving bone metabolism of a peptide consisting of the sequences of Ile-Arg of Example 2, Ile-Gln of Example 1, and Leu-Lys of Example 5 was investigated. .. After removing the tibia and femur of a 5-day-old rabbit and removing soft tissue, 500,000 cells / of total bone marrow cells including osteoclasts mechanically chopped in α-MEM medium containing 5% FBS. It was sprinkled on the wells of a crystalline calcium phosphate plate (manufactured by Corning) so as to form a well, and cultured. After 2 hours of culturing, after exchanging with a new medium, the peptide Ile-Arg of the present invention was added to a concentration of 2 mM, and Ile-Gln and Leu-Lys were added to a concentration of 4 mM and cultured for 72 hours. Then, after removing the cells by adding a 5% sodium hypochlorite solution, the bone resorption pits (pits) formed on the wells of the calcium phosphate plate were photographed under a stereoscopic microscope, and the area was measured by image analysis. The effect of suppressing bone resorption by osteoclasts was investigated (Takeshi Seno et al., Research Theme-Specific Animal Culture Cell Manual, p. 199-200, 1993). The results are shown in Table 1.
（試験例２）
ウサギの全骨髄細胞を用いて、実施例品４のＳｅｒ−Ｐｒｏ、実施例品５のＬｙｓ−Ｇｌｙ、実施例品４のＴｈｒ−Ｐｒｏ、実施例品６のＶａｌ−Ｌｅｕ、実施例品２のＬｅｕ−Ｇｌｕ、及び実施例品３のＡｓｎ−Ｐｒｏ−Ｔｈｒの配列からなるペプチドの、骨代謝改善効果を調べた。５日齢のウサギの脛骨及び大腿骨を摘出し、軟組織を除去した後、５％ＦＢＳを含むα‐ＭＥＭ培地中で機械的に細切した破骨細胞を含む全骨髄細胞を５００，０００ｃｅｌｌｓ／ｗｅｌｌになるように結晶性リン酸カルシウムプレート（Ｃｏｒｎｉｎｇ社製）のウェル上に撒き込み、培養した。培養２時間後に、新しい培地へと交換した後、本発明のペプチドＳｅｒ−Ｐｒｏ、Ｌｙｓ−Ｇｌｙ、Ｔｈｒ−Ｐｒｏ、Ｖａｌ−Ｌｅｕ、Ｌｅｕ−Ｇｌｕ、及びＡｓｎ−Ｐｒｏ−Ｔｈｒを１０ｍＭの濃度となるように添加して７２時間培養した。そして、５％次亜塩素酸ナトリウム溶液を添加することで細胞を取り除いた後、リン酸カルシウムプレートのウェル上にできた骨吸収窩（ピット）を実体顕微鏡下で撮影し、画像解析によってその面積を測定することにより破骨細胞による骨吸収を抑制する効果を調べた（瀬野悍二ら，研究テーマ別動物培養細胞マニュアル，ｐ．１９９−２００，１９９３）。その結果を表２に示す。
(Test Example 2)
Using rabbit whole bone marrow cells, Ser-Pro of Example product 4, Lys-Gly of Example product 5, Thr-Pro of Example product 4, Val-Leu of Example product 6, and Example product 2. The bone marrow metabolism improving effect of the peptide consisting of the sequence of Leu-Glu and Asn-Pro-Thr of Example product 3 was investigated. After removing the tibia and femur of a 5-day-old rabbit and removing soft tissue, 500,000 cells / of total bone marrow cells including osteoclasts mechanically chopped in α-MEM medium containing 5% FBS. It was sprinkled on the wells of a crystalline calcium phosphate plate (manufactured by Corning) so as to form a well, and cultured. After 2 hours of culturing, after exchanging with a new medium, the peptides of the present invention Ser-Pro, Lys-Gly, Thr-Pro, Val-Leu, Leu-Glu, and Asn-Pro-Thr were adjusted to a concentration of 10 mM. Was added to and cultured for 72 hours. Then, after removing the cells by adding a 5% sodium hypochlorite solution, the bone resorption pits (pits) formed on the wells of the calcium phosphate plate were photographed under a stereoscopic microscope, and the area was measured by image analysis. The effect of suppressing bone resorption by osteoclasts was investigated (Takeshi Seno et al., Research Theme-Specific Animal Culture Cell Manual, p. 199-200, 1993). The results are shown in Table 2.
（実施例７）
（骨強化用カプセル剤の調製）
表３に示す配合で原材料を混合後、常法により造粒し、カプセルに充填して、本発明の骨強化用カプセル剤を製造した。
(Example 7)
(Preparation of capsules for bone strengthening)
The raw materials were mixed according to the formulations shown in Table 3, granulated by a conventional method, and filled into capsules to produce the bone-strengthening capsule of the present invention.
(実施例８）
（骨強化用錠剤の調製）
表４に示す配合で原材料を混合後、常法により１ｇに成型、打錠して本発明の骨強化用錠剤を製造した。
(Example 8)
(Preparation of bone strengthening tablets)
After mixing the raw materials with the formulations shown in Table 4, they were molded into 1 g by a conventional method and tableted to produce the bone strengthening tablet of the present invention.
(実施例９）
（骨強化用液状栄養組成物の調製）
Ｌｅｕ−Ｌｙｓ（実施例品５）２．５ｇを４９９７．５ｇの脱イオン水に溶解し、４０℃まで加熱後、ＴＫホモミクサー（ＴＫ ＲＯＢＯ ＭＩＣＳ；特殊機化工業社製）にて、６，０００ｒｐｍで１０分間撹拌混合して２．５ｇ／５ｋｇのＬｅｕ−Ｌｙｓ溶液を得た。このＬｅｕ−Ｌｙｓ溶液５．０ｋｇに、カゼイン５．０ｋｇ、大豆タンパク質５．０ｋｇ、魚油１．０ｋｇ、シソ油３．０ｋｇ、デキストリン１７．０ｋｇ、ミネラル混合物６．０ｋｇ、ビタミン混合物１．９５ｋｇ、乳化剤２．０ｋｇ、安定剤４．０ｋｇ、香料０．０５ｋｇを配合し、２００ｍｌのレトルトパウチに充填し、レトルト殺菌機(第１種圧力容器、ＴＹＰＥ: ＲＣＳ−４ＣＲＴＧＮ、日阪製作所製)で１２１℃、２０分間殺菌して、本発明の骨強化用液状栄養組成物５０ｋｇを製造した。なお、本発明の骨強化用液状栄養組成物２００ｇには、Ｌｅｕ−Ｌｙｓが１０ｍｇ含まれていた。
(Example 9)
(Preparation of liquid nutritional composition for bone strengthening)
2.5 g of Leu-Lys (Example product 5) is dissolved in 4997.5 g of deionized water, heated to 40 ° C., and then 6,000 rpm with a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). The mixture was stirred and mixed for 10 minutes to obtain a 2.5 g / 5 kg Leu-Lys solution. 5.0 kg of this Leu-Lys solution, 5.0 kg of casein, 5.0 kg of soybean protein, 1.0 kg of fish oil, 3.0 kg of perilla oil, 17.0 kg of dextrin, 6.0 kg of mineral mixture, 1.95 kg of vitamin mixture, emulsifier Mix 2.0 kg, stabilizer 4.0 kg, and fragrance 0.05 kg, fill a 200 ml retort pouch, and use a retort sterilizer (Type 1 pressure vessel, TYPE: RCS-4CRTGN, manufactured by Nisaka Seisakusho) at 121 ° C. , 20 minutes of sterilization to produce 50 kg of the liquid nutritional composition for bone strengthening of the present invention. In addition, 200 g of the liquid nutritional composition for bone strengthening of this invention contained 10 mg of Leu-Lys.
（実施例１０）
（骨強化用飲料の調製）
実施例品４のＳｅｒ−Ｐｒｏ０．５ｇを６９９．５ｇの脱イオン水に溶解した後、４０℃まで加熱後、ウルトラディスパーサー（ＵＬＴＲＡ−ＴＵＲＲＡＸ Ｔ−２５；ＩＫＡジャパン社製）にて、９，５００ｒｐｍで２０分間撹拌混合した。マルチトール１００ｇ、酸味料２ｇ、還元水２０ｇ、香料２ｇ、脱イオン水１７６ｇを添加した後、１００ｍｌのガラス瓶に充填し、９５℃、１５秒間殺菌後、密栓し、本発明の骨強化用飲料１０本（１００ｍｌ入り）を調製した。なお、本発明の骨強化用飲料１００ｇには、Ｓｅｒ−Ｐｒｏが５０ｍｇ含まれていた。
(Example 10)
(Preparation of beverage for bone strengthening)
After dissolving 0.5 g of Ser-Pro of Example product 4 in 699.5 g of deionized water, heating to 40 ° C., and then using an ultra disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan), 9, The mixture was stirred and mixed at 500 rpm for 20 minutes. After adding 100 g of maltitol, 2 g of acidulant, 20 g of reduced water, 2 g of flavor and 176 g of deionized water, the bottle is filled in a 100 ml glass bottle, sterilized at 95 ° C. for 15 seconds, and then sealed to seal the bone-strengthening beverage 10 of the present invention. A book (containing 100 ml) was prepared. In addition, 50 mg of Ser-Pro was contained in 100 g of the bone-strengthening beverage of the present invention.
（実施例１１）
（イヌ用骨強化用飼料の調製）
実施例品３のＬｙｓ−Ｇｌｙ２ｇを３，９９８ｇの脱イオン水に溶解し、４０℃まで加熱後、ＴＫホモミクサー（ＭＡＲＫ ＩＩ １６０型；特殊機化工業社製）にて、３，６００ｒｐｍで２０分間撹拌混合して２ｇ／４ｋｇのＬｙｓ−Ｇｌｙ溶液を得た。このＬｙｓ−Ｇｌｙ溶液２ｋｇに大豆粕１ｋｇ、脱脂粉乳１ｋｇ、大豆油０．４ｋｇ、コーン油０．２ｋｇ、パーム油２.３ｋｇ、トウモロコシ澱粉１ｋｇ、小麦粉０．９ｋｇ、ふすま０．２ｋｇ、ビタミン混合物０．５ｋｇ、セルロース０．３ｋｇ、ミネラル混合物０．２ｋｇを配合し、１２０℃、４分間加熱殺菌して、本発明の骨強化用飼料１０ｋｇを製造した。なお、本発明の骨強化用飼料１００ｇには、Ｌｙｓ−Ｇｌｙが１０ｍｇ含まれていた。
(Example 11)
(Preparation of feed for bone strengthening for dogs)
2 g of Lys-Gly of Example product 3 is dissolved in 3,998 g of deionized water, heated to 40 ° C., and then used with a TK homomixer (MARK II 160 type; manufactured by Tokushu Kika Kogyo Co., Ltd.) at 3,600 rpm for 20 minutes. The mixture was stirred and mixed to obtain a 2 g / 4 kg Lys-Gly solution. 2 kg of this Lys-Gly solution contains 1 kg of soybean meal, 1 kg of skim milk powder, 0.4 kg of soybean oil, 0.2 kg of corn oil, 2.3 kg of palm oil, 1 kg of corn starch, 0.9 kg of wheat flour, 0.2 kg of bran, and 0 vitamin mixture. 5.5 kg, 0.3 kg of cellulose and 0.2 kg of a mineral mixture were blended and sterilized by heating at 120 ° C. for 4 minutes to produce 10 kg of the feed for bone strengthening of the present invention. In addition, 100 g of the bone strengthening feed of this invention contained 10 mg of Lys-Gly.
（実施例１２）
(骨強化用粉乳の調製)
実施例品１のＴｈｒ−Ｐｒｏ１ｇ、脱脂粉乳９．９９９ｋｇ、脱イオン水９０ｋｇを混合し、４０℃まで加熱後、ＴＫホモミクサー（ＴＫ ＲＯＢＯ ＭＩＣＳ；特殊機化工業社製）にて、６，０００ｒｐｍで１０分間撹拌混合した。この溶液を噴霧乾燥して本発明の骨強化用粉乳１０ｋｇを製造した。なお、本発明の骨強化粉乳１０ｇにはＴｈｒ−Ｐｒｏが１ｍｇ含まれていた。
(Example 12)
(Preparation of milk powder for bone strengthening)
Mix 1 g of Thr-Pro of Example product 1, 9.999 kg of skim milk powder, and 90 kg of deionized water, heat to 40 ° C., and then use a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kagaku Kogyo Co., Ltd.) at 6,000 rpm. The mixture was stirred and mixed for 10 minutes. This solution was spray-dried to produce 10 kg of milk powder for bone strengthening of the present invention. In addition, 1 mg of Thr-Pro was contained in 10 g of the bone-strengthening milk powder of the present invention.
（実施例１３）
（骨強化用乳飲料の調製）
実施例品６のＶａｌ−Ｌｅｕ１ｇ、牛乳９．９９９ｋｇを混合し、４０℃まで加熱後、ＴＫホモミクサー（ＴＫ ＲＯＢＯ ＭＩＣＳ；特殊機化工業社製）にて、６，０００ｒｐｍで１０分間撹拌混合した。１３０℃で２秒間、加熱殺菌した後、１０℃以下まで冷却して本発明の骨強化用乳飲料１０ｋｇを製造した。なお、本発明の骨強化用乳飲料２００ｇにはＶａｌ−Ｌｅｕが２０ｍｇ含まれていた。
(Example 13)
(Preparation of milk drink for bone strengthening)
1 g of Val-Leu of Example 6 and 9.999 kg of milk were mixed, heated to 40 ° C., and then stirred and mixed at 6,000 rpm for 10 minutes with a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.). After heat sterilization at 130 ° C. for 2 seconds, the mixture was cooled to 10 ° C. or lower to produce 10 kg of the milk beverage for bone strengthening of the present invention. In addition, 20 mg of Val-Leu was contained in 200 g of the milk beverage for bone strengthening of the present invention.
（実施例１４）
（骨強化用発酵乳の調製）
実施例品５のＬｅｕ−Ｇｌｕ０．０１ｇ、脱脂粉乳１７００ｇ、グルコース３００ｇ、脱イオン水７６９９．９９ｇを混合し、９５℃で２時間保持することで加熱殺菌した。これを３７℃まで冷却し、乳酸菌スターター（Ｌｂ．ｃａｓｅｉ）を３００ｇ植菌し、攪拌混合後、３７℃に保持したインキュベーター内でｐＨ４．０まで発酵させた。ｐＨ４．０到達後１０℃以下まで冷却し、本発明の骨強化用発酵乳１０ｋｇを製造した。なお、本発明の骨強化用発酵乳２００ｇには、Ｌｅｕ−Ｇｌｕが０．２ｍｇ含まれていた。
(Example 14)
(Preparation of fermented milk for bone strengthening)
Leu-Glu 0.01 g, skim milk powder 1700 g, glucose 300 g, and deionized water 7699.99 g of Example Product 5 were mixed and sterilized by heating by holding at 95 ° C. for 2 hours. This was cooled to 37 ° C., 300 g of a lactic acid bacterium starter (Lb. Casei) was inoculated, stirred and mixed, and then fermented to pH 4.0 in an incubator maintained at 37 ° C. After reaching pH 4.0, the mixture was cooled to 10 ° C. or lower to produce 10 kg of fermented milk for bone strengthening of the present invention. In addition, 200 g of fermented milk for bone strengthening of the present invention contained 0.2 mg of Leu-Glu.
（実施例１５）
（骨強化用乳酸菌飲料の調製）
脱脂粉乳１７００ｇ、グルコース３００ｇ、脱イオン水７７００ｇを混合し、９５℃で２時間保持することで加熱殺菌した。これを３７℃まで冷却し、乳酸菌スターター（Ｌｂ．ｃａｓｅｉ）を３００ｇ植菌し、攪拌混合後、３７℃に保持したインキュベーター内でｐＨ４．０まで発酵させた。ｐＨ４．０到達後、攪拌しながら１０℃以下まで冷却し、発酵ベースを得た。また、実施例品３のＡｓｎ−Ｐｒｏ−Ｔｈｒ４ｇ、上白糖１８００ｇ、酸味料２０ｇ、香料１０ｇ、脱イオン水８１６６ｇを混合し、９０℃で１０分間殺菌後１０℃以下まで冷却し、糖液を得た。前述の発酵ベース２０００ｇと糖液８０００ｇを混和し、均質機で組織を滑らかにし、２００ｍｌ入り紙容器５０本に分注後、アルミ蓋で密封し、本発明の骨強化用乳酸菌飲料１０ｋｇを製造した。なお、本発明の骨強化用乳酸菌飲料２００ｍｌには、Ａｓｎ−Ｐｒｏ−Ｔｈｒが６４ｍｇ含まれていた。
(Example 15)
(Preparation of lactic acid bacteria drink for bone strengthening)
1700 g of skim milk powder, 300 g of glucose, and 7700 g of deionized water were mixed and sterilized by heating by holding at 95 ° C. for 2 hours. This was cooled to 37 ° C., 300 g of a lactic acid bacterium starter (Lb. Casei) was inoculated, stirred and mixed, and then fermented to pH 4.0 in an incubator maintained at 37 ° C. After reaching pH 4.0, the mixture was cooled to 10 ° C. or lower with stirring to obtain a fermentation base. Further, 4 g of Asn-Pro-Thr of Example product 3, 1800 g of white sugar, 20 g of acidulant, 10 g of flavor and 8166 g of deionized water were mixed and sterilized at 90 ° C. for 10 minutes and then cooled to 10 ° C. or lower to obtain a sugar solution. It was. 2000 g of the above-mentioned fermentation base and 8000 g of sugar solution were mixed, the structure was smoothed with a homogenizer, dispensed into 50 paper containers containing 200 ml, and sealed with an aluminum lid to produce 10 kg of the lactic acid bacterium beverage for bone strengthening of the present invention. .. In addition, 64 mg of Asn-Pro-Thr was contained in 200 ml of the lactic acid bacterium drink for bone strengthening of this invention.
（実施例１６）
（骨強化用清涼飲料水の調製）
Ｉｌｅ−Ａｒｇ（ＧＬ Ｂｉｏｃｈｅｍ社製）３ｇ、５０％乳酸０．７５ｋｇ、エリスリトール５．７ｋｇ、香料１ｋｇ、脱イオン水４２．５４７ｋｇを混合し、４０℃まで加熱後、ＴＫホモミクサー（ＴＫ ＲＯＢＯ ＭＩＣＳ；特殊機化工業社製）にて、６，０００ｒｐｍで１０分間撹拌混合した。この溶液を９０℃で１０分間殺菌後１０℃以下まで冷却することで、本発明の骨強化用清涼飲料水５０ｋｇを製造した。なお、本発明の骨強化用清涼飲料水２００ｍｌには、Ｉｌｅ−Ａｒｇが１２ｍｇ含まれていた。
(Example 16)
(Preparation of soft drinks for bone strengthening)
Ile-Arg (manufactured by GL Biochem) 3 g, 50% lactic acid 0.75 kg, erythritol 5.7 kg, fragrance 1 kg, deionized water 42.547 kg are mixed, heated to 40 ° C, and then TK homomixer (TK ROBO MICS; special). The mixture was stirred and mixed at 6,000 rpm for 10 minutes at (manufactured by Kika Kogyo Co., Ltd.). This solution was sterilized at 90 ° C. for 10 minutes and then cooled to 10 ° C. or lower to produce 50 kg of the soft drink for bone strengthening of the present invention. In addition, 12 mg of Ile-Arg was contained in 200 ml of the soft drink for strengthening bone of the present invention.
（実施例１７）
（骨強化用チーズの調製）
ゴーダチーズ９．５ｋｇ、チェダーチーズ９．５ｋｇ、Ｉｌｅ−Ｇｌｎ（ＧＬ Ｂｉｏｃｈｅｍ社製）４ｇ、クエン酸ナトリウム２００ｇ、脱イオン水７９６ｇを混合し、８５℃で乳化した。乳化後にチーズをカルトンに充填して２昼夜、５℃で冷却して、本発明の骨強化用チーズ２０ｋｇを製造した。なお、本発明の骨強化用チーズ１００ｇには、Ｉｌｅ−Ｇｌｎが２０ｍｇ含まれていた。
(Example 17)
(Preparation of cheese for bone strengthening)
9.5 kg of Gouda cheese, 9.5 kg of cheddar cheese, 4 g of Ile-Gln (manufactured by GL Biochem), 200 g of sodium citrate, and 796 g of deionized water were mixed and emulsified at 85 ° C. After emulsification, the carton was filled with cheese and cooled at 5 ° C. for 2 days and nights to produce 20 kg of the bone-strengthening cheese of the present invention. In addition, 100 g of the bone strengthening cheese of the present invention contained 20 mg of Ile-Gln.
（実施例１８）
（骨強化用マーガリンの調製）
大豆硬化油２ｋｇ、大豆白絞油４ｋｇ、パーム油２．５ｋｇ、グリセリン脂肪酸エステル５０ｇを混合して油層を調製した。次に、Ｌｅｕ−Ｌｙｓ（ＧＬ Ｂｉｏｃｈｅｍ社製）２０ｇ、乳酸１０ｇ、脱イオン水１４２０ｇを混合し、油層へ添加して油中水型乳化物を得た、この乳化物をマーガリン製造機で冷却、固化および練圧して、本発明の骨強化用マーガリン１０ｋｇを製造した。なお本発明の骨強化マーガリン１０ｇには、Ｌｅｕ−Ｌｙｓが２０ｍｇ含まれていた。
(Example 18)
(Preparation of margarine for bone strengthening)
An oil layer was prepared by mixing 2 kg of hydrogenated soybean oil, 4 kg of soybean white squeezed oil, 2.5 kg of palm oil, and 50 g of glycerin fatty acid ester. Next, 20 g of Leu-Lys (manufactured by GL Biochem), 10 g of lactic acid, and 1420 g of deionized water were mixed and added to an oil layer to obtain a water-in-oil emulsion. The emulsion was cooled by a margarine maker. By solidifying and kneading, 10 kg of the margarine for bone strengthening of the present invention was produced. In addition, 20 mg of Leu-Lys was contained in 10 g of the bone-strengthening margarine of the present invention.
（実施例１９）
（骨強化用クリームの調製）
ナタネ硬化油４．５ｋｇ、レシチン４０ｇ、モノグリセリン脂肪酸エステル１０ｇ、ソルビタン脂肪酸エステル１０ｇを混合し、油相を調製した。次に、Ｓｅｒ−Ｐｒｏ（ジェンスクリプト社製）４０ｇ、脱脂粉乳４００ｇ、カゼインナトリウム１０ｇ、シュガーエステル２０ｇ、リン酸塩１０ｇ、キサンタンガム５ｇ、脱イオン水４．９５５ｋｇを混合して、水相を調製した。水相を６５℃に加温し、７０℃に加温した油相を少量ずつ攪拌しながら添加し、ＴＫホモミクサー（ＴＫ ＲＯＢＯ ＭＩＣＳ；特殊機化工業社製）にて、６，０００ｒｐｍで１０分間撹拌混合した。これを均質機で均質処理して本発明の骨強化クリーム１０ｋｇを製造した。なお、本発明の骨強化クリーム１０ｇには、Ｓｅｒ−Ｐｒｏが４０ｍｇ含まれていた。
(Example 19)
(Preparation of bone strengthening cream)
An oil phase was prepared by mixing 4.5 kg of hydrogenated rapeseed oil, 40 g of lecithin, 10 g of monoglycerin fatty acid ester, and 10 g of sorbitan fatty acid ester. Next, 40 g of Ser-Pro (manufactured by Genscript), 400 g of skim milk powder, 10 g of casein sodium, 20 g of sugar ester, 10 g of phosphate, 5 g of xanthan gum, and 4.955 kg of deionized water were mixed to prepare an aqueous phase. .. The aqueous phase is heated to 65 ° C., and the oil phase heated to 70 ° C. is added little by little with stirring, and a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kika Kogyo Co., Ltd.) is used at 6,000 rpm for 10 minutes. Stirred and mixed. This was homogenized with a homogenizing machine to produce 10 kg of the bone strengthening cream of the present invention. In addition, 40 mg of Ser-Pro was contained in 10 g of the bone strengthening cream of the present invention.
（実施例２０）
（骨強化用プリンの調製）
はちみつ２０００ｇ、Ｌｙｓ−Ｇｌｙ（ＧＬ Ｂｉｏｃｈｅｍ社製）４ｇ、脱脂粉乳８００ｇ、マスカルポーネ３００ｇ、液状水７００ｇ、グラニュー糖５００ｇ、生クリーム２５０ｇ、バター２００ｇ、加糖卵黄４００ｇ、ゼラチン４０ｇ、寒天１５ｇ、ローカストビーンガム１２０ｇ、脱イオン水４６７１ｇを混合して、プリンミックスとした。このプリンミックスをＴＫホモミクサー（ＴＫ ＲＯＢＯ ＭＩＣＳ；特殊機化工業社製）にて、６，０００ｒｐｍで１０分間撹拌混合し、６０℃に加熱して溶解した後、１００ｇずつ容器へ充填して冷却することで、本発明の骨強化用プリン１００個を製造した。なお、本発明の骨強化プリン１００ｇには、Ｌｙｓ−Ｇｌｙが４０ｍｇ含まれていた。
(Example 20)
(Preparation of pudding for bone strengthening)
2000g of honey, 4g of Lys-Gly (manufactured by GL Biochem), 800g of skim milk powder, 300g of mascarpone, 700g of liquid water, 500g of granulated sugar, 250g of fresh cream, 200g of butter, 400g of sweetened egg yolk, 40g of gelatin, 15g of agar, 120g of locust bean gum , 4671 g of deionized water was mixed to prepare a pudding mix. This pudding mix is stirred and mixed at 6,000 rpm for 10 minutes with a TK homomixer (TK ROBO MICS; manufactured by Tokushu Kagaku Kogyo Co., Ltd.), heated to 60 ° C. to dissolve, and then filled in a container in 100 g portions and cooled. As a result, 100 bone-strengthening puddings of the present invention were produced. In addition, 40 mg of Lys-Gly was contained in 100 g of the bone-strengthening purine of the present invention.
（実施例２１）
（骨強化用ゼリーの調製）
Ｔｈｒ−Ｐｒｏ（ジェンスクリプト社製）４．４ｇ、果糖２０００ｇ、グラニュー糖１５００ｇ、水５００ｇ、寒天１００ｇ、香料１０ｇ、脱イオン水５８８５．６ｇを混合し、ＴＫホモミクサー（ＴＫ ＲＯＢＯ ＭＩＣＳ；特殊機化工業社製）にて、６，０００ｒｐｍで１０分間撹拌混合し、５０℃に加熱して溶解した後、１００ｇずつ容器へ充填して冷却することで、本発明の骨強化用ゼリー１００個を製造した。なお、本発明の骨強化用ゼリー１００ｇには、Ｔｈｒ−Ｐｒｏが４４ｍｇ含まれていた。
(Example 21)
(Preparation of jelly for bone strengthening)
Thr-Pro (manufactured by Jenscript) 4.4 g, fructose 2000 g, granulated sugar 1500 g, water 500 g, agar 100 g, flavor 10 g, deionized water 5885.6 g are mixed, and TK homomixer (TK ROBO MICS; The 100 g jelly for bone strengthening of the present invention was produced by stirring and mixing at 6,000 rpm for 10 minutes, heating to 50 ° C. to dissolve the jelly, and then filling a container with 100 g each and cooling. .. In addition, 44 mg of Thr-Pro was contained in 100 g of the bone strengthening jelly of the present invention.
（実施例２２）
（骨強化用ウエハースの調製）
Ｖａｌ−Ｌｅｕ（ＧＬ Ｂｉｏｃｈｅｍ社製）９．２ｇ、小麦粉８．５ｋｇ、コーンスターチ１．２１ｋｇ、パーム油０．２２ｋｇ、膨張剤０．０５ｋｇを混合した後、脱イオン水を適量加えてバッターを調製した後、ウエハース焼成機で焼成して、本発明の骨強化用ウエハース１０ｋｇを製造した。なお、本発明の骨強化用ウエハース５０ｇには、Ｖａｌ−Ｌｅｕが４６ｍｇ含まれていた。
(Example 22)
(Preparation of wafers for bone strengthening)
Val-Leu (manufactured by GL Biochem) 9.2 g, wheat flour 8.5 kg, cornstarch 1.21 kg, palm oil 0.22 kg, leavening agent 0.05 kg were mixed, and then an appropriate amount of deionized water was added to prepare a batter. After that, it was fired with a wafer baking machine to produce 10 kg of the wafer for bone strengthening of the present invention. In addition, 46 mg of Val-Leu was contained in 50 g of the wafer for bone strengthening of this invention.
本発明によれば長期摂取が可能で、安全性が高く、破骨細胞の分化ならびに該細胞による骨吸収を抑制する作用を介した骨強化作用が顕著な骨強化用組成物を提供することが可能である。したがって、本発明の摂取により骨粗鬆症や骨折、リウマチ、関節炎などの種々の骨疾患の予防や治療が可能である。 According to the present invention, it is possible to provide a composition for bone strengthening which can be ingested for a long period of time, has high safety, and has a remarkable bone strengthening action through an action of suppressing the differentiation of osteoclasts and bone resorption by the cells. It is possible. Therefore, by ingesting the present invention, it is possible to prevent or treat various bone diseases such as osteoporosis, bone fracture, rheumatism, and arthritis.
Claims (2)
A food or drink for bone strengthening, a nutritional composition for bone strengthening, a feed for bone strengthening, or a pharmaceutical product for bone strengthening, which comprises the bone strengthening composition according to claim 1.
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Non-Patent Citations (3)
| Title |
|---|
| HYPERTENSION RESEARCH, vol. 32, JPN6023000122, 2009, pages 786 - 790, ISSN: 0004960676 * |
| J. BONE MINER. METAB., vol. 29, JPN6023000120, 2011, pages 149 - 158, ISSN: 0004960677 * |
| PROCESS BIOCHEMISTRY, vol. 54, JPN6023000121, 2017, pages 180 - 187, ISSN: 0004960675 * |
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